Sandor Szabo

Neutralizing Anti-Vascular Endothelial Growth Factor (VEGF) Antibody Reduces Severity of Experimental Ulcerative Colitis in Rats: Direct Evidence for the Pathogenic Role of VEGF

In ulcerative colitis (UC), an increased expression of vascular endothelial growth factor (VEGF) correlates with disease activity, but a causal relationship is unknown. We tested the hypothesis that VEGF plays a mechanistic role in the pathogenesis of experimental UC and that VEGF neutralization may exert therapeutic effect. UC was induced in Sprague-Dawley rats by 6% iodoacetamide given intracolonically. Neutralizing anti-VEGF antibody (50 μg/rat), nonspecific IgG, or saline (0.1 ml/rat) was injected intramuscularly on the 3rd and 5th days after iodoacetamide enema. Rats were euthanized on the 7th day. We examined the extent of macroscopic, histologic, and clinical features of colitis and colonic vascular permeability. Colonic VEGF mRNA and protein expressions increased as early as 0.5 h after iodoacetamide enema and remained elevated in the active phase of colitis. Treatment with anti-VEGF antibody markedly improved the clinical and morphologic features of UC. Colonic lesion area was significantly reduced from 370 ± 140 or 311 ± 170 mm2 in saline- or IgG-treated groups to 122 ± 57 mm2 in the anti-VEGF-group (p < 0.05). Increased colonic vascular permeability was decreased by the anti-VEGF antibody (p < 0.05) and the Src inhibitor PP1 [pyrazolopyrimidine, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine] (p < 0.01). The number of acute and chronic inflammatory cells in the lesion area was significantly reduced in anti-VEGF-treated rats. In the anti-VEGF-treated group, mucosal levels of VEGF, platelet-derived growth factor, and basic fibroblast growth factor were also reduced. In conclusion: 1) Neutralizing anti-VEGF antibody significantly ameliorates experimental UC in rats in part by reducing excessive vascular permeability and decreasing inflammatory cells infiltration; and 2) VEGF seems to mediate increased colonic vascular permeability in experimental UC via the Src-dependent mechanism.

Mesalamine Restores Angiogenic Balance in Experimental Ulcerative Colitis by Reducing Expression of Endostatin and Angiostatin: Novel Molecular Mechanism for Therapeutic Action of Mesalamine

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 α (TNF-α), 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 ∼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-α 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 TNFα. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.

Early endothelial damage and increased colonic vascular permeability in the development of experimental ulcerative colitis in rats and mice.

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.

New molecular mechanisms of duodenal ulceration.

Abstract:  Stress is a major etiologic factor in the pathogenesis of gastric and duodenal ulceration, as first described in rats by Hans Selye. In patients with “peptic ulcers” duodenal ulcers are more frequent than gastric ulcers (except in Japan). Thus, our research during the last three decades focused on the molecular mechanisms of duodenal ulcer in rodent models of chemically induced duodenal ulceration, and here we review our three recent findings: Endothelins (ET‐1), the immediate early gene egr‐1 and imbalance of angiogenic/antiangiogenic molecules. Namely, we found an enhanced expression and release of ET‐1 within 15–30 min after the administration of duodenal ulcerogen cysteamine, resulting in local ischemia that triggers the expression of hypoxia‐inducible factors (HIF‐1α). Our gene expression studies also revealed an early (0.5–2 h) increase in the expression of egr‐1 that is followed (12–24 h) by upregulation of angiogenic growth factors (e.g., VEGF, bFGF, PDGF). Surprisingly, this event is also associated with an enhanced production of angiostatin and endostatin that probably counteract the beneficial effect of angiogenic molecules. Thus, the initial injury to endothelial and epithelial cells in duodenal ulceration seems to be aggravated (and not initiated) by HCl and proteolytic enzymes. The resulting mucosal necrosis does not rapidly heal because of the imbalance of VEGF and angiostatin/endostatin, hence duodenal ulcers develop. The experimental ulcers Selye described morphologically are now characterized at the molecular and genome level, involving unexpected mediators like ET‐1, egr‐1 and angiogenesis‐related molecules.

Cosegregation of Gastrointestinal Ulcers and Schizophrenia in a Large National Inpatient Discharge Database: Revisiting the “Brain-Gut Axis” Hypothesis in Ulcer Pathogenesis

The lifetime prevalence of duodenal ulcer in the United States is 8 to 10%, whereas another 1% of the population is affected by gastric ulcer. Both central and peripheral dopamine pathways may influence ulcer pathogenesis. Dopamine agonists prevent whereas antagonists augment stress- and chemically induced gastrointestinal ulcers in preclinical models. The dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,36-tetrahydropyridine (MPTP) depletes central dopamine and induces lesions in the substantia nigra, and, if given in high doses, MPTP induces a Parkinson disease-like syndrome and gastric ulcers. Because schizophrenia is attributed, in part, to an overactive dopaminergic system, persons with schizophrenia may display a reduced susceptibility toward gastrointestinal ulcers. A case-control study was conducted in patients represented in the 2002 National Inpatient Sample, the largest all-payer inpatient care database in the United States, consisting of 5 to 8 million inpatient hospital stays per year, which approximates a 20% sample of community hospitals. A significant association was observed between schizophrenia and diminished risk for duodenal (odds ratio [OR] 0.55; 95% confidence interval [CI] 0.45-0.67) and gastric (OR 0.54; 95% CI 0.46-0.63) (p < .01) ulcers but not for gastrojejunal ulcers (OR 0.44; 95% CI 0.16-1.20) (p = .11). Potential confounders such as age, gender, race, tobacco or alcohol dependence, and Helicobacter pylori infection were controlled in multivariate analyses. This observational study in a large sample of patients in community hospitals suggests that schizophrenia and attendant neurobiologic mechanisms (eg, variability in dopamine pathways) may act in concert to modify the composite risk for gastrointestinal ulcers. Dopamine pathways warrant further prospective research as new potential drug targets in ulcer disease.

Role of anti-angiogenic factor endostatin in the pathogenesis of experimental ulcerative colitis

AIMS Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC). MAIN METHODS We used three animal models of UC: (1) induced by 6% iodoacetamide (IA) in rats, or (2) by 3% dextran sulfate sodium (DSS) in matrix metalloproteinase-9 (MMP-9) knockout (KO) and wild-type mice, and (3) interleukin-10 (IL-10) KO mice. Groups of MMP-9 KO mice with DSS-induced UC were treated with endostatin or water for 5days. KEY FINDINGS We found concomitant upregulation of VEGF, PDGF, MMP-9 and endostatin in both rat and mouse models of UC. A positive correlation between the levels of endostatin or VEGF and the sizes of colonic lesions was seen in IA-induced UC. The levels and activities of MMP-9 were also significantly increased during UC induced by IA and IL-10 KO. Deletion of MMP-9 decreased the levels of endostatin in both water- and DSS-treated MMP-9 KO mice. Treatment with endostatin significantly improved DSS-induced UC in MMP-9 KO mice. SIGNIFICANCE 1) Concomitantly increased endostatin is a defensive response to the increased VEGF in UC, 2) MMP-9 is a key enzyme to generate endostatin which may modulate the balance between VEGF and endostatin during experimental UC, and 3) endostatin treatment plays a beneficial role in UC. Thus, anti-angiogenesis seems to be a new therapeutic option for UC.

Molecular Mechanisms of Basic Fibroblast Growth Factor Effect on Healing of Ulcerative Colitis in Rats

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.

Angiogenic and Anti-Angiogenic Therapy for Gastrointestinal Ulcers: New Challenges for Rational Therapeutic Predictions and Drug Design

Gastrointestinal (GI) ulcers are essentially internal wounds that resist normal healing processes. Since their pathogenesis is poorly understood, and the etiologic (e.g., gastric acid, aspirin-like drugs, stress) and aggravating factors (e.g., H. pylori) are not well characterized, the remaining therapeutic option is to accelerate healing. Superficial mucosal lesions, i.e., erosions usually heal by epithelial regeneration and restitution, but when ulcers involve the muscularis propria, smooth muscle cells do not divide/regenerate. These deep lesions are filled by granulation tissue, i.e., angiogenesis followed by proliferation of connective tissue fibroblasts that deposit collagen over which adjacent surviving and dividing epithelial cells migrate to complete the healing. Our laboratory was the first to postulate that stimulation of angiogenesis alone might be sufficient to accelerate ulcer healing in the GI tract. Indeed, daily treatment of rats with bFGF, PDGF or VEGF markedly improved the healing of cysteamine-induced chronic duodenal ulcers, without any reduction in gastric acid secretion. These results were reproduced by a single dose of gene therapy by adenoviral vectors encoding PDGF or VEGF genes. The molar potency of angiogenic growth factors was 2-7 million times better than the antiulcerogenic effect of antisecretory H2 antagonists. Since histologically & pathologically gastroduodenal ulcers look similar to ulcers in the lower GI tract, we also predicted that the healing of experimental ulcerative colitis might be also improved by these angiogenic growth factors. Rectal enemas containing bFGF or PDGF indeed accelerated the healing of chemically induced ulcerative colitis in rats. VEGF, also known as VPF (vascular permeability factor), however, had no effect or slightly aggravated the colonic lesions. Injection of anti-VEGF neutralizing antibodies, however, counteracted the increased vascular permeability in the early stages of experimental ulcerative colitis and subsequently decreased the number of inflammatory cells in colonic ulcers in rats, resulting in significantly improved healing in the lower GI tract lesions. Thus, the three angiogenic growth factors tested exerted beneficial effect on gastroduodenal ulcers, and rectal enemas with bFGF or PDGF also accelerated the healing of experimental ulcerative colitis. Surprisingly, we achieved the latter effect with anti-VEGF antibodies, most likely because of the pro-inflammatory actions of VEGF in the pathogenesis of ulcerative colitis.

New molecular mechanisms of the unexpectedly complex role of VEGF in ulcerative colitis

The effects of VEGF on endothelial cells are mediated by different intracellular signaling cascades (e.g., Erk1/2, Akt, Src). VEGF plays a recently recognized role in ulcerative colitis (UC) pathogenesis, mostly by increasing vascular permeability and promoting the infiltration of inflammatory cells. We hypothesized that the excessive activation of signal transduction pathways, which is responsible for VEGF/VEGFR-2-mediated endothelial permeability (Src, Akt), is a new element in the pathogenesis of chronic UC. We demonstrated increased expression of pro-angiogenic growth factor VEGF and its receptor VEGFR-2 in colonic tissue during acute 6% iodoacetamide-induced UC in rats and chronic spontaneously developed UC in IL-10 knockout mice (IL-10 KO). Development of acute 6% iodoacetamide-induced UC in rats was accompanied by activation of Erk1/2 and Src kinase, while expression of total proteins Erk1/2 and Src was unchanged. During chronic colitis phosphorylation (i.e., activation) of Erk1/2 was significantly decreased in IL-10 KO mice vs. wild-type mice. Levels of total Erk1/2 proteins were unchanged, but the expression of total Src protein as well as its phosphorylated form was significantly increased in IL-10 KO vs. wild-type mice. There were no changes in total Akt proteins, while levels of activated Akt (pAkt) were slightly increased in IL-10 KO vs. wild-type mice. We conclude that VEGF/VEGFR-2-associated signal transduction pathways, that mediate increased vascular permeability (Src, Akt), might play a central role in perpetuation of chronic experimental UC.

Inappropriate Angiogenic Response as a Novel Mechanism of Duodenal Ulceration and Impaired Healing

BackgroundDespite recent advances and better understanding of the etiology and the pathogenesis of gastrointestinal ulcer diseases, e.g., duodenal ulcer, the molecular events leading to ulcer development, delayed healing, and recurrence remain poorly elucidated.AimsAfter we found that duodenal ulcers did not heal despite increased levels of vascular endothelial growth factor (VEGF), we tested the hypothesis that an imbalance in angiogenic VEGF and anti-angiogenic endostatin and angiostatin might be important in the development and delayed healing of experimental duodenal ulcers.MethodsLevels of VEGF, endostatin, and angiostatin, and the expression and activity of related matrix metalloproteinases (MMP) 2 and 9 were measured in scrapings of rat proximal duodenal mucosa in the early and late stages of chemically induced duodenal ulceration. Furthermore, animals were treated with recombinant endostatin and MMP 2 inhibitor to test the relationship between MMP2 and endostatin and their involvement in healing of experimental duodenal ulcers.ResultsA concurrent increase of duodenal VEGF, endostatin, and angiostatin was noted during duodenal ulceration. Endostatin treatment aggravated duodenal ulcer. Levels of MMP2, but not MMP9, were increased. Inhibition of MMP2 reduced levels of endostatin and angiostatin, and attenuated duodenal ulcers.ConclusionsIncreased levels of endostatin and angiostatin induced by MMP2 delayed healing of duodenal ulcers despite concurrently increased VEGF. Thus, an inappropriate angiogenic response or “angiogenic imbalance” may be an important new mechanism in ulcer development and impaired healing.