Transforming growth factor-β1 signalling triggers vascular endothelial growth factor resistance and monocyte dysfunction in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) leads to monocyte dysfunction associated with atherogenesis and defective arteriogenesis. Transforming growth factor (TGF)-β1, placenta growth factor (PlGF)-1 and vascular endothelial growth factor (VEGF)A play important roles in atherogenesis and arteriogenesis. VEGF-receptor (VEGFR)-mediated monocyte migration is inhibited in T2DM (VEGFA resistance), while TGF-β1-induced monocyte migration is fully functional. Therefore, we hypothesize that TGF-β antagonises the VEGFA responses in human monocytes. We demonstrate that monocytes from T2DM patients have an increased migratory response towards low concentrations of TGF-β1, while PlGF-1/VEGFA responses are mitigated. Mechanistically, this is due to increased expression of type II TGF-β receptor in monocytes under high-glucose conditions and increased expression of soluble (s)VEGFR1, which is known to interfere with VEGFA signalling. VEGFA resistance in monocytes from T2DM patients can be rescued by either experimental down-regulation of TGF-β receptor expression in vitro or by functional blocking of TGF-β signalling using either a TGF-β receptor kinase inhibitor or a TGF-β neutralizing antibody. Our data demonstrate that both T2DM and high-glucose potentiate the TGF-β pathway. TGF-β signalling impairs VEGFR-mediated responses in T2DM monocytes and in this way contributes to mononuclear cell dysfunction, provide novel insights into T2DM vascular dysfunction.     

Retinal pigment epithelium-derived transforming growth factor-β2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor-2 expression

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-β secretion, particularly TGF-β2. However, it is largely unclear whether and how TGF-β2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-β2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-β2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-β2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-β2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-β2 expression in RPE cells under pathologic conditions.     

Betulinic acid inhibits the expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in human endometrial adenocarcinoma cells

Although betulinic acid (BA) is known to induce apoptosis and antiangiogenic response in tumor cells, the underlying mechanism of its action is unknown. Deregulation of tissue collagen metabolism is one of the consequences of neoplastic transformation. The final step of collagen degradation is mediated by prolidase [E.C.3.4.13.9] which may play a role in angiogenesis. The formation of new blood vessels is regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of vascular endothelial cell growth factor (VEGF). Since BA evokes anticancer activity, its effect on collagen biosynthesis, HIF-1α and VEGF expressions, as well as prolidase activity and expression was studied in cultured endometrial adenocarcinoma (EA) cells. It was found that BA inhibits collagen biosynthesis in EA cells (5[³H] proline incorporation assay). It was accompanied by a parallel decrease in prolidase activity and expression and decrease in expressions of α₁ and α₂ integrins, HIF-1α, and VEGF (western immunoblot analysis) in cultured human EA cells. The data suggest that BA may have anti-angiogenic potential by inhibition of prolidase, HIF-1α and VEGF expressions, and inhibition of collagen biosynthesis.     

Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1α and vascular endothelial growth factor in pressure-overloaded rat heart

Summary The use of -blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1 (HIF-1) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1 in cardiac hypertrophy due to pressure overload and after treatment with -blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1 expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 ± 9 mmHg in the sham group to 94 ±5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor- (NGF-) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1 and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1, VEGF and BNP increased and mRNA of NGF- decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1, VEGF, BNP, and NGF- to the baseline values. Increased immunohistochemical labeling of HIF-1, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF- mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1,VEGF, BNP, and NGF- in the hypertrophic myocardium.     

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-β

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A₂, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-κβ signaling pathway.     

Fluoxetine protects against monocrotaline-induced pulmonary arterial remodeling by inhibition of hypoxia-inducible factor-1α and vascular endothelial growth factor

The selective serotonin re-uptake inhibitor fluoxetine has been shown to protect against monocrotaline (MCT)-induced pulmonary hypertension in rats. To investigate the possible role of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in mediating this protective effect, MCT-treated rats were administered fluoxetine by gavage, at doses of 2?mg/kg body mass or 10?mg/kg once daily for 3 weeks. Changes in pulmonary hemodynamic parameters, pulmonary artery morphologies, and expressions of HIF-1α and VEGF were assessed. Fluoxetine at the 10?mg/kg dose, but not at the 2?mg/kg dose, attenuated the effects of MCT on pulmonary artery pressure, right ventricle index, and medial wall thickness. In addition, 10?mg/kg fluoxetine mitigated the MCT-induced up-regulation of HIF-1α and VEGF protein and reactive oxygen species (ROS) in the lungs. This dosage also decreased pERK1/2 levels and inhibited proliferation of pulmonary arterial smooth muscle cells in MCT-treated rats. In conclusion, fluoxetine can protect against MCT-induced pulmonary arterial remodeling, which linked to reduced ROS generation and decreased HIF-1α and VEGF protein levels via the ERK1/2 phosphorylation pathway.     

Expressions of vascular endothelial growth factor and CD34 in oral aphthous lesions of Behçet's disease

OBJECTIVE: Beh?et's disease (BD) is an immunoin-flammatory vasculitis with an unknown etiopathogenesis. Vascular endothelial growth factor (VEGF) is a cytokine-stimulating angiogenesis. It has been suggested to play a role in inflammation and pathogenesis of vasculitic processes. STUDY DESIGN: VEGF and CD34 expressions were assessed in samples taken from oral aphthous lesions. The patients were evaluated for disease activity, duration of lesions, serum C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR). RESULTS: Twenty-six patients were included. Fourteen (53.9%) had increased CRP levels and 12 (46.1%) had increased ESR levels. Positive VEGF and CD34 staining were detected in 46.2% and 69.2% of biopsy samples, respectively. There was good positive correlation between them. The frequency of positive VEGF and CD34 staining was statistically higher in lesions with a duration of more than 6 days. No correlation was found between positive VEGF staining and serum CRP level, ESR and disease activity. CONCLUSION: Increased VEGF expression in correlation with CD34 positivity in oral aphthous lesions may show the role of VEGF in pathogenesis of these vasculitic lesions. We have concluded that VEGF may play a role during the course of oral aphthous lesions in BD.     

Immunolocalization of interleukin-1α in rat mandibular molars and its enhancement after in vivo injection of epidermal growth factor

Immunolocalization of interleukin-1 in the first mandibular molars of rats from day 0–12 postnatally showed that the protein was localized in the epithelial stellate reticulum adjacent to the dental follicle. Staining of the stellate reticulum was most prominent in the early days postnatally and was absent by postnatal day 11. Injection of epidermal growth factor into rats at day 0 greatly increased the intensity of the staining for interleukin-1 in the stellate reticulum. Epidermal growth factor (EGF) enhanced the gene expression of interleukin-1 in stellate reticulum cells in vitro, and this study suggests there is enhanced translation of interleukin-1 messenger RNA in the stellate reticulum following EGF injection. In turn, the interleukin-1 may exert its effect on the dental follicle cells adjacent to the stellate reticulum because EGF also enhanced expression of the interleukin-1 receptor type I messenger RNA in cultured dental follicle cells as well as enhancing its expression in vivo. In view of the fact that injection of EGF will stimulate precocious eruption of teeth, its stimulus of interleukin-1 synthesis in the stellate reticulum may be the mechanism by which EGF initiates a cascade of molecular events to signal the onset of tooth eruption.     

Role of PPARα and HNF4α in Stress-Mediated Alterations in Lipid Homeostasis

Stress is a risk factor for several cardiovascular pathologies. PPARα holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPARα agonists are effective in raising HDL-cholesterol and lowering triglycerides, properties that reduce the risk for cardiovascular diseases. This study investigated the role of stress and adrenergic receptor (AR)-related pathways in PPARα and HNF4α regulation and signaling in mice following repeated restraint stress or treatment with AR-antagonists administered prior to stress to block AR-linked pathways. Repeated restraint stress up-regulated Pparα and its target genes in the liver, including Acox, Acot1, Acot4, Cyp4a10, Cyp4a14 and Lipin2, an effect that was highly correlated with Hnf4α. In vitro studies using primary hepatocyte cultures treated with epinephrine or AR-agonists confirmed that hepatic AR/cAMP/PKA/CREB- and JNK-linked pathways are involved in PPARα and HNF4α regulation. Notably, restraint stress, independent of PPARα, suppressed plasma triglyceride levels. This stress-induced effect could be attributed in part to hormone sensitive lipase activation in the white adipose tissue, which was not prevented by the increased levels of perilipin. Overall, this study identifies a mechanistic basis for the modification of lipid homeostasis following stress and potentially indicates novel roles for PPARα and HNF4α in stress-induced lipid metabolism.     

A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Vascular endothelial growth factor receptor 2 (VEGFR2) has been reported to play an important role in angiogenesis and tumorigenesis. A murine anti-VEGFR2 mAb (A8H1) has been established in a previous study. To reduce the incompatibility of the murine mAb for human use, the chimeric anti-VEGFR2-IgG was developed by genetic recombination of the variable regions of the A8H1 antibody and the constant regions of human IgG, and was expressed in Sp2/0 cells transfected with the two recombinant vectors containing the heavy chain and the light chain regions. After screening, clone 2F12 was selected and was found to stably secrete the murine–human chimeric anti-VEGFR2-IgG (coded 2F12). This chimeric IgG maintained the specificity and the affinity of the parental murine antibody against VEGFR2, and effectively identified VEGFR2 expressed on the surface of HUVECs and BEL-7402 cells. Furthermore, the 2F12 antibody demonstrated inhibition of angiogenesis in vitro, such as proliferation, migration, invasion and tube formation of HUVECs. This murine–human chimeric IgG may be considered for further development as an anti-angiogenesis and anti-tumor agent.     

