Karla B. Neves

Downregulation of Nuclear Factor Erythroid 2–Related Factor and Associated Antioxidant Genes Contributes to Redox-Sensitive Vascular Dysfunction in Hypertension

Oxidative stress is implicated in vascular dysfunction in hypertension. Although mechanisms regulating vascular pro-oxidants are emerging, there is a paucity of information on antioxidant systems, particularly nuclear factor erythroid 2–related factor (Nrf2), a master regulator of antioxidants enzymes. We evaluated the vascular regulatory role of Nrf2 in hypertension and examined molecular mechanisms, whereby Nrf2 influences redox signaling in small arteries and vascular smooth muscle cells from Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Cells were stimulated with angiotensin II in the absence/presence of Nrf2 activators (bardoxolone/L-sulforaphane). Increased vascular reactive oxygen species production (chemiluminescence and amplex red) was associated with reduced Nrf2 activity in arteries (18%) and vascular smooth muscle cells (48%) in SHRSP (P<0.05 versus WKY). Expression of antioxidant enzymes, including superoxide dismutase-1 (64%), catalase (60%), peroxiredoxin 1 (75%), and glutathione peroxidase (54%), was reduced in SHRSP. L-sulforaphane reversed these effects. Angiotensin II increased nuclear accumulation of Nrf2 in vascular smooth muscle cells from WKY (197% versus vehicle), with blunted effects in SHRSP (44% versus vehicle). These responses were associated with increased antioxidant expression (superoxide dismutase-1, 32%; catalase, 42%; thioredoxin, 71%; peroxiredoxin, 1%–90%; quinone oxidoreductase, 84%; P<0.05 versus vehicle) and increased activity of superoxide dismutase-1, catalase, and thioredoxin in WKY but not in SHRSP, which exhibited increased Bach1 expression. Nrf2 activators blocked angiotensin II–induced reactive oxygen species generation. Vascular function demonstrated increased contractility (Emax WKY 113.4±5.6 versus SHRSP 159.0±8.3) and decreased endothelial-dependent relaxation (Emax WKY 88.6±3.1 versus SHRSP 74.6±3.2, P<0.05) in SHRSP, effects corrected by L-sulforaphane. Our findings suggest that Nrf2 downregulation contributes to redox-sensitive vascular dysfunction in hypertension.

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 and Vascular Function in Aging

Androgen receptors are widely distributed in several tissues, including vascular endothelial and smooth muscle cells. Through classic cytosolic androgen receptors or membrane receptors, testosterone induces genomic and non-genomic effects, respectively. Testosterone interferes with the vascular function by increasing the production of pro-inflammatory cytokines and arterial thickness. Experimental evidence indicates that sex steroid hormones, such as testosterone modulate the synthesis and bioavailability of NO and, consequently, endothelial function, which is key for a healthy vasculature. Of interest, aging itself is accompanied by endothelial and vascular smooth muscle dysfunction. Aging-associated decline of testosterone levels is accompanied by age-related diseases, such as metabolic and cardiovascular diseases, indicating that very low levels of androgens may contribute to cardiovascular dysfunction observed in these age-related disorders or, in other words, that testosterone may have beneficial effects in the cardiovascular system. However, testosterone seems to play a negative role in the severity of renal disease. In this mini-review, we briefly comment on the interplay between aging and testosterone levels, the vascular actions of testosterone and its implications for vascular aging. Renal effects of testosterone and the use of testosterone to prevent vascular dysfunction in elderly are also addressed.

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway.

AIMS Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. MAIN METHODS Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24h), and isometric contraction was recorded. Protein expression was determined by Western blotting. KEY FINDINGS Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1h with chemerin. Chemerin incubation for 24h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. SIGNIFICANCE Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases.

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.

NLRP3 Inflammasome Mediates Aldosterone-Induced Vascular Damage

Background: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. Methods: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout (NLRP3−/−), caspase-1 knockout (Casp-1−/−), and interleukin-1 receptor knockout (IL-1R−/−) mice treated with vehicle or aldosterone (600 µg·kg−1·d−1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. Results: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1&bgr; levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1&bgr; secretion by bone marrow–derived macrophages by activating nuclear factor-&kgr;B signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1&bgr; in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. Conclusions: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Cholesteryl Ester-Transfer Protein Inhibitors Stimulate Aldosterone Biosynthesis in Adipocytes through Nox-Dependent Processes

Hyperaldosteronism and hypertension were unexpected side effects observed in trials of torcetrapib, a cholesteryl ester-transfer protein (CETP) inhibitor that increases high-density lipoprotein. Given that CETP inhibitors are lipid soluble, accumulate in adipose tissue, and have binding sites for proteins involved in adipogenesis, and that adipocytes are a source of aldosterone, we questioned whether CETP inhibitors (torcetrapib, dalcetrapib, and anacetrapib) influence aldosterone production by adipocytes. Studies were performed using human adipocytes (SW872), which express CETP, and mouse adipocytes (3T3-L1), which lack the CETP gene. Torcetrapib, dalcetrapib, and anacetrapib increased expression of CYP11B2, CYP11B1, and steroidogenic acute regulatory protein, enzymes involved in mineralocorticoid and glucocorticoid generation. These effects were associated with increased reactive oxygen species formation. Torcetrapib, dalcetrapib, and anacetrapib upregulated signal transducer and activator of transcription 3 (STAT3) and peroxisome proliferation-activated receptor-γ, important in adipogenesis, but only torcetrapib stimulated production of chemerin, a proinflammatory adipokine. To determine mechanisms whereby CETP inhibitors mediate effects, cells were pretreated with inhibitors of Nox1/Nox4 [GKT137831; 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione], Nox1 (ML171 [2-acetylphenothiazine]), mitochondria (rotenone), and STAT3 (S3I-201 [2-hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid]). In torcetrapib-stimulated cells, Nox inhibitors, rotenone, and S3I-201 downregulated CYP11B2 and steroidogenic acute regulatory protein and reduced aldosterone. Dalcetrapib and anacetrapib effects on aldosterone were variably blocked by GKT137831, ML171, rotenone, and S3I-201. In adipocytes, torcetrapib, dalcetrapib, and anacetrapib inhibit enzymatic pathways responsible for aldosterone production through Nox1/Nox4- and mitochondrial-generated reactive oxygen species and STAT3. CETP inhibitors also influence adipokine production. These processes may be CETP independent. Our findings identify novel adipocyte-related mechanisms whereby CETP inhibitors increase aldosterone production. Such phenomena may contribute to hyperaldosteronism observed in CETP inhibitor clinical trials.

Adipokine Chemerin Bridges Metabolic Dyslipidemia and Alveolar Bone Loss in Mice

Chemerin is an adipokine that regulates adipogenesis and metabolic functions of mature adipocytes mainly through the activation of chemokine‐like receptor 1 (CMKLR1). Elevated levels of chemerin have been found in individuals with obesity, type 2 diabetes, and osteoporosis. This adipokine was identified as an inflammatory and metabolic syndrome marker. Considering that the association between metabolic syndrome and bone health remains unclear, the present study aimed to clarify the role of chemerin in the pathophysiology of bone loss induced by dyslipidemia, particularly modulating osteoclastogenesis. In vitro analyses showed a downregulation of CMKLR1 at the early stage of differentiation and a gradual increase at late stages. Strikingly, chemerin did not modify osteoclast differentiation markers or osteoclast formation; however, it increased the actin‐ring formation and bone resorption activity in mature osteoclasts. The increased bone resorption activity induced by chemerin was effectively inhibited by CMKLR1 antagonist (CCX832). Chemerin boosting mature osteoclast activity involves ERK5 phosphorylation. Moreover, two models of dyslipidemia (high‐fat diet [HFD]‐treated C57/BL6 and db/db mice) exhibited significantly increased level of chemerin in the serum and gingival tissue. Morphometric analysis showed that HFD‐treated and db/db mice exhibited increased alveolar bone loss compared to respective control mice, which was associated with an up‐regulation of chemerin, CMKLR1 and cathepsin K mRNA expression in the gingival tissue. The treatment of db/db mice with CCX832 effectively inhibited bone loss. Antagonism of chemerin receptor also inhibited the expression of cathepsin K in the gingival tissue. Our results show that chemerin not only increases osteoclasts activity in vitro, but also that increased level of chemerin in dyslipidemic mice plays a critical role in bone homeostasis.

Internal Pudental Artery Dysfunction in Diabetes Mellitus Is Mediated by NOX1-Derived ROS-, Nrf2-, and Rho Kinase–Dependent Mechanisms

Oxidative stress plays an important role in diabetes mellitus (DM)–associated vascular injury. DM is an important risk factor for erectile dysfunction. Functional and structural changes in internal pudendal arteries (IPA) can lead to erectile dysfunction. We hypothesized that downregulation of nuclear factor E2–related factor 2 (Nrf2), consequent to increased nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1)-derived reactive oxygen species (ROS), impairs IPA function in DM. IPA and vascular smooth muscle cells from C57BL/6 (control) and NOX1 knockout mice were used. DM was induced by streptozotocin in C57BL/6 mice. Functional properties of IPA were assessed using a myograph, protein expression and peroxiredoxin oxidation by Western blot, RNA expression by polymerase chain reaction, carbonylation by oxyblot assay, ROS generation by lucigenin, nitrotyrosine, and amplex red, and Rho kinase activity and nuclear accumulation of Nrf2 by ELISA. IPA from diabetic mice displayed increased contractions to phenylephrine (control 138.5±9.5 versus DM 191.8±15.5). ROS scavenger, Nrf2 activator, NOX1 and Rho kinase inhibitors normalized vascular function. High glucose increased ROS generation in IPA vascular smooth muscle cell. This effect was abrogated by Nrf2 activation and not observed in NOX1 knockout vascular smooth muscle cell. High glucose also increased levels of nitrotyrosine, protein oxidation/carbonylation, and Rho kinase activity, but reduced Nrf2 activity and expression of Nrf2-regulated genes (catalase [25.6±0.05%], heme oxygenase-1 [21±0.1%], and NAD(P)H:quinone oxidoreductase 1 [22±0.1%]) and hydrogen peroxide levels. These effects were not observed in vascular smooth muscle cell from NOX1 knockout mice. In these cells, high glucose increased hydrogen peroxide levels. In conclusion, Rho kinase activation, via NOX1-derived ROS and downregulation of Nrf2 system, impairs IPA function in DM. These data suggest that Nrf2 is vasoprotective in DM-associated erectile dysfunction.

Off-target vascular effects of cholesteryl ester transfer protein inhibitors involve redox-sensitive and signal transducer and activator of transcription 3-dependent pathways

Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50: 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which may contribute to the adverse cardiovascular effects of these drugs such as hypertension.