Phenol exposure promotes tumor-related signaling and blood vessel formation through the extracellular signal-regulated kinase/p38/hypoxia-inducible factor-1α pathway in cellular and zebrafish models.

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

Purpose: To investigate the role played by E26 transformation-specific-1 (Ets-1), a transcription factor, and extracellular signal-regulated kinase 1/2 (ERK1/2) in the expression of vascular endothelial growth factor (VEGF), and the interaction of Ets-1 and ERK1/2 in the retina of diabetic rats. Methods: Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). To follow the time course in the expression of Ets-1, phosphorylated ERK1/2 (pERK1/2), and VEGF, rats were killed at 1, 2, 4, and 8 weeks after the injection of STZ, and total proteins were extracted from the isolated retinas. An adenovirus vector encoding dominant-negative Ets-1 and an inhibitor of PD98059 was injected intravitreally to investigate the effects of Ets-1 blockade and ERK1/2 inhibition on the expression of VEGF. Four weeks after the first intravitreal injection, total proteins and total RNA were extracted from the retinas for Western blot and Northern blot analyses. Results: The expression of Ets-1, pERK1/2, and VEGF in the retina increased in a time-dependent manner after STZ injection. The phosphorylation of ERK1/2 and protein level of VEGF were significantly reduced following intravitreal Ets-1. Inhibition of ERK1/2 phosphorylation resulted in a significant reduction in the expression of Ets-1 and the level of VEGF protein. Conclusions: These results indicate that in the retina of STZ-induced diabetic rats: (1) the alterations of Ets-1, pERK1/2, and VEGF are approximately synchronized; (2) the phosphorylation of ERK1/2 is regulated by the expression of Ets-1; (3) the production of Ets-1 protein is dependent on the ERK1/2 pathway, and (4) the protein level of VEGF is regulated by both Ets-1 expression and ERK1/2 phosphorylation. We propose that VEGF, Ets-1, and ERK1/2 act synergistically in the development of diabetic retinopathy.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.

Vascular Endothelial Growth Factor Localization in the Adult

Etiology of OHSS and use of dopamine agonists

Diabetic retinopathy is one of the most common causes of blindness in North America. Several signaling mechanisms are activated secondary to hyperglycemia in diabetes, leading to activation of vasoactive factors. We investigated a novel pathway, namely extracellular signal regulated kinase 5 (ERK5) mediated signaling, in modulating glucose-induced vascular endothelial growth factor (VEGF) expression. Human microvascular endothelial cells (HMVEC) were exposed to glucose. In parallel, retinal tissues from streptozotocin-induced diabetic rats were examined after 4 months of follow-up. In HMVECs, glucose caused initial activation followed by deactivation of ERK5 and its downstream mediators myocyte enhancing factor 2C (MEF2C) and Kruppel-like factor 2 (KLF2) mRNA expression. ERK5 inactivation further led to augmented VEGF mRNA expression. Furthermore, siRNA mediated ERK5 gene knockdown suppressed MEF2C and KLF2 expression and increased VEGF expression and angiogenesis. On the other hand, constitutively active MEK5, an activator of ERK5, increased ERK5 activation and ERK5 and KLF2 mRNA expression and attenuated basal- and glucose-induced VEGF mRNA expression. In the retina of diabetic rats, depletion of ERK5, KLF2 and upregulation of VEGF mRNA were demonstrated. These results indicated that ERK5 depletion contributes to glucose induced increased VEGF production and angiogenesis. Hence, ERK5 may be a putative therapeutic target to modulate VEGF expression in diabetic retinopathy.

Vascular endothelial growth factor (VEGF) is a potent mitogen and permeability factor for endothelial cells that plays a central role in angiogenesis, vascular maintenance, inflammation, and cancer. VEGF also mediates the homeostatic adaptation to hypoxic conditions by promoting an increase in vascular density to compensate for decreased oxygenation. This process is triggered by an oxygen-sensitive transcription factor, hypoxia-inducible factor-1 (HIF1alpha), which becomes active in hypoxic tissues, leading to the synthesis and secretion of VEGF. The role of HIF1alpha in other processes that involve angiogenesis such as in inflammation is less clear. Of interest, endothelial cells not only respond to but also store and secrete VEGF, which is required for the maintenance of the integrity of the vascular system. How this intracellular pool of VEGF is regulated is still not understood. Here, we found that CXCL8/IL8, a potent proangiogenic and inflammatory chemokine, up-regulates VEGF mRNA and protein levels in endothelial cells by acting on its cognate receptor, CXCR2, and that this results in the autocrine activation of VEGFR2. Surprisingly, this process does not involve HIF1alpha but instead requires the activation of the transcription factor NFkappaB. Furthermore, we identified the components of the CBM complex, Carma3, Bcl10, and Malt1, as key mediators of the CXCL8/IL8-induced NFkappaB activation and VEGF up-regulation. Together, these findings support the existence of an NFkappaB-mediated pathway by which the proinflammatory chemokine CXCL8/IL8 controls the expression of VEGF in endothelial cells, thereby promoting the activation of VEGF receptors in an autocrine fashion.

Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a). evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b). compare the prognostic significance of VEGF isoforms; and (c). analyze the combined effects of HER-2/neu and VEGF on clinical outcome. HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF(121-206) ELISA uses antibodies that bind to VEGF(121) and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF(165-206) ELISA uses antibodies that bind to VEGF(165) and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF(121-206) and VEGF(165-206) were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF(121-206) and VEGF(165-206) expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (chi(2) test: P < 0.001 for both VEGF(121-206) and VEGF(165-206)). VEGF(121-206) and VEGF(165-206) demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF(121-206), P = 0.0068; VEGF(165-206), P = 0.0046; multivariate analysis: VEGF(121-206), P = 0.1475; VEGF(165-206), P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF(121-206) and VEGF(165-206) were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF(121-206), P = 0.0003; VEGF(165-206), P = 0.0038; multivariate analysis: VEGF(121-206), P = 0.0103; VEGF(165-206), P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF(121-206), P = 0.0280; VEGF(165-206,) P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF(121-206)/VEGF(165-206) results in additional prognostic information for survival (VEGF(121-206), P = 0.0133; VEGF(165-206), P = 0.0092). The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Bone healing depends on vascular endothelial growth factor (VEGF) secretion from osteoblasts to promote angiogenesis. We examined the influence of the tyrosine 321 site of G protein-coupled receptor kinase interacting protein 1 (GIT1) on platelet-derived growth factor (PDGF)-induced VEGF synthesis in vitro and on bone healing in vivo. Cultured osteoblasts were prepared from calvaria of 1-2-day-old rats. The phospho-activation of extracellular signal-regulated kinases 1/2 (ERK1/2), GIT1, the interaction between GIT1 and ERK1/2, and VEGF mRNA expression were measured in response to PDGF. In addition, PDGF was applied following pretreatment with the MEK1/2 inhibitor PD98059 or the Src inhibitor PP2. We mutated tyrosines 293 or 321 of GIT1 individually to phenylalanine (mutants GIT1Y293F and GIT1Y321F) and incorporated these mutants and native GIT1 into lentivirus vectors. The relationship between GIT1 and ERK1/2, and VEGF mRNA expression in cultured osteoblasts were detected after infection with GIT1WT-, GIT1Y293F- and GIT1Y321F-expressing lentivirus in response to PDGF. Bone healing and expression of VEGF and the angiogenic marker PECAM-1 were evaluated after infection at the fracture site. Activation of ERK1/2 by phosphorylation, GIT1 tyrosine phosphorylation, GIT1-ERK1/2 interaction, and VEGF mRNA expression were all significantly increased in osteoblasts after PDGF stimulation, but all responses were dramatically inhibited by pretreatment with PD98059. Tyrosine phosphorylation, GIT1 interaction with ERK1/2, and VEGF mRNA expression were dramatically inhibited by pretreatment with PP2 or infection with GIT1Y321F-expressing lentivirus. Expression of VEGF and PECAM-1 was significantly lower at the fracture sites infected with GIT1Y321F-expressing lentivirus and bone healing was significantly delayed compared to fracture sites infected with GIT1WT. In conclusion, tyrosine 321 of GIT1 is a critical phosphorylation site for GIT1 interaction with ERK1/2, regulation of ERK1/2 activation, VEGF expression and angiogenesis at the fracture site. Furthermore, GIT1 is a seminal signaling protein regulating bone healing.

Different Regulation of Vascular Endothelial Growth Factor Expression by the ERK and p38 Kinase Pathways in v-ras, v-raf, and v-myc Transformed Cells

Effect of inflammatory cytokines on the expression of the vascular endothelial growth factor-C.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

Objective: To investigate the expression of von Hippel-Lindau (VHL), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) in endolymphatic sac tumor (ELST) and its clinical significance, and to analyze its association with VHL gene mutation. Methods: Twenty-four cases of ELST, which were surgically resected and diagnosed by pathological examination in Beijing Tongren Hospital Affiliated to Capital Medical University, Beijing, China during 2012-2020, were recruited as the ELST group, and 24 cases of otitis media diagnosed in the same hospital were selected as the control group. The expression of VHL, VEGF, and HIF-1α was assessed using EnVision immunohistochemical staining and compared between the ELST and control groups. Sanger sequencing was performed to detect the VHL mutation status in 24 ELSTs. The correlations among VHL, VEGF and HIF-1α expression were analyzed. The associations of VHL, VEGF and HIF-1α expression with age of onset, gender, tumor size, bone invasion and clinical stage in ELST were also analyzed. Results: The expression rate of VHL in the ELST group was significantly lower than that in the control group (P<0.05), but the expression rates of VEGF and HIF-1α in the ELST group were significantly higher than those in the control group (P<0.05). VHL expression was inversely correlated with VEGF and HIF-1α expression. The expression of VEGF and HIF-1α was associated with bone invasion and clinical stage (P<0.05), but the expression of VHL, VEGF and HIF-1α had no significant associations with the age of onset, gender, or tumor size of ELST (P>0.05). Conclusions: The expression of VHL is decreased while that of VEGF and HIF-1α increased in ELST. Expression of VHL is inversely correlated with that of VEGF and HIF-1α. The expression of VEGF and HIF-1α is correlated with bone invasion and clinical stage. Thus, VEGF and HIF-1α may be therapeutic targets of ELST.

n-3 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1alpha induction pathway.

Objective To observe the expressions of microRNA-126 (miR-126) and vascular endothelial growth factor (VEGF) in gastric carcinoma tissues, and their associations with clinicopathologic features in gastric carcinoma. Methods Fifty cases of gastric carcinoma and matched tumor adjacent tissue specimens were collected from January 2012 to March 2014 in Shangqiu First People′s Hospital of He′nan Province. Expression levels of miR-126 and VEGF were examined using reverse transcriptase-polymerase chain reaction. Their correlations and the relationships between them and the clinicopathologic features in gastric carcinoma were analyzed. Results The expression levels of miR-126 in the tumor tissues and in the adjacent tissues were 0.652±0.102 and 0.379±0.069, and the expression levels of VEGF in the two kinds of tissues were 1.523±0.142 and 0.604±0.152, with statistical differences (t=8.374, P=0.001; t=29.508, P=0.000). The expressions of miR-126 and VEGF were significantly positively correlated (r=0.735, P=0.001). The expre-ssions of miR-126 and VEGF were associated with TNM stage (t=22.981, P=0.001; t=18.053, P=0.001), depth of tumor invasion (t=26.792, P=0.001; t=20.234, P=0.001) and tumor node metastasis (t=26.668, P=0.001; t=14.857, P=0.001). Conclusion miR-126 is high expressed in the gastric carcinoma tissue, which may up-regulate the expression of VEGF, and lead to the formation of new blood vessels, then to promote gastric carcinogenesis and the development process. Key words: Stomach neoplasms; MicroRNAs; Vascular endothelial growth factors