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Tarnawski AS, Ahluwalia A. Endothelial cells and blood vessels are major targets for COVID-19-induced tissue injury and spreading to various organs. World J Gastroenterol 2022; 28:275-289. [PMID: 35110950 PMCID: PMC8771611 DOI: 10.3748/wjg.v28.i3.275] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Revised: 12/02/2021] [Accepted: 01/11/2022] [Indexed: 02/06/2023] [Imported: 06/25/2023] Open
Abstract
The coronavirus disease 2019 (COVID-19) infected so far over 250 million people and caused the death of over 5 million worldwide. Aging, diabetes, and cardiovascular diseases, conditions with preexisting impaired endothelial functions predispose to COVID-19. While respiratory epithelium is the main route of virus entry, the endothelial cells (ECs) lining pulmonary blood vessels are also an integral part of lung injury in COVID-19 patients. COVID-19 not only affects the lungs and respiratory system but also gastrointestinal (GI) tract, liver, pancreas, kidneys, heart, brain, and skin. Blood vessels are likely conduits for the virus dissemination to these distant organs. Importantly, ECs are also critical for vascular regeneration during injury/lesions healing and restoration of vascular network. The World Journal of Gastroenterology has published in last two years over 67 outstanding papers on COVID-19 infection with a focus on the GI tract, liver, pancreas, etc., however, the role of the endothelial and vascular components as major targets for COVID-19-induced tissue injury, spreading to various organs, and injury healing have not been sufficiently emphasized. In the present article, we focus on these subjects and on current treatments including the most recent oral drugs molnupiravir and paxlovid that show a dramatic, significant efficacy in controlling severe COVID-19 infection.
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Affiliation(s)
- Andrzej S Tarnawski
- Gastroenterology Research Department, University of California Irvine and the Veterans Administration Long Beach Healthcare System, Long Beach, CA 90822, United States
| | - Amrita Ahluwalia
- Research Service, Veterans Administration Long Beach Healthcare System, Long Beach, CA 90822, United States
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Ahluwalia A, Patel K, Hoa N, Brzozowska I, Jones MK, Tarnawski AS. Melatonin ameliorates aging-related impaired angiogenesis in gastric endothelial cells via local actions on mitochondria and VEGF-survivin signaling. Am J Physiol Gastrointest Liver Physiol 2021; 321:G682-G689. [PMID: 34668398 DOI: 10.1152/ajpgi.00101.2021] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Accepted: 10/12/2021] [Indexed: 02/06/2023]
Abstract
Tissue injury healing is impaired in aging, and this impairment is caused in part by reduced angiogenesis. Melatonin, a neuroendocrine hormone that regulates sleep and circadian rhythm, is also produced in the gastrointestinal tract. The expression of melatonin receptors MT1 and MT2 in gastric endothelial cells and their roles in aging-related impairment of gastric angiogenesis have not been examined. We hypothesized that MT1 and MT2 expression is reduced in gastric endothelial cells of aging rats and that melatonin treatment can upregulate their expression and improve angiogenesis. We examined the expression of MT1 and MT2 in gastric endothelial cells (GECs) isolated from young and aging rats. We also examined the effects of melatonin treatment on angiogenesis, GEC mitochondrial function, expression of vascular endothelial growth factor (VEGF), its signaling receptor (VEGFR-2), and the inhibitor of apoptosis protein, survivin. Young and aging GECs expressed MT1 (in the cytoplasm and mitochondria) and MT2 (in nucleus and mitochondria). In aging GECs, MT1 and MT2 levels, in vitro angiogenesis, and mitochondrial membrane potential were significantly reduced (by 1.5-fold, 1.9-fold, 3.1-fold, and 1.63-fold, respectively) compared with young GECs. Melatonin treatment of aging GECs significantly increased MT1 and MT2 expression compared with the controls, induced nuclear translocation of MT1, and significantly ameliorated the aging-related impairment of angiogenesis and mitochondrial function. Aging GECs have significantly reduced MT1 and MT2 expression, angiogenesis, and mitochondrial membrane potential compared with young GECs. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function.NEW & NOTEWORTHY This study showed reduced expression of melatonin receptors MT1 and MT2, angiogenesis, and mitochondrial function in gastric endothelial cells (GECs) isolated from aging rats. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function. These studies provide new insight into the mechanisms of the aging-related impairment of angiogenesis and delayed tissue injury healing and provide a rationale for melatonin treatment to reverse these abnormalities.
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MESH Headings
- Age Factors
- Angiogenesis Inducing Agents/pharmacology
- Animals
- Cells, Cultured
- Endothelial Cells/drug effects
- Endothelial Cells/metabolism
- Gastric Mucosa/blood supply
- Melatonin/pharmacology
- Mitochondria/drug effects
- Mitochondria/metabolism
- Neovascularization, Physiologic/drug effects
- Rats, Inbred F344
- Receptor, Melatonin, MT1/agonists
- Receptor, Melatonin, MT1/metabolism
- Receptor, Melatonin, MT2/agonists
- Receptor, Melatonin, MT2/metabolism
- Signal Transduction
- Survivin/metabolism
- Vascular Endothelial Growth Factor A/metabolism
- Rats
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Affiliation(s)
- Amrita Ahluwalia
- Research Service, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
| | - Khushin Patel
- Research Service, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
| | - Neil Hoa
- Research Service, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
| | - Iwona Brzozowska
- Department of Anatomy, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland
| | - Michael K Jones
- Research Service, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
- Department of Medicine, University of California, Irvine, California
| | - Andrzej S Tarnawski
- Research Service, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
- Department of Medicine, University of California, Irvine, California
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Tarnawski AS, Ahluwalia A. The Critical Role of Growth Factors in Gastric Ulcer Healing: The Cellular and Molecular Mechanisms and Potential Clinical Implications. Cells 2021; 10:cells10081964. [PMID: 34440733 PMCID: PMC8392882 DOI: 10.3390/cells10081964] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Revised: 07/25/2021] [Accepted: 07/29/2021] [Indexed: 02/06/2023] Open
Abstract
In this article we review the cellular and molecular mechanisms of gastric ulcer healing. A gastric ulcer (GU) is a deep defect in the gastric wall penetrating through the entire mucosa and the muscularis mucosae. GU healing is a regeneration process that encompasses cell dedifferentiation, proliferation, migration, re-epithelialization, formation of granulation tissue, angiogenesis, vasculogenesis, interactions between various cells and the matrix, and tissue remodeling, all resulting in scar formation. All these events are controlled by cytokines and growth factors (e.g., EGF, TGFα, IGF-1, HGF, bFGF, TGFβ, NGF, VEGF, angiopoietins) and transcription factors activated by tissue injury. These growth factors bind to their receptors and trigger cell proliferation, migration, and survival pathways through Ras, MAPK, PI3K/Akt, PLC-γ, and Rho/Rac/actin signaling. The triggers for the activation of these growth factors are tissue injury and hypoxia. EGF, its receptor, IGF-1, HGF, and COX-2 are important for epithelial cell proliferation, migration, re-epithelialization, and gastric gland reconstruction. VEGF, angiopoietins, bFGF, and NGF are crucial for blood vessel regeneration in GU scars. The serum response factor (SRF) is essential for VEGF-induced angiogenesis, re-epithelialization, and blood vessel and muscle restoration. Local therapy with cDNA of human recombinant VEGF165 in combination with angiopoietin1, or with the NGF protein, dramatically accelerates GU healing and improves the quality of mucosal restoration within ulcer scars. The future directions for accelerating and improving healing include local gene and protein therapies with growth factors, their combinations, and the use of stem cells and tissue engineering.
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Affiliation(s)
- Andrzej S. Tarnawski
- Medical Research Service, VA Long Beach Healthcare System Long Beach, 5901 East Seventh Street, Long Beach, CA 90822, USA
- Division of Gastroenterology, Department of Medicine and Digestive Health Institute, The University of California-Irvine, Irvine, CA 92697, USA
- Correspondence: (A.S.T.); (A.A.); Tel.: +1-(562)-826-5813 (A.A.); Fax: +1-(562)-826-5675 (A.A.)
| | - Amrita Ahluwalia
- Medical Research Service, VA Long Beach Healthcare System Long Beach, 5901 East Seventh Street, Long Beach, CA 90822, USA
- Correspondence: (A.S.T.); (A.A.); Tel.: +1-(562)-826-5813 (A.A.); Fax: +1-(562)-826-5675 (A.A.)
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Ahluwalia A, Hoa N, Jones MK, Tarnawski AS. NSAID-induced injury of gastric epithelial cells is reversible: roles of mitochondria, AMP kinase, NGF, and PGE 2. Am J Physiol Gastrointest Liver Physiol 2019; 317:G862-G871. [PMID: 31545918 PMCID: PMC6962499 DOI: 10.1152/ajpgi.00192.2019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2019] [Revised: 09/03/2019] [Accepted: 09/16/2019] [Indexed: 02/06/2023]
Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1-4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR.NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Neil Hoa
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Michael K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
- Department of Medicine, University of California, Irvine, California
| | - Andrzej S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
- Department of Medicine, University of California, Irvine, California
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Ahluwalia A, Jones MK, Hoa N, Tarnawski AS. Mitochondria in gastric epithelial cells are the key targets for NSAIDs-induced injury and NGF cytoprotection. J Cell Biochem 2019; 120:11651-11659. [PMID: 30790334 DOI: 10.1002/jcb.28445] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2018] [Revised: 12/05/2018] [Accepted: 12/10/2018] [Indexed: 02/05/2023]
Abstract
Gastric epithelial cells are important components of mucosal protection and targets of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced injury. Diclofenac (DFN) is one of the most widely used NSAIDs; however, even its short-term use can induce gastric erosions and ulcers. Nerve growth factor (NGF) has been reported to act not only on neuronal cells but also on endothelial cells; however, its action on gastric epithelial cells is unknown. This study was aimed to determine, whether NGF can protect gastric epithelial cells against DFN-induced injury, and to determine the underlying molecular mechanisms with a focus on mitochondria, survivin, and insulin-like growth factor 1 (IGF-1). Cultured normal rat gastric mucosal epithelial cells 1 (RGM1) were treated with phosphate-buffered saline (PBS; control), NGF (100 ng/mL) and/or DFN (0.25-1.00 mM) for 4 hours. We examined: (1) cell injury by confocal microscopy; (2) cell death/survival using Calcein AM live cell tracking dye; (3) mitochondrial structure and membrane potential function using MitoTracker in live cells; and (4) expression of NGF, its receptor - tropomyosin receptor kinase A (TrkA), survivin and IGF-1 by immunostaining. DFN treatment of RGM1 cells for 4 hours caused extensive cell injury, mitochondrial disintegration, reduced cell viability (from 94 ± 3% in controls to 14 ± 4% in 0.5 mM DFN-treated cells; P < 0.001), and expression of survivin and IGF-1. NGF treatment significantly increased survivin and IGF-1 expression by 41% and 75%, respectively versus PBS controls. Pretreatment with NGF before DFN treatment reduced mitochondrial damage and cell death by 73% and 82%, respectively versus treatment with DFN alone (all P < 0.001). This study also showed the presence of high-affinity TrkA receptors in the plasma membrane and mitochondria of RGM1 cells indicating novel actions of NGF.
