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Multiple organ perfusion is impaired in sepsis. Clinical studies suggest that persistent perfusion disturbances are prognostic of fatal outcome in sepsis. Pyroptosis occurs upon activation of caspases and their subsequent cleavage of gasdermin D (Gsdmd), resulting in Gsdmd-N (activated NH2-terminal fragment of Gsdmd) that form membrane pores to induce cell death in sepsis. In addition, Gsdmd-/- mice are protected from a lethal dose of lipopolysaccharide (LPS). However, how Gsdmd-mediated pyroptosis occurs in endothelial cells and leads to impaired perfusion remain unexplored in endotoxemia. We used transgenic mice with ablation of Gsdmd and determined that mice lacking Gsdmd exhibited reduced breakdown of endothelial barrier, improved organ perfusion, as well as increased survival in endotoxemia. Phospholipase Cγ1 (PLCγ1) contributed to Gsdmd-mediated endothelial pyroptosis in a calcium-dependent fashion, without affecting Gsdmd-N production. Cytosolic calcium signaling promoted Gsdmd-N translocation to the plasma membrane, enhancing endothelial pyroptosis induced by LPS. We used adeno-associated virus (AAV9) vectors carrying a short hairpin RNA (shRNA) against murine PLCγ1 mRNA under control of the tie1 core promoter (AAV-tie1-sh-PLCγ1) to uniquely downregulate PLCγ1 expression in the endothelial cells. Here, we showed that unique inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, and improved perfusion disturbances. Moreover, unique downregulate endothelial PLCγ1 expression markedly decreased mortality of mice in endotoxemia. Thus, we establish that endothelial injury as an important trigger of fatal outcome in endotoxemia. Additionally, these findings suggest that interfering with Gsdmd and PLCγ1-calcium pathway may represent a new treatment strategy for critically ill patients sustaining endotoxemia. [ABSTRACT FROM AUTHOR]

Endothelial cell senescence is regarded as a vital characteristic of cardiovascular diseases. Elevated palmitate (PA) is an independent risk factor of cardiovascular diseases, but its role in endothelial cell senescence is currently unknown. During the course of studying the prosenescent role of PA, we discovered a key role of dsRNA-dependent protein kinase [protein kinase R (PKR)] in endothelial senescence. Exposure of human umbilical vein endothelial cells (HUVECs) to PA-induced cell senescence is characterized by increased levels of senescence-associated β-galactose glucosidase activity, excessive production of reactive oxygen species production, impaired cellular proliferation, and G1 phase arrest. This phenomenon is associated with an increase of PKR autophosphorylation and decreased activity of sirtuin 1 (Sirt1), a pivotal antisenescent factor. PKR inactivation by PKR siRNA or its phosphorylation inhibitor 2-aminopurine significantly attenuated PAinduced HUVEC senescence by reversing Sirt1 activity and its downstream signaling. Moreover, to study the regulatory mechanism between PKR and Sirt1, we found that PKR promotes JNK activation to inhibit Sirt1 activity and that this effect could be reversed by the JNK inhibitor SP600125. These findings provide evidence that PKR mediates PA-induced HUVEC senescence by inhibiting Sirt1 signaling. Our study provides novel insights into the actions and mechanisms of PKR in endothelial senescence. [ABSTRACT FROM AUTHOR]

Bone marrow-derived progenitor cells (BMPCs) are potential candidates for autologous cell therapy in tissue repair and regeneration because of their high angiogenic potential. However, increased progenitor cell apoptosis in diabetes directly limits their success in the clinic. MicroRNAs are endogenous noncoding RNAs that regulate gene expression at the posttranscriptional level, but their roles in BMPC-mediated angiogenesis are incompletely understood. In the present study, we tested the hypothesis that the proangiogenic miR-27b inhibits BMPC apoptosis in Type 2 diabetes. Bone marrowderived EPCs from adult male Type 2 diabetic db/db mice and their normal littermates db/+ mice were used. MiR-27b expression (realtime PCR) in EPCs was decreased after 24 h of exposure to methylglyoxal (MGO) or oxidized low-density lipoprotein but not high glucose, advanced glycation end products, the reactive oxygen species generator LY83583, or H2O2. The increase in BMPC apoptosis in the diabetic mice was rescued following transfection with a miR-27b mimic, and the increased apoptosis induced by MGO was also rescued by the miR-27b mimic. p53 protein expression and the Bax/Bcl-2 ratio in EPCs (Western blot analyses) were significantly higher in db/db mice, both of which were suppressed by miR-27b. Furthermore, mitochondrial respiration, as measured by oxygen consumption rate, was enhanced by miR-27b in diabetic BMPCs, with concomitant decrease of mitochondrial Bax/Bcl-2 ratio. The 3= UTR binding assays revealed that both Bax, and its activator RUNX1, were direct targets of miR-27b, suggesting that miR-27b inhibits Bax expression in both direct and indirect manners. miR-27b prevents EPC apoptosis in Type 2 diabetic mice, at least in part, by suppressing p53 and the Bax/Bcl-2 ratio. These findings may provide a mechanistic basis for rescuing BMPC dysfunction in diabetes for successful autologous cell therapy. [ABSTRACT FROM AUTHOR]

Peripheral artery disease (PAD) is a common clinical problem, and its pathophysiological mechanisms are incompletely understood. Double-stranded RNA-activated protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. Although PKR has been reported in antivirus and the immune system, the role of PKR in vascular function, especially in angiogenesis, is still unclear. PKR-/- mice were used in our experiments. Blood flow recovery was significantly delayed in PKR-/- vs. WT mice (Laser Doppler detection, n = 9, P < 0.01), accompanied by 34% reduced CD31-positive stain in ischemic muscle 28 days after procedure (immunohistochemistry, n = 9, P < 0.05). PKR expression decreased in the first 12 h and increased to peak at 24 h in human umbilical vein endothelial cells (HUVECs) in response to hypoxia (Western blot analyses, n = 3, P < 0.05). Accordingly, phospho-PKR expression increased in HUVECs 24 h after treatment with hypoxia (Western blot analyses, n = 3, P < 0.05). Inhibition of PKR (siRNA transfection) reduced microtubule formation (Matrigel tube formation, n = 3, vs. control siRNA, P < 0.05) and migration (wound healing, n = 3, vs. control siRNA, P < 0.05) by 33 and 59%, respectively. Vascular endothelial growth factor (VEGF) expression in ischemic muscle from PKR-/- mice was significantly decreased by 54% 1 day after procedure (n = 3, P < 0.05, vs. WT) and by 63% 7 days after procedure (n = 3, P < 0.01, vs. WT), respectively. At the same time, VEGF expression in HUVECs decreased by 21% (n = 3, P < 0.05, PKR siRNA vs. control siRNA). These findings demonstrate that PKR mediates angiogenesis through a VEGF pathway, which may form the basis for future intervention of PAD. [ABSTRACT FROM AUTHOR]

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-L-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds and accelerated wound healing significantly, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were reduced significantly in STZ-WT and hph-1 mice, paralleled by increased superoxide anion levels; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice at least partly through the attenuation of superoxide and TSP-1 levels and augmentation of NO level. [ABSTRACT FROM AUTHOR]

Refractory wounds in diabetic patients present a significant clinical problem. Sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes. Regulation of SHH in wound healing is poorly understood. We hypothesize that thrombospondin-1 (TSP-1), through its receptor CD36, contributes to the SHH signaling defect in bone marrow-derived angiogenic cells (BMACs) in type 1 diabetic mice. Isolated BMACs from TSP-1-knockout mice demonstrated improved tube formation, migration, and adhesion in parallel with active SHH signaling. BMACs from STZ-induced type 1 diabetic mice showed significantly impaired Matrigel tube formation (n = 5; P < 0.05 vs. control), which was rescued by TSP-1 depletion (n = 5; P < 0.05 STZ-TSP-1/ vs. STZ-WT) or exogenous SHH (20 mg/l, 24 h, n = 4; P < 0.05 vs. STZ-control). The expression of CD36 was elevated in BMACs from STZ mice (n = 4; P < 0.05). SHH signaling was significantly higher in BMACs from TSP-1-/- mice and TSP-1 receptor CD36-knockout mice (n = 6; P < 0.05 vs. WT) but not CD47-knockout mice (n = 3; P > 0.05 vs. WT). The impairment of recombinant human TSP-1 (2.2 nM, 24 h) on BMAC Matrigel tube formation was delayed significantly by CD36 deletion (n = 5; P < 0.05). CD36-/- BMACs demonstrated better tube formation under both normal and diabetic conditions with active SHH signaling (n=4; P<0.05 vs. WT BMACs). In conclusion, The TSP-1/CD36 pathway contributes to the SHH signaling defect, resulting in BMAC dysfunction in type 1 diabetic mice. [ABSTRACT FROM AUTHOR]

Within the paraventricular nucleus (PVN), there is a balance between the excitatory and inhibitory neurotransmitters that regulate blood pressure; in hypertension, the balance shifts to enhanced excitation. Nitric oxide (NO) is an atypical neurotransmitter that elicits inhibitory effects on cardiovascular function. We hypothesized that reduced PVN NO led to elevations in blood pressure during both the onset and sustained phases of hypertension due to decreased NO synthase (NOS) and increased asymmetrical dimethylarginine (ADMA; an endogenous NOS inhibitor) and symmetric dimethylarginine (SDMA). Elevated blood pressure, in response to PVN bilateral microinjections of a NO inhibitor, nitro-L-arginine methyl ester, was blunted in renal wrapped rats during the onset of hypertension (day 7) and sustained renal wrap hypertension (day 28) compared with sham-operated rats. Adenoviruses (Ad) encoding endothelial NOS (eNOS) or LacZ microinjected into the PVN [1 ≤ 109 plaque-forming units, bilateral (200 nl/site)] reduced mean arterial pressure compared with control (Day 7, Ad LacZ wrap: 144 ± 7 mmHg and Ad eNOS wrap: 117 ± 5 mmHg, P ≤ 0.05) throughout the study (Day 28, Ad LacZ wrap: 123 ± 1 mmHg and Ad eNOS wrap: 108 ± 4 mmHg, P ≤ 0.05). Western blot analyses of PVN NOS revealed significantly lower PVN neuronal NOS during the onset of hypertension but not in sustained hypertension. Reduced SDMA was found in the PVN during the onset of hypertension; however, no change in ADMA was observed. In conclusion, functional indexes of NO activity indicated an overall downregulation of NO in renal wrap hypertension, but the mechanism by which this occurs likely differs throughout the development of hypertension. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPCs) play an essential role in angiogenesis but are functionally impaired in diabetes. We recently reported that decreased expression of manganese superoxide dismutase (MnSOD) critically contributes to diabetic EPC dysfunction. AMP-activated protein kinase (AMPK) activation has been shown to induce MnSOD and suppress hyperglycemia-induced mitochondrial ROS production in endothelial cells. However, whether AMPK protects EPCs from oxidative stress in diabetes is unknown. We tested the hypothesis that AMPK activation rescues impaired EPC functions through MnSOD induction in type 1 diabetes. Bone marrow-derived EPCs from adult male streptozotocin-induced diabetic mice and normal controls were used. AMPK activity was decreased in diabetic EPCs, indicated by reduced AMPK and acetyl-CoA carboxylase phosphorylation. AMPK activation by treating diabetic EPCs with its selective agonist AICAR rescued their in vitro functions, including Matrigel tube formation, adhesion, and migration. Furthermore, AICAR restored the decreased MnSOD protein and enzymatic activity and suppressed the mitochondrial superoxide level in diabetic EPCs, indicated by MitoSOX flow cytometry. These beneficial effects of AICAR on MnSOD and EPC functions were significantly attenuated by silencing MnSOD or AMPK antagonist compound C pretreatment. Finally, the expression of protein phosphatase 2A, a key enzyme for AMPK dephosphorylation and inactivation, was increased in diabetic EPCs, and its inhibition by siRNA or okadaic acid reversed the deficient AMPK activation and MnSOD level in diabetic EPCs. These findings demonstrate for the first time that AMPK activation rescues impaired EPC functions and suppresses mitochondrial superoxide by inducing MnSOD in type 1 diabetes. [ABSTRACT FROM AUTHOR]

S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H2S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to NG-nitro-l-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H2S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H2S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an NG-nitro-l-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H2S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia (HHcy) has been associated with impaired vascular endothelial function. Our previous study demonstrated significantly higher secretion of the chemokine monocyte chemoattractant protein-1 from monocytes in response to lipopolysaccharide in patients with HHcy. In the present study, we investigated whether coronary endothelial function was damaged in patients with chronic HHcy (plasma level of homocysteine >15 μmol/l) and, if so, whether this impaired endothelial function is induced by the uncoupling of endothelial nitric oxide synthase (eNOS). When tetrahydrobiopterin levels are inadequate, eNOS is no longer coupled to L-arginine oxidation, which results in reactive oxygen species rather than nitric oxide production, thereby inducing vascular endothelial dysfunction. The 71 participants were divided into two groups, control (n = 50) and HHcy (n = 21). Quantification of coronary flow velocity reserve (CFVR) was after rest and after adenosine administration done by noninvasive Doppler echocardiography. Plasma levels of nitric oxide and tetrahydrobiopterin were significantly lower in patients with HHcy than in controls (99.54 ± 32.23 vs. 119.50 ± 37.68 μmol/l and 1.43 ± 0.46 vs. 1.73 ± 0.56 pmol/ml, all P < 0.05). Furthermore, CFVR was significantly lower in the HHcy than the control group (2.76 ± 0.49 vs. 3.09 ± 0.52, P < 0.05). In addition, plasma level of homocysteine was negatively correlated with CFVR. Chronic HHcy may contribute to coronary artery disease by inducing dysfunction of the coronary artery endothelium. The uncoupling of eNOS induced by HHcy in patients with chronic HHcy may explain this adverse effect in part. [ABSTRACT FROM AUTHOR]

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and increased mortality and morbidity associated with pulmonary and cardiovascular diseases. However, a precise understanding of the biological mechanism underlying PM-associated toxicity and pathogenesis remains elusive. Here, we investigated the impact of PM exposure in intracellular stress signaling pathways with animal models and cultured cells. Inhalation exposure of the mice to environmentally relevant fine particulate matter (aerodynamic diameter < 2.5 µm, PM2.5) induces endoplasmic reticulum (ER) stress and activation of unfolded protein response (UPR) in the lung and liver tissues as well as in the mouse macrophage cell line RAW264.7. Ambient PM2.5 exposure activates double-strand RNA-activated protein kinase-like ER kinase (PERK), leading to phosphorylation of translation initiation factor eIF2α and induction of C/EBP homologous transcription factor CHOP/GADDI53. Activation of PERK-mediated UPR pathway relies on the production of reactive oxygen species (ROS) and is critical for PM2.5-induced apoptosis. Furthermore, PM2.5 exposure can activate ER stress sensor IRE1α, but it decreases the activity of IRE1α in splicing the mRNA encoding the UPR trans-activator X-box binding protein 1 (XBP1). Together, our study suggests that PM2.5 exposure differentially activates the UPR branches, leading to ER stress-induced apoptosis through the PERK-eIF2α-CHOP UPR branch. This work provides novel insights into the cellular and molecular basis by which ambient PM2.5 exposure elicits its cytotoxic effects that may be related to air pollution-associated pathogenesis. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPCs) play an important role in angiogenesis, which is essential for numerous physiological processes as well as tumor growth. Several microRNAs (miRNAs) have been reported to be involved in angiogenesis. MiR-34a, recently reported as a tumor suppressor, has been found to target silent information regulator 1 (Sirt1), leading to cell cycle arrest or apoptosis. However, the role of miR-34a in EPC-mediated angiogenesis was unknown. The present study tested the hypothesis that miR-34a inhibits EPC-mediated angiogenesis by inducing senescence via suppressing Sirt1. Bone marrow-derived EPCs from adult male Spraque-Dawley rats were used. Results of flow cytometry showed that EPCs after 7 days of culture expressed both stem cell markers CD34 and CD133 and endothelial cell markers VEGFR-2 (flk-1) and VE-cadherin. MiR-34a was expressed in normal EPCs, and overexpression of miR-34a via its mimic transfection significantly increased its expression and impaired in vitro EPC angiogenesis. MiR-34a overexpression led to a significantly increased EPC senescence, paralleled with an ~40% Sirt1 reduction. Furthermore, knockdown of Sirt1 by its siRNA resulted in diminished EPC angiogenesis and increased senescence. Finally, overexpression of miR-34a increased the level of Sirt1 effector-acetylated forkhead box O transcription factors 1 (FoxO1), an effect mimicked in EPCs following Sirt1 knockdown. In conclusion, miR-34a impairs EPC-mediated angiogenesis by induction of senescence via inhibiting Sirt1. [ABSTRACT FROM AUTHOR]

Sonic hedgehog (SHH) plays an important role in postnatal tissue repair. The present study tested the hypothesis that impaired SHH pathway results in delayed wound healing by suppressing cutaneous nitric oxide (NO) function in type 1 diabetes. Adult male C57/B6 mice and streptozotocin (STZ)-induced type 1 diabetic mice were used. Although cutaneous SHH and Patched-i (Ptc-1 encoded by PTCH, PTCH 1) proteins were increased significantly on day 4 after wounding compared with day 0 in normal mice, both were decreased significantly in STZ-induced diabetic mice. Topical application of SHH restored wound healing delay in STZ-induced diabetic mice, with a concomitant augmentation of both cutaneous constitutive nitric oxide synthase (NOS) activity and nitrite level. The effects of SHH on wound healing and cutaneous NO function were markedly inhibited by SHH receptor inhibitor cyclopamine. After 24-h treatment in vitro, SHH (5-20 μg/ml) significantly increased cutaneous endothelial NOS protein expression, NOS activity and NO level in normal mice and STZ-induced diabetic mice in a concentration-dependent manner, an effect that was blunted by cyclopamine and NOS inhibitor Nω-nitro-L-arginine methyl ester. The phosphatidylinositol 3-kinase inhibitor LY-294002 significantly blunted the increase of NOS activity and NO level induced by SHH treatment in human umbilican vein endothelial cells. These results demonstrate that the SHH pathway is activated in a normal wound, and its reduction results in impaired NO function and wound healing in diabetes. Strategies aimed at augmenting the endogenous SHH pathway may provide an effective means in ameliorating delayed diabetic wound healing. [ABSTRACT FROM AUTHOR]

To determine the mechanism(s) underlying enhanced oxidative stress in kidneys of salt-sensitive hypertension, neonatal Wistar rats were given vehicle or capsaicin (CAP, 50 mg/kg Sc) on the first and second days of life. After being weaned, male rats were assigned into four groups and treated for 2 wk with the following: vehicle + a normal sodium diet (NS, 0.4%, CON-NS), vehicle + a high-sodium diet (HS, 4%, CON-HS), CAP + NS (CAP-NS), and CAP + HS (CAP-HS). Systolic blood pressure was significantly increased in CAP-HS but not CAP-NS or CON-HS rats. Plasma and urinary 8-iso-prostaglandin F2α levels increased by ~40% in CON-HS and CAP-HS rats compared with their respective controls fed a NS diet (P < 0.05), and these parameters were higher in CAP-HS compared with CON-HS rats. Superoxide (O2-·) levels in the renal cortex and medulla increased by ~45% in CAP-HS compared with CON-HS, CON-NS, and CAP-NS rats (P < 0.05). Enhanced O2-· levels in the cortex and medulla in CAP-HS rats were prevented by preincubation of renal tissues with apocynin, a selective NAD(P)H oxidase inhibitor. Protein expression of NAD(P)H oxidase subunits, including p47phox and gp91phox in the renal cortex and medulla, was significantly increased in CAP-HS compared with CON-HS, CON-NS, and CAP-NS rats. In contrast, protein expression and activities of Cu/Zn SOD and Mn SOD were significantly increased in the renal medulla in both CAP-HS and CON-HS but in the cortex in CAP-HS rats only. Creatinine clearance decreased by ~45% in CAP-HS rats compared with CON-HS, CON-NS, and CAP-NS rats (P < 0.05). O2-· levels in the renal cortex of CAP-HS rats negatively correlated with creatinine clearance (r = -0.76; P < 0.001). Therefore, regardless of enhanced SOD activity to suppress oxidative stress, increased oxidative stress in the kidney of CAP-treated rats fed a HS diet is likely the result of increased expression and activities of NAD(P)H oxidase, which may contribute to decreased renal function and increased blood pressure in these rats. Our results suggest that sensory nerves may play a compensatory role in attenuating renal oxidative stress during HS intake. [ABSTRACT FROM AUTHOR]

Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg Sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazohum chloride staining. NADPH oxidase enzymatic activity arid superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 µM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91phox and membrane-translocated subunit p47phox and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91phox, and prevented translocation of cytoplasmic subunit p47phox to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. [ABSTRACT FROM AUTHOR]

To test the hypothesis that activation of the endothelin type A (ETA) receptor contributes to decreased renal excretory function and increased blood pressure in sensory nerve-degenerated rats fed a high-salt diet, neonatal Wistar rats were given vehicle or capsaicin (CAP, 50 mg/kg sc) on the first and second day of life. After being weaned, vehicle or CAP-treated rats were fed a normal (NS, 0.5%) or a high- (HS, 4%) sodium diet for 2 wk with or without ABT-627 (5 mg·kg-1·day-1, a selective ETA receptor antagonist). Systolic blood pressure increased in CAP-treated rats fed a HS diet (CAP-HS) compared with vehicle-treated rats fed a HS diet (CON-HS, 145 ± 7 vs. 89 ± 5 mmHg, P < 0.05). Creatinine clearance and fractional sodium excretion (FENa) decreased in CAP-HS rats compared with CON-HS rats (creatinine clearance, 0.54 ± 0.05 vs. 0.81 ± 0.09 ml·min-1·100 g body wt-1; FENa, 8.68 ± 0.99 vs. 12.53 ± 1.47%, respectively; P < 0.05). Water and sodium balance increased in CAP-HS rats compared with CON-HS (water balance, 20.2 ± 1.5 vs. 15.5 ± 1.9 ml/day; sodium balance, 11.9 ± 3.1 vs. 2.4 ± 0.3 meq/day, respectively; P < 0.05). The endothelin (ET)-1 levels in plasma and isolated glomeruli increased by about twofold in CAP-HS rats compared with CON-HS rats (P < 0.05). ABT-627 prevented the decrease in creatinine clearance and FENa, the increase in water and sodium balance, and the increase in blood pressure in CAP-HS rats (P < 0.05). Therefore, the blockade of the ETA receptor ameliorates the impairment of renal excretory function and prevents the elevation in blood pressure in salt-sensitive hypertension induced by degeneration of sensory nerves, indicating that the activation of the ETA receptor impairs renal function and contributes to the development of a salt-induced increase in blood pressure in this model. [ABSTRACT FROM AUTHOR]

Vascular capacitance is reduced by endothelin-1 (ET-1) in deoxycorticosterone (DOCA)-salt hypertensive rats. This may contribute to hypertension development. Because the splanchnic blood vessels (especially veins) are important in determining vascular capacitance, we tested the hypothesis that ET-1 levels in the splanchnic vasculature are elevated in hypertensive DOCA-salt compared with normotensive rats. Tissue ET-1 content was measured by ELISA in aorta, vena cava, superior mesenteric artery and vein, and small mesenteric arteries and veins from normotensive sham-operated (sham) and 4-wk DOCA-salt rats. We also determined ET-1 concentration in aortic and portal venous blood (draining the nonhepatic splanchnic organs) in anesthetized and conscious sham and DOCA-salt rats before and after acute blockade of ETB receptor-mediated plasma clearance of ET-1. Results showed a higher ET-1 content in veins than in arteries of similar size. However, ET-1 content was similar in vessels from sham and DOCA-salt rats, except in aorta and superior mesenteric artery, where ET-1 content was greater in DOCA-salt rats. ET-1 concentration was significantly higher in portal venous than in aortic blood, indicating net nonhepatic splanchnic release (nNHSR) of ET-1. However, nNHSR of ET-1 was similar in sham and DOCA-salt rats. Although nNHSR of ET-1 increased significantly after ETB receptor blockade in sham rats, it was completely unchanged in DOCA-salt rats. These data suggest that, despite the absence of ETB receptor-mediated plasma clearance of ET-1, neither the venous peptide content nor the net release of ET-1 is increased in the splanchnic vasculature of DOCA-salt rats. These results argue against the hypothesis that increased venomotor tone in DOCA-salt hypertension is caused by increased ET-1 concentration around splanchnic venous smooth muscle cells. [ABSTRACT FROM AUTHOR]

Examines whether delivery of an adenoviral vector encoding endothelial nitric oxide synthase (eNOS) gene to liver affects vasomotor function in vivo and the mechanism of nitric oxide production in vitro. Agent causing increase in liver beta-galactosidase activity; Effects of eNOS overexpression; Successful delivery of the recombinant eNOS gene to the liver.

The article offers a brief overview of the complex interactions among cardiac sympathetic afferent, chemoreflex and baroreflex and its role in regulating the sympathetic outflow in a congestive heart failure (CHF) state. CHF is a syndrome characterized by the reduction in pump function of the heart, while the three cardiovascular reflexes controls the circulation of blood. Evidence shows that augmented excitatory reflexes in CHF may contribute to activation of sympathetic outflow.

Multiple organ perfusion is impaired in sepsis. Clinical studies suggest that persistent perfusion disturbances are prognostic of fatal outcome in sepsis. Pyroptosis occurs upon activation of caspases and their subsequent cleavage of gasdermin D (Gsdmd), resulting in Gsdmd-N (activated NH 2 -terminal fragment of Gsdmd) that form membrane pores to induce cell death in sepsis. In addition, Gsdmd -/- mice are protected from a lethal dose of lipopolysaccharide (LPS). However, how Gsdmd-mediated pyroptosis occurs in endothelial cells and leads to impaired perfusion remain unexplored in endotoxemia. We used transgenic mice with ablation of Gsdmd and determined that mice lacking Gsdmd exhibited reduced breakdown of endothelial barrier, improved organ perfusion, as well as increased survival in endotoxemia. Phospholipase Cγ1 (PLCγ1) contributed to Gsdmd-mediated endothelial pyroptosis in a calcium-dependent fashion, without affecting Gsdmd-N production. Cytosolic calcium signaling promoted Gsdmd-N translocation to the plasma membrane, enhancing endothelial pyroptosis induced by LPS. We used adeno-associated virus (AAV9) vectors carrying a short hairpin RNA (shRNA) against murine PLCγ1 mRNA under control of the tie1 core promoter (AAV-tie1-sh-PLCγ1) to uniquely downregulate PLCγ1 expression in the endothelial cells. Here, we showed that unique inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, and improved perfusion disturbances. Moreover, unique downregulate endothelial PLCγ1 expression markedly decreased mortality of mice in endotoxemia. Thus, we establish that endothelial injury as an important trigger of fatal outcome in endotoxemia. Additionally, these findings suggest that interfering with Gsdmd and PLCγ1-calcium pathway may represent a new treatment strategy for critically ill patients sustaining endotoxemia. NEW & NOTEWORTHY Our study newly reveals that Phospholipase Cγ1 (PLCγ1) contributes to gasdermin D (Gsdmd)-mediated endothelial pyroptosis in a calcium-dependent fashion. Cytosolic calcium signaling promotes activated NH 2 -terminal fragment of Gsdmd (Gsdmd-N) to translocate to the plasma membrane, enhancing endothelial pyroptosis induced by cytoplasmic LPS. Genetic or pharmacologic inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, improve perfusion disturbances, and decrease mortality of mice in endotoxemia.

Endothelial inflammation plays an important role in hyperhomocysteinemia (HHcy)-associated vascular diseases. High mobility group box 1 (HMGB1) is a pro-inflammatory danger molecule produced by endothelial cells. However, whether HMGB1 is involved in vascular endothelial inflammation of HHcy is poorly understood. Neuropilin-1 (NRP1) mediates inflammatory response and activates mitogen-activated protein kinases (MAPKs) pathway that has been reported to be involved in regulation of HMGB1. The aim of this study was to determine the alteration of HMGB1 in HHcy, and the role of NRP1 in regulation of endothelial HMGB1 under high homocysteine (Hcy) condition. In the present study, we first observed that the plasma level of HMGB1 was elevated in HHcy patients and an experimental rat model, and increased HMGB1 was also observed in the thoracic aorta of an HHcy rat model. HMGB1 was induced by Hcy accompanied with upregulated NRP1 in vascular endothelial cells. Overexpression of NRP1 promoted expression and secretion of HMGB1 and endothelial inflammation; knockdown of NRP1 inhibited HMGB1 and endothelial inflammation induced by Hcy, which partially regulated through p38 MAPK pathway. Furthermore, NRP1 inhibitor ATWLPPR reduced plasma HMGB1 level and expression of HMGB1 in the thoracic aorta of HHcy rats. In conclusion, our data suggested that Hcy requires NRP1 to regulate expression and secretion of HMGB1. The present study provides the evidence for inhibition of NRP1 and HMGB1 to be the novel therapeutic targets of vascular endothelial inflammation in HHcy in the future. NEW & NOTEWORTHY This study shows for the first time to our knowledge that the plasma level of high mobility group box 1 (HMGB1) is elevated in hyperhomocysteinemia (HHcy) patients, and homocysteine promotes expression and secretion of HMGB1 partially regulated by neuropilin-1 in endothelial cells, which is involved in endothelial inflammation. Most importantly, these new findings will provide a potential therapeutic strategy for vascular endothelial inflammation in HHcy.

Endothelial cell senescence is regarded as a vital characteristic of cardiovascular diseases. Elevated palmitate (PA) is an independent risk factor of cardiovascular diseases, but its role in endothelial cell senescence is currently unknown. During the course of studying the prosenescent role of PA, we discovered a key role of dsRNA-dependent protein kinase [protein kinase R (PKR)] in endothelial senescence. Exposure of human umbilical vein endothelial cells (HUVECs) to PA-induced cell senescence is characterized by increased levels of senescence-associated β-galactose glucosidase activity, excessive production of reactive oxygen species production, impaired cellular proliferation, and G 1 phase arrest. This phenomenon is associated with an increase of PKR autophosphorylation and decreased activity of sirtuin 1 (Sirt1), a pivotal antisenescent factor. PKR inactivation by PKR siRNA or its phosphorylation inhibitor 2-aminopurine significantly attenuated PA-induced HUVEC senescence by reversing Sirt1 activity and its downstream signaling. Moreover, to study the regulatory mechanism between PKR and Sirt1, we found that PKR promotes JNK activation to inhibit Sirt1 activity and that this effect could be reversed by the JNK inhibitor SP600125. These findings provide evidence that PKR mediates PA-induced HUVEC senescence by inhibiting Sirt1 signaling. Our study provides novel insights into the actions and mechanisms of PKR in endothelial senescence. NEW & NOTEWORTHY This study first provides a novel observation that dsRNA-dependent protein kinase (PKR) mediates palmitate-induced sirtuin 1 inactivation and subsequent human umbilical vein endothelial cell senescence. Most importantly, these new findings will provide a potential therapeutic strategy to improve free fatty acid-induced endothelial senescence by targeting PKR in cardiovascular diseases.

Bone marrow-derived progenitor cells (BMPCs) are potential candidates for autologous cell therapy in tissue repair and regeneration because of their high angiogenic potential. However, increased progenitor cell apoptosis in diabetes directly limits their success in the clinic. MicroRNAs are endogenous noncoding RNAs that regulate gene expression at the posttranscriptional level, but their roles in BMPC-mediated angiogenesis are incompletely understood. In the present study, we tested the hypothesis that the proangiogenic miR-27b inhibits BMPC apoptosis in Type 2 diabetes. Bone marrow-derived EPCs from adult male Type 2 diabetic db/db mice and their normal littermates db /+ mice were used. MiR-27b expression (real-time PCR) in EPCs was decreased after 24 h of exposure to methylglyoxal (MGO) or oxidized low-density lipoprotein but not high glucose, advanced glycation end products, the reactive oxygen species generator LY83583, or H 2 O 2 The increase in BMPC apoptosis in the diabetic mice was rescued following transfection with a miR-27b mimic, and the increased apoptosis induced by MGO was also rescued by the miR-27b mimic. p53 protein expression and the Bax/Bcl-2 ratio in EPCs (Western blot analyses) were significantly higher in db/db mice, both of which were suppressed by miR-27b. Furthermore, mitochondrial respiration, as measured by oxygen consumption rate, was enhanced by miR-27b in diabetic BMPCs, with concomitant decrease of mitochondrial Bax/Bcl-2 ratio. The 3' UTR binding assays revealed that both Bax, and its activator RUNX1, were direct targets of miR-27b, suggesting that miR-27b inhibits Bax expression in both direct and indirect manners. miR-27b prevents EPC apoptosis in Type 2 diabetic mice, at least in part, by suppressing p53 and the Bax/Bcl-2 ratio. These findings may provide a mechanistic basis for rescuing BMPC dysfunction in diabetes for successful autologous cell therapy.

Peripheral artery disease (PAD) is a common clinical problem, and its pathophysiological mechanisms are incompletely understood. Double-stranded RNA-activated protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. Although PKR has been reported in antivirus and the immune system, the role of PKR in vascular function, especially in angiogenesis, is still unclear. PKR(-/-) mice were used in our experiments. Blood flow recovery was significantly delayed in PKR(-/-) vs. WT mice (Laser Doppler detection, n = 9, P < 0.01), accompanied by 34% reduced CD31-positive stain in ischemic muscle 28 days after procedure (immunohistochemistry, n = 9, P < 0.05). PKR expression decreased in the first 12 h and increased to peak at 24 h in human umbilical vein endothelial cells (HUVECs) in response to hypoxia (Western blot analyses, n = 3, P < 0.05). Accordingly, phospho-PKR expression increased in HUVECs 24 h after treatment with hypoxia (Western blot analyses, n = 3, P < 0.05). Inhibition of PKR (siRNA transfection) reduced microtubule formation (Matrigel tube formation, n = 3, vs. control siRNA, P < 0.05) and migration (wound healing, n = 3, vs. control siRNA, P < 0.05) by 33 and 59%, respectively. Vascular endothelial growth factor (VEGF) expression in ischemic muscle from PKR(-/-) mice was significantly decreased by 54% 1 day after procedure (n = 3, P < 0.05, vs. WT) and by 63% 7 days after procedure (n = 3, P < 0.01, vs. WT), respectively. At the same time, VEGF expression in HUVECs decreased by 21% (n = 3, P < 0.05, PKR siRNA vs. control siRNA). These findings demonstrate that PKR mediates angiogenesis through a VEGF pathway, which may form the basis for future intervention of PAD., (Copyright © 2015 the American Physiological Society.)

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature., (Copyright © 2014 the American Physiological Society.)

Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds and accelerated wound healing significantly, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were reduced significantly in STZ-WT and hph-1 mice, paralleled by increased superoxide anion levels; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice at least partly through the attenuation of superoxide and TSP-1 levels and augmentation of NO level., (Copyright © 2014 the American Physiological Society.)

Refractory wounds in diabetic patients present a significant clinical problem. Sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes. Regulation of SHH in wound healing is poorly understood. We hypothesize that thrombospondin-1 (TSP-1), through its receptor CD36, contributes to the SHH signaling defect in bone marrow-derived angiogenic cells (BMACs) in type 1 diabetic mice. Isolated BMACs from TSP-1-knockout mice demonstrated improved tube formation, migration, and adhesion in parallel with active SHH signaling. BMACs from STZ-induced type 1 diabetic mice showed significantly impaired Matrigel tube formation (n = 5; P < 0.05 vs. control), which was rescued by TSP-1 depletion (n = 5; P < 0.05 STZ-TSP-1(-/-) vs. STZ-WT) or exogenous SHH (20 mg/l, 24 h, n = 4; P < 0.05 vs. STZ-control). The expression of CD36 was elevated in BMACs from STZ mice (n = 4; P < 0.05). SHH signaling was significantly higher in BMACs from TSP-1(-/-) mice and TSP-1 receptor CD36-knockout mice (n = 6; P < 0.05 vs. WT) but not CD47-knockout mice (n = 3; P > 0.05 vs. WT). The impairment of recombinant human TSP-1 (2.2 nM, 24 h) on BMAC Matrigel tube formation was delayed significantly by CD36 deletion (n = 5; P < 0.05). CD36(-/-) BMACs demonstrated better tube formation under both normal and diabetic conditions with active SHH signaling (n = 4; P < 0.05 vs. WT BMACs). In conclusion, The TSP-1/CD36 pathway contributes to the SHH signaling defect, resulting in BMAC dysfunction in type 1 diabetic mice.

Endothelial progenitor cells (EPCs) play an essential role in angiogenesis but are functionally impaired in diabetes. We recently reported that decreased expression of manganese superoxide dismutase (MnSOD) critically contributes to diabetic EPC dysfunction. AMP-activated protein kinase (AMPK) activation has been shown to induce MnSOD and suppress hyperglycemia-induced mitochondrial ROS production in endothelial cells. However, whether AMPK protects EPCs from oxidative stress in diabetes is unknown. We tested the hypothesis that AMPK activation rescues impaired EPC functions through MnSOD induction in type 1 diabetes. Bone marrow-derived EPCs from adult male streptozotocin-induced diabetic mice and normal controls were used. AMPK activity was decreased in diabetic EPCs, indicated by reduced AMPK and acetyl-CoA carboxylase phosphorylation. AMPK activation by treating diabetic EPCs with its selective agonist AICAR rescued their in vitro functions, including Matrigel tube formation, adhesion, and migration. Furthermore, AICAR restored the decreased MnSOD protein and enzymatic activity and suppressed the mitochondrial superoxide level in diabetic EPCs, indicated by MitoSOX flow cytometry. These beneficial effects of AICAR on MnSOD and EPC functions were significantly attenuated by silencing MnSOD or AMPK antagonist compound C pretreatment. Finally, the expression of protein phosphatase 2A, a key enzyme for AMPK dephosphorylation and inactivation, was increased in diabetic EPCs, and its inhibition by siRNA or okadaic acid reversed the deficient AMPK activation and MnSOD level in diabetic EPCs. These findings demonstrate for the first time that AMPK activation rescues impaired EPC functions and suppresses mitochondrial superoxide by inducing MnSOD in type 1 diabetes.

S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H(2)S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N(G)-nitro-L-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H(2)S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H(2)S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N(G)-nitro-L-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H(2)S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.

Hyperhomocysteinemia (HHcy) has been associated with impaired vascular endothelial function. Our previous study demonstrated significantly higher secretion of the chemokine monocyte chemoattractant protein-1 from monocytes in response to lipopolysaccharide in patients with HHcy. In the present study, we investigated whether coronary endothelial function was damaged in patients with chronic HHcy (plasma level of homocysteine >15 μmol/l) and, if so, whether this impaired endothelial function is induced by the uncoupling of endothelial nitric oxide synthase (eNOS). When tetrahydrobiopterin levels are inadequate, eNOS is no longer coupled to l-arginine oxidation, which results in reactive oxygen species rather than nitric oxide production, thereby inducing vascular endothelial dysfunction. The 71 participants were divided into two groups, control (n = 50) and HHcy (n = 21). Quantification of coronary flow velocity reserve (CFVR) was after rest and after adenosine administration done by noninvasive Doppler echocardiography. Plasma levels of nitric oxide and tetrahydrobiopterin were significantly lower in patients with HHcy than in controls (99.54 ± 32.23 vs. 119.50 ± 37.68 μmol/l and 1.43 ± 0.46 vs. 1.73 ± 0.56 pmol/ml, all P < 0.05). Furthermore, CFVR was significantly lower in the HHcy than the control group (2.76 ± 0.49 vs. 3.09 ± 0.52, P < 0.05). In addition, plasma level of homocysteine was negatively correlated with CFVR. Chronic HHcy may contribute to coronary artery disease by inducing dysfunction of the coronary artery endothelium. The uncoupling of eNOS induced by HHcy in patients with chronic HHcy may explain this adverse effect in part.

Endothelial progenitor cells (EPCs) play an important role in angiogenesis, which is essential for numerous physiological processes as well as tumor growth. Several microRNAs (miRNAs) have been reported to be involved in angiogenesis. MiR-34a, recently reported as a tumor suppressor, has been found to target silent information regulator 1 (Sirt1), leading to cell cycle arrest or apoptosis. However, the role of miR-34a in EPC-mediated angiogenesis was unknown. The present study tested the hypothesis that miR-34a inhibits EPC-mediated angiogenesis by inducing senescence via suppressing Sirt1. Bone marrow-derived EPCs from adult male Sprague-Dawley rats were used. Results of flow cytometry showed that EPCs after 7 days of culture expressed both stem cell markers CD34 and CD133 and endothelial cell markers VEGFR-2 (flk-1) and VE-cadherin. MiR-34a was expressed in normal EPCs, and overexpression of miR-34a via its mimic transfection significantly increased its expression and impaired in vitro EPC angiogenesis. MiR-34a overexpression led to a significantly increased EPC senescence, paralleled with an approximately 40% Sirt1 reduction. Furthermore, knockdown of Sirt1 by its siRNA resulted in diminished EPC angiogenesis and increased senescence. Finally, overexpression of miR-34a increased the level of Sirt1 effector-acetylated forkhead box O transcription factors 1 (FoxO1), an effect mimicked in EPCs following Sirt1 knockdown. In conclusion, miR-34a impairs EPC-mediated angiogenesis by induction of senescence via inhibiting Sirt1.

Refractory wound is a severe complication that leads to limb amputation in diabetes. Endothelial nitric oxide synthase (eNOS) plays a key role in normal wound repair but is uncoupled in streptozotocin (STZ)-induced type 1 diabetes because of reduced cofactor tetrahydrobiopterin (BH(4)). We tested the hypothesis that overexpression of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for de novo BH(4) synthesis, retards NOS uncoupling and accelerates wound healing in STZ mice. Blood glucose levels were significantly increased in both male endothelium-specific GTPCH I transgenic mice (Tg-GCH; via a tie-2 promoter) and wild-type (WT) littermates 5 days after STZ regimen. A full-thickness excisional wound was created on mouse dorsal skin by a 4-mm punch biopsy. Wound closure was delayed in STZ mice, which was rescued in STZ Tg-GCH mice. Cutaneous BH(4) level was significantly reduced in STZ mice vs. WT mice, which was maintained in STZ Tg-GCH mice. In STZ mice, constitutive NOS (cNOS) activity and nitrite levels were decreased compared with WT mice, paralleled by increased superoxide anion (O(2)(-)) level and inducible NOS (iNOS) activity. In STZ Tg-GCH mice, nitrite level and cNOS activity were potentiated and O(2)(-) level and iNOS activity were suppressed compared with STZ mice. Thus endothelium-specific BH(4) overexpression accelerates wound healing in type 1 diabetic mice by enhancing cNOS activity and suppressing oxidative stress.

To determine the mechanism(s) underlying enhanced oxidative stress in kidneys of salt-sensitive hypertension, neonatal Wistar rats were given vehicle or capsaicin (CAP, 50 mg/kg sc) on the first and second days of life. After being weaned, male rats were assigned into four groups and treated for 2 wk with the following: vehicle + a normal sodium diet (NS, 0.4%, CON-NS), vehicle + a high-sodium diet (HS, 4%, CON-HS), CAP + NS (CAP-NS), and CAP + HS (CAP-HS). Systolic blood pressure was significantly increased in CAP-HS but not CAP-NS or CON-HS rats. Plasma and urinary 8-iso-prostaglandin F(2alpha) levels increased by approximately 40% in CON-HS and CAP-HS rats compared with their respective controls fed a NS diet (P < 0.05), and these parameters were higher in CAP-HS compared with CON-HS rats. Superoxide (O(2)(-)*) levels in the renal cortex and medulla increased by approximately 45% in CAP-HS compared with CON-HS, CON-NS, and CAP-NS rats (P < 0.05). Enhanced O(2)(-)* levels in the cortex and medulla in CAP-HS rats were prevented by preincubation of renal tissues with apocynin, a selective NAD(P)H oxidase inhibitor. Protein expression of NAD(P)H oxidase subunits, including p47(phox) and gp91(phox) in the renal cortex and medulla, was significantly increased in CAP-HS compared with CON-HS, CON-NS, and CAP-NS rats. In contrast, protein expression and activities of Cu/Zn SOD and Mn SOD were significantly increased in the renal medulla in both CAP-HS and CON-HS but in the cortex in CAP-HS rats only. Creatinine clearance decreased by approximately 45% in CAP-HS rats compared with CON-HS, CON-NS, and CAP-NS rats (P < 0.05). O(2)(-)* levels in the renal cortex of CAP-HS rats negatively correlated with creatinine clearance (r = -0.76; P < 0.001). Therefore, regardless of enhanced SOD activity to suppress oxidative stress, increased oxidative stress in the kidney of CAP-treated rats fed a HS diet is likely the result of increased expression and activities of NAD(P)H oxidase, which may contribute to decreased renal function and increased blood pressure in these rats. Our results suggest that sensory nerves may play a compensatory role in attenuating renal oxidative stress during HS intake.

Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazolium chloride staining. NADPH oxidase enzymatic activity and superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 microM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91(phox) and membrane-translocated subunit p47(phox) and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91(phox), and prevented translocation of cytoplasmic subunit p47(phox) to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia.

To test the hypothesis that activation of the endothelin type A (ET(A)) receptor contributes to decreased renal excretory function and increased blood pressure in sensory nerve-degenerated rats fed a high-salt diet, neonatal Wistar rats were given vehicle or capsaicin (CAP, 50 mg/kg s.c.) on the first and second day of life. After being weaned, vehicle or CAP-treated rats were fed a normal (NS, 0.5%) or a high- (HS, 4%) sodium diet for 2 wk with or without ABT-627 (5 mg x kg(-1) x day(-1), a selective ET(A) receptor antagonist). Systolic blood pressure increased in CAP-treated rats fed a HS diet (CAP-HS) compared with vehicle-treated rats fed a HS diet (CON-HS, 145 +/- 7 vs. 89 +/- 5 mmHg, P < 0.05). Creatinine clearance and fractional sodium excretion (FE(Na)) decreased in CAP-HS rats compared with CON-HS rats (creatinine clearance, 0.54 +/- 0.05 vs. 0.81 +/- 0.09 ml x min(-1) x 100 g body wt(-1); FE(Na), 8.68 +/- 0.99 vs. 12.53 +/- 1.47%, respectively; P < 0.05). Water and sodium balance increased in CAP-HS rats compared with CON-HS (water balance, 20.2 +/- 1.5 vs. 15.5 +/- 1.9 ml/day; sodium balance, 11.9 +/- 3.1 vs. 2.4 +/- 0.3 meq/day, respectively; P < 0.05). The endothelin (ET)-1 levels in plasma and isolated glomeruli increased by about twofold in CAP-HS rats compared with CON-HS rats (P < 0.05). ABT-627 prevented the decrease in creatinine clearance and FE(Na), the increase in water and sodium balance, and the increase in blood pressure in CAP-HS rats (P < 0.05). Therefore, the blockade of the ET(A) receptor ameliorates the impairment of renal excretory function and prevents the elevation in blood pressure in salt-sensitive hypertension induced by degeneration of sensory nerves, indicating that the activation of the ET(A) receptor impairs renal function and contributes to the development of a salt-induced increase in blood pressure in this model.