Differential effects of cyclo-oxygenase 1 and 2 inhibition on angiogenesis inhibitor-induced hypertension and kidney damage.

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.

Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species.

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar-Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.

Reduced Lymphatic Reserve in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Microvascular dysfunction plays an important role in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, no mechanistic link between systemic microvasculature and congestion, a central feature of the syndrome, has yet been investigated. OBJECTIVES: This study aimed to investigate capillary-interstitium fluid exchange in HFpEF, including lymphatic drainage and the potential osmotic forces exerted by any hypertonic tissue Na+ excess. METHODS: Patients with HFpEF and healthy control subjects of similar age and sex distributions (n = 16 per group) underwent: 1) a skin biopsy for vascular immunohistochemistry, gene expression, and chemical (water, Na+, and K+) analyses; and 2) venous occlusion plethysmography to assess peripheral microvascular filtration coefficient (measuring capillary fluid extravasation) and isovolumetric pressure (above which lymphatic drainage cannot compensate for fluid extravasation). RESULTS: Skin biopsies in patients with HFpEF showed rarefaction of small blood and lymphatic vessels (p = 0.003 and p = 0.012, respectively); residual skin lymphatics showed a larger diameter (p = 0.007) and lower expression of lymphatic differentiation and function markers (LYVE-1 [lymphatic vessel endothelial hyaluronan receptor 1]: p < 0.05; PROX-1 [prospero homeobox protein 1]: p < 0.001) compared with control subjects. In patients with HFpEF, microvascular filtration coefficient was lower (calf: 3.30 [interquartile range (IQR): 2.33 to 3.88] l × 100 ml of tissue-1 × min-1 × mm Hg-1 vs. 4.66 [IQR: 3.70 to 6.15] µl × 100 ml of tissue-1 × min-1 × mm Hg-1; p < 0.01; forearm: 5.16 [IQR: 3.86 to 5.43] l × 100 ml of tissue-1 × min-1 × mm Hg-1 vs. 5.66 [IQR: 4.69 to 8.38] µl × 100 ml of tissue-1 × min-1 × mm Hg-1; p > 0.05), in keeping with blood vascular rarefaction and the lack of any observed hypertonic skin Na+ excess, but the lymphatic drainage was impaired (isovolumetric pressure in patients with HFpEF vs. control subjects: calf 16 ± 4 mm Hg vs. 22 ± 4 mm Hg; p < 0.005; forearm 17 ± 4 mm Hg vs. 25 ± 5 mm Hg; p < 0.001). CONCLUSIONS: Peripheral lymphatic vessels in patients with HFpEF exhibit structural and molecular alterations and cannot effectively compensate for fluid extravasation and interstitial accumulation by commensurate drainage. Reduced lymphatic reserve may represent a novel therapeutic target.

Chemerin receptor blockade improves vascular function in diabetic obese mice via redox-sensitive and Akt-dependent pathways.

Chemerin and its G protein-coupled receptor [chemerin receptor 23 (ChemR23)] have been associated with endothelial dysfunction, inflammation, and insulin resistance. However, the role of chemerin on insulin signaling in the vasculature is still unknown. We aimed to determine whether chemerin reduces vascular insulin signaling and whether there is interplay between chemerin/ChemR23, insulin resistance, and vascular complications associated with type 2 diabetes (T2D). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMCs) from C57BL/6J, nondiabetic lean db/m, and diabetic obese db/db mice as well as in human microvascular endothelial cells (HMECs). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, an effect prevented by CCX832 (ChemR23 antagonist) treatment. In VSMCs, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced Akt phosphorylation, glucose transporter 4 translocation to the membrane, and glucose uptake. In HMECs, chemerin decreased insulin-activated nitric oxide signaling. AMP-activated protein kinase phosphorylation was reduced by chemerin in both HMECs and VSMCs. CCX832 treatment of db/db mice decreased body weight, insulin, and glucose levels as well as vascular oxidative stress. CCX832 also partially restored vascular insulin responses in db/db and high-fat diet-fed mice. Our novel in vivo findings highlight chemerin/ChemR23 as a promising therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes. NEW & NOTEWORTHY Our novel findings show that the chemerin/chemerin receptor 23 axis plays a critical role in diabetes-associated vascular oxidative stress and altered insulin signaling. Targeting chemerin/chemerin receptor 23 may be an attractive strategy to improve insulin signaling and vascular function in obesity-associated diabetes.

Upregulation of Nrf2 and Decreased Redox Signaling Contribute to Renoprotective Effects of Chemerin Receptor Blockade in Diabetic Mice.

Chemerin, acting through its receptor ChemR23, is an adipokine associated with inflammatory response, glucose and lipid metabolism and vascular function. Although this adipokine has been associated with the development and progression of kidney disease, it is not clear whether the chemerin/ChemR23 system plays a role in renal function in the context of diabetes. Therefore, we sought to determine whether ChemR23 receptor blockade prevents the development and/or progression of diabetic nephropathy and questioned the role of oxidative stress and Nrf2 in this process. Renal redox state and function were assessed in non-diabetic lean db/m and diabetic obese db/db mice treated with vehicle or CCX832 (ChemR23 antagonist). Renal reactive oxygen species (ROS) production, which was increased in diabetic mice, was attenuated by CCX832. This was associated with an increase in Nox 4 expression. Augmented protein oxidation in db/db mice was not observed when mice were treated with CCX832. CCX832 also abrogated impaired Nrf2 nuclear activity and associated downregulation in antioxidants expression in kidneys from db/db mice. Our in vivo findings highlight the role of the redox signaling and Nrf2 system as renoprotective players during chemerin receptor blockade in diabetic mice. The chemerin/ChemR23 system may be an important target to limit renal dysfunction associated with obesity-related diabetes.

Glycosylation with O-linked ß-N-acetylglucosamine induces vascular dysfunction via production of superoxide anion/reactive oxygen species.

Overproduction of superoxide anion (•O2-) and O-linked ß-N-acetylglucosamine (O-GlcNAc) modification in the vascular system are contributors to endothelial dysfunction. This study tested the hypothesis that increased levels of O-GlcNAc-modified proteins contribute to •O2- production via activation of NADPH oxidase, resulting in impaired vasodilation. Rat aortic segments and vascular smooth muscle cells (VSMCs) were incubated with vehicle (methanol) or O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc) (100 µM). PUGNAc produced a time-dependent increase in O-GlcNAc levels in VSMC and decreased endothelium-dependent relaxation, which was prevented by apocynin and tiron, suggesting that •O2- contributes to endothelial dysfunction under augmented O-GlcNAc levels. Aortic segments incubated with PUGNAc also exhibited increased levels of reactive oxygen species, assessed by dihydroethidium fluorescence, and augmented •O2- production, determined by lucigenin-enhanced chemiluminescence. Additionally, PUGNAc treatment increased Nox-1 and Nox-4 protein expression in aortas and VSMCs. Translocation of the p47phox subunit from the cytosol to the membrane was greater in aortas incubated with PUGNAc. VSMCs displayed increased p22phox protein expression after PUGNAc incubation, suggesting that NADPH oxidase is activated in conditions where O-GlcNAc protein levels are increased. In conclusion, O-GlcNAc levels reduce endothelium-dependent relaxation by overproduction of •O2- via activation of NADPH oxidase. This may represent an additional mechanism by which augmented O-GlcNAc levels impair vascular function.

TNF-α induces vascular insulin resistance via positive modulation of PTEN and decreased Akt/eNOS/NO signaling in high fat diet-fed mice.

BACKGROUND: High fat diet (HFD) induces insulin resistance in various tissues, including the vasculature. HFD also increases plasma levels of TNF-α, a cytokine that contributes to insulin resistance and vascular dysfunction. Considering that the enzyme phosphatase and tension homologue (PTEN), whose expression is increased by TNF-α, reduces Akt signaling and, consequently, nitric oxide (NO) production, we hypothesized that PTEN contributes to TNF-α-mediated vascular resistance to insulin induced by HFD. Mechanisms underlying PTEN effects were determined. METHODS: Mesenteric vascular beds were isolated from C57Bl/6J and TNF-α KO mice submitted to control or HFD diet for 18 weeks to assess molecular mechanisms by which TNF-α and PTEN contribute to vascular dysfunction. RESULTS: Vasodilation in response to insulin was decreased in HFD-fed mice and in ex vivo control arteries incubated with TNF-α. TNF-α receptors deficiency and TNF-α blockade with infliximab abolished the effects of HFD and TNF-α on insulin-induced vasodilation. PTEN vascular expression (total and phosphorylated isoforms) was increased in HFD-fed mice. Treatment with a PTEN inhibitor improved insulin-induced vasodilation in HFD-fed mice. TNF-α receptor deletion restored PTEN expression/activity and Akt/eNOS/NO signaling in HFD-fed mice. CONCLUSION: TNF-α induces vascular insulin resistance by mechanisms that involve positive modulation of PTEN and inhibition of Akt/eNOS/NO signaling. Our findings highlight TNF-α and PTEN as potential targets to limit insulin resistance and vascular complications associated with obesity-related conditions.