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Michael K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
- Department of Medicine, University of California, Irvine, California
| | - Neil Hoa
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Andrzej S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
- Department of Medicine, University of California, Irvine, California
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Tarnawski AS, Ahluwalia A. Application of artificial intelligence for the assessment of mucosal healing and inflammation. J Med Artif Intell 2019. [DOI: 10.21037/jmai.2019.03.02] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
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Tarnawski AS, Ahluwalia A. Increased susceptibility of aging gastric mucosa to injury and delayed healing: Clinical implications. World J Gastroenterol 2018; 24:4721-4727. [PMID: 30479459 PMCID: PMC6235800 DOI: 10.3748/wjg.v24.i42.4721] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2018] [Revised: 10/12/2018] [Accepted: 10/21/2018] [Indexed: 02/06/2023] Open
Abstract
In this editorial we comment on the article by Fukushi K et al published in the recent issue of the World Journal of Gastroenterology 2018; 24(34): 3908-3918. We focus specifically on the mechanisms of the anti-thrombotic action of aspirin, gastric mucosal injury and aging-related increased susceptibility of gastric mucosa to injury. Aspirin is widely used not only for the management of acute and chronic pain and arthritis, but also importantly for the primary and secondary prevention of cardiovascular events such as myocardial infarcts and strokes. Clinical trials have consistently shown that antiplatelet therapy with long term, low dose aspirin (LDA) - 75 to 325 mg daily, dramatically reduces the risk of non-fatal myocardial infarcts, stroke and mortality in patients with established arterial diseases. However, such treatment considerably increases the risk of gastrointestinal (GI) ulcerations and serious bleeding by > 2-4 fold, especially in aging individuals. This risk is further increased in patients using LDA together with other antiplatelet agents, other nonsteroidal anti-inflammatory agents (NSAIDs) and/or alcohol, or in patients with Helicobacter pylori (H. pylori) infection. Previous studies by our group and others have demonstrated prominent structural and functional abnormalities in gastric mucosa of aging individuals (which we refer to as aging gastric mucosa or “aging gastropathy”) compared to the gastric mucosa of younger individuals. Aging gastric mucosa has impaired mucosal defense, increased susceptibility to injury by a variety of noxious agents such as aspirin, other NSAIDs and ethanol, and delayed and impaired healing of injury. The mechanism underlying these abnormalities of aging gastric mucosa include reduced mucosal blood flow causing hypoxia, upregulation of PTEN, activation of pro-apoptotic caspase-3 and caspase-9, and reduced survivin (anti-apoptosis protein), importin-α (nuclear transport protein), vascular endothelial growth factor, and nerve growth factor. The decision regarding initiation of a long-term LDA therapy should be made after a careful consideration of both cardiovascular and GI risk factors. The latter include a previous history of GI bleeding and/or ulcers, age ≥ 70, male gender, concurrent use of other NSAIDs, alcohol consumption and H. pylori infection. Furthermore, the incidence of GI ulcers and bleeding can be reduced in patients on long term LDA treatment by several measures. Clinicians treating such patients should test for and eradicate H. pylori, instruct patients to avoid alcohol and non-aspirin NSAIDs, including cyclooxygenase-2-selective NSAIDs, and prescribe proton pump inhibitors in patients on LDA therapy. In the future, clinicians may be able to prescribe one of several potential new drugs, which include aspirin associated with phosphatidylcholine (PL2200), which retains all property of aspirin but reduces by approximately 50% LDA-induced GI ulcerations.
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Affiliation(s)
- Andrzej S Tarnawski
- Department of Gastroenterology Research, University of California Irvine and the Veterans Administration Long Beach Healthcare System, Long Beach, CA 90822, United States
| | - Amrita Ahluwalia
- Department of Gastroenterology Research, University of California Irvine and the Veterans Administration Long Beach Healthcare System, Long Beach, CA 90822, United States
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Tarnawski AS. To our readers: Important questions from the editors/editorial board. World J Gastroenterol 2018; 24:4615-4616. [PMCID: PMC6209571 DOI: 10.3748/wjg.v24.i40.4615] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/05/2018] [Revised: 10/12/2018] [Accepted: 10/15/2018] [Indexed: 02/01/2023] Open
Abstract
This letter proposes updating the World Journal of Gastroenterology (WJG) with several new items and subsections and seeks the readers’ input for these suggested changes. In order to further enhance the scope of the WJG, to make it more interactive, and to accommodate our readers’ interest, the editors ask the readers for their input regarding potential new proposed changes and new subsections listed below: (1) new subsection - selected highlights from other related Baishideng Publishing Group world series journals; (2) new subsection - challenging images of the week - a quiz with an answer and references on another page; (3) to re-introduce or restore for some issues the cover page on which selected illustration; (4) new subsection - new hypotheses, new concepts and revisiting old concepts in a short, commentary format; (5) preview of the forthcoming issue articles (title and authors); (6) suggestions submitted by the readers - new topics and initiatives; and (7) additional topic area - molecular basis of gastric/liver diseases as an additional area for articles submitted to the journal.
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Affiliation(s)
- Andrzej S Tarnawski
- Gastroenterology Research Department, University of California Irvine and the Veterans Administration Long Beach Healthcare System, Long Beach, CA 90822, United States
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Ahluwalia A, Brzozowska IM, Hoa N, Jones MK, Tarnawski AS. Melatonin signaling in mitochondria extends beyond neurons and neuroprotection: Implications for angiogenesis and cardio/gastroprotection. Proc Natl Acad Sci U S A 2018; 115:E1942-E1943. [PMID: 29440384 PMCID: PMC5834732 DOI: 10.1073/pnas.1722131115] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, CA 90822
| | - Iwona M Brzozowska
- Department of Anatomy, Jagiellonian University Medical College, 31-008 Cracow, Poland
| | - Neil Hoa
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, CA 90822
| | - Michael K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, CA 90822
- Department of Medicine, University of California, Irvine, CA 92617
| | - Andrzej S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, CA 90822;
- Department of Medicine, University of California, Irvine, CA 92617
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Ahluwalia A, Jones MK, Hoa N, Zhu E, Brzozowski T, Tarnawski AS. Reduced NGF in Gastric Endothelial Cells Is One of the Main Causes of Impaired Angiogenesis in Aging Gastric Mucosa. Cell Mol Gastroenterol Hepatol 2018; 6:199-213. [PMID: 29992182 PMCID: PMC6037903 DOI: 10.1016/j.jcmgh.2018.05.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2017] [Accepted: 05/10/2018] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS Aging gastric mucosa has increased susceptibility to injury and delayed healing owing to impaired angiogenesis, but the mechanisms are not fully known. We examined whether impairment of angiogenesis in aging gastric mucosa is caused by deficiency of nerve growth factor (NGF) in gastric endothelial cells (ECs), and whether NGF therapy could reverse this impairment. METHODS In gastric mucosal ECs (GECs) isolated from young and aging rats we examined the following: (1) in vitro angiogenesis, (2) NGF expression, and (3) the effect of NGF treatment on angiogenesis, GEC proliferation and migration, and dependence on serum response factor. In in vivo studies in young and aging rats, we examined NGF expression in gastric mucosa and the effect of NGF treatment on angiogenesis and gastric ulcer healing. To determine human relevance, we examined NGF expression in gastric mucosal biopsy specimens of aging (≥70 y) and young (≤40 y) individuals. RESULTS In cultured aging GECs, NGF expression and angiogenesis were reduced significantly by 3.0-fold and 4.1-fold vs young GECs. NGF therapy reversed impairment of angiogenesis in aging GECs, and serum response factor silencing completely abolished this response. In gastric mucosa of aging rats, NGF expression in GECs was reduced significantly vs young rats. In aging rats, local NGF treatment significantly increased angiogenesis and accelerated gastric ulcer healing. In aging human subjects, NGF expression in ECs of gastric mucosal vessels was 5.5-fold reduced vs young individuals. CONCLUSIONS NGF deficiency in ECs is a key mechanism underlying impaired angiogenesis and delayed ulcer healing in aging gastric mucosa. Local NGF therapy can reverse these impairments.
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Key Words
- Aging
- Akt, serine threonine kinase signaling protein
- Angiogenesis
- BrdU, bromodeoxyuridine
- EC, endothelial cell
- Endothelial Cells
- FITC, fluorescein isothiocyanate
- GEC, gastric mucosal microvascular endothelial cells isolated from rats
- GU, gastric ulcer
- Gene Therapy
- LV-GFP, lentiviral green fluorescent protein
- LV-NGF, lentiviral nerve growth factor
- NGF, nerve growth factor
- NSAID, nonsteroidal anti-inflammatory drug
- Nerve Growth Factor
- PBS, phosphate-buffered saline
- PCNA, proliferating cell nuclear antigen
- PCR, polymerase chain reaction
- PI3, phosphoinositide-3
- SRF, serum response factor
- Ulcer Healing
- VEGF, vascular endothelial growth factor
- mRNA, messenger RNA
- mTOR, mammalian target of rapamycin
- siRNA, small interfering RNA
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Michael K. Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
- Department of Medicine, University of California, Irvine, California
| | - Neil Hoa
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Ercheng Zhu
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
| | - Tomasz Brzozowski
- Department of Physiology, Jagiellonian University Medical College, Krakow, Poland
| | - Andrzej S. Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California
- Department of Medicine, University of California, Irvine, California
- Correspondence Address correspondence to: Andrzej S. Tarnawski, MD, PhD, AGAF, FACG, Veterans Affairs Long Beach Healthcare System, 5901 East 7th Street, 09/151, Long Beach, California 90822. fax: (562) 826-5675.
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Ahluwalia A, Jones MK, Brzozowska I, Tarnawski AS. In vitro model of vasculo-angiogenesis: demonstration that bone marrow derived endothelial progenitor cells form new hybrid capillary blood vessels jointly with gastric endothelial cells. J Physiol Pharmacol 2017; 68:841-846. [PMID: 29550796] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Received: 07/25/2017] [Accepted: 11/20/2017] [Indexed: 02/06/2023]
Abstract
Regeneration of blood vessels (neovascularization) is critical for tissue injury healing. The contribution of bone marrow-derived endothelial progenitor cells (BMD-EPCs) to neovascularization during tissue injury healing is not fully elucidated and it is not clear whether BMD-EPCs can form new capillary blood vessels independently or jointly with fully differentiated endothelial cells (ECs). The aim of this study was to establish an in vitro model of vasculogenesis/angiogenesis by co-culture of BMD-EPCs and gastric endothelial cells (GECs) on Matrigel, examine direct interactions of these cells; and, identify the mechanisms involved. We isolated BMD-EPCs and GECs from bone marrow and stomach of rats, respectively. In these cells, we examined the expression of CD34, CD133, CD31, VEGF-R2, stromal derived factor 1 (SDF-1) and CXCR4, and, their ability to form capillary-like tubes when cultured separately or when co-cultured (1:5 ratio) on growth factor-reduced Matrigel. Fluorescence-labeled BMD-EPCs seeded alone on Matrigel formed capillary-like tubes reflecting in vitro vasculogenesis, and when co-cultured with GECs on Matrigel, formed 'hybrid' tubes containing BMD-EPCs nested between GECs thus reflecting in vitro angio-vasculogenesis. These 'hybrid' tubes were 1.5-fold wider (P < 0.001) and had more extensive (5.1-fold increase) loops (P < 0.01) at the junctions of BMD-EPCs and GECs versus tubes formed by GECs alone. GECs expressed SDF-1 that likely mediated homing of BMD-EPCs (which expressed the SDF-1 receptor, CXCR4) and their incorporation during neovascularization. BMD-EPCs can independently form capillary-like tubes on Matrigel, and when co-cultured with fully differentiated ECs on Matrigel, form capillary-like 'hybrid' tubes comprised of both cell types. Both BMD-EPCs and GECs express SDF-1 and CXCR4, which indicate direct paracrine interactions between these cells during neovascularization.
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Affiliation(s)
- A Ahluwalia
- Medical and Research Services, Veterans Affairs Medical Center, Long Beach, CA, USA
| | - M K Jones
- Medical and Research Services, Veterans Affairs Medical Center, Long Beach, CA, USA
- Department of Medicine, University of California, Irvine, CA, USA
| | - I Brzozowska
- Department of Anatomy, Jagiellonian University Medical College, Cracow, Poland
| | - A S Tarnawski
- Medical and Research Services, Veterans Affairs Medical Center, Long Beach, CA, USA. ;
- Department of Medicine, University of California, Irvine, CA, USA
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Ahluwalia A, Jones MK, Hoa N, Tarnawski AS. NGF protects endothelial cells from indomethacin-induced injury through activation of mitochondria and upregulation of IGF-1. Cell Signal 2017; 40:22-29. [PMID: 28843696 DOI: 10.1016/j.cellsig.2017.08.006] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Revised: 08/02/2017] [Accepted: 08/20/2017] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS Endothelial cells (ECs) lining blood vessels are critical for delivery of oxygen and nutrients to all tissues and organs and play a crucial role in the regeneration of blood vessel following tissue injury. ECs are also major targets of injury by a variety of noxious factors [e.g., ethanol and nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin, diclofenac], especially in gastric mucosa that has direct exposure to these agents. In this study, we investigated whether nerve growth factor (NGF) can protect gastric microvascular ECs (GECs) from injury by indomethacin (INDO) and the mechanisms involved. METHODS GECs were isolated from rat gastric mucosa and pre-treated with either vehicle or NGF (100ng/ml) for 30min to 4h followed by treatment with vehicle or 0.25mM INDO for 4h. STUDIES 1) cell viability using Calcein AM live cell tracking dye, 2) mitochondrial structure and function using MitoTracker, molecular probe that stains mitochondria in live cells in a manner dependent on mitochondrial membrane potential (MMP), 3) in vitro angiogenesis - endothelial tube formation on Matrigel, 4) expression and subcellular localization of NGF receptor, TrkA, and 5) expression of IGF-1 protein. RESULTS Treatment with INDO reduced GEC viability and in vitro angiogenesis and induced mitochondrial injury and MMP depolarization. NGF pre-treatment protected GECs from INDO-induced injury preventing both INDO-induced MMP depolarization and reduced in vitro angiogenesis. The NGF high affinity receptor, TrkA, was localized in GECs to both cell membrane and mitochondria. NGF treatment of GECs also resulted in increased IGF-1 protein expression. CONCLUSIONS 1) NGF protects GECs against IND-induced injury. 2) Mitochondria are major targets of both INDO-induced injury and NGF afforded protection of GECs. 3) TrkA expression in the mitochondria of GECs indicates that the protection afforded by NGF is partly mediated by its direct action on mitochondria. 4) NGF prevents MMP depolarization and increases expression of IGF-1 protein in GECs. These studies indicate that NGF may play a protective role against injury to GECs; and, that maintenance of mitochondrial structure and function is one of the mechanisms.
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, CA, USA
| | - Michael K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA
| | - Neil Hoa
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, CA, USA
| | - Andrzej S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA.
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Ahluwalia A, Brzozowski T, Jones MK, Ichikawa Y, Tarnawski AS. Formation of new blood vessels during gastric ulcer healing. Role of bone marrow derived endothelial progenitor cells. J Physiol Pharmacol 2017; 68:585-589. [PMID: 29151075] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 08/21/2017] [Indexed: 02/06/2023]
Abstract
Regeneration of blood vessels (neovascularization) is critical for gastric ulcer (GU) healing. The contributions of bone marrow-derived endothelial progenitor cells (BMD-EPCs) to neovascularization during GU healing are not fully elucidated. Our specific aims were to determine whether in GU, BMD-EPCs are incorporated into blood vessels of GU granulation tissue jointly with ECs, thus forming hybrid vessels; or, form separate vessels consisting of only BMD-EPCs. GUs were induced in rats by serosal application of acetic acid. Vascular cast studies were performed at 7, 21 and 60 days after GU induction and tissue specimens were immunostained for CD34, CD133, VEGFR2, and SDF-1 at 14 days. Human relevance was determined using archival human GU specimens. In rat GU granulation tissue BMD-EPCs constituted 28 ± 3% of all cells lining newly formed blood vessels, and were nested between fully differentiated ECs. In rat GU granulation tissue, expression of stromal derived factor-1 (SDF-1) - the major chemoattractant for BMD-EPCs was strongly upregulated. In human GU specimens, BMD-EPCs were also present in granulation tissue constituting 34 ± 3% of all cells lining blood vessels and jointly formed hybrid vessels with differentiated ECs. Our study uncovered that BMD-EPCs incorporate into newly formed blood vessels in GU granulation tissue; and, together with ECs of pre-existing vessels, contribute to and support neovascularization through vasculogenesis. This study is the first demonstration that vasculogenesis occurs during GU healing in both humans and in rats.
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Affiliation(s)
- A Ahluwalia
- Medical and Research Services, Veterans Affairs Medical Center, Long Beach, California, USA
| | - T Brzozowski
- Department of Physiology, Jagiellonian University Medical College, Cracow, Poland
| | - M K Jones
- Medical and Research Services, Veterans Affairs Medical Center, Long Beach, California, USA
- Department of Medicine, University of California, Irvine, CA, USA
| | - Y Ichikawa
- Department of Oncology, Yokohama City University, Graduate School of Medicine,Yokohama, Japan
| | - A S Tarnawski
- Medical and Research Services, Veterans Affairs Medical Center, Long Beach, California, USA. ;
- Department of Medicine, University of California, Irvine, CA, USA
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Tarnawski AS, Ahluwalia A, Jones MK, Brzozowski T. Expression of nerve growth factor in rat stomach. Implications for interactions between endothelial, neural and epithelial cells. J Physiol Pharmacol 2016; 67:879-883. [PMID: 28195068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Received: 08/26/2016] [Accepted: 12/22/2016] [Indexed: 02/06/2023]
Abstract
UNLABELLED This study was aimed to determine the expression and localization of nerve growth factor (NGF) in the gastric mucosa. Transmural gastric specimens were obtained from euthanized rats. STUDIES 1) expression of NGF and TrkA receptor by Western blotting; 2) histological evaluation of gastric wall architecture; 3) expression of NGF using immunostaining. Immunostaining showed strong and differential expression of NGF in neural elements of gastric myenteric and submucosal plexuses; in epithelial cells: mainly in chief and progenitor cells, in enterochromaffin-like (ECL) cells; and, in endothelial cells (ECs) lining blood vessels. We concluded that NGF expression in neural elements, epithelial cells and endothelial cells of blood vessels indicated a complex local interaction between neural, epithelial and endothelial cells that regulated gastric mucosal homeostasis and, likely, the protection against gastric injury and ulcer healing.
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Affiliation(s)
- A S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, USA.
- Department of Medicine, University of California, Irvine, California, USA
| | - A Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, USA
| | - M K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, USA
- Department of Medicine, University of California, Irvine, California, USA
| | - T Brzozowski
- Department of Physiology, Jagiellonian University Medical College, Cracow, Poland
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Ahluwalia A, Jones MK, Brzozowski T, Tarnawski AS. Nerve growth factor is critical requirement for in vitro angiogenesis in gastric endothelial cells. Am J Physiol Gastrointest Liver Physiol 2016; 311:G981-G987. [PMID: 27742705 DOI: 10.1152/ajpgi.00334.2016] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/08/2016] [Accepted: 10/10/2016] [Indexed: 02/06/2023]
Abstract
Angiogenesis is critical for the healing of gastric mucosal injury and is considered to be primarily regulated by vascular endothelial growth factor (VEGF), the fundamental proangiogenic factor. The role of nerve growth factor (NGF) in gastric angiogenesis is unknown. We examined the expression of NGF and its TrkA receptor in endothelial cells (ECs) isolated from gastric mucosa of rats (GMECs), the effect of NGF treatment on in vitro angiogenesis in GMECs, and, the mechanisms underlying NGF's proangiogenic actions. Isolated GMECs from Fisher rats were treated with vehicle, NGF (10-1,000 ng/ml), VEGF (20 ng/ml), or NGF+VEGF. To determine whether and to what extent NGF is critical for angiogenesis in GMECs, we silenced NGF expression using specific siRNA and examined in vitro angiogenesis with and without treatment with exogenous NGF and/or VEGF. Treatment of GMECs with NGF significantly increased in vitro angiogenesis similar to that seen in GMECs treated with VEGF. Silencing of NGF in GMECs abolished angiogenesis, and this effect was reversed only by exogenous NGF but not VEGF, which indicates a direct proangiogenic action of NGF on GMECs that is, at least in part, distinct and independent of VEGF. NGF's proangiogenic action on GMECs was mediated via PI3-K/Akt signaling. This study showed for the first time that gastric mucosal ECs express NGF and its receptor TrkA and that NGF is critical for angiogenesis in these cells.
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California, and the Department of Medicine, University of California, Irvine, California; and
| | - Michael K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California, and the Department of Medicine, University of California, Irvine, California; and
| | | | - Andrzej S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System, Long Beach, California, and the Department of Medicine, University of California, Irvine, California; and
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Samarasena JB, Ahluwalia A, Shinoura S, Choi KD, Lee JG, Chang KJ, Tarnawski AS. In vivo imaging of porcine gastric enteric nervous system using confocal laser endomicroscopy &molecular neuronal probe. J Gastroenterol Hepatol 2016; 31:802-7. [PMID: 26482711 DOI: 10.1111/jgh.13194] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/16/2015] [Revised: 10/05/2015] [Accepted: 10/14/2015] [Indexed: 02/05/2023]
Abstract
BACKGROUND AND AIM The gastric enteric nervous system (GENS) is organized into the submucosal plexus and the myenteric plexus that regulate muscle activity and mucosal functions, respectively. A non-invasive, in vivo visualization of GENS was not possible until recent introduction of needle-based confocal laser endomicroscopy (nCLE). Our aim was to determine the feasibility of in vivo visualization of GENS in the porcine stomach using endoscopic ultrasound (EUS) guided nCLE and local injection of molecular neuronal probe NeuroTrace. METHODS In anesthetized pigs during endoscopy, NeuroTrace was injected into the submucosa and muscularis propria of distal, and proximal stomach under EUS guidance and nCLE imaging was performed using the Cellvizio AQ Flex probe. After euthanasia, transmural gastric specimens from the areas of NeuroTrace injection were obtained for histology. We performed quantitative analysis of nCLE images recorded during in vivo studies: histologic evaluation of unstained specimens under fluorescence microscope for NeuroTrace localization. We also performed immunostaining of these specimens for nerve growth factor (NGF). In in vitro studies, we examined the uptake of NeuroTrace by glial cells. RESULTS The nCLE imaging successfully visualized neuronal cells and nerve fibers in distinctive image patterns. Fluorescence microscopy of mucosal sections showed that in vivo-injected NeuroTrace was retained in GENS components. NGF was strongly expressed in neural and glial cells, and the pattern of NGF staining was similar to that of NeuroTrace staining. CONCLUSIONS This study demonstrates for the first time that combined use of EUS-guided nCLE and NeuroTrace is capable to visualize GENS.
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Affiliation(s)
- Jason B Samarasena
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange
| | - Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
| | - Susumu Shinoura
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange
| | - Kee Don Choi
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange
| | - John G Lee
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange
| | - Kenneth J Chang
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange
| | - Andrzej S Tarnawski
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California
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Samarasena JB, Tarnawski AS, Ahluwalia A, Shinoura S, Choi KD, Lee JG, Chang KJ. EUS-guided in vivo imaging of the porcine esophageal enteric nervous system by using needle-based confocal laser endomicroscopy. Gastrointest Endosc 2015; 82:1116-20. [PMID: 26318831 DOI: 10.1016/j.gie.2015.06.048] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/15/2015] [Accepted: 06/18/2015] [Indexed: 02/06/2023]
Abstract
BACKGROUND AND AIMS The GI tract is innervated by the autonomic enteric nervous system, mainly composed of submucosal Meissner's plexus and myenteric Auerbach's plexus, which is essential for motility, blood flow regulation, and secretory functions. In vivo visualization of the esophageal enteric nervous system (EENS) during endoscopy has not been possible without invasive mucosal resection. This study aimed to visualize the EENS without mucosal resection, in vivo by using the novel probe, needle-based confocal laser-induced endomicroscopy (nCLE) with a fluorescence neuronal probe, NeuroTrace, under EUS guidance and to evaluate the feasibility of ex vivo imaging of the neuronal network in submucosal biopsy samples acquired at endoscopy. METHODS Four Yorkshire pigs were anesthetized and examined. In vivo experiment: During endoscopy, NeuroTrace was injected into the submucosa and muscularis propria of the middle and distal esophagus under EUS guidance, and nCLE imaging was performed. Ex vivo experiment: Submucosal tissue biopsy specimens from the porcine esophagus were obtained for ex vivo evaluation by using a "through-the-needle" forceps technique. After incubation of the samples in NeuroTrace solution, pCLE was used to visualize the EENS elements in the tissue. RESULTS Imaging of the EENS network by using EUS-guided nCLE was successful, both within the submucosa and the muscularis propria, and clearly visualized neuronal cells, glial cells, nerve bundles, and nerve fibers provided distinctive image patterns with excellent imaging quality. The use of the "through-the-needle" forceps technique achieved ex vivo images similar to those acquired in vivo. CONCLUSIONS EUS-guided in vivo imaging of the enteric nervous system is feasible without mucosal resection and provides a novel ex vivo imaging alternative for human application. These novel, minimally invasive imaging approaches could be of tremendous diagnostic value to better characterize and explore the EENS of the GI tract.
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Affiliation(s)
- Jason B Samarasena
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California-Irvine, Orange, California, USA
| | - Andrzej S Tarnawski
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California-Irvine, Orange, California, USA; Division of Gastroenterology and Hepatology, Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California, USA
| | - Amrita Ahluwalia
- Division of Gastroenterology and Hepatology, Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California, USA
| | - Susumu Shinoura
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California-Irvine, Orange, California, USA
| | - Kee Don Choi
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California-Irvine, Orange, California, USA
| | - John G Lee
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California-Irvine, Orange, California, USA
| | - Kenneth J Chang
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California-Irvine, Orange, California, USA
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18
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Tolstanova G, Deng X, Ahluwalia A, Paunovic B, Prysiazhniuk A, Ostapchenko L, Tarnawski A, Sandor Z, Szabo S. Role of Dopamine and D2 Dopamine Receptor in the Pathogenesis of Inflammatory Bowel Disease. Dig Dis Sci 2015; 60:2963-75. [PMID: 25972152 DOI: 10.1007/s10620-015-3698-5] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Accepted: 04/29/2015] [Indexed: 02/06/2023]
Abstract
BACKGROUND VEGF-induced vascular permeability and blood vessels remodeling are key features of inflammatory bowel disease (IBD) pathogenesis. Dopamine through D2 receptor (D2R) inhibits VEGF/VPF-mediated vascular permeability and angiogenesis in tumor models. In this study, we tested the hypothesis that pathogenesis of IBD is characterized by the disturbance of dopaminergic system and D2R activity. METHODS IL-10 knockout (KO) mice and rats with iodoacetamide-induced ulcerative colitis (UC) were treated intragastrically with D2R agonists quinpirole (1 mg/100 g) or cabergoline (1 or 5 µg/100 g). Macroscopic, histologic, and clinical features of IBD, colonic vascular permeability, and angiogenesis were examined. RESULTS Although colonic D2R protein increased, levels of tyrosine hydroxylase and dopamine transporter DAT decreased in both models of IBD. Treatment with quinpirole decreased the size of colonic lesions in rats with iodoacetamide-induced UC (p < 0.01) and reduced colon wet weight in IL-10 KO mice (p < 0.05). Quinpirole decreased colonic vascular permeability (p < 0.001) via downregulation of c-Src and Akt phosphorylation. Cabergoline (5 µg/100 g) reduced vascular permeability but did not affect angiogenesis and improved signs of iodoacetamide-induced UC in rats (p < 0.05). CONCLUSIONS Treatment with D2R agonists decreased the severity of UC in two animal models, in part, by attenuation of enhanced vascular permeability and prevention of excessive vascular leakage. Hence, the impairment dopaminergic system seems to be a feature of IBD pathogenesis.
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MESH Headings
- Animals
- Biopsy, Needle
- Blotting, Western
- Cabergoline
- Capillary Permeability/drug effects
- Colitis, Ulcerative/chemically induced
- Colitis, Ulcerative/drug therapy
- Colitis, Ulcerative/pathology
- Disease Models, Animal
- Dopamine/metabolism
- Ergolines/pharmacology
- Female
- Humans
- Immunohistochemistry
- Inflammation Mediators/metabolism
- Inflammatory Bowel Diseases/chemically induced
- Inflammatory Bowel Diseases/drug therapy
- Inflammatory Bowel Diseases/pathology
- Interleukin-10/metabolism
- Iodoacetamide/pharmacology
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Quinpirole/pharmacology
- Random Allocation
- Rats
- Rats, Sprague-Dawley
- Receptors, Dopamine D2/metabolism
- Statistics, Nonparametric
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Affiliation(s)
- Ganna Tolstanova
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA.
- Educational-Scientific Center "Institute of Biology", Taras Shevchenko National University of Kyiv, Kiev, Ukraine.
| | - Xiaoming Deng
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA
| | - Amrita Ahluwalia
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA
| | - Brankica Paunovic
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA
| | - Alona Prysiazhniuk
- Educational-Scientific Center "Institute of Biology", Taras Shevchenko National University of Kyiv, Kiev, Ukraine
| | - Lyudmyla Ostapchenko
- Educational-Scientific Center "Institute of Biology", Taras Shevchenko National University of Kyiv, Kiev, Ukraine
| | - Andrzej Tarnawski
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA
| | - Zsuzsanna Sandor
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA
| | - Sandor Szabo
- VA Long Beach Healthcare System, Departments of Medicine, Pathology and Pharmacology, VA Medical Center (05/113), University of California-Irvine, 5901 East 7th Street, Long Beach, CA, 90822, USA.
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Tanigawa T, Ahluwalia A, Watanabe T, Arakawa T, Tarnawski AS. Nerve growth factor injected into the gastric ulcer base incorporates into endothelial, neuronal, glial and epithelial cells: implications for angiogenesis, mucosal regeneration and ulcer healing. J Physiol Pharmacol 2015; 66:617-21. [PMID: 26348086] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 03/05/2015] [Accepted: 07/20/2015] [Indexed: 02/06/2023]
Abstract
A previous study has demonstrated that locally administered growth factors such as epidermal growth factor, basic fibroblast growth factor and hepatocyte growth factor can accelerate healing of experimental gastric ulcers in rats. That study indicates that locally administered growth factors can exert potent biological effects resulting in enhanced gastric ulcers healing. However, the fate of injected growth factors, their retention and localization to specific cellular compartments have not been examined. In our preliminary study, we demonstrated that local injection of nerve growth factor to the base of experimental gastric ulcers dramatically accelerates ulcer healing, increases angiogenesis - new blood vessel formation, and improves the quality of vascular and epithelial regeneration. Before embarking on larger, definitive and time sequence studies, we wished to determine whether locally injected nerve growth factor is retained in gastric ulcer's tissues and taken up by specific cells during gastric ulcer healing. Gastric ulcers were induced in anesthetized rats by local application of acetic acid using standard methods; and, 60 min later fluorescein isothiocyanate-labeled nerve growth factor was injected locally to the ulcer base. Rats were euthanized 2, 5 and 10 days later. Gastric specimens were obtained and processed for histology. Unstained paraffin sections were examined under a fluorescence microscope, and the incorporation of fluorescein isothiocyanate-labeled nerve growth factor into various gastric tissue cells was determined and quantified. In addition, we performed immunostaining for S100β protein that is expressed in neural components. Five and ten days after ulcer induction labeled nerve growth factor (injected to the gastric ulcer base) was incorporated into endothelial cells of blood vessels, neuronal, glial and epithelial cells, myofibroblasts and muscle cells. This study demonstrates for the first time that during gastric ulcer healing locally administered exogenous nerve growth factor is retained in gastric tissue and is taken up by endothelial, neural, muscle and epithelial cells. This is likely the basis for the therapeutic action of locally administered nerve growth factor and its stimulation of angiogenesis, tissue regeneration and gastric ulcer healing.
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Affiliation(s)
- T Tanigawa
- Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California and the University of California-Irvine, CA, USA
- Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan
| | - A Ahluwalia
- Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California and the University of California-Irvine, CA, USA
| | - T Watanabe
- Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan
| | - T Arakawa
- Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan
| | - A S Tarnawski
- Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California and the University of California-Irvine, CA, USA.
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Samarasena JB, Ahluwalia A, Tarnawski AS, Shinoura S, Choi KD, Lee JG, Chang KJ. Expression of nerve growth factor, its TrkA receptor, and several neuropeptides in porcine esophagus. Implications for interactions between neural, vascular and epithelial components of the esophagus. J Physiol Pharmacol 2015; 66:415-20. [PMID: 26084223] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 03/20/2015] [Accepted: 04/16/2015] [Indexed: 02/06/2023]
Abstract
UNLABELLED This study was aimed to determine the expression and localization of nerve growth factor (NGF) and several neural peptides in porcine esophagus. Transmural esophageal specimens were obtained from euthanized pigs. STUDIES 1) histologic evaluation, 2) expressions of NGF and its tropomyosin receptor kinase A (TrkA) receptor, calcitonin generelated peptide (CGRP), neuronal nitric oxide synthase (nNOS), and neuronal enolase using immunostaining and quantification of signal distribution and intensity. Immunostaining for NGF, CGRP, nNOS and neuronal specific enolase (NSE) showed their strong and differential expression and localization in the neuronal network. NGF was strongly expressed in the majority of neurons and nerves, distribution of TrkA was complementary; its signal was 1.5-fold weaker P < 0.001 than NGF). Quantitatively the signal intensity was: CGRP > nNOS > NGF > NES > TrkA. In addition to neural structures, nNOS, NGF and TrkA were expressed in keratinocyte progenitor cells of esophageal mucosa and in endothelial cells of blood vessels. We conclude that a strong expression of NGF in majority of esophageal neurons and nerves indicates important, but previously unrecognized regulatory roles in the esophagus; 2) This study showed expression of NGF and some of the neuropeptides in neural elements, keratinocyte progenitor cells and endothelial cells of blood vessels, which indicates local interactions between neural, epithelial and endothelial cells.
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Affiliation(s)
- J B Samarasena
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange, California, USA
| | - A Ahluwalia
- Division of Gastroenterology, Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California, USA
| | - A S Tarnawski
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange, California, USA
- Division of Gastroenterology, Department of Medicine, Long Beach Veterans Affairs Healthcare System, Long Beach, California, USA
| | - S Shinoura
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange, California, USA
| | - K D Choi
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange, California, USA
| | - J G Lee
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange, California, USA
| | - K J Chang
- Division of Gastroenterology and Hepatology, Department of Medicine, University of California - Irvine, Orange, California, USA.
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21
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Macé V, Ahluwalia A, Coron E, Le Rhun M, Boureille A, Bossard C, Mosnier JF, Matysiak-Budnik T, Tarnawski AS. Confocal laser endomicroscopy: a new gold standard for the assessment of mucosal healing in ulcerative colitis. J Gastroenterol Hepatol 2015; 30 Suppl 1:85-92. [PMID: 25827810 DOI: 10.1111/jgh.12748] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND AND AIM Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. METHODS Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). RESULTS In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). CONCLUSIONS (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC.
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Affiliation(s)
- Vincent Macé
- Institut des Maladies de l'Appareil Digestif, CIC INSERM 04 et Service d'Hépato-Gastroentérologie, CHU de Nantes, France
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Tarnawski AS, Ahluwalia A, Jones MK. Angiogenesis in gastric mucosa: an important component of gastric erosion and ulcer healing and its impairment in aging. J Gastroenterol Hepatol 2014; 29 Suppl 4:112-23. [PMID: 25521743 DOI: 10.1111/jgh.12734] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Angiogenesis (also referred to as neovascularization-formation of new blood vessels from existing vessels) is a fundamental process essential for healing of tissue injury and ulcers because regeneration of blood microvessels is a critical requirement for oxygen and nutrient delivery to the healing site. This review article updates the current views on angiogenesis in gastric mucosa following injury and during ulcer healing, its sequential events, the underlying mechanisms, and the impairment of angiogenesis in aging gastric mucosa. We focus on the time sequence and ultrastructural features of angiogenesis, hypoxia as a trigger, role of vascular endothelial growth factor signaling (VEGF), serum response factor, Cox2 and prostaglandins, nitric oxide, and importin. Recent reports indicate that gastric mucosa of aging humans and experimental animals exhibits increased susceptibility to injury and delayed healing. Gastric mucosa of aging rats has increased susceptibility to injury by a variety of damaging agents such as ethanol, aspirin, and other non-steroidal anti-inflammatory drugs because of structural and functional abnormalities including: reduced gastric mucosal blood flow, hypoxia, reduced expression of vascular endothelial growth factor and survivin, and increased expression of early growth response protein 1 (egr-1) and phosphatase and tensin homolog (PTEN). Until recently, postnatal neovascularization was assumed to occur solely through angiogenesis sprouting of endothelial cells and formation of new blood vessels from pre-existing blood vessels. New studies in the last decade have challenged this paradigm and indicate that in some tissues, including gastric mucosa, the homing of bone marrow-derived endothelial progenitor cells to the site of injury can also contribute to neovascularization by a process termed vasculogenesis.
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Affiliation(s)
- Andrzej S Tarnawski
- Veterans Administration Long Beach Healthcare System, 5901 E. Seventh Street, Long Beach, CA, 90822, USA; The University of California, Irvine, CA, USA
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Ahluwalia A, Baatar D, Jones MK, Tarnawski AS. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB. Am J Physiol Gastrointest Liver Physiol 2014; 307:G602-10. [PMID: 25059824 PMCID: PMC4166721 DOI: 10.1152/ajpgi.00177.2014] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, and the Department of Medicine/Gastroenterology, University of California, Irvine, California
| | - Dolgor Baatar
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, and the Department of Medicine/Gastroenterology, University of California, Irvine, California
| | - Michael K Jones
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, and the Department of Medicine/Gastroenterology, University of California, Irvine, California
| | - Andrzej S Tarnawski
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS), Long Beach, California, and the Department of Medicine/Gastroenterology, University of California, Irvine, California
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Tarnawski AS, Ahluwalia A, Jones MK. Increased susceptibility of aging gastric mucosa to injury: The mechanisms and clinical implications. World J Gastroenterol 2014; 20:4467-4482. [PMID: 24782600 PMCID: PMC4000484 DOI: 10.3748/wjg.v20.i16.4467] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Accepted: 04/03/2014] [Indexed: 02/06/2023] Open
Abstract
This review updates the current views on aging gastric mucosa and the mechanisms of its increased susceptibility to injury. Experimental and clinical studies indicate that gastric mucosa of aging individuals-“aging gastropathy”-has prominent structural and functional abnormalities vs young gastric mucosa. Some of these abnormalities include a partial atrophy of gastric glands, impaired mucosal defense (reduced bicarbonate and prostaglandin generation, decreased sensory innervation), increased susceptibility to injury by a variety of damaging agents such as ethanol, aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), impaired healing of injury and reduced therapeutic efficacy of ulcer-healing drugs. Detailed analysis of the above changes indicates that the following events occur in aging gastric mucosa: reduced mucosal blood flow and impaired oxygen delivery cause hypoxia, which leads to activation of the early growth response-1 (egr-1) transcription factor. Activation of egr-1, in turn, upregulates the dual specificity phosphatase, phosphatase and tensin homologue deleted on chromosome ten (PTEN) resulting in activation of pro-apoptotic caspase-3 and caspase-9 and reduced expression of the anti-apoptosis protein, survivin. The imbalance between pro- and anti-apoptosis mediators results in increased apoptosis and increased susceptibility to injury. This paradigm has human relevance since increased expression of PTEN and reduced expression of survivin were demonstrated in gastric mucosa of aging individuals. Other potential mechanisms operating in aging gastric mucosa include reduced telomerase activity, increase in replicative cellular senescence, and reduced expression of vascular endothelial growth factor and importin-α-a nuclear transport protein essential for transport of transcription factors to nucleus. Aging gastropathy is an important and clinically relevant issue because of: (1) an aging world population due to prolonged life span; (2) older patients have much greater risk of gastroduodenal ulcers and gastrointestinal complications (e.g., NSAIDs-induced gastric injury) than younger patients; and (3) increased susceptibility of aging gastric mucosa to injury can be potentially reduced or reversed pharmacologically.
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Ahluwalia A, Jones MK, Szabo S, Tarnawski AS. Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival. J Physiol Pharmacol 2014; 65:209-15. [PMID: 24781730] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 02/05/2014] [Accepted: 04/03/2014] [Indexed: 02/06/2023]
Abstract
In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.
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Affiliation(s)
- A Ahluwalia
- Veterans Affairs Long Beach Healthcare System, Long Beach, CA, USA.
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Ahluwalia A, Jones MK, Tarnawski AS. Key role of endothelial importin-α in VEGF expression and gastric angiogenesis: novel insight into aging gastropathy. Am J Physiol Gastrointest Liver Physiol 2014; 306:G338-45. [PMID: 24356884 DOI: 10.1152/ajpgi.00382.2013] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Recent in vivo studies demonstrated that aging gastric mucosa has impaired angiogenesis and reduced expression of vascular endothelial growth factor (VEGF). Angiogenesis is triggered by hypoxia and VEGF gene activation, and the latter requires transport of transcription factor(s) into endothelial cell nuclei. We focused on gastric mucosal endothelial cells (GMEC), which are key targets and effectors of gastric angiogenesis, and determined whether and to what extent importin-α, a nuclear transport protein, regulates VEGF gene activation and gastric angiogenesis and the possible role of importin-α in aging gastropathy. GMEC were isolated from rats 3 and 24 mo of age, young (YGEC) and aging (AGEC), respectively. We examined in these cells 1) in vitro angiogenesis, 2) expression of VEGF and importin-α, 3) nuclear transport of hypoxia-inducible factor (HIF)-1α by importin-α, 4) binding of HIF-1α to the VEGF gene promoter, and 5) effects of importin-α silencing in YGEC and its upregulation in AGEC on angiogenesis and VEGF expression. AGEC exhibited significantly impaired in vitro angiogenesis by fourfold and decreased expression of VEGF, importin-α, and nuclear HIF-1α by 1.4-fold, 1.6-fold, and 2.9-fold, respectively, vs. YGEC. Upregulation of importin-α in AGEC significantly reversed all these abnormalities. In YGEC, knockdown of importins-α1 and -α3 significantly reduced in vitro angiogenesis by 93% and 73% and VEGF expression by 48% and 52%, respectively. The above findings demonstrate that importin-α is a novel and critical regulator of gastric angiogenesis. Its reduced expression in AGEC is the key mechanism for impaired angiogenesis and reduced VEGF.
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Affiliation(s)
- Amrita Ahluwalia
- Medical and Research Services, Veterans Affairs Long Beach Healthcare System (VALBHS) and Southern California Institute for Research and Education
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Khomenko T, Deng X, Ahluwalia A, Tarnawski A, Patel KN, Sandor Z, Szabo S. STAT3 and importins are novel mediators of early molecular and cellular responses in experimental duodenal ulceration. Dig Dis Sci 2014; 59:297-306. [PMID: 24385009 DOI: 10.1007/s10620-013-2807-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2013] [Accepted: 07/15/2013] [Indexed: 02/06/2023]
Abstract
OBJECTIVES Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directly upregulates VEGF, Ref-1, p21, and anti-apoptotic genes such as Bcl-xL. In this study, we hypothesized that STAT3 signaling is activated and provides a critical protective role that is required for enterocyte survival during the early phases of cysteamine-induced duodenal ulcers. METHODS We studied the effect of inhibition of STAT3 activity on cysteamine-induced duodenal ulcers in rats and egr-1 knockout mice using STAT3/DNA binding assay, immunohistochemistry, immunoblot, and quantitative reverse transcriptase PCR analyses. RESULTS We found that G-quartet oligodeoxynucleotides T40214, a specific inhibitor of STAT3/DNA binding, aggravated cysteamine-induced duodenal ulcers in rats 2.8-fold (p < 0.05). In the pre-ulcerogenic stage, cysteamine induced STAT3 tyrosine phosphorylation, its translocation to nuclei, an increased expression and nuclear translocation of importin α and β in the rat duodenal mucosa. Cysteamine enhanced the binding of STAT3 to its DNA consensus sequences at 6, 12, and 24 h after cysteamine by 1.5-, 1.8-, and 3.5-fold, respectively, and activated the expression of STAT3 target genes such as VEGF, Bcl-xL, Ref-1, and STAT3-induced feedback inhibitor, a suppressor of cytokine signaling 3. We also demonstrated that egr-1 knockout mice, which are more susceptible to cysteamine-induced duodenal ulcers, had lower levels of STAT3 expression, its phosphorylation, expression of importin α or β, and STAT3/DNA binding than wild-type mice in response to cysteamine. CONCLUSIONS Thus, STAT3 represents an important new molecular mechanism in experimental duodenal ulceration.
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Affiliation(s)
- Tetyana Khomenko
- VA Medical Center, (05/113) 5901 East 7th Street, Long Beach, CA, 90822-5201, USA
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Ahluwalia A, Jones MK, Matysiak-Budnik T, Tarnawski AS. VEGF and colon cancer growth beyond angiogenesis: does VEGF directly mediate colon cancer growth via a non-angiogenic mechanism? Curr Pharm Des 2014; 20:1041-4. [PMID: 23755727 DOI: 10.2174/1381612819999131218175905] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2013] [Accepted: 06/07/2013] [Indexed: 02/06/2023]
Abstract
In this article we review the role of vascular endothelial growth factor (VEGF) in colon cancer growth and the underlying mechanisms. Angiogenesis, the growth of new capillary blood vessels in the body, is critical for tissue injury healing and cancer growth. In 1971, Judah Folkman proposed the concept that tumor growth beyond 2 mm is critically dependent on angiogenesis. Tumors including colon cancers release angiogenic growth factors that stimulate blood vessels to grow into the tumors thus providing oxygen and nutrients that enable exponential growth. VEGF is the most potent angiogenic growth factor. Several studies have highlighted the role of VEGF in colon cancer, specifically in the stimulation of angiogenesis. This role of VEGF is strongly supported by studies showing that inhibition of VEGF using the blocking antibody, bevacizumab, results in decreased angiogenesis and abrogation of cancer growth. In the United States, bevacizumab in combination with chemotherapy is FDA approved for the treatment of metastatic colon cancer. However, the source of VEGF in colon cancer tissue, the mechanisms of VEGF generation in colon cancer cells and the molecular pathways involved in VEGF mediated angiogenesis in colon cancer are not fully known. The possibility that VEGF directly stimulates cancer cell growth in an autocrine manner has not been explored in depth.
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Affiliation(s)
| | | | | | - Andrzej S Tarnawski
- Veterans Administration Long Beach Healthcare System, 5901 E. 7th Street, Long Beach, CA 90822- 5201, USA.
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Ahluwalia A, Jones MK, Deng X, Sandor Z, Szabo S, Tarnawski AS. An imbalance between VEGF and endostatin underlies impaired angiogenesis in gastric mucosa of aging rats. Am J Physiol Gastrointest Liver Physiol 2013; 305:G325-32. [PMID: 23788612 DOI: 10.1152/ajpgi.00127.2013] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Gastric mucosa of aging individuals exhibits increased susceptibility to injury and delayed healing. Our previous studies in young rats showed that healing of mucosal injury depends on and is critically dependent on VEGF and angiogenesis. Since angiogenesis in aging gastric mucosa has not been examined before, in this study we examined the extent to which angiogenesis is impaired in gastric mucosa of aging vs. young rats and determined the underlying mechanisms with a focus on mucosal expression of VEGF (proangiogenic factor) and endostatin (antiangiogenic factor). Aging rats had significantly impaired gastric angiogenesis by ~12-fold, 5-fold, 4-fold, and 3-fold, respectively (vs. young rats; all P < 0.001) at 24, 48, 72, and 120 h following ethanol-induced gastric injury and reduced and delayed healing of mucosal erosions. In gastric mucosa of aging (vs. young) rats at baseline, VEGF expression was significantly reduced, whereas endostatin levels were significantly increased (P < 0.05 and P < 0.01, respectively). In contrast to young rats, gastric mucosal VEGF levels did not increase following ethanol-induced injury in aging rats. MMP-9 enzyme activity was significantly higher in gastric mucosa of aging vs. young rats both at baseline (2.7-fold) and 24 h (3.8-fold) after ethanol injury (both P < 0.001). Since endostatin is generated from collagen XVIII by MMP-9, this finding can explain the mechanism of increased endostatin expression in aging gastric mucosa. The above findings demonstrate that reduced VEGF and increased endostatin result in the impaired angiogenesis and delayed injury healing in gastric mucosa of aging rats.
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Affiliation(s)
- Amrita Ahluwalia
- Veterans Affairs Long Beach Healthcare System, and Univ. of California, Irvine, 5901 E. 7th St., 09/151, Bldg. 162, Rm. 115, Long Beach, CA 90822. or
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Ahluwalia A, Jones MK, Szabo S, Tarnawski AS. Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism. Biochem Biophys Res Commun 2013; 437:515-20. [PMID: 23831629 DOI: 10.1016/j.bbrc.2013.06.096] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2013] [Accepted: 06/25/2013] [Indexed: 02/06/2023]
Abstract
UNLABELLED Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. MATERIAL AND METHODS We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n=43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. RESULTS Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. CONCLUSIONS Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is independent of its primary function in the induction of angiogenesis.
