Intestinal Vascular Endothelial Growth Factor Research Articles

Recombinant IGF-1/BP3 protects against intestinal injury in a neonatal mouse NEC model.

Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP3) is currently being tested in phase II clinical trials in premature infants to prevent bronchopulmonary dysplasia, but its impact on the neonatal intestine remains unclear. The aim of this study was to determine whether rhIGF-1/BP3 protects against necrotizing enterocolitis (NEC) in mice and to investigate the mechanisms involved. Neonatal mice were dam fed or injected intraperitoneally with rhIGF-1/BP3 (or vehicle) and submitted to an experimental NEC model. Serum IGF-1 was assessed by ELISA and intestinal vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression by Western blot. Intestinal endothelial cell proliferation, and enterocyte proliferation and migration were examined by immunofluorescence. Pup survival and histological intestinal injury were determined. In pups exposed to experimental NEC, serum IBP3-bound IGF-1 level was decreased. Exogenous rhIGF-1/BP3 preserved VEGF and VEGFR2 protein expression, decreased vascular permeability, and preserved endothelial cell proliferation in the small intestine. Furthermore, rhIGF-1/BP3 promoted enterocyte proliferation and migration, which effects were attenuated by inhibiting VEGFR2 signaling, decreased enterocyte apoptosis and decreased systemic and intestinal inflammation. rhIGF-1/BP3 improved survival and reduced the incidence of severe intestinal injury in experimental NEC. Exogenous rhIGF-1/BP3 protects neonatal mice against experimental NEC via multiple mechanisms. Exogenous rhIGF-1/BP3 preserves intestinal microvascular development and integrity, promotes enterocyte proliferation and migration, decreases local and systemic inflammation, and protects neonatal mice against NEC. The article adds pre-clinical evidence of a protective role for rhIGF-1/BP3 on the premature gut. It provides evidence supporting the use of rhIGF1/BP3 in premature neonates to protect against NEC.

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.

Interleukin-6 Therapy Improves Intestinal Recovery Following Ischemia

BackgroundInterleukin-6 (IL6) has both proinflammatory and anti-inflammatory pathways, but its effects on intestinal recovery following ischemia are unknown. We hypothesized that administration of IL6 following intestinal ischemia would improve mesenteric perfusion and mucosal injury. MethodsAdult male C57Bl6J mice were anesthetized, and a laparotomy was performed. Baseline intestinal perfusion was assessed by laser Doppler imaging. Intestinal ischemia was induced for 60 min by temporarily occluding the superior mesenteric artery. After ischemia, treatments were administered intraperitoneally before closure (Vehicle: 250 μL phosphate-buffered-saline, IL6 low dose (20 ng), IL6 medium dose (200 ng), or IL6 high dose (2 μg)). Animals were allowed to recover for 24 h, were reanesthetized, and their mesenteric perfusion was reassessed. Perfusion was expressed as percentage of baseline. Animals were then sacrificed, and the intestines were explanted for histological analysis. Separate frozen samples were homogenized and analyzed by ELISA for vascular endothelial growth factor (VEGF) and interferon gamma-induced protein 10. ResultsIL6 increased mesenteric perfusion in low dose groups only, whereas it improved postischemic mucosal injury scores in both low and medium dose groups. No differences in perfusion or histology were seen when high dose IL6 was utilized. Intestinal VEGF was higher in the low dose IL6 group compared to vehicle, whereas IP-10 levels were lower in low and medium dose groups compared to vehicle. No differences were noted compared to vehicle in intestinal VEGF and IP-10 with high dose IL6 therapy. ConclusionsLower doses of IL6 may serve as effective therapy to decrease intestinal injury after ischemia. Further studies are needed to elucidate the downstream mechanisms before widespread clinical use.

Human mesenchymal stromal cells decrease mortality after intestinal ischemia and reperfusion injury

BackgroundCellular therapy is a novel treatment option for intestinal ischemia. Bone marrow–derived mesenchymal stromal cells (BMSCs) have previously been shown to abate the damage caused by intestinal ischemia/reperfusion (I/R) injury. We therefore hypothesized that (1) human BMSCs (hBMSCs) would produce more beneficial growth factors and lower levels of proinflammatory mediators compared to differentiated cells, (2) direct application of hBMSCs to ischemic intestine would decrease mortality after injury, and (3) decreased mortality would be associated with an altered intestinal and hepatic inflammatory response. MethodsAdult hBMSCs and keratinocytes were cultured on polystyrene flasks. For in vitro experiments, cells were exposed to tumor necrosis factor, lipopolysaccharides, or 2% oxygen for 24 h. Supernatants were then analyzed for growth factors and chemokines by multiplex assay. For in vivo experiments, 8- to 12-wk-old male C57Bl6J mice were anesthetized and underwent a midline laparotomy. Experimental groups were exposed to temporary superior mesenteric artery occlusion for 60 min. Immediately after ischemia, 2 × 106 hBMSCs or keratinocytes in phosphate-buffered saline were placed into the peritoneal cavity. Animals were then closed and allowed to recover for 6 h (molecular/histologic analysis) or 7 d (survival analysis). After 6-h reperfusion, animals were euthanized. Intestines and livers were harvested and analyzed for inflammatory chemokines, growth factors, and histologic changes. ResultshBMSCs expressed higher levels of human interleukin (IL) 6, IL-8, vascular endothelial growth factor (VEGF), and epidermal growth factor and lower levels of IL-1, IL-3, IL-7, and granulocyte-monocyte colony-stimulating factor after stimulation. In vivo, I/R resulted in significant mortality (70% mortality), whereas application of hBMSCs after ischemia decreased mortality to 10% in a dose-dependent fashion (P = 0.004). Keratinocyte therapy offered no improvements in mortality above I/R. Histologic profiles were equivalent between ischemic groups, regardless of the application of hBMSCs or keratinocytes. Cellular therapy yielded significantly decreased murine intestinal levels of soluble activin receptor-like kinase 1, betacellulin, and endothelin, whereas increasing levels of eotaxin, monokine induced by gamma interferon (MIG), monocyte chemoattractant protein 1, IL-6, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein 10 (IP-10) from ischemia were appreciated. hBMSC therapy yielded significantly higher expression of murine intestinal VEGF and lower levels of intestinal MIG compared to keratinocyte therapy. Application of hBMSCs after ischemia yielded significantly lower murine levels of hepatic MIG, IP-10, and G-CSF compared to keratinocyte therapy. ConclusionsHuman BMSCs produce multiple beneficial growth factors. Direct application of hBMSCs to the peritoneal cavity after intestinal I/R decreased mortality by 60%. Improved outcomes with hBMSC therapy were not associated with improved histologic profiles in this model. hBMSC therapy was associated with higher VEGF in intestines and lower levels of proinflammtory MIG, IP-10, and G-CSF in liver tissue after ischemia, suggesting that reperfusion with hBMSC therapy may alter survival by modulating the systemic inflammatory response to ischemia.

Prenatal Exposure to Inflammation Decreases Endothelial Cell Proliferation in the Mouse Intestinal Mucosa

Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal disease affecting premature infants. Chorioamnionitis has been shown to increase its incidence. However, the mechanism by which prenatal infection increases susceptibility to NEC remains elusive. Our lab has shown that VEGF is decreased in human NEC. VEGF may play a role in NEC pathogenesis by compromising vascular network integrity. Here we hypothesized that antenatal inflammation decreases the expression of vascular endothelial growth factor (VEGF) in intestine tissues and mucosal vascular endothelial cell proliferation. To test our hypothesis, pregnant mice were injected i.p. at E16 with LPS or normal saline. VEGF protein of Day 0 pup intestines was analyzed by western blot and band density values normalized to b‐actin. To quantify endothelial cell (E.C.) proliferation, pups were injected with BrdU i.p. 4 hours prior to euthanasia, and the number of BrdU positive cells co‐staining for CD31, an endothelial cell marker, were counted in the villi (3 fields/ sample at 10x magnification). We found that intestinal VEGF expression was significantly lower in pups from LPS‐treated dams compared to controls (mean±SEM: 1.40 ± 0.14 v.s 2.80 ± 0.15; p<0.0005). Also, the number of proliferating endothelial cells was significantly decreased in newborn pups from LPS‐treated dams compared to controls (8.0 ± 0.88 v.s 26.0 ± 1.60; p<0.0001). In conclusion, prenatal exposure to inflammation decreases intestinal VEGF protein and intestinal mucosal endothelial cell proliferation. This suggests that antenatal inflammation may predispose to intestinal injury and NEC by affecting mucosal microvasculature (supported by NIH grant RO1HD060876).

Intestinal Vascular Endothelial Growth Factor Is Decreased in Necrotizing Enterocolitis

Background: Decreased intestinal perfusion may contribute to the development of necrotizing enterocolitis (NEC). Vascular endothelial growth factor (VEGF) is an angiogenic protein necessary for the development and maintenance of capillary networks. Whether VEGF is dysregulated in NEC remains unknown. Objectives: The objective of this study was to determine whether intestinal VEGF expression is altered in a neonatal mouse model of NEC and in human NEC patients. Methods: We first assessed changes of intestinal VEGF mRNA and protein in a neonatal mouse NEC model before significant injury occurs. We then examined whether exposure to formula feeding, bacterial inoculation, cold stress and/or intermittent hypoxia affected intestinal VEGF expression. Last, we visualized VEGF protein in intestinal tissues of murine and human NEC and control cases by immunohistochemistry. Results: Intestinal VEGF protein and mRNA were significantly decreased in pups exposed to the NEC protocol compared to controls. Hypoxia, cold stress and commensal bacteria, when administered together, significantly downregulated intestinal VEGF expression, while they had no significant effect when given alone. VEGF was localized to a few single intestinal epithelial cells and some cells of the lamina propria and myenteric plexus. VEGF staining was decreased in murine and human NEC intestines when compared to control tissues. Conclusion: Intestinal VEGF protein is reduced in human and experimental NEC. Decreased VEGF production might contribute to NEC pathogenesis.

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

ObjectiveBurn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.MethodsRats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO2 cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.ResultsBurn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.ConclusionsThese results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.

Enhancing apoptotic cell clearance mitigates bacterial translocation and promotes tissue repair after gut ischemia-reperfusion injury

A key aspect of intestinal ischemia/reperfusion (I/R) injury is the increased occurrence of apoptotic cell death in the gut. Insufficient clearance of apoptotic cells leads to increased inflammation and impaired tissue repair. Our recent studies have shown that administration of milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a crucial molecule for apoptotic cell clearance, reduces apoptosis and inflammation under various disease conditions. The purpose of this study was to determine whether MFG-E8 reduces bacterial translocation and promotes tissue repair in a mouse model of gut I/R. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 90 min in male adult mice. After removing the clip, recombinant murine MFG-E8 (rmMFG-E8) (0.4 μg/20 g BW) or normal saline (Vehicle) was intraperitoneally injected. At 4 h after reperfusion, apoptosis in the gut was measured by TUNEL staining. The mesenteric lymph node (MLN) complex was homogenized and plated on chocolate agar plates for bacterial culture. Neutrophil infiltration was assessed by examining myeloperoxidase (MPO) activity in the gut. Vascular endothelial growth factor (VEGF) levels in the gut, an indicator of tissue repair, were measured by western blotting. Out results showed that TUNEL-positive staining in the gut increased significantly in gut I/R vehicle-treated mice. Treatment with rmMFG-E8 markedly suppressed the number of apoptotic cells. Bacterial translocation to the MLN was minimal in sham mice, but was extensive in gut I/R vehicle-treated mice. rmMFG-E8 treatment significantly reduced bacterial translocation to the MLN. Similarly, gut I/R induced a significant increase in intestinal MPO activities in vehicle-treated mice. rmMFG-E8 treatment markedly reduced the increase in intestinal MPO activities after gut I/R. Intestinal levels of VEGF decreased significantly at 4 h after gut I/R. rmMFG-E8 treatment significantly increased intestinal VEGF levels. Thus, enhancing apoptotic cell clearance by rmMFG-E8 mitigates bacterial translocation, inhibits neutrophil infiltration and promotes tissue repair after gut I/R. Enhancing apoptotic cell clearance can be a novel concept in the treatment of gut I/R injury.

Vascular Endothelial Growth Factor Protein is Decreased in a Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a severe intestinal disease affecting premature infants. Its pathogenesis remains poorly understood. Vascular endothelial growth factor (VEGF) is an angiogenic protein that plays a role in several pathologic processes. While VEGF polymorphisms have been associated with NEC, the role of VEGF in the pathogenesis of NEC remains unknown. Here, we examined the developmental expression of VEGF in the mouse intestine and in a neonatal mouse NEC model. To determine VEGF developmental expression, pups were sacrificed at embryonic day (E) 16, postnatal day (D) 0 or 3. To examine intestinal VEGF in our NEC model, animals were submitted to the NEC protocol for 48 hours or allowed to be dam fed. Intestinal VEGF protein was assessed by western blot followed by band densitometry (values were normalized to β‐actin). Compared to D0, only a low amount of VEGF was detected in the mouse intestine at E16 (2.6±0.42 % of D0 value; p<0.05). At D3, there was a marginal increase in VEGF compared to D0 (194 ±20 %), although this was not statistically significant. In pups exposed to the NEC protocol, intestinal VEGF was not detectable while it was strongly present in dam fed controls. In conclusion, we found that intestinal VEGF is increased after birth in dam fed pups, but undetectable at 48 hours in our neonatal mouse NEC model. Therefore, we speculate that VEGF plays a role in the pathogenesis of this disease.

P306 - Targeting angiogenesis in colitis-associated cancer

P306 - Targeting angiogenesis in colitis-associated cancer

P307 - Increased intestinal vascular endothelial growth factor expression and mucosal vascularization in patients with spondylarthropathy

P307 - Increased intestinal vascular endothelial growth factor expression and mucosal vascularization in patients with spondylarthropathy

Implication of STAT3 Signaling in Human Colonic Cancer Cells during Intestinal Trefoil Factor 3 (TFF3) – and Vascular Endothelial Growth Factor–Mediated Cellular Invasion and Tumor Growth

Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr(705) phosphorylation of both STAT3alpha and STAT3beta isoforms. Blockade of STAT3 signaling by STAT3beta, depletion of the STAT3alpha/beta isoforms by RNA interference, and pharmacologic inhibition of STAT3alpha/beta phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3beta down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF(165) exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.