Hypoxia-inducible factor 1 alpha expression correlates with angiogenesis and unfavorable prognosis in bladder cancer

INTRODUCTION AND OBJECTIVES: Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is a critical regulatory protein of cellular response to hypoxia and is closely related to the triggering of the angiogenic process. We examined the relationship between hypoxia and angiogenesis, as well as their prognostic impact in patients with urothelial bladder cancer. METHODS: The immunohistochemical expression of HIF-1 alpha was evaluated in 93 formalin-fixed paraffin-embedded primary transitional cell carcinoma tissue samples. HIF-1 alpha was recognized through nuclear staining of positive cells. The angiogenic profile was individually assessed immunohistochemically using a monoclonal antibody to vascular endothelial growth factor (VEGF) and microvessel density (MVD) was calculated with immunohistochemical staining of the adhesion molecule CD31 of the endothelial cells. RESULTS: A significant positive association between HIF-1 alpha immunoreactivity and histological grade (p=0.009) was found. VEGF and MVD were closely related to tumor grade (p=0.06 and p<0.001) and clinical stage (p=0.04 and p<0.01, respectively). HIF-1 alpha was significantly correlated with VEGF expression (p=0.01) and MVD (p<0.001). Patients characterized by HIF-1 alpha overexpression had significantly worse overall (p=0.009) and disease-free survival (p=0.03). When HIF-1 alpha, histologic grade and stage were included in multivariate Cox regression analysis, HIF-1 alpha emerged as an independent prognostic factor (p=0.02) along with grade and stage, but lost its independent prognostic value after the inclusion of angiogenic factors in the multivariate model. In the subgroup of patients with T1 disease, HIF-1 alpha emerged as a significant negative predictor of the time to first recurrence. CONCLUSIONS: HIF-1 alpha and angiogenesis markers may play an important predictive and prognostic role in patients with bladder cancer. HIF-1 alpha may be of biologic and clinical value as its overexpression is related to up-regulation of VEGF, the stimulation of angiogenesis and worse prognosis.     

Expression of hypoxia-inducible angiogenic proteins (hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and E26 transformation-specific-1) and plaque hemorrhage in human carotid atherosclerosis

OBJECT: Plaque hemorrhage in carotid atherosclerosis promotes plaque progression, resulting in cerebrovascular disease. Hypoxia inducible factor-1alpha (HIF-1alpha) induces angiogenesis via the expression of vascular endothelial growth factor (VEGF) and E26 transformation-specific-1 (Ets-1). The authors investigated human carotid plaques to determine whether these hypoxia-inducible angiogenic proteins play a major role in intraplaque angiogenesis and hemorrhage. METHODS: The expression of HIF-1alpha, VEGF, and Ets-1 was analyzed using immunohistochemistry and Western blotting in 29 human carotid plaques obtained at carotid endarterectomy. The authors investigated the relationship between plaque characteristics and clinical symptoms. RESULTS: A higher incidence of plaque hemorrhage was observed in plaques associated with symptoms than in those without symptoms (p = 0.03). Hypoxia-inducible factor-1alpha, VEGF, and Ets-1 coexisted in the deep layer of plaque, where angiogenesis was remarkably developed; the expression levels of HIF-1alpha, VEGF, and Ets-1 were significantly enhanced in the main lesion of the plaque (p < 0.01). Symptomatic plaques showed higher expression of VEGF (p = 0.04) than asymptomatic plaques. Plaques with hemorrhage showed a higher incidence of plaque ulcer (p = 0.001) and higher expression of Ets-1 (p = 0.03) than those without hemorrhage. Moreover, significantly increased expressions of VEGF (p = 0.01) and Ets-1 (p = 0.006) were observed in plaques with not only hemorrhages but also ulcers and severe stenosis. CONCLUSIONS: The findings in this study suggest that hypoxia-inducible angiogenic proteins in human carotid atherosclerosis promote intraplaque angiogenesis, which can induce plaque hemorrhage and progression.     

Caveolin-1在一氧化氮介导的三维纤维蛋白凝胶血管新生模型中的作用

目的 观察Caveolin-1( Cav-1)在一氧化氮(N0)介导的血管新生中的作用.方法 建立人脐静脉内皮细胞(HUVECs)三维纤维蛋白凝胶血管新生模型.通过管腔形成指数(TFI)来定量分析毛细血管管腔样结构,以亚硝酸盐来反映NO的产生量.根据是否添加血管内皮生长因子(VEGF)和N-硝基-L-精氨酸(L-NAME)分为VEGF组、VEGF+L-NAME组和对照组.HUVECs转染反义寡核苷酸( ASON)和无义寡核苷酸(NSON),并将其分为ASON组、NSON组和对照组.结果 VEGF组TFI和亚硝酸盐分别为(3.15±0.46)、(0.36±0.05) mmol/L,明显高于对照组(0.89±0.09)、(0.18 ±0.02) mmol/L(P＜0.05).VEGF+ L-NAME组TFI和亚硝酸盐分别为(1.06±0.13)、(0.21±0.02) mmol/L,与对照组比较差异无统计学意义(P＞0.05).HUVECs在三维纤维蛋白凝胶内培养后Cav-1的表达水平逐渐升高.ASON组TFI(1.28±0.15)明显低于对照组(3.27 ±0.33)和NSON组(3.05±0.31) (P ＜0.05),转染ASON的HUVECs添加VEGF和L-NAME后TFI和亚硝酸盐无明显改变,差异有统计学意义(P＞0.05).结论 Cav-1对NO介导的血管新生起非常重要的调节作用.     

The correlation between nitric oxide and vascular endothelial growth factor in spinal cord injury

STUDY DESIGN: Prospective, randomized, placebo-controlled, experimental study. OBJECTIVES: The issue of whether nitric oxide (NO) production is beneficial or deleterious on ischemic injuries of the central nervous system still remains doubtful. Vascular endothelial growth factor (VEGF) is known to induce the release of NO from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. We aimed to determine the effects of L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) on VEGF synthesis and free radicals in a rat model of spinal cord ischemia-reperfusion (IR) injury. SETTING: Surgical Research Laboratory of a Medical School. MATERIAL AND METHODS: Twenty-eight Wistar rats were divided into four groups as follows (n=7): Sham, IR injury, L-arginine, and L-NAME. Infrarenal abdominal aorta was occluded to induce spinal cord ischemia. L-Arginine (100 mg/kg) and L-NAME (10 mg/kg) were given before aortic occlusion. Biochemical assays of malondialdehyde (MDA), NO and VEGF were carried out in spinal cord specimens. RESULTS: L-Arginine treatment significantly increased MDA and NO, but decreased VEGF levels in spinal cord. However, nonselective inhibition of NOS with L-NAME significantly decreased MDA and NO, but increased VEGF levels. Besides, the positive linear correlation between MDA and NO, and negative linear correlations between MDA, NO and VEGF levels have also been demonstrated. CONCLUSION: Nonselective inhibition of NO synthase activity with L-NAME attenuated free radical formation and increased VEGF level when compared with NO precursor L-arginine in a rat model of spinal cord ischemia. We suggest that inhibition of NO synthase, as well as induction of VEGF, may be a therapeutic option in spinal cord IR injury.     

The effect of chemical hemodynamic regulation on the survival of arterialized venous flaps

The role of nitric oxide (NO) on the microcirculation of arterialized venous flaps (AVFs) remains controversial. We aimed to investigate the effect of hemodynamic regulation using nitric oxide synthase (NOS) inhibitor and its agonist as a chemical intervention on the survival of AVF. A 10?×?8?cm arterialized venous flap was designed symmetrically on the rabbit abdomen. Thirty-six rabbits were used and randomly divided into three groups: control group, L-arg group and L-NAME group, respectively. The L-arg group and the L-NAME group received intraperitoneal injections of L-arginine (a NOS agonist, 1?g/kg/d) and L-NAME (nitro-L-arginine-methyl ester, a NOS inhibitor, 50?mg/kg/d) respectively, whereas the control group received intraperitoneal injections of the same amount of saline. Flap viability, water content, status of vascular perfusion and gene expression of eNOS and HIF-1α in each group were observed and analyzed. The average value of water content (venous congestion) in the L-arg group was the highest in comparison with the control group and the L-NAME group with a statistically significant difference (all p?相似文献   

VEGF and VEGF receptors are differentially expressed in chondrocytes

During long bone development, cartilage replacement by bone is governed in part by angiogenesis. Although it has been demonstrated that vascular endothelial growth factor (VEGF-A) is crucial during endochondral ossification, little is known about the involvement of the other VEGF family members. Thus, we examined the expression and production of these members on primary chondrocytes and ATDC5 chondrogenic cells. VEGF-A, VEGF-B, VEGF-C and VEGF-D were shown to be expressed and synthesized demonstrating that numerous angiogenic factors can be produced by chondrocytes. In ATDC5 VEGF-A, VEGF-B and VEGF-C were over-expressed in the presence of chondrogenic and bone morphogenetic protein (BMP)-2 treatment suggesting that these factors play an important role during chondrogenesis. In addition, neuropilin-1, VEGF receptor-2 and VEGF receptor-3 gene expression were observed with an increase in VEGF-R2 expression under chondrogenic and BMP-2 treatment, suggesting that VEGF proteins could act in an autocrine/paracrine manner in addition to their angiogenic function. In conclusion, we demonstrated for the first time that chondrocytes secreted the four members of the VEGF family. We also showed that VEGF-B, VEGF-C and VEGF-D were secreted as processed proteins. The up-regulation of VEGF-B and VEGF-C at the mRNA and protein levels under chondrogenic stimulation strongly suggests a major role for these proteins in growth plate physiology.     