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Tarnawski A, Ahluwalia A, Jones MK. Gastric cytoprotection beyond prostaglandins: cellular and molecular mechanisms of gastroprotective and ulcer healing actions of antacids. Curr Pharm Des 2013. [PMID: 22950493 DOI: 10.2174/1381612811306010126] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
This article updates current views on gastric mucosal defense, injury, protection and ulcer healing with a focus on mucosal protective and ulcer healing actions of antacids. The gastric mucosa is continuously exposed to a variety of noxious factors, both endogenous such as: 0.1N hydrochloric acid, pepsin, bile acids, lysolecithin, H. pylori toxins and exogenous such as NSAIDs, ethanol and others. Gastric mucosal integrity is maintained by pre-epithelial, epithelial and post-epithelial defense mechanisms permitting the mucosa to withstand exposure to the above damaging factors. When mucosal defense is weakened or overwhelmed by injurious factors, injury develops in the form of erosions or ulcers. In the late 1970s Andre Robert and coworkers discovered that microgram amounts of a prostaglandin E2 analog protects the gastric mucosa against a variety of ulcerogenic and necrotizing agents - even such strong inducers of injury as 100% ethanol and boiling water. They proposed a new concept of cytoprotection. Subsequently, other compounds, such as sulfhydryls, sucralfate and epidermal growth factor were shown to exert protective action on gastric mucosa. Additionally, some antacids have been shown to exert a potent mucosal protective action against a variety of injurious factors and accelerate healing of erosions and gastric ulcers. These actions of antacids, especially hydrotalcite - the newest and the most extensively studied antacid - are due to activation of prostaglandin synthesis; binding to and inactivation of pepsin, bile acids and H. pylori toxins; induction of heat shock proteins; and, activation of genes encoding growth factors and their receptors.
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Affiliation(s)
- Andrzej Tarnawski
- University of California, Irvine, Chief, Gastroenterology, VA Long Beach Healthcare System, 5901 E. 7th Street, Long Beach, CA 90822-5201, USA.
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Tarnawski A, Ahluwalia A, Jones MK. Gastric cytoprotection beyond prostaglandins: cellular and molecular mechanisms of gastroprotective and ulcer healing actions of antacids. Curr Pharm Des 2013; 19:126-32. [PMID: 22950493 DOI: 10.2174/13816128130117] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2012] [Accepted: 07/30/2012] [Indexed: 02/06/2023]
Abstract
This article updates current views on gastric mucosal defense, injury, protection and ulcer healing with a focus on mucosal protective and ulcer healing actions of antacids. The gastric mucosa is continuously exposed to a variety of noxious factors, both endogenous such as: 0.1N hydrochloric acid, pepsin, bile acids, lysolecithin, H. pylori toxins and exogenous such as NSAIDs, ethanol and others. Gastric mucosal integrity is maintained by pre-epithelial, epithelial and post-epithelial defense mechanisms permitting the mucosa to withstand exposure to the above damaging factors. When mucosal defense is weakened or overwhelmed by injurious factors, injury develops in the form of erosions or ulcers. In the late 1970s Andre Robert and coworkers discovered that microgram amounts of a prostaglandin E2 analog protects the gastric mucosa against a variety of ulcerogenic and necrotizing agents - even such strong inducers of injury as 100% ethanol and boiling water. They proposed a new concept of cytoprotection. Subsequently, other compounds, such as sulfhydryls, sucralfate and epidermal growth factor were shown to exert protective action on gastric mucosa. Additionally, some antacids have been shown to exert a potent mucosal protective action against a variety of injurious factors and accelerate healing of erosions and gastric ulcers. These actions of antacids, especially hydrotalcite - the newest and the most extensively studied antacid - are due to activation of prostaglandin synthesis; binding to and inactivation of pepsin, bile acids and H. pylori toxins; induction of heat shock proteins; and, activation of genes encoding growth factors and their receptors.
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Affiliation(s)
- Andrzej Tarnawski
- University of California, Irvine, Chief, Gastroenterology, VA Long Beach Healthcare System, 5901 E. 7th Street, Long Beach, CA 90822-5201, USA.
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Nakai Y, Shinoura S, Ahluwalia A, Tarnawski AS, Chang KJ. In vivo visualization of epidermal growth factor receptor and survivin expression in porcine pancreas using endoscopic ultrasound guided fine needle imaging with confocal laser-induced endomicroscopy. J Physiol Pharmacol 2012; 63:577-80. [PMID: 23388473] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 10/29/2012] [Accepted: 12/03/2012] [Indexed: 02/06/2023]
Abstract
UNLABELLED The aims of this pilot study were to establish a principle of molecular imaging of the pancreas and determine in vivo expression of epidermal growth factor receptor (EGF-R) and survivin using a novel endoscopic ultrasound-guided fine needle imaging (EUS-FNI) technique, which incorporates needle based confocal laser-induced endomicroscopy (nCLE) after intrapancreatic injection of FTIC-labeled antibodies. Studies were performed in anesthetized pigs. FITC-labeled specific antibodies against EGF-R and survivin were injected into the tail and neck of the pancreas using a 19 gauge needle introduced under EUS guidance. Thirty minutes later, nCLE was performed using a prototype needle-based confocal laser-induced endomicroscopy probe (Cellvizio AQ-Flex-19, Mauna Kea Technologies, Paris, France) to determine cellular and tissue localization of EGF-R and survivin in the pancreas. Then pigs were euthanized and specimens of pancreas from areas injected with antibodies were obtained for histologic examination under epifluorescence microscope. RESULTS EUS-guided nCLE enabled visualization of EGF-R and survivin in pancreatic tissue. Expression of EGF-R and survivin in pancreas was confirmed by histology. EGF-R immunoreactivity was localized to majority of duct-lining cells and to the surface and cytoplasm of many acinar cells. Survivin was localized mainly to the acinar cells. This study demonstrated the feasibility of in vivo, real time visualization of EGF-R and survivin in the pancreas by local injection of FITC-labeled antibodies via EUS-guided fine needle injection, followed by EUS-guided needle based confocal laser-induced endomicroscopy.
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Affiliation(s)
- Y Nakai
- Division of Gastroenterology and Hepatology, H.H. Chao Comprehensive Digestive Disease Center, University of California, Irvine School of Medicine, CA, USA
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Wyle FA, Tarnawski AS. Helicobacter pylori from an Infectious Disease Viewpoint. Clin Drug Investig 1990; 2:40-5. [DOI: 10.1007/bf03259180] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Nakai Y, Shinoura S, Ahluwalia A, Tarnawski AS, Chang KJ. Molecular imaging of epidermal growth factor-receptor and survivin in vivo in porcine esophageal and gastric mucosae using probe-based confocal laser-induced endomicroscopy: proof of concept. J Physiol Pharmacol 2012; 63:303-7. [PMID: 22791645] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 05/10/2012] [Accepted: 06/20/2012] [Indexed: 02/06/2023]
Abstract
UNLABELLED Confocal laser-induced endomicroscopy (CLE) enables in vivo, real time visualization of the subsurface cells and tissue structures in gastrointestinal mucosa at a subcellular resolution of ≈1000x magnification. The aims of this pilot study were to establish a principle of molecular imaging and determine in vivo expression of epidermal growth factor receptor (EGF-R) and survivin in porcine esophageal and gastric mucosa using probe-based CLE (pCLE) and topically applied FITC-labeled antibodies. Studies were performed in anesthetized pigs. During endoscopy FITC-labeled antibodies against EGF-R and survivin were either sprayed onto esophageal and gastric mucosa in preselected areas or administered via submucosal injection. Thirty minutes later pCLE was performed using a through-the-scope probe (GastroFlex UHD, Cellvizio, Mauna Kea Technologies, Paris, France) to determine cellular and tissue localization of EGF-R and survivin. Then the pigs were euthanized and esophageal and gastric walls from the areas sprayed or injected with antibodies were collected for histologic examination under epifluorescence microscopy. RESULTS CLE enabled visualization of EGF-R and survivin in esophageal and gastric mucosa and this was confirmed by histology. In the esophagus both EGF-R and survivin were localized predominantly to the keratinocyte progenitor cells. In the stomach, EGF-R was localized to progenitor zone cells and some epithelial cells. Localization of survivin was similar, but involved more surface epithelial cells. This study demonstrated feasibility of using CLE and topical administration of FITC labeled antibodies for in vivo localization of EGF-R and survivin in esophageal and gastric mucosa.
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Affiliation(s)
- Y Nakai
- Division of Gastroenterology and Hepatology, H.H. Chao Comprehensive Digestive Disease Center, University of California, Irvine, School of Medicine, CA, USA
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Coron E, Mosnier JF, Ahluwalia A, Le Rhun M, Galmiche JP, Tarnawski AS, Matysiak-Budnik T. Colonic mucosal biopsies obtained during confocal endomicroscopy are pre-stained with fluorescein in vivo and are suitable for histologic evaluation. Endoscopy 2012; 44:148-53. [PMID: 22271025 DOI: 10.1055/s-0031-1291534] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND AND STUDY AIMS Confocal laser endomicroscopy (CLE) with intravenous infusion of fluorescein allows noninvasive, real-time in vivo visualization of gastrointestinal mucosa at ~ × 1000 magnification ("virtual biopsy"). Conventional biopsies obtained during these procedures serve as the reference and established diagnostic standard. The aim of the present study was to assess whether the standard histologic biopsies that are obtained during CLE retain fluorescein in the tissues and allow the visualization of mucosal structures without any additional staining. PATIENTS AND METHODS CLE optical imaging of the mucosa was performed in 16 patients who were undergoing CLE colonoscopy. Standard conventional biopsies were also obtained from both normal colonic mucosa and colonic polyps. De-paraffinized mucosal sections were examined under a fluorescence microscope for the presence and distribution of fluorescein, and then underwent immunostaining for expression of vascular endothelial growth factor (VEGF). RESULTS Standard mucosal biopsy sections from patients undergoing CLE displayed a strong fluorescence and showed well-delineated mucosal structures. In colonic adenomas, there was a 4.6-fold increased vascular permeability compared with normal mucosa (P<0.001), indicated by fluorescein leakage to the extravascular space. Immunostaining demonstrated an aberrantly increased expression of VEGF in the epithelium of colonic adenomas but not in the epithelium of normal mucosa or hyperplastic polyps. CONCLUSIONS This study shows for the first time that standard colonic biopsies obtained during CLE retain fluorescein, show excellent delineation of mucosal structures without additional staining, allow the evaluation of mucosal microvasculature and vascular permeability, and are suitable for immunostaining.
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Affiliation(s)
- E Coron
- Institut des Maladies de l'Appareil Digestif - INSERM U913, CIC 04 et Service d'Hépato-Gastroentérologie, Hôtel Dieu, CHU de Nantes, France
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Ahluwalia A, Tarnawski AS. Critical role of hypoxia sensor--HIF-1α in VEGF gene activation. Implications for angiogenesis and tissue injury healing. Curr Med Chem 2012; 19:90-7. [PMID: 22300081 DOI: 10.2174/092986712803413944] [Citation(s) in RCA: 466] [Impact Index Per Article: 38.8] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2011] [Revised: 11/07/2011] [Accepted: 11/10/2011] [Indexed: 02/06/2023]
Abstract
Vascular injury of esophageal and gastrointestinal mucosa caused by injurious and ulcerogenic factors leads to the cessation of blood flow, ischemia, and hypoxia and tissue necrosis in form of erosions or ulcers. The re-establishment of blood vessels through the process of angiogenesis--formation of new blood vessels--is critical for healing of tissue injury because is essential for delivery of oxygen and nutrients to the healing site. Hypoxia increases expression of hypoxia inducible factor (HIF-1), which serves as hypoxia sensor and activates compensatory and adaptive mechanisms. However, the molecular mechanisms and the role of HIF-1α in hypoxia-driven cellular and molecular events of angiogenesis in gastrointestinal injury healing have not been fully explored. The review discusses the novel molecular mechanisms of angiogenesis in gastric and esophageal mucosa with focus on HIF1α and VEGF interactions during healing of gastric mucosal injury and esophageal ulcers. HIF-1α is upregulated by gastric mucosal injury and esophageal ulcers; this upregulation correlates with VEGF gene activation and initiation of angiogenesis. The non-steroidal anti-inflammatory drugs (NSAIDs) interfere with hypoxia-induced HIF-1α accumulation, VEGF gene activation and angiogenesis through upregulation of von Hippel- Lindau (VHL) tumor suppressor, which activates degradation of HIF-1α protein. HIF-1α is a transcription factor that under hypoxic conditions, accumulates in endothelial cells and can bind to VEGF gene promoter and induce VEGF gene expression. In order to activate the VEGF gene, HIF-1α must be transported to the nucleus. Recent evidence implicates importins as key mechanism in this process.
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Affiliation(s)
- A Ahluwalia
- VA Long Beach Healthcare System & the University of California, Irvine, 5901 E. 7th Street, Long Beach, CA 90822-5201, USA.
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40
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Tarnawski AS, Ahluwalia A. Molecular mechanisms of epithelial regeneration and neovascularization during healing of gastric and esophageal ulcers. Curr Med Chem 2012; 19:16-27. [PMID: 22300072 DOI: 10.2174/092986712803414088] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2011] [Revised: 11/03/2011] [Accepted: 11/07/2011] [Indexed: 02/06/2023]
Abstract
In this paper we reviewed and updated current views on the cellular and molecular mechanisms of gastric and esophageal ulcer healing. Gastric ulcer healing encompasses inflammation, cell proliferation, epithelial regeneration, gland reconstruction, formation of granulation tissue, neovascularization (new blood vessel formation), interactions between various cells and the matrix and tissue remodeling, resulting in scar formation. All these events are controlled by the cytokines and growth factors, GI hormones including gastrin, CCK, and orexigenic peptides such as ghrelin, orexin-A and obestatin as well as Cox2 generated prostaglandins. These growth factors and hormones trigger cell proliferation, migration, and survival utilizing Ras, MAPK, PI-3K/AKT, PLC-γ and Rho/Rac/actin signaling pathways. Hypoxia triggers activation of some of these genes (e.g., VEGF) via hypoxia inducible factor (HIF). Growth factors: EGF, HGF, IGF-1, their receptors and Cox2 are important for epithelial cell proliferation, migration, re-epithelialization and regeneration of gastric glands during gastric ulcer healing. Serum response factor (SRF) is also essential for re-epithelialization and muscle restoration. VEGF, bFGF, angiopoietins, nitric oxide, endothelin, prostaglandins and metalloproteinases are important for angiogenesis, vascular remodeling and mucosal regeneration within gastric ulcer scar. SRF is critical limiting factor for VEGF-induced angiogenesis. Esophageal ulcer healing follows similar pattern to gastric ulcer, but KGF and its receptor are the key players in regeneration of the epithelium. In addition to local mucosal cells from viable mucosa bordering necrosis, circulating bone marrow derived stem and progenitor cells are potentially important for ulcer healing, contributing to the regeneration of epithelial and connective tissue components and neovascularization.
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Affiliation(s)
- A S Tarnawski
- Veterans Administration Long Beach Healthcare System, 5901 E. 7th Street, Long Beach, CA 90822-5201, USA.
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41
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Tolstanova G, Deng X, French SW, Lungo W, Paunovic B, Khomenko T, Ahluwalia A, Kaplan T, Dacosta-Iyer M, Tarnawski A, Szabo S, Sandor Z. Early endothelial damage and increased colonic vascular permeability in the development of experimental ulcerative colitis in rats and mice. J Transl Med 2012; 92:9-21. [PMID: 21894149 DOI: 10.1038/labinvest.2011.122] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The role of endothelial damage and increased vascular permeability (VP) in the pathogenesis of ulcerative colitis (UC) has not been investigated. We examined using functional, morphologic, and molecular biologic studies whether and to what extent the endothelial barrier dysfunction precedes enhanced epithelial permeability (EP) and the development of mucosal lesions during the early stages of experimental UC. We showed that in rats with iodoacetamide (IA)-induced UC increased colonic VP occurs early (ie, 2.6-fold increase at 15 min, P<0.01) preceding changes in epithelial barrier permeability. EP was unchanged at 15 and 30 min after IA administration and was increased 1.9-fold at 1 h and 6.7-fold at 2 h (both P<0.001) after IA. In the dextran sodium sulfate-induced slowly developing UC, colonic VP was significantly increased in 2 days (P<0.05) and EP only in 4 days (P<0.05). Mucosal endothelial injury led to hypoxia (P<0.05) of colonic surface epithelial cells 30 min after IA administration that was associated with increased expressions of transcription factors hypoxia-inducible factor-1α and early growth response-1. Electron and light microscopy demonstrated areas of colonic mucosa with perivascular edema covered by intact layer of surface epithelial cells in both rat and mouse models of UC. This is the first demonstration in four models of UC that endothelial damage, increased colonic VP, perivascular edema, and epithelial hypoxia precede epithelial barrier dysfunction that is followed by erosions, ulceration, and inflammation in UC.
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Affiliation(s)
- Ganna Tolstanova
- Diagnostic and Molecular Medicine, VA Long Beach Healthcare System, Long Beach, CA 90822, USA
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Tarnawski AS, Ahluwalia A, Jones MK. The mechanisms of gastric mucosal injury: focus on microvascular endothelium as a key target. Curr Med Chem 2012; 19:4-15. [PMID: 22300071 DOI: 10.2174/092986712803414079] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2011] [Revised: 11/04/2011] [Accepted: 11/07/2011] [Indexed: 02/06/2023]
Abstract
This paper reviews and updates current views on gastric mucosal injury with a focus on the microvascular endothelium as the key target and the role of the anti-apoptosis protein survivin. Under normal conditions, mucosal integrity is maintained by well structured and mutually amplifying defense mechanisms, which include pre-epithelial "barrier"--the first line of defense; and, an epithelial "barrier". Other important defense mechanisms of gastric mucosa include: continuous epithelial cell renewal, blood flow through mucosal microvessels (providing oxygen and nutrients), an endothelial microvascular "barrier," sensory innervation, and generation of PGs, nitric oxide and hydrogen sulfide. The microvascular endothelium lining gastric mucosal blood microvessels severs not only as a barrier but is a biologically active tissue involved in many synthetic and metabolic functions. It allows transport of oxygen and nutrients, and produces prostaglandins and leukotriens, procoagulant factors, nitric oxide, endothelin, ghrelin, HSP, growth factors such VEGF, bFGF, angiopoietin 2 and others, specific types of collagen, plasminogen activator, and can also actively contract. Accumulating evidence indicates that the gastric microvascular endothelium is a critical target for injury by ethanol, NSAIDs, free radicals, ischemia-reperfusion and other damaging factors. The injury--microvessel rupture, plasma and erythrocyte extravasation, platelet aggregation and fibrin deposition caused by these damaging factors--occurs early (1-5 min), precedes glandular epithelial cell injury and results in cessation of blood flow, ischemia, hypoxia and impaired oxygen and nutrient transport. As a consequence, mucosal necrosis develops. One of the main reasons for the increased susceptibility of gastric microvascular endothelial (vs. epithelial) cells to injury is reduced expression levels of survivin, an anti-apoptosis protein, which is a regulator of both proliferation and cell survival.
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Affiliation(s)
- A S Tarnawski
- Veterans Administration Long Beach Healthcare System, 5901 E. 7th Street, Long Beach, CA 90822-5201, USA.
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43
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Paunovic B, Deng X, Khomenko T, Ahluwalia A, Tolstanova G, Tarnawski A, Szabo S, Sandor Z. Molecular mechanisms of basic fibroblast growth factor effect on healing of ulcerative colitis in rats. J Pharmacol Exp Ther 2011; 339:430-7. [PMID: 21841041 DOI: 10.1124/jpet.111.183665] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
We demonstrated previously that basic fibroblast growth factor (bFGF) accelerated the healing of experimental duodenal ulcers, and we now hypothesize that bFGF might also accelerate the healing of experimental ulcerative colitis (UC). We also explored the potential molecular mechanisms involved in the accelerated healing of UC in rats treated with bFGF. The results demonstrated that colonic lesions were significantly reduced by bFGF treatment, whereas neutralization of bFGF aggravated iodoacetamide-induced UC. Protein expression of bFGF was increased during the healing stage of UC. Tumor necrosis factor-α levels and myeloperoxidase activity were significantly decreased in UC rats treated with bFGF, whereas they increased in rats treated with anti-bFGF antibody. Real-time polymerase chain reaction and immunohistochemistry showed decreased levels of p27 in the UC rats compared with the healthy controls, which was reversed by bFGF treatment in a dose-dependent manner. By immunohistochemistry and double labeling of Ki-67 and CD34, prominent positive staining of Ki-67 and CD34 was seen after bFGF treatment, indicating the enhanced proliferation of fibroblasts and epithelial and endothelial cells, i.e., angiogenesis. We conclude that bFGF plays a beneficial role in the healing of UC in rats. The molecular mechanisms of bFGF in UC healing not only involve the expected increased cell proliferation, especially angiogenesis, but also encompass the reduction of inflammatory cytokines and infiltration of inflammatory cells. Thus, bFGF enema may be a new therapeutic option for UC.
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Affiliation(s)
- Brankica Paunovic
- Diagnostic and Molecular Medicine Health Care Group, VA Long Beach Healthcare System, Long Beach, CA 90822, USA
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44
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Ahluwalia A, Tarnawski AS. Activation of the metabolic sensor-AMP activated protein kinase reverses impairment of angiogenesis in aging myocardial microvascular endothelial cells. Implications for the aging heart. J Physiol Pharmacol 2011; 62:583-7. [PMID: 22204807] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 08/26/2011] [Accepted: 09/16/2011] [Indexed: 02/06/2023]
Abstract
Impairment of angiogenesis - new capillary blood vessel formation from pre-existing vessels, is frequent in aging tissues and cells. Reduced angiogenesis in aging individuals is associated with increased incidence of myocardial infarctions and other cardiovascular diseases. Therefore there is a need to develop novel strategies to enhance angiogenesis in aging individuals. Our previous study demonstrated aging-related impairment of angiogenesis in aging (vs. young) rat myocardial microvascular endothelial cells (MMEC), and identified reduced activation of the vascular endothelial growth factor (VEGF, the most potent stimulator of angiogenesis) gene as the main underlying mechanism. In the present study we examined the possibility of increasing angiogenesis and activating VEGF gene expression in aging MMECs using a chemical activator of the metabolic sensor - AMP activated protein kinase (AMPK). We hypothesized that activation of VEGF gene in aging MMECs by AMPK would stimulate angiogenesis and reverse the impairment in angiogenesis seen in these cells. We used MMECs isolated from aging (24 months old) Fisher F-344 rats and treated them with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a specific pharmacological stimulator of AMPK. We examined: 1) in vitro angiogenesis; and 2) the expression of phosphorylated AMPK, VEGF, and P-MAPK/Erk1/2. Treatment of aging MMECs with AICAR increased in vitro angiogenesis and VEGF mRNA expression by 2.1-fold and 3.7-fold, respectively. Furthermore, AICAR treatment resulted in phosphorylation of MAPK/Erk1/2. This study demonstrated the successful use AICAR to reverse aging-related impairment of angiogenesis in aging MMECs by enhancing VEGF gene expression and also identified phosphorylation of MAPK/Erk1/2 as a likely mechanism of these changes.
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Affiliation(s)
- A Ahluwalia
- Southern California Intitute for Research and Education, VA Long Beach Health Care System, CA 90822, USA.
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45
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Florkiewicz RZ, Ahluwalia A, Sandor Z, Szabo S, Tarnawski AS. Gastric mucosal injury activates bFGF gene expression and triggers preferential translation of high molecular weight bFGF isoforms through CUG-initiated, non-canonical codons. Biochem Biophys Res Commun 2011; 409:494-9. [PMID: 21600881 DOI: 10.1016/j.bbrc.2011.05.033] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2011] [Accepted: 05/05/2011] [Indexed: 02/06/2023]
Abstract
Basic fibroblast growth factor (bFGF or FGF-2) is a pleiotropic growth factor that promotes growth of mesenchymal and epithelial cells, stimulates angiogenesis and neuroprotection. Moreover, exogenous bFGF by stimulating angiogenesis promotes healing of gastroduodenal ulcers and cardiac and brain injury. All these actions were demonstrated in regard to 18kDa bFGF isoform that is secreted by cells via an ER/Golgi-independent pathway and activates FGF receptors. However in some transformed and stressed cells and in some tissues (e.g. brain) the single copy bFGF gene encodes multiple gene products: 18 kDa and also higher molecular weight (HMW) bFGF isoforms: ∼21 and ∼22 kDa in rodents, and ∼22, ∼23 and ∼24 kDa in humans. The biologic roles of these HMW bFGF isoforms in vivo remain unknown. In this study we demonstrated that in normal, uninjured gastric mucosa, bFGF is almost exclusively expressed as 18kDa isoform translated through a classical AUG (methionine) codon. In contrast, in injured gastric mucosa of rat, bFGF gene is preferentially translated to HMW bFGF isoforms through alternative CUG (leucine) initiation codon. Gastric mucosal injury caused in rats a significant increase in bFGF mRNA at 8 and 24h vs. normal mucosa and a significant increase in bFGF protein at 24-72h, mainly due to increased expression of ∼21 and ∼22 kDa HMW bFGF isoforms. This is first demonstration that gastric mucosal injury and repair triggers local activation of bFGF gene with preferential translation of HMW bFGF isoforms through a non-canonical CUG codon. This study uncovered CUG-initiated HMW bFGF translation as a novel regulatory mechanism operating in vivo during gastric injury repair.
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Affiliation(s)
- Robert Z Florkiewicz
- Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
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46
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Deng X, Szabo S, Chen L, Paunovic B, Khomenko T, Tolstanova G, Tarnawski AS, Jones MK, Sandor Z. New cell therapy using bone marrow-derived stem cells/endothelial progenitor cells to accelerate neovascularization in healing of experimental ulcerative colitis. Curr Pharm Des 2011; 17:1643-51. [PMID: 21548863 DOI: 10.2174/138161211796197007] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2011] [Accepted: 04/27/2011] [Indexed: 02/06/2023]
Abstract
Inflammatory bowel disease (IBD): ulcerative colitis (UC) and Crohn disease (CD) are characterized by recurrent inflammation and ulceration of intestinal and/or colonic mucosa and an inappropriate and delayed healing. Current therapies with, e.g., anti-TNFα antibody (infliximab) and other anti-inflammatory drugs (e.g., mesalamine) do not induce sustained remission, complete healing or prevent recurrence of UC. Although the pathogenesis of UC is not fully understood, pathologic angiogenesis has been postulated as a critical pathogenic component in UC. Recent studies demonstrated that the poor healing, chronic inflammation in colon of UC could be the result of microvascular dysfunction and endothelial barrier defect, resulting in sustained tissue hypoperfusion and ischemia in the colon. Previously, regeneration of injured endothelium and neovascularization were believed to rely solely on the migration and proliferation of neighboring endothelial cells from existing blood vessels. However, accumulating evidence shows that additional mechanisms may exist, and may be mediated by the circulating pool of bone marrow-derived endothelial progenitor cells (BMD-EPC). Furthermore, stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 have been demonstrated to play an important role in the "homing" of BMD-EPC to injured sites and neovascularization in tissue repair. Recent studies by others and us showed reduced BMD-EPC levels in the circulation of IBD patients and rats with experimental UC. However, the potential therapeutic effect of BMD-EPC on neovascularization and colonic mucosal repair in UC has not been elucidated. In this review, we discussed the possibility that impaired contribution of BMD-EPC (i.e., decreased release of BMD-EPC from bone marrow to circulation and/or blocked/impaired homing of BMD-EPC to colonic lesions) may be a critical component of mechanisms in the incomplete/delayed healing of UC, and may offer a novel form of cell therapy for IBD.
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Affiliation(s)
- Xiaoming Deng
- Diagnostic & Molecular Medicine HCG, VA Long Beach Healthcare System, Long Beach, CA 90822, USA
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47
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Tarnawski AS, Pai R, Tanigawa T, Matysiak-Budnik T, Ahluwalia A. PTEN silencing reverses aging-related impairment of angiogenesis in microvascular endothelial cells. Biochem Biophys Res Commun 2010; 394:291-6. [PMID: 20193662 DOI: 10.1016/j.bbrc.2010.02.161] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2010] [Accepted: 02/24/2010] [Indexed: 02/06/2023]
Abstract
Aging is associated with impaired angiogenesis (new blood vessels formation from the endothelial cells of pre-existing vessels) in a variety of tissues. The precise mechanisms of aging-related impairment of angiogenesis are not known. PTEN is a dual-specificity phosphatase that antagonizes in some cells the PI3K/Akt signaling pathway, important for cell survival, function and angiogenesis. PTEN's role in aging-related impairment of angiogenesis is not known. In this study, we investigated whether expression of PTEN in endothelial cells may play a mechanistic role in aging-related impairment of angiogenesis. We demonstrated that human microvascular endothelial cells (HMVEC) derived from aging individuals (Aged-HMVEC) have: (1) significantly increased PTEN mRNA and protein levels and (2) impaired in vitro angiogenesis vs. neonatal derived HMVEC (Neo-HMVEC), and that (3) downregulation of PTEN using specific siRNA restores angiogenesis in Aged-HMVEC to normal. This is the first demonstration of increased PTEN expression in human microvascular endothelial cells derived from aging tissues and that elevated PTEN is a major factor responsible for aging-related impairment of in vitro angiogenesis.
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Affiliation(s)
- Andrzej S Tarnawski
- Medical Service, VALBHS and Department of Medicine, University of California Irvine, Long Beach, CA 90822, USA.
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48
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Ahluwalia A, Narula J, Jones MK, Deng X, Tarnawski AS. Impaired angiogenesis in aging myocardial microvascular endothelial cells is associated with reduced importin alpha and decreased nuclear transport of HIF1 alpha: mechanistic implications. J Physiol Pharmacol 2010; 61:133-9. [PMID: 20436213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 09/21/2009] [Accepted: 03/19/2010] [Indexed: 02/06/2023]
Abstract
Aging is associated with increased incidence of myocardial infarctions and impaired angiogenesis - new capillary blood vessel formation from preexisting vessels. The molecular mechanism(s) of aging-related impairment of angiogenesis are unknown. In the present study we focused on the mechanism of activation of the gene for vascular endothelial growth factor (VEGF - the most potent stimulator of angiogenesis) in young and aging myocardial microvascular endothelial cells (MMEC). Activation of VEGF gene in the cell nucleus is mediated in part by the transcription factor hypoxia-inducible factor 1 alpha (HIF1 alpha). In order to activate VEGF gene, HIF1 alpha must first be transported to the nucleus, but the mechanisms of this transport are unknown. We hypothesized that reduced VEGF gene activation and impaired angiogenesis in myocardium during aging can result from downregulation of the nuclear transport receptor - importin alpha that leads to decreased transport of HIF1 alpha to the nucleus. We examined in MMEC isolated from young (3 months of age) and aging (24 months old) Fisher F-344 rats: 1) in vitro angiogenesis; and 2) the expression of VEGF, importin alpha and HIF1 alpha. Aging MMEC exhibited a 3.7-fold reduction in angiogenesis and a corresponding reduction in VEGF (by 3-fold) and importin alpha (by 1.9-fold) levels compared to young MMEC. Aging MMEC also exhibited cytoplasmic accumulation (by 1.8-fold) of HIF1 alpha protein, reduced HIF1 alpha transport to the nucleus and decreased binding of HIF1 alpha protein to the VEGF gene promoter. This study is the first demonstration of the downregulation of importin alpha in aging MMEC and reduced nuclear transport of HIF1 alpha, which likely lead to decreased VEGF gene activation and impaired angiogenesis.
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Affiliation(s)
- A Ahluwalia
- VA Long Beach Healthcare System, Long Beach, CA, USA
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49
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Deng X, Tolstanova G, Khomenko T, Chen L, Tarnawski A, Szabo S, Sandor Z. Mesalamine restores angiogenic balance in experimental ulcerative colitis by reducing expression of endostatin and angiostatin: novel molecular mechanism for therapeutic action of mesalamine. J Pharmacol Exp Ther 2009; 331:1071-8. [PMID: 19762547 DOI: 10.1124/jpet.109.158022] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Mesalamine (5-aminosalicylate acid, 5-ASA) is an effective treatment for ulcerative colitis (UC). The mechanisms of its actions are not fully understood. Because angiogenesis is critical for healing UC, we examined whether 5-ASA alters the angiogenic balance between angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and antiangiogenic factors (e.g., endostatin and angiostatin) in the colon in experimental UC. Rats were treated with saline or 5-ASA (100 mg/kg) twice daily and euthanized 3 or 7 days after iodoacetamide-induced UC. Clinical signs (e.g., lethargy, diarrhea) and UC lesions were measured. Expression of VEGF, endostatin, angiostatin, tissue necrosis factor alpha (TNF-alpha), and matrix metalloproteinases (MMPs) 2 and 9 was determined by Western blots, enzyme-linked immunosorbent assay, and zymography in the distal colon. 5-ASA treatment reduced lethargy and diarrhea and significantly decreased colonic lesions (by approximately 50%) compared with saline treatment in UC (both, P < 0.05). 5-ASA did not reverse the increased levels of VEGF, but it significantly reduced expression of endostatin and angiostatin in UC compared with vehicle treatment (both, P < 0.05). Furthermore, 5-ASA treatment significantly diminished increased activity of TNF-alpha and MMP9 in UC. This is the first demonstration that 5-ASA treatment reverses an imbalance between the angiogenic factor VEGF and antiangiogenic factors endostatin and angiostatin in experimental UC. The effect of 5-ASA in UC may be caused by the down-regulation of expression of endostatin and angiostatin by modulation of MMP2 and MMP9 via inhibition of TNFalpha. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.
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MESH Headings
- Angiostatins/antagonists & inhibitors
- Angiostatins/biosynthesis
- Animals
- Anti-Inflammatory Agents, Non-Steroidal/administration & dosage
- Anti-Inflammatory Agents, Non-Steroidal/pharmacology
- Anti-Inflammatory Agents, Non-Steroidal/therapeutic use
- Blotting, Western
- Colitis, Ulcerative/drug therapy
- Colitis, Ulcerative/enzymology
- Colitis, Ulcerative/physiopathology
- Colon/blood supply
- Colon/drug effects
- Colon/enzymology
- Electrophoresis, Polyacrylamide Gel
- Endostatins/antagonists & inhibitors
- Endostatins/biosynthesis
- Enzyme-Linked Immunosorbent Assay
- Female
- Matrix Metalloproteinase 2/metabolism
- Matrix Metalloproteinase 9/metabolism
- Mesalamine/administration & dosage
- Mesalamine/pharmacology
- Mesalamine/therapeutic use
- Neovascularization, Physiologic/drug effects
- Rats
- Rats, Sprague-Dawley
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Affiliation(s)
- Xiaoming Deng
- Diagnostic and Molecular Medicine, Health Care Groups, VA Medical Center, Long Beach, California 90822, USA
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Ahluwalia A, Li A, Cheng G, Deng X, Tarnawski AS. Reduced ghrelin in endothelial cells plays important mechanistic role in aging-related impairment of angiogenesis. J Physiol Pharmacol 2009; 60:29-34. [PMID: 19617642] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [What about the content of this article? (0)] [Affiliation(s)] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 01/20/2009] [Accepted: 04/30/2009] [Indexed: 02/06/2023]
Abstract
Ghrelin, a hormone produced mainly by gastric mucosal cells stimulates growth hormone (GH) release. Ghrelin is also expressed in the endothelial cells of blood vessels suggesting its physiological role and a function in these cells. We recently demonstrated that ghrelin induces angiogenesis--new capillary blood vessel formation- in neonatal human microvascular endothelial cells (HMVECs). Angiogenesis is impaired in aging individuals both in vitro and in vivo, but the precise mechanism(s) of this phenomenon is unknown. We examined whether HMVECs derived from aging individuals (66 years and 90 years old), 66-HMVECs and 90-HMVECs have reduced ghrelin levels vs. neonatal (Neo) HMVECs and whether treatment with exogenous ghrelin can restore impaired in vitro angiogenesis on matrigel in aged HMVECs. Ghrelin levels were reduced in the aged HMVECs by 3.2-fold (p<0.05) compared to Neo-HMVECs. Angiogenesis was significantly decreased in the aged 66- and 90-HMVECs by 39.7% (p = 0.003) and 62.4% (p = 0.003), respectively compared to Neo-HMVECs. Treatment with exogenous ghrelin significantly reversed impaired angiogenesis in aged HMVECs with the EC(50) 0.05 nM. Ghrelin induced angiogenesis in Neo-HMVECs mainly through ERK2 activation. This study is the first demonstration that reduced ghrelin is one of the factors responsible for aging-related impairment of angiogenesis.
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Affiliation(s)
- A Ahluwalia
- VA Long Beach Healthcare System, Long Beach, CA 90822, USA
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