Impaired Acetylcholine-Induced Endothelium-Dependent Aortic Relaxation by Caveolin-1 in Angiotensin II-Infused Apolipoprotein-E (ApoE?/?) Knockout Mice

Objective

The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE^−/−) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE^−/− mice and the underlying mechanism involved.

Methods

ApoE^−/− mice were infused with vehicle (distilled water) or AngII subcutaneously for 14 days. Blood pressure and heart rate were measured using the non-invasive tail cuff method. Aortic vascular reactivity and expression of key proteins (endothelial nitric oxide synthase (eNOS), phospho-eNOS and caveolin-1) were assessed using tension myography and Western blotting respectively. Plasma nitric oxide (NO) level was estimated using a colorimetric assay.

Results

AngII infusion caused a time-dependent increase in blood pressure (P<0.001). Aortas from AngII-infused mice were significantly less responsive to acetylcholine-induced endothelium-dependent relaxation when compared to aortas from mice infused with vehicle control (P<0.05). Contractile responses to phenylephrine (P<0.01) and potassium chloride (P<0.001) were significantly enhanced in aortas from AngII-infused mice. eNOS phosphorylation was significantly decreased in the aorta of AngII-infused mice (P<0.05). Aortic caveolin-1 protein expression was significantly increased in AngII-infused mice (P<0.05). Plasma nitrate/nitrite level was significantly reduced in AngII-infused mice (P<0.05). Pharmacological disruption of caveolae using methyl-β-cyclodextrin (MβCD) in isolated aortas from AngII-infused mice caused a significant leftward shift of the acetylcholine-induced relaxation concentration-response curve when compared to vehicle control (P<0.05).

Conclusion

Upregulation of caveolin-1 protein expression and reduced NO bioavailability contributes to aortic endothelial dysfunction in AngII-infused ApoE^−/− mice.     

Adenovirus-Mediated Prothