Abstract 010: Adipocyte-derived Aldosterone And Cortisol Are Nox1/4 Dependent: Implications In Obesity-associated Vascular Dysfunction.

We previously demonstrated that aldosterone (aldo) is produced by adipocytes, an effect associated with reactive oxygen species (ROS) production and adipokine production, which influences vascular function. These processes are exaggerated in obesity. Whether ROS themselves play a role in adipocyte-derived aldo is unclear. Studies were performed in db/m (lean) and db/db (obese) mice, treated with low (20mg/kg/day) or high dose (60mg/kg/day) GKT137831 (GKT, Nox1/4 inhibitor, 16 weeks). Epididymal (EVAT) and perivascular (PVAT) fat were collected. Human adipocytes (SW872) were also studied. Aldo and corticosterone levels were measured by ELISA. Gene expression was assessed by qPCR. ROS generation was assessed by chemiluminescence and amplex red. Plasma aldo levels in db/db (pg/mL: 518 vs. 272g) and aldo levels in culture media from db/db adipocytes were increased (pg/mL/μg RNA: 1964 vs. 388), p<0.05. All effects were decreased by high dose GKT. In PVAT, CYP11B2 gene expression was increased in db/db (2.6±0.8 vs control 1.1±0.1, p<0.05), an effect blocked by Nox1/4 inhibition. Corticosterone levels in culture media from db/db adipocytes were also increased. Gene expression of adipocyte differentiation marker, AP2, was increased (3.5±1.1 vs control 1.4±0.4) in obese mice. GKT decreased AP2 levels. In human adipocytes, AngII stimulation increased aldo (6 fold) and cortisol (4 fold) production, as well as superoxide (1 fold) and H2O2 (2 fold) levels (p<0.05 vs vehicle). Increased levels of superoxide by Ang II were blocked by GKT and ML171 (Nox1 inhibitor); while Ang II-induced H2O2 production was inhibited only by GKT. Ang II-induced aldo production was blocked by tempol (SOD mimetic), GKT and ML171. In contrast, cortisol was only blocked by tempol and GKT. In conclusion, aldo production in adipocytes is dependent on ROS formation and involves Nox1 and Nox4. Nox4 also influences adipocyte-derived cortisol. These data suggest that Nox1/4 may play a role in adipocyte-derived aldosterone and cortisol production, effects that are amplified in obesity. Our findings suggest that adipocyte Nox1/4 may be a putative therapeutic target in obesity-associated hyperaldosteronism and cardiovascular damage.

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Abstract 74: The Nox4 Inhibitor GKT137831, Prevents Aldosterone Production by Adipocytes and Protects Against Adipose Tissue Inflammation and Fibrosis in Obese Mice

Hypertension ◽

10.1161/hyp.62.suppl_1.a74 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Sarah Even ◽

Aurelie Nguyen Dinh Cat ◽

Francisco J Rios ◽

Antunes T Tayze ◽

Ying He ◽

...

Keyword(s):