Chemerin Regulates Crosstalk Between Adipocytes and Vascular Cells Through Nox.

Adipocytes produce adipokines, including chemerin, a chemoattractant that mediates effects through its ChemR23 receptor. Chemerin has been linked to endothelial dysfunction and vascular injury in pathological conditions, such as obesity, diabetes mellitus, and hypertension. Molecular mechanisms underlying this are elusive. Here we assessed whether chemerin through redox-sensitive signaling influences molecular processes associated with vascular growth, apoptosis, and inflammation. Human microvascular endothelial cells and vascular smooth muscle cells were stimulated with chemerin (50 ng/mL). Chemerin increased generation of reactive oxygen species and phosphorylation of mitogen-activated protein kinases, effects that were inhibited by ML171, GKT137831 (Nox inhibitors), and N-acetylcysteine (reactive oxygen species scavenger). Chemerin increased mRNA expression of proinflammatory mediators in vascular cells and increased monocyte-to-endothelial cell attachment. In human vascular smooth muscle cells, chemerin induced phosphorylation of mitogen-activated protein kinases and stimulated proliferation (increased proliferating cell nuclear antigen expression [proliferation marker] and BrdU incorporation [proliferation assay]). Chemerin decreased phosphatidylinositol 3-kinase/protein kinase B activation and increased TUNEL-positive human vascular smooth muscle cells. In human microvascular endothelial cells, chemerin reduced endothelial nitric oxide synthase activity and nitric oxide production. Adipocyte-conditioned medium from obese/diabetic mice (db/db), which have elevated chemerin levels, increased reactive oxygen species generation in vascular smooth muscle cells, whereas adipocyte-conditioned medium from control mice had no effect. Chemerin actions were blocked by CCX 832, a ChemR23 inhibitor. Our data demonstrate that chemerin, through Nox activation and redox-sensitive mitogen-activated protein kinases signaling, exerts proapoptotic, proinflammatory, and proliferative effects in human vascular cells. These findings elucidate some molecular mechanisms through chemerin, which is increased in obesity, whereby adipocytes may influence vascular function. We identify chemerin as a novel vasoactive adipokine, which may be important in obesity-related vascular injury.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement.

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

Chemerin reduces vascular nitric oxide/cGMP signalling in rat aorta: a link to vascular dysfunction in obesity?

The adipokine chemerin has been implicated in cardiovascular complications associated with obesity and the metabolic syndrome. Chemerin has direct effects on the vasculature, augmenting vascular responses to contractile stimuli. As NO/cGMP signalling plays a role in vascular dysfunction associated with obesity and the metabolic syndrome, we hypothesized that chemerin induces vascular dysfunction by decreasing NO/cGMP signalling. Aortic rings from male Wistar rats (10-12 weeks of age) were incubated with chemerin (0.5 or 5 ng/ml for 1 h) or vehicle and isometric tension was recorded. Vasorelaxation in response to ACh (acetylcholine), SNP (sodium nitroprusside) and BAY 412272 [an sGC (soluble guanylate cyclase) stimulator] were decreased in chemerin-treated vessels. The NOS (NO synthase) cofactor BH4 (tetrahydrobiopterin), an O2- (superoxide anion) scavenger (tiron) and a SOD (superoxide dismutase) mimetic (tempol) abolished the effects of chemerin on ACh-induced vasodilation. eNOS (endothelial NOS) phosphorylation, determined by Western blotting, was increased in chemerin-treated vessels; however, the enzyme was mainly in the monomeric form, with decreased eNOS dimer/monomer ratio. Chemerin decreased the mRNA levels of the rate-limiting enzyme for BH4 biosynthesis GTP cyclohydrolase I. Chemerin-incubated vessels displayed decreased NO production, along with increased ROS (reactive oxygen species) generation. These effects were abrogated by BH4, tempol and L-NAME (NG-nitro-L-arginine methyl ester). sGC protein expression and cGMP levels were decreased in chemerin-incubated vessels. These results demonstrate that chemerin reduces NO production, enhances NO breakdown and also decreases NO-dependent cGMP signalling, thereby reducing vascular relaxation. Potential mechanisms mediating the effects of chemerin in the vasculature include eNOS uncoupling, increased O2- generation and reduced GC activity.