Uncoupling of vascular endothelial growth factor with nitric oxide as a mechanism for diabetic vasculopathy

The role of VEGF in vascular disease is complicated. Vascular endothelial growth factor (VEGF) expression can be deleterious in diabetic vasculopathy, especially in kidney and retina. In contrast, VEGF seems to be renoprotective in nondiabetic renal disease. VEGF exerts it biologic effects in association with nitric oxide (NO), yet it is known that NO bioavailability is reduced in diabetes. Thus, it was hypothesized that this diverse biologic effect of VEGF on diabetic vasculopathy is due to uncoupling of VEGF with NO. VEGF stimulated NO production in a dose-dependent manner in bovine aortic endothelial cells (BAEC), and this was inhibited by either high glucose or Nomega-nitro-l-arginine methyl ester (L-NAME) treatment. Endothelial NO synthase phosphorylation by VEGF was also inhibited by high glucose. It is interesting that both high glucose and L-NAME enhanced the proliferative response of endothelial cells, which was prevented by an NO donor. Furthermore, high glucose as well as L-NAME stimulated VEGF and kinase-insert domain receptor (KDR) (VEGF receptor 2) mRNA expression in BAEC. These data suggest that the uncoupling of VEGF with NO enhances endothelial cell proliferation via the KDR pathway. Compatible with these findings, a KDR antagonist blocked this response. In addition, a VEGF mutant, which binds only KDR, induced extracellular signal-regulated kinase (ERK) activation, and inhibition of ERK completely blocked endothelial cell proliferation under this condition, suggesting a role of the KDR-ERK1/2 pathway on endothelial cell proliferation. In conclusion, high glucose causes an uncoupling of VEGF with NO, which enhances endothelial cell proliferation via activation of the KDR-ERK1/2 pathway. These results may provide new insights into the understanding of the mechanism of diabetic vascular disease.     

Nitric oxide modulates vascular endothelial growth factor and receptors in chronic cyclosporine nephrotoxicity

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing, and inflammation and exerts its effect via tyrosine kinase receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1 or KDR). We have previously shown that VEGF is up-regulated in a model of chronic cyclosporine (CsA) nephrotoxicity and that l-arginine (l-Arg) improved while N-nitro-l-arginine-methyl ester (L-NAME) worsened fibrosis. We examined the role of nitric oxide modulation on VEGF in this model. METHODS: Pair-fed salt-depleted rats were administered CsA, CsA + L-NAME, CsA +l-Arg, vehicle (VH), VH + L-NAME or VH +l-Arg and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot and the protein expression of VEGF by Western blot and immunohistochemical staining. RESULTS: While L-NAME worsened renal function and histology, l-Arg had the opposite beneficial effect in CsA-treated rats. VEGF mRNA and protein expressions increased with CsA, further increased with L-NAME and became significantly reduced with L-Arg. Flt-1 expression was similar in all groups. On the other hand, KDR/Flk-1 mRNA expression was modulated in a fashion similar to VEGF. Also, nitric oxide modulation did not have an effect on VH-treated rats. CONCLUSIONS: VEGF expression in chronic CsA nephrotoxicity is increased by nitric oxide blockade and decreased by nitric oxide enhancement. Moreover, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but it is probable that VEGF plays a role, either independently or through nitric oxide, in CsA-induced fibrosis.     

Decreased Hypoxia-Inducible Factor-1α in Gastrocnemius Muscle in Rats with Chronic Kidney Disease

Background/Aims: Hypoxia-inducible factor (HIF)-1α is responsible for increased expression of genes engaged in angiogenesis. Our previous study indicated capillary rarefaction and atrophy of glycolytic fibers, mainly in locomotor muscles of uremic animals. Perhaps these changes are secondary to disturbances of HIF-1α in skeletal muscles. Methods: Expression of HIF-1α at mRNA and protein levels, as well as mRNA of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), in gastrocnemius muscle (MG) and longissimus thoracic muscle (ML) were measured by RT-PCR and Western blot. Rats were randomized to subtotal nephrectomy (CKD5/6), uninephrectomy (CKD1/2) or sham operation (controls). Results: For CKD5/6 versus controls, mRNA levels for HIF-1α, VEGF-A, VEGFR-1 and VEGFR-2 were significantly reduced only in MG, while eNOS was significantly decreased and iNOS was significantly increased only in ML. Western blot analysis indicated significantly increased HIF-1α protein levels in MG and ML from CKD1/2 animals versus controls, whereas in the CKD5/6 group, the level of HIF-1α protein decreased significantly in MG and increased significantly in ML versus controls and CKD1/2. Conclusion: The reduced expression of HIF-1α mRNA and protein in locomotor muscle from CKD5/6 animals may be involved in the pathogenesis of uremic myopathy. Increased expression of iNOS in the postural muscles may act as a protective factor through HIF-1α stabilization.