Gene Expression ◽

Adipose Tissue ◽

Culture Media ◽

Inflammatory Marker ◽

High Dose ◽

Obese Mice ◽

Cardiovascular Damage ◽

Aldosterone Production ◽

Inflammatory Phenotype ◽

Sirius Red

Aldosterone (aldo) plays an important role in obesity-associated cardiovascular risk. We demonstrated that aldo is produced by adipocytes, an effect associated with increased generation of reactive oxygen species (ROS). These processes are exaggerated in obesity. The relationship between adipocyte aldosterone and ROS is unclear. We postulated that Nox4-derived ROS is important for aldo production in adipocytes and leads to a pro-inflammatory phenotype in obesity. Studies were performed in db/m (lean) and db/db (obese) mice, treated with low (20mg/kg/day) or high dose (60mg/kg/day) GKT137831 (GKT, Nox4 inhibitor, 16 weeks). Epididymal (EVAT) and perivascular (PVAT) fat were collected. Plasma and adipocyte aldo were measured by ELISA. Adipose tissue fibrosis was evaluated by picro Sirius red staining and inflammatory mediators by immunostudies. Body weight was increased in db/db mice (61.8g vs control 33.5g), with no effect of GKT. Epididymal adiposity was increased in db/db mice (0.098g vs. 0.067g, p<0.05). Plasma aldo levels in db/db (pg/mL: 518 vs. 272g) and aldo levels in culture media from db/db adipocytes were increased (pg/mL/μg RNA: 1964 vs. 388), p<0.05. All effects decreased by high dose GKT. In PVAT, CYP11B2 gene expression was increased in db/db (2.6±0.8 vs control 1.1±0.1, p<0.05), an effect blocked by Nox4 inhibition. Gene expression of adipocyte differentiation marker, AP2, was increased (3.5±1.1 vs control 1.4±0.4) while anti-inflammatory marker adiponectin was decreased (0.7±0.1 vs control 1.3±0.2, p<0.05)) in obese mice. GKT decreased AP2 levels. Adipocyte-derived TNFα was increased in db/db (4.9±1.8 vs control 1.6±0.6, p<0.05), an effect blocked by GKT. Pro-collagen I, marker of fibrosis, was increased in db/db mice (132±11 vs control 87±4, p<0.05). Sirius red staining was exaggerated in EVAT from db/db mice, and decreased by Nox4 inhibition. In conclusion, Nox4 plays a role in regulating adipocyte-derived aldosterone and promotes a pro-inflammatory and profibrotic adipose phenotype in obese db/db mice. These findings suggest that adipocyte Nox4 links hyperaldosteronism and inflammation/fibrosis in adiposity and as such may be a putative therapeutic target for obesity-associated cardiovascular damage.

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Abstract P006: Liraglutide Attenuates Cardiac Fibrosis and Vascular Dysfunction in a Non-diabetic Angiotensin II-infusion Model via Anti-inflammatory and Anti-oxidant Mechanisms

Hypertension ◽

10.1161/hyp.66.suppl_1.p006 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Huey Wen Lee ◽

Melita Brdar ◽

Robert Widdop ◽

Anthony Dear ◽

Tracey Gaspari

Keyword(s):

Blood Pressure ◽

Vascular Function ◽

Cardiac Fibrosis ◽

Vascular Dysfunction ◽

Cardiomyocyte Hypertrophy ◽

High Dose ◽

Protective Effects ◽

Ang Ii ◽

Treatment Protocols ◽

Glucose Levels

Glucagon-like peptide-1 (GLP-1) based therapies are used to treat type II diabetes via increasing insulin secretion and inhibiting glucagon production. Recent evidence suggests that activating the GLP-1 receptor may also mediate direct vaso-protective effects. Therefore the objective of the study was to determine whether GLP-1R stimulation conferred cardio- and vaso-protection in a non-diabetic setting using the angiotensin (Ang) II infusion model of hypertension and cardiovascular dysfunction. Male C57Bl/6J mice (4-6 months) were assigned to one of the following 4 week treatment protocols: 1) vehicle (saline), 2) Ang II (800ng/kg/day), 3) Ang II + liraglutide (30μg/kg/day), 4) Ang II + liraglutide (300μg/kg/day). All treatments were administered via osmotic mini-pumps (s.c). After 4 weeks the effect of liraglutide treatment on blood pressure, vascular function and cardiac remodelling was examined. Liraglutide (both doses) attenuated Ang II-induced increase in systolic blood pressure (Ang II: 175.3 ± 8.6mmHg vs Ang II+Lirag (30) 150.2 ± 6.4 mmHg or Ang II+Lirag (300): 145.4 ± 6.9 mmHg) without affecting blood glucose levels. Liraglutide (both doses) completely prevented Ang II-induced endothelial dysfunction (% maximum relaxation: Ang II=50.7 ± 7.8%; Ang II+Lirag (30)=82.7 ± 5.8; Ang II+Lirag (300)=81.5 ± 6.1%). In the heart, liraglutide prevented Ang II-induced cardiomyocyte hypertrophy (n=7-10; p<0.05) and reduced collagen deposition (% collagen expression: Ang II=4.4 ± 0.5 vs Ang II+Lirag(300)=2.9 ± 0.3; n=7-9; p<0.01). This anti-fibrotic effect was attributed to reduced fibroblast/myofibroblast expression as well as decreased inflammation with reduced NFκB and MCP-1 expression and decreased oxidative stress with a significant reduction in superoxide production using high dose of liraglutide. Overall, stimulation of GLP-1R in a non-diabetic setting protected against Ang II-mediated cardiac hypertrophy, cardiac fibrosis and vascular dysfunction, indicating potential for use of GLP-1 based therapies in treatment of cardiovascular disease independent of diabetes.

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Abstract P090: Nox5 Induces Vascular Damage Through C-src Activation In Human Hypertension

Hypertension ◽

10.1161/hyp.76.suppl_1.p090 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Livia L Camargo ◽

Augusto C Montezano ◽

Misbah Hussain ◽

Yu Wang ◽

Zhiguo Zou ◽

...

Keyword(s):

Vascular Function ◽

Actin Polymerization ◽

Vascular Dysfunction ◽

Redox Signaling ◽

Ros Generation ◽

Ang Ii ◽

Human Hypertension ◽

Src Signaling ◽

Specific Effects ◽

And Migration

Nox5 is the major ROS-generating Nox isoform in human vascular smooth muscle cells (VSMC). The role of Nox5 in oxidative stress and redox signaling underlying vascular dysfunction in hypertension is unclear. We examined molecular processes that regulate VSMC Nox5-induced ROS generation, focusing on c-Src. VSMC isolated from small arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Nox5 expression and phosphorylation (immunoblotting, immunoprecipitation); ROS generation (chemiluminescence); activation of contractile signaling pathways (immunoblotting), Ca 2+ influx (Cal-520AM fluorescence), reversible protein oxidation (cysteine sulfenic acid probe BCN-E-BCN), actin polymerization (phalloidin staining) and migration (wound healing assay) were assessed in absence/presence of Nox5 (melittin) and Src (PP2) inhibitors. To study Nox5-specific effects, we used p22phox-silenced VSMCs (siRNA). Vascular function in VSMC-specific Nox5 transgenic mice was studied by wire myography. In HT, ROS levels (139±27%), Nox5 expression (103±23%) and phosphorylation were increased (77±17.93%) (p<0.05, vs NT). Activation of c-Src (101±26%), PKC (96±33%), MLC 20 (416±71%) and Ang II-induced Ca 2+ influx (574±44 vs NT:451±26) were also increased in HT (p<0.05, vs NT). Melittin reduced Ang II-induced ROS generation in both groups (p<0.05 vs Ctl). In contrast, p22phox silencing increased ROS in both groups, an effect blocked by melittin (p<0.05 vs Ctl). Nox5 inhibition reduced Ang II-induced c-Src phosphorylation and oxidation. In HT, p22phox silencing was associated with sustained Ang II-induced PKC (83±21% vs Ctl) and MLC 20 (89±22% vs Ctl) phosphorylation, effects blocked by melittin and PP2 (p<0.05 vs Ctl). Nox5 and c-Src inhibition reduced Ca 2+ influx, actin polymerization and migration in HT. Hypercontractility observed in Nox5 mice was abolished by melittin and PP2. Our findings demonstrate that Nox5 is upregulated in human hypertension. This is associated with activation of c-Src, increased redox signaling and VSMC cytoskeletal reorganization, migration and vascular contraction. We define a novel Nox5-ROS-c-Src signaling pathway that may play a role in vascular remodeling/dysfunction in hypertension.

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Abstract 3203: The role of Neprilysin in the Development of Insulin Resistance, Type 2 Diabetes and Cardiovascular Disease

Circulation ◽

10.1161/circ.118.suppl_18.s_1112 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Kristina F Standeven ◽

Angela M Carter ◽

Anthony J Balmforth ◽

Stephen B Wheatcroft ◽

Nigel M Hooper ◽

...

Keyword(s):

Insulin Resistance ◽

Metabolic Syndrome ◽

Vascular Function ◽

Animal Studies ◽

Obese Mice ◽

Human Adipocytes ◽

The Metabolic Syndrome ◽

Mesenteric Fat ◽

Order Of Magnitude ◽

Average Gene

Neprilysin (NEP) cleaves several bioactive peptides involved in the regulation of vascular function. In human microvascular endothelial cells, fatty acids and glucose increase NEP activity, and inhibition of NEP in animal studies results in increased insulin sensitivity, suggesting that NEP may be related to the metabolic syndrome. We tested this hypothesis in cell, animal and human based models. Microarray analysis of mRNA expression in differentiated human adipocytes (Affymetrix Human Genome U133 Plus 2.0 arrays) showed NEP expression to be an order of magnitude higher than the average gene signal, suggesting that human adipocytes express high endogenous levels of NEP mRNA. Real time PCR confirmed high levels of NEP mRNA in preadipocytes which increased 28 fold during differentiation and reached levels equivalent to the endogenous control, GAPDH, by 14 days. We created a diet induced model of obesity by feeding male C57BL/6J mice a high-fat diet, which resulted in decreased glucose tolerance and insulin resistance in obese mice. Plasma NEP levels measured after 15 weeks of feeding were significantly higher in obese mice (1642 [± 529]) pg/μl) compared to lean mice (820 [± 487] pg/μl) (p < 0.01). NEP levels increased 4- and 9-fold in epididymal and mesenteric fat in obese, compared to lean, mice. In a study of 318 healthy white European males, plasma NEP measured by activity assay was significantly higher in subjects with the metabolic syndrome (MetS) and levels increased progressively with increasing number of MetS components, being ~8-fold higher in those with 5 MetS components compared with those with none. NEP correlated with insulin, HOMA and BMI in all subjects. In conclusion, we have generated cell, murine and human data which suggest that NEP may have an important role in cardio-metabolic risk associated with insulin resistance, with the adipocyte as a major source of NEP. These findings indicate that NEP is a novel adipokine that links insulin resistance to vascular risk.

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Adipocyte Specific HO-1 Gene Therapy Is Effective in Antioxidant Treatment of Insulin Resistance and Vascular Function in an Obese Mice Model

Antioxidants ◽

10.3390/antiox9010040 ◽

2020 ◽

Vol 9 (1) ◽

pp. 40 ◽

Cited By ~ 11

Author(s):

Shailendra P. Singh ◽

Menachem Greenberg ◽

Yosef Glick ◽

Lars Bellner ◽

Gaia Favero ◽

...

Keyword(s):

Insulin Resistance ◽

Gene Therapy ◽

Adipose Tissue ◽

Vascular Function ◽

Therapeutic Approach ◽

Vascular Dysfunction ◽

Cellular Respiration ◽

Heme Oxygenase 1 ◽

Obese Mice ◽

Mice Model

Obesity is a risk factor for vascular dysfunction and insulin resistance. The study aim was to demonstrate that adipocyte-specific HO-1 (heme oxygenase-1) gene therapy is a therapeutic approach for preventing the development of obesity-induced metabolic disease in an obese-mice model. Specific expression of HO-1 in adipose tissue was achieved by using a lentiviral vector expressing HO-1 under the control of the adiponectin vector (Lnv-adipo-HO-1). Mice fed a high-fat diet (HFD) developed adipocyte hypertrophy, fibrosis, decreased mitochondrial respiration, increased levels of inflammatory adipokines, insulin resistance, vascular dysfunction, and impaired heart mitochondrial signaling. These detrimental effects were prevented by the selective expression of HO-1 in adipocytes. Lnv-adipo-HO-1-transfected mice on a HFD display increased cellular respiration, increased oxygen consumption, increased mitochondrial function, and decreased adipocyte size. Moreover, RNA arrays confirmed that targeting adipocytes with HO-1 overrides the genetic susceptibility of adiposopathy and correlated with restoration of the expression of anti-inflammatory, thermogenic, and mitochondrial genes. Our data demonstrate that HO-1 gene therapy improved adipose tissue function and had positive impact on distal organs, suggesting that specific targeting of HO-1 gene therapy is an attractive therapeutic approach for improving insulin sensitivity, metabolic activity, and vascular function in obesity.

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Abstract P341: Osteoprotegerin-induced ROS Production and Redox Signalling is Exacerbated in Hypertension and Involves Syndecan-1 and TRPM2 Activation

Hypertension ◽

10.1161/hyp.68.suppl_1.p341 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Augusto C Montezano ◽

Ross Hepburn ◽

Delyth Graham ◽

Rhian M Touyz

Keyword(s):

Vascular Function ◽

Molecular Mechanisms ◽

Metabolic Diseases ◽

Vascular Dysfunction ◽

Ros Generation ◽

Ros Production ◽

Redox Signalling ◽

Wky Rats ◽

Amplex Red ◽

Src Activation

Osteoprotegerin (OPG) levels are increased in metabolic diseases, and are a biomarker of vascular dysfunction and cardiovascular risk. Mechanisms related to OPG-induced vascular dysfunction and its role in hypertension are not fully understood, but we previously demonstrated that OPG induces vascular dysfunction through ROS-dependent mechanisms. Here we assessed the molecular mechanisms whereby OPG regulates ROS and vascular function, with a focus on syndecan-1. VSMCs from normotensive (WKY) and hypertensive (SHRSP) rats were stimulated with OPG (50 ng/mL). ROS production was measured by lucigenin, amplex red and ELISA. In VSMCs from WKY rats, OPG increased ROS generation (158±15% vs veh, p<0.05). This effect was blocked by the syndecan-1 inhibitor (synstatin) and by removal of syndecan-1 sulfate proteoglycans side chains, chondroitinase and heparinase. OPG also increased H 2 O 2 (2 fold) and ONOO - (1.5 fold) levels in VSMCs (p<0.05). H 2 O 2 further stimulates ROS levels and redox signalling through activation of TRPM2, a redox-sensitive Ca 2+ channel. TRPM2 inhibitors, 8-bromo-ADPR (8Br) and N-(p-amylcinnamoyl)anthranilic acid (ACA), did not block OPG-induced ROS generation in VSMCs from WKY rats. Syndecan-1 activation leads to FAK and c-Src activation, which are redox-sensitive signalling proteins. FAK, but not c-Src, activation (117±2%, p<0.05) was observed after OPG stimulation of WKY VSMCs. In VSMCS from SHRSP rats, OPG effects on ROS generation were exacerbated (230±40%, p<0.05) and inhibited by synstatin, 8Br and ACA. OPG also increased FAK (118±2) and c-Src (113±1) activation (p<0.05) in VSMCs from SHRSP rats. In conclusion, OPG regulation of oxidative stress is increased in hypertension and involves not only syndecan-1, but also TRPM2 channels, which may lead to activation of redox-sensitive proteins and vascular damage.

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Abstract 348: Inhibition Of Phosphodiesterase 5 Attenuates Angiotensin II-dependent Hypertension And Renal Vascular Dysfunction In C57bl/6 Mice But Not In Enos-Ko Mice

Hypertension ◽

10.1161/hyp.64.suppl_1.348 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Manuel Thieme ◽

Sema Sivritas ◽

Sebastian A Potthoff ◽

Evanthia Mergia ◽

Lars C Rump ◽

...

Keyword(s):

Vascular Function ◽

Vascular Tone ◽

Vascular Dysfunction ◽

Pivotal Role ◽

Ang Ii ◽

Cgmp Signaling ◽

Renal Vascular ◽

The Impact

The kidney plays an outstanding role in the blood pressure (BP) regulation. The renal vasoconstrictor response to angiotensin (Ang) II is balanced by the NO/cGMP-signalling cascade. Ang II causes hypertension and vascular dysfunction by reducing cGMP sensitivity. Ang II is able to increase cGMP degradation by activating phosphodiesterase (PDE)1 and PDE5. The aim of the present study was to identify the predominant PDE subunit regulating renal blood flow (RBF) and vascular tone during hypertension. Therefore, we tested in vivo effects of acute PDE1 (vinpocetine) and PDE5 (sildenafil) inhibition at baseline and during acute Ang II infusion (200ng/kg/min). Furthermore, we examined the impact of PDE-inhibition on Ang II dependent hypertension (500ng/kg/min; 14 days) and on renal vascular function in the isolated perfused kidney. Acute vinpocetine administration (0.8-800μg/kg BW) showed almost no effect on systemic BP and RBF at baseline and during acute Ang II infusion. In contrast, sildenafil (0.8-800μg/kg BW) significantly decreased BP under baseline conditions. During acute Ang II infusion, BP reduction and RBF increase induced by sildenafil was even more pronounced suggesting a pivotal role of the PDE5 in the regulation of renal vascular tone. Based on these results, we tested whether inhibition of the PDE5 protects from hypertension and vascular dysfunction. Indeed, chronic sildenafil treatment significantly attenuated Ang II dependent hypertension in C57BL/6 (vehicle vs. sil: 156±4 vs. 139±7; p<0.05). Moreover, Sildenafil treatment significantly improved NO-dependent vasorelaxation in kidneys of Ang II- treated C57BL/6. To confirm that PDE5 is activated by an increased NO/cGMP signaling, we used eNOS-KO mice, a model known for decreased NO dependent cGMP generation. In eNOS-KO mice, sildenafil failed to reduce Ang II dependent hypertension (172,4 ± 4,3 mmHg vs. 166,1 ± 3,8 mmHg, p=0,2753) and did not improve vascular dysfunction in Ang II treated kidneys. In summary, the PDE5 is the predominant PDE regulating RBF. Inhibition of PDE5 by sildenafil ameliorates chronic Ang II dependent hypertension and improves vascular dysfunction. This study reveals new evidence for the pivotal role of PDE5 in the pathogenesis of AngII-induced hypertension.

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Context-dependent effects of SOCS3 in angiotensin II-induced vascular dysfunction and hypertension in mice: mechanisms and role of bone marrow-derived cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00204.2016 ◽

2016 ◽

Vol 311 (1) ◽

pp. H146-H156 ◽

Cited By ~ 7

Author(s):

Ying Li ◽

Dale A. Kinzenbaw ◽

Mary L. Modrick ◽

Lecia L. Pewe ◽

Frank M. Faraci

Keyword(s):

Bone Marrow ◽

Angiotensin Ii ◽

Endothelial Dysfunction ◽

Vascular Function ◽

Carotid Artery Disease ◽

Vascular Dysfunction ◽

Cytokine Signaling ◽

Ang Ii ◽

Direct Effects ◽

Bone Marrow Derived Cells

Carotid artery disease is a major contributor to stroke and cognitive deficits. Angiotensin II (Ang II) promotes vascular dysfunction and disease through mechanisms that include the IL-6/STAT3 pathway. Here, we investigated the importance of suppressor of cytokine signaling 3 (SOCS3) in models of Ang II-induced vascular dysfunction. We examined direct effects of Ang II on carotid arteries from SOCS3-deficient (SOCS3+/−) mice and wild-type (WT) littermates using organ culture and then tested endothelial function with acetylcholine (ACh). A low concentration of Ang II (1 nmol/l) did not affect ACh-induced vasodilation in WT but reduced that of SOCS3+/−mice by ∼50% ( P < 0.05). In relation to mechanisms, effects of Ang II in SOCS3+/−mice were prevented by inhibitors of STAT3, IL-6, NF-κB, or superoxide. Systemic Ang II (1.4 mg/kg per day for 14 days) also reduced vasodilation to ACh in WT. Surprisingly, SOCS3 deficiency prevented most of the endothelial dysfunction. To examine potential underlying mechanisms, we performed bone marrow transplantation. WT mice reconstituted with SOCS3+/−bone marrow were protected from Ang II-induced endothelial dysfunction, whereas reconstitution of SOCS3+/−mice with WT bone marrow exacerbated Ang II-induced effects. The SOCS3 genotype of bone marrow-derived cells did not influence direct effects of Ang II on vascular function. These data provide new mechanistic insight into the influence of SOCS3 on the vasculature, including divergent effects depending on the source of Ang II. Bone marrow-derived cells deficient in SOCS3 protect against systemic Ang II-induced vascular dysfunction.

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Abstract P097: Molecular Mechanisms of VEGFR Inhibition-induced Endothelial Cell Damage

Hypertension ◽

10.1161/hyp.66.suppl_1.p097 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Francisco J Rios ◽

Augusto C Montezano ◽

Lucas Van Der Mey ◽

Heather Y Small ◽

Carmine Savoia ◽

...

Keyword(s):

Endothelial Cells ◽

Mrna Expression ◽

Vascular Function ◽

Molecular Mechanisms ◽

Cell Damage ◽

Vascular Dysfunction ◽

Endothelial Damage ◽

Mrna Levels ◽

Ang Ii ◽

Vegf Inhibitors

VEGF/VEGFR inhibitors, used as anti-angiogenic drugs to treat cancer, induce severe hypertension. Molecular mechanisms whereby VEGF inhibitors cause hypertension are unclear, but nitric oxide (NO) and oxidative stress may be involved. We questioned whether reactive oxygen species (ROS) and Ang II, important regulators of vascular function in hypertension, also play a role in VEGF inhibitor-induced vascular dysfunction. Human microvascular endothelial cells (HMECs) were stimulated with vatalanib (VAT-VEGFR inhibitor) and gefitinib (GEF-EGFR inhibitor) in the absence/presence of Ang II. Activation of eNOS and MAPKs were assessed by immunoblotting. Antioxidant enzyme mRNA was analysed by qPCR. Microparticle levels were measured by flow cytometry. Endothelial microparticles, biomarkers of endothelial damage, tend to increase in subjects treated with VEGFR inhibitors. Phosphorylation of eNOS activation site (Ser1177) (28.3% ± 7.1) was decreased by VAT, while no changes were observed after exposure of HMECs to GEF (p<0.05). VAT decreased mRNA expression of Nox4 (0.5 ± 0.2) and H2O2-regulating antioxidants enzymes such as catalase (0.4 ± 0.1) and glutathione peroxidase (0.4 ± 0.1), while increased mRNA levels of Nox5 (3.35±1.1) (p<0.05 vs. veh). Ang II stimulation increased eNOS (171.2% ± 17.4) and ERK1/2 (177.5% ± 38.5) activation (p<0.05); all effects were blocked only by GEF. Inhibition of VEGFR also blocked Ang II effects on SOD1 (1.33 ± 0.1), HO-1 (1.6 ± 0.3) and NQO1 (1.6 ± 0.3) mRNA levels (p<0.05). In addition, Ang II increased Nox4 mRNA expression through VEGFR-dependent mechanisms. VEGFR1/2 and AT2R, but not AT1R, were expressed in HMEC. Ang II effects on eNOS phosphorylation were inhibited by PD123319 (AT2R antagonist) but not by losartan (AT1R antagonist). In conclusion, our data identify novel mechanisms whereby AngII, possibly through AT2R-dependent VEGFR transactivation, regulates eNOS activation, MAPK signalling and H2O2-related antioxidant enzymes. In addition to changes in NO availability, VEGFR inhibition may interfere with the redox status of endothelial cells, leading to vascular dysfunction and hypertension.

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Abstract 057: Nox5 Induces Vascular Dysfunction and Arterial Remodelling Independently of Blood Pressure Elevation in Ang II-infused Nox5-expressing Mice

Hypertension ◽

10.1161/hyp.68.suppl_1.057 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Augusto C Montezano ◽

Adam P Harvey ◽

Francisco J Rios ◽

Maria Dulak-Lis ◽

Wendy Beatie ◽

...

Keyword(s):

Blood Pressure ◽

Vascular Function ◽

Kinase Inhibitor ◽

Vascular Dysfunction ◽

Rho Kinase ◽

Lumen Diameter ◽

Ang Ii ◽

Cross Sectional ◽

Blood Pressure Elevation ◽

Endothelium Dependent Relaxation

Nox5 is a unique Ca 2+ -sensitive Nox isoform that is expressed in human vascular smooth muscle cells (VSMC). Although Nox5 has been implicated in diabetic nephropathy, its role in vascular function and development of hypertension remain unclear. Nox5 is not expressed in rodents, and accordingly we generated humanised Nox5 mice with Nox5 expressed in a VSMC-specific manner (Nox5SM22). Control (wild-type) and Nox5SM22 mice were infused with Ang II (600 ng/Kg/day). Blood pressure (BP) was assessed by tail-cuff. Vascular function and structure of resistance arteries were measured by myography. Ang II increased BP in WT (182.5±10 mmHg) and Nox5SM22 mice (173.1±5 mmHg) with no significant differences. Arteries from Nox5SM22 mice exhibited reduced endothelium-dependent relaxation versus WT controls (%ACh relaxation: 55.1±4 vs ctl: 81.6±7%). Fasudil (Rho kinase inhibitor)-induced relaxation was reduced in Nox5SM22 mice versus controls (%Fas: 111.3±11 vs ctl: 166.6±8%) (p<0.05). Ang II increased the maximal contraction to U46619 (thromboxane A2 mimetic) in WT (115.8±2 vs untreated: 101.4±2%) and Nox5SM22 (121.3±3 vs untreated: 99.1±2) (p<0.05) and induced endothelial dysfunction in all groups. Fasudil-induced relaxation was impaired by Ang II in WT (102.7±6 vs untreated: 166.6±8%, p<0.05) but not further impaired in Nox5SM22 mice (114.9±6 vs untreated: 111.3±11%). Ang II increased cross-sectional area (CSA) and lumen diameter; while in Nox5SM22 mice, Ang II increased wall thickness, wall-to-lumen ratio, CSA and decreased lumen diameter, with associated increased vascular stiffness. Our findings indicate that in mice expressing human Nox5 in VSMCs, endothelium-dependent relaxation is impaired, fasudil-mediated vasodilation is attenuated and vessels undergo exaggerated hypertrophic inward remodelling with increased stiffness; processes that occur independently of BP elevation. These data suggest an important role for Nox5 in Ang II-induced vascular dysfunction and remodeling, but not in the development of hypertension. Moreover, we identify Rho kinase as a putative target for Nox5-induced vascular injury. We provide novel insights into Nox5 vascular biology and demonstrate that vascular Nox5 actions are dissociated from BP effects.

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