γ-Secretase and presenilin mediate cleavage and phosphorylation of vascular endothelial growth factor receptor-1

We have reported previously that pigment epithelium-derived factor (PEDF) can, via γ-secretase-mediated events, inhibit VEGF-induced angiogenesis in microvascular endothelial cells by both (a) cleavage and intracellular translocation of a C-terminal fragment of VEGF receptor-1 (VEGFR1) and (b) inhibition of VEGF-induced phosphorylation of VEGFR1. Using site-direct mutagenesis and transfection of wild type and mutated receptors into endothelial cells, we showed that transmembrane cleavage of VEGFR1 occurs at valine 767 and that a switch from valine to alanine at this position prevented cleavage and formation of a VEGFR1 intracellular fragment. Using siRNA to selectively knock down protein-tyrosine phosphatases (PTPs) in endothelial cells, we demonstrated that vascular endothelial PTP is responsible for dephosphorylation of activated VEGFR1. PEDF up-regulation of full-length presenilin 1 (Fl.PS1) facilitated the association of vascular endothelial PTP and VEGFR1. Knockdown of Fl.PS1 prevented dephosphorylation of VEGFR1, whereas up-regulation of Fl.PS1 stimulated VEGFR1 dephosphorylation. Fl.PS1 associated with VEGFR1 within 15 min after PEDF treatment. In conclusion, we determined the PEDF-mediated events responsible for VEGFR1 signaling and identified full-length presenilin as a critical adaptor molecule in the dephosphorylation of VEGFR1. This greater understanding of the regulation of VEGFR1 signaling will help identify novel anti-VEGF therapeutic strategies.     

The roles of integrin β4 in vascular endothelial cells

Integrin heterodimers play diverse and important roles in physiological and pathological processes, such as cell adhesion, migration, proliferation, differentiation, angiogenesis, and tumor progression, via the outside-in and/or inside-out signaling pathways. Aberrant functions of integrins have been implicated in the causation and intervention of multiple diseases. Integrin β(4), a laminin-5 (LN5) receptor, mainly locates in the adhesion structure of hemidesmosome (HD). Most of the previous researches concentrated on the role of integrin β(4) in cancer and cancer therapy, and a few focused on the physiological roles of normal mammalian cells. Recently, accumulating data reveal that integrin β(4) participates in cell death, macroautophagy (hereafter autophagy), senescence, and differentiation regulations in various cell types including human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells, and mouse neural cells, implying the key roles of integrin β(4) in the physiological alteration of mammalian cells. Thus, the elucidation of integrin β(4)-mediated signaling may undoubtedly contribute to novel therapeutic strategies for various human diseases, such as vascular and neural disorders. We have reviewed the roles of integrin β(4) in neural cells. In the present review we will discuss the recent research progress in the inherent functions and pharmacological modulation of integrin β(4) in vascular endothelial cells.     

Vascular endothelial growth factor: an angiogenic factor reflecting airway inflammation in healthy smokers and in patients with bronchitis type of chronic obstructive pulmonary disease?

BackgroundPatients with bronchitis type of chronic obstructive pulmonary disease (COPD) have raised vascular endothelial growth factor (VEGF) levels in induced sputum. This has been associated with the pathogenesis of COPD through apoptotic and oxidative stress mechanisms. Since, chronic airway inflammation is an important pathological feature of COPD mainly initiated by cigarette smoking, aim of this study was to assess smoking as a potential cause of raised airway VEGF levels in bronchitis type COPD and to test the association between VEGF levels in induced sputum and airway inflammation in these patients.Methods14 current smokers with bronchitis type COPD, 17 asymptomatic current smokers with normal spirometry and 16 non-smokers were included in the study. VEGF, IL-8, and TNF-α levels in induced sputum were measured and the correlations between these markers, as well as between VEGF levels and pulmonary function were assessed.ResultsThe median concentrations of VEGF, IL-8, and TNF-α were significantly higher in induced sputum of COPD patients (1,070 pg/ml, 5.6 ng/ml and 50 pg/ml, respectively) compared to nonsmokers (260 pg/ml, 0.73 ng/ml, and 15.4 pg/ml, respectively, p < 0.05) and asymptomatic smokers (421 pg/ml, 1.27 ng/ml, p < 0.05, and 18.6 pg/ml, p > 0.05, respectively). Significant correlations were found between VEGF levels and pack years (r = 0.56, p = 0.046), IL-8 (r = 0.64, p = 0.026) and TNF-α (r = 0.62, p = 0.031) levels both in asymptomatic and COPD smokers (r = 0.66, p = 0.027, r = 0.67, p = 0.023, and r = 0.82, p = 0.002, respectively). No correlation was found between VEGF levels in sputum and pulmonary function parameters.ConclusionVEGF levels are raised in the airways of both asymptomatic and COPD smokers. The close correlation observed between VEGF levels in the airways and markers of airway inflammation in healthy smokers and in smokers with bronchitis type of COPD is suggestive of VEGF as a marker reflecting the inflammatory process that occurs in smoking subjects without alveolar destruction.     

TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts

The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-α and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iα)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-α and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iα). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth.