STAT3 and importins are novel mediators of early molecular and cellular responses in experimental duodenal ulceration.

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 ß 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 ß, 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.

Novel pharmacologic approaches to the prevention and treatment of ulcerative colitis.

Ulcerative colitis (UC) is a chronic, relapsing inflammatory disorder of the colonic mucosa followed by poor quality of healing and recurring lesions. Recent studies demonstrated that the poor healing and 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. Long before angiogenesis became a popular research topic, our laboratory was the first to postulate that stimulation of angiogenesis alone might be sufficient to accelerate ulcer healing in the gastrointestinal tract. Our earlier studies demonstrated that therapy with genes or peptides of angiogenic growth factors, e.g., bFGF, PDGF and VEGF significantly accelerated healing of experimental duodenal ulcers (DU), while blockade of these angiogenic factors resulted in impaired healing of DU. However, unlike the angiogenesis in DU, increasing evidences from us and others indicate that angiogenesis plays a pathogenic role in UC, e.g., VEGF induces an abnormal "pathologic" angiogenesis which interferes with UC healing. Recently, another angiogenic factor, placental growth factor (PlGF), has also been suggested to be a marker of pathologic angiogenesis and may play a critical role in pathogenesis of UC. Although inhibition of pathologic angiogenesis by, e.g., anti-VEGF or -PlGF, was demonstrated to be a new approach to attenuate UC development, additional data of our and others showed that stimulating angiogenesis by administration of PDGF or bFGF significantly accelerated healing of UC. Also, activation of Rac1, a small GTPase, markedly improved VEGF-induced neovessel architecture defect and reduced vascular permeability (VP) in an angiogenic model. Thus, it seems that both angiogenic and anti-angiogenic therapies may be used in various stages of UC. More recently, we demonstrated that increased VP in colonic mucosa is an early and essential element in the initiation and progression of UC. The increased VP is initiated by early release of histamine and maintained/aggravated by VEGF, leading to perivascular edema, vascular stasis, hypoxia, inflammatory cell infiltration, and colonic erosions/ulcers. Inhibition of increased VP prevents or reduces development and progression of UC. In this review, we discuss novel pharmacologic approaches to prevent UC, differential actions of angiogenic growth factors in UC pathogenesis and blocking the early increase in VP in UC development, these new findings may provide new insights into the regulation of angiogenesis in UC and may lead to development of VP-related drugs to accelerate the healing of UC.

Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (∼1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (∼6.8pmol/g fresh tissue).

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

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.

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.

Inappropriate angiogenic response as a novel mechanism of duodenal ulceration and impaired healing.

BACKGROUND: Despite 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. AIMS: After 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. METHODS: Levels 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. RESULTS: A 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. CONCLUSIONS: Increased 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.

New cell therapy using bone marrow-derived stem cells/endothelial progenitor cells to accelerate neovascularization in healing of experimental ulcerative colitis.

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.

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.

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.

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.

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 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.

Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism.

An HPLC method with coulometric detection is presented for the quantitation of cysteamine, cystamine, thialysine, glutathione, glutathione disulfide and an oxidized metabolite of thialysine [S-(2-aminoethyl)-L-cysteine ketimine decarboxylated dimer (AECK-DD)]. The advantage of coulometric detection is that derivatization is unnecessary if the analyte is redox sensitive. The method was used to quantitate several sulfur-containing compounds in plasma and brain following gavage feeding of cysteamine to rats. Cysteamine, cystamine, thialysine and AECK-DD were detected in the brains of these animals. Interestingly, cysteamine treatment resulted in greatly elevated levels of cerebral methionine, despite the fact that cysteamine is not a precursor of methionine.

Role of iron in the pathogenesis of cysteamine-induced duodenal ulceration in rats.

Cysteamine induces perforating duodenal ulcers in rats within 24-48 h. This reducing aminothiol generates hydrogen peroxide in the presence of transition metals (e.g., ferric iron), producing oxidative stress, which may contribute to organ-specific tissue damage. Since most intestinal iron absorption takes place in the proximal duodenum, we hypothesized that cysteamine may disrupt regulation of mucosal iron transport, and iron may facilitate cysteamine-induced duodenal ulceration. We show here that cysteamine-induced ulceration was aggravated by pretreatment of rats with Fe(3+) or Fe(2+) compounds, which elevated iron concentration in the duodenal mucosa. In contrast, feeding rats an iron-deficient diet was associated with a 4.6-fold decrease in ulcer formation, accompanied by a 34% decrease (P < 0.05) in the duodenal mucosal iron concentration. Administration of deferoxamine inhibited ulceration by 65%. We also observed that the antiulcer effect of H2 receptor antagonist cimetidine included a 35% decrease in iron concentration in the duodenal mucosa. Cysteamine-induced duodenal ulcers were also decreased in iron-deficient Belgrade rats (P < 0.05). In normal rats, cysteamine administration increased the iron concentration in the proximal duodenal mucosa by 33% in the preulcerogenic stage but at the same time decreased serum iron (P < 0.05). Cysteamine also enhanced activation of mucosal iron regulatory protein 1 and increased the expression of divalent metal transporter 1 mRNA and protein. Transferrin receptor 1 protein expression was also increased, although mucosal ferroportin and ferritin remained almost unchanged. These results indicate an expansion of the intracellular labile iron pool in the duodenal mucosa, increasing its susceptibility to oxidative stress, and suggest a role for iron in the pathogenesis of organ-specific tissue injury such as duodenal ulcers.

Neutralizing anti-vascular endothelial growth factor (VEGF) antibody reduces severity of experimental ulcerative colitis in rats: direct evidence for the pathogenic role of VEGF.

UNLABELLED: 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 microg/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 mm(2) in saline- or IgG-treated groups to 122 +/- 57 mm(2) 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.

Detection of duodenal ulcer-associated genes in rats.

UNLABELLED: We assessed the expression of about 8,000 known or unknown genes in the preulcerogenic stages of cysteamine-induced duodenal ulceration in rats, in comparison with the toxic but nonulcerogen ethanolamine. The most prominent gene changes were confirmed by custom gene blots, reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, radio-immunoassay, Western blot, or enzyme-linked immunosorbent assay (ELISA), and the levels of their expression in other gastrointestinal organs such as ileum and colon were identified by real-time PCR. The time-course study after cysteamine showed 40 genes with marked changes, belonging to cell surface antigens, transcription factors, DNA binding proteins, ion channels, transport proteins, cellular receptors, and expressed sequence tags (i.e., unknown genes). In comparison with ethanolamine, these 40 genes changed by cysteamine only may represent ulcer-associated genes, such as endothelin receptor B, endothelin 1, caspase 3, transcription factors egr-1, Sp1, the angiogenic growth factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and especially egr-1 and endothelin receptor B (ETRB) showed no changes in ileum and colon. CONCLUSIONS: (1) These data suggest that duodenal ulcerogenesis may require the interaction of several genes leading to endothelial and epithelial cell injury, mucosal erosion, and ulcer; (2) these new findings may offer a new approach to the identification of potential ulcerogenic genes and provide new insights into the molecular mechanisms of duodenal ulceration.

Aging gastropathy-novel mechanisms: hypoxia, up-regulation of multifunctional phosphatase PTEN, and proapoptotic factors.

BACKGROUND & AIMS: Aging gastric mucosa has impaired mucosal defense and increased susceptibility to injury. Our aims were to determine the mechanisms responsible for above abnormalities. METHODS: We used Fisher F-344 rats, 3 and 24 months of age. We measured gastric mucosal blood flow; visualized mucosal hypoxia; examined expression of early growth response-1 transcription factor and phosphatase and tensin homologue deleted on chromosome 10 (PTEN); assessed apoptosis; and determined expression of caspase-3, caspase-9, and survivin. We also examined susceptibility of gastric mucosa of young and aging rats to ethanol injury and whether down-regulation of PTEN affects susceptibility of aging gastric mucosa to injury. To determine human relevance, we examined expression of PTEN and survivin in human gastric specimens of young and aging individuals. RESULTS: Gastric mucosa of aging (vs young) rats has a 60% reduction in mucosal blood flow; prominent hypoxia; and increased early growth response-1 transcription factor and PTEN messenger RNAs, and proteins. It also has increased expression of proapoptotic proteins caspase-3 and capase-9, reduced survivin, and a 6-fold increased apoptosis vs mucosa of young rats. Ethanol-induced gastric mucosal injury in aging (vs young) rats was significantly increased. The down-regulation of PTEN in gastric mucosa of aging rats completely reversed its increased susceptibility to ethanol injury. In aging human gastric mucosa, PTEN expression was significantly increased, whereas survivin was significantly reduced. CONCLUSIONS: (1) Gastric mucosa of aging rats has significantly reduced blood flow, tissue hypoxia, activation of Egr-1, PTEN; increased caspases; and reduced survivin. (2) These changes increase susceptibility of aging gastric mucosa to injury.

New molecular mechanisms of duodenal ulceration.

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-1alpha). 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.

Suppression of early growth response factor-1 with egr-1 antisense oligodeoxynucleotide aggravates experimental duodenal ulcers.

Previously, we demonstrated that cysteamine releases endothelin-1 in the rat duodenal mucosa, followed by increased expression of early growth response factor-1 (egr-1). We hypothesized that egr-1 is a key mediator gene in the multifactorial mechanisms of duodenal ulcer development and healing because its protein, transcription factor product Egr-1, regulates the expression of angiogenic growth factors. We wanted to determine the effect of egr-1 antisense oligonucleotide on cysteamine-induced duodenal ulcers as well as on the expression of bFGF, PDGF, and VEGF, of which synthesis is modulated by Egr-1. An antisense oligonucleotide to egr-1 was used to inhibit the synthesis of Egr-1 and to determine its effect on ulcer formation in the rat model of cysteamine-induced duodenal ulceration. Real-time RT-PCR and Western blot analysis were used to assess the expression of Egr-1 mRNA and protein as well as ERK, bFGF, PDGF, and VEGF. The antisense Egr-1 oligonucleotide inhibited the expression of egr-1 mRNA and protein and increased the duodenal ulcer size from 8.1 +/- 1.8 mm(2) in controls to 20.7 +/- 4.0 mm(2) (P < 0.01). Cysteamine induced phosphorylation of ERK1/2 and enhanced the synthesis of bFGF, PDGF, and VEGF in the preulcerogenic stages of duodenal ulceration, whereas egr-1 antisense oligonucleotide markedly decreased the expression of these growth factors in the duodenal mucosa. We also demonstrated that Egr-1 expression relates to the ulcerogenic effect of cysteamine because these actions were not exerted by the toxic analog ethanolamine. Thus Egr-1 seems to play a critical role in duodenal ulceration because Egr-1 downregulation aggravates experimental duodenal ulcers, most likely through the transcriptional inhibition of bFGF, PDGF, and VEGF synthesis.

Antiangiogenic drugs synergize with a membrane macrophage colony-stimulating factor-based tumor vaccine to therapeutically treat rats with an established malignant intracranial glioma.

Combining a T9/9L glioma vaccine, expressing the membrane form of M-CSF, with a systemic antiangiogenic drug-based therapy theoretically targeted toward growth factor receptors within the tumor's vasculature successfully treated >90% of the rats bearing 7-day-old intracranial T9/9L gliomas. The antiangiogenic drugs included (Z)-3-[4-(dimethylamino)benzylidenyl]indolin-2-one (a platelet-derived growth factor receptor beta and a fibroblast growth factor receptor 1 kinase inhibitor) and oxindole (a vascular endothelial growth factor receptor 2 kinase inhibitor). A total of 20-40% of the animals treated with the antiangiogenic drugs alone survived, while all nontreated controls and tumor vaccine-treated rats died within 40 days. In vitro, these drugs inhibited endothelial cells from proliferating in response to the angiogenic factors produced by T9/9L glioma cells and prevented endothelial cell tubulogenesis. FITC-labeled tomato lectin staining demonstrated fewer and constricted blood vessels within the intracranial tumor after drug therapy. Magnetic resonance imaging demonstrated that the intracranial T9 glioma grew much slower in the presence of these antiangiogenic drugs. These drugs did not affect in vitro glioma cell growth nor T cell mitogenesis. Histological analysis revealed that the tumor destruction occurred at the margins of the tumor, where there was a heavy lymphocytic infiltrate. Real-time PCR showed more IL-2-specific mRNA was present within the gliomas in the vaccinated rats treated with the drugs. Animals that rejected the established T9/9L glioma by the combination therapy proved immune against an intracranial rechallenge by T9/9L glioma, but showed no resistance to an unrelated MADB106 breast cancer.