Vascular endothelial growth factor increases pulmonary vascular permeability in cystic fibrosis patients undergoing lung transplantation.

Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood. The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1alpha, beta, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation. VEGF-A165 and Flt-1 mRNA expression (P<0.05), VEGF-A (P<0.05), and HIF-1alpha (P<0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P<0.001) and extravascular lung water content (P<0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P<0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P<0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P<0.001). These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.

Krenn K ，Klepetko W ，Taghavi S ，Paulus P ，Aharinejad S ... -  《EUROPEAN JOURNAL OF CARDIO-THORACIC SURGERY》

Upregulated hypoxia-inducible factor-1 DNA binding activity to the vascular endothelial growth factor-A promoter mediates increased vascular permeability in donor lung grafts.

Transplantation-induced hypoxia results in enhanced vascular permeability and tissue vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) overexpression in donor lung grafts. Promoter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF; and ET-1 potently stimulates VEGF-A production. We hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in lung grafts. We studied the mRNA and protein expression of HIF-1 and its protein-binding capacity to the HBS of the VEGF gene in biopsies of preserved donor and control lungs, using real-time reverse transcription-polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay. Wet-to-dry lung weight ratio was measured in donor and control lungs. While HIF-1 alpha mRNA expression was unchanged, HIF-1 beta was downregulated (p < 0.05) in donor versus control lungs. Protein expression of both, HIF-1 alpha and -beta was significantly upregulated in donor lung grafts. HIF-1 binding to the HBS of the VEGF promoter as well as tissue fluid content were increased in donor lung biopsies versus controls (p < 0.05). These data indicate that upregulated HIF-1 DNA binding activity to the HBS of VEGF-A most likely contributes to elevated VEGF levels in preserved lung grafts. Unchanged HIF-1 alpha mRNA expression did not affect HIF-1 alpha protein levels. Endothelin-1 increases HIF-1 alpha accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1 alpha protein stability most likely leads to transcriptional activation of VEGF during lung graft preservation. Targeting HIF might be of benefit to counteract edema formation in preserved lung grafts.

Abraham D ，Krenn K ，Seebacher G ，Paulus P ，Klepetko W ，Aharinejad S ... -  《ANNALS OF THORACIC SURGERY》

Effect of endothelial PAS domain protein 1 and hypoxia inducible factor 1alpha on vascular endothelial growth factor expression in human pancreatic carcinoma.

Transcription factors hypoxia inducible factor 1alpha (HIF 1alpha) and endothelial PAS domain protein 1 (EPAS1) promote the transcription of vascular endothelial growth factor (VEGF). VEGF enhances angiogenesis and vascular permeability of tumours, which promotes tumour growth and facilitates entry of cancer cells into blood circulation and metastasizing. This study examined whether HIF 1alpha and EPAS1 stimulated angiogenesis through activation of VEGF in human pancreatic carcinoma. Specimens from pancreatic carcinoma and healthy parts of same pancreas were taken from 60 patients. Real time quantitative reverse transcription polymerase chain reaction estimated expression of HIF 1alpha, EPAS1, and VEGF mRNAs. Western blotting and immunohistochemical, streptavidin peroxidase method assessed expression of HIF 1alpha, EPAS1, and VEGF proteins. Microvessel density (MVD) was assessed. Highly significant increases in expression of EPAS1, VEGF, and MVD were found in pancreatic carcinoma tissue but not in normal pancreatic tissue: VEGF at mRNA and protein levels (t = 17.32, P = 0.0001; t = 98.41, P = 0.0001); EPAS1 protein level (t = 22.51, P = 0.0001). Expression of HIF 1alpha was similar in pancreatic carcinoma and normal pancreatic tissues at both mRNA and protein levels. Significant correlations were observed between EPAS1 and VEGF (r = 0.736, P = 0.0041), between VEGF and MVD (r = 0.858, P = 0.0001), and between EPAS1 and MVD (r = 0.641, P = 0.0003). No significant correlations were observed between HIF 1alpha and VEGF, or between HIF 1alpha and MVD. MVD and expression of EPAS1 and VEGF were significantly related with TNM staging, so was EPASI and VEGF with size of tumour. EPAS1 and VEGF, but not HIF1alpha, are overexpressed in pancreatic carcinoma. The expression of EPAS1 is correlated with that of VEGF and MVD. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF. Targeting EPAS1 may be a new method of antiangiogenic tumour therapy for pancreatic carcinoma.

Zhu DM ，Li DC ，Zhang ZX ，Zhang XY ... -  《-》

[Expression of hypoxia-inducible factor-1alpha, vascular endothelial growth factor and sFlt-1 in preeclampsia placenta].

To investigate the expression and correlation of hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and sFlt-1 in the preeclampsia placenta, and discuss their significance in the pathogenesis of preeclampsia. Placentas were collected from 20 pregnant women with preeclampsia as study group and 15 normal pregnant women as control group. The expressions of HIF-1alpha, VEGF and sFlt-1 protein were semi-quantitatively analyzed with immunohistochemical assay and mRNA level was determined using reverse transcription polymerase chain reaction (RT-PCR) technique. (1) the expression of HIF-1alpha and sFlt-1 protein in preeclampsia group obviously increased. Strong (+++) positive expression was observed in 9 and 11 cases respectively, significantly higher than in control group (2 and 3 cases) (P < 0.05), however, VEGF expression obviously reduced in preeclampsia group (P < 0.01). (2) the level of HIF-1alpha and sFlt-1 mRNA in preeclamptic placenta was 0.604 +/- 0.013, 0.898 +/- 0.041, significantly higher than 0.208 +/- 0.007 and 0.559 +/- 0.244 in normal placenta (P < 0.05). Although the level of VEGF mRNA increased in preeclampsia placenta, it was not significantly different from that in normal placenta (P > 0.05). The ratio of VEGF mRNA/sFlt-1mRNA obviously reduced in preeclampsia group and was significantly lower than in control group (P < 0.05). (3) in preeclampsia group, HIF-1alpha mRNA expression was positively correlated with the expression of sFlt-1 mRNA (r = 0.577, P < 0.05), and negatively correlated with the ratio of VEGF mRNA/sFlt-1 mRNA (r = -0.376, P < 0.05). Abnormal high HIF-1alpha expression in preeclampsia placenta indicates that HIF-1alpha might play an important role in the pathogenesis of preeclampsia, possibly through affecting the cytotrophoblastic invasion and placental vascular reconstruction via the modulation of VEGF and sFlt-1 gene transcription.

Sun SG ，Shen N ，Zheng YH ，Shang T ... -  《-》

Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease.

Ongoing inflammatory processes resulting in airway and vascular remodelling characterise chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) could play a role in tissue remodelling and angiogenesis in COPD. The cellular expression pattern of VEGF, Flt-1, and KDR/Flk-1 was examined by immunohistochemistry in central and peripheral lung tissues obtained from ex-smokers with COPD (forced expiratory volume in 1 second (FEV(1)) <75% predicted; n = 14) or without COPD (FEV(1) >85% predicted; n = 14). The immunohistochemical staining of each molecule was quantified using a visual scoring method with grades ranging from 0 (no) to 3 (intense). VEGF, Flt-1, and KDR/Flk-1 immunostaining was localised in vascular and airway smooth muscle (VSM and ASM) cells, bronchial, bronchiolar and alveolar epithelium, and macrophages. Pulmonary endothelial cells expressed Flt-1 and KDR/Flk-1 abundantly but not VEGF. Bronchial VEGF expression was higher in microvascular VSM cells and ASM cells of patients with COPD than in patients without COPD (1.7 and 1.6-fold, p<0.01, respectively). VEGF expression in intimal and medial VSM (1.7 and 1.3-fold, p<0.05) of peripheral pulmonary arteries associated with the bronchiolar airways was more intense in COPD, as was VEGF expression in the small pulmonary vessels in the alveolar region (1.5 and 1.7-fold, p<0.02). In patients with COPD, KDR/Flk-1 expression was enhanced in endothelial cells and in intimal and medial VSM (1.3, 1.9 and 1.5-fold, p<0.02) while endothelial Flt-1 expression was 1.7 times higher (p<0.03). VEGF expression was significantly increased in bronchiolar and alveolar epithelium as well as in bronchiolar macrophages (1.5-fold, p<0.001). The expression of VEGF in bronchial VSM and mucosal microvessels as well as bronchiolar epithelium was inversely correlated with FEV(1) (r<-0.45; p<0.01). VEGF and its receptors Flt-1 and KDR/Flk-1 may be involved in peripheral vascular and airway remodelling processes in an autocrine and/or paracrine manner. This system may also be associated with epithelial cell viability during airway wall remodelling in COPD.

Kranenburg AR ，de Boer WI ，Alagappan VK ，Sterk PJ ，Sharma HS ... -  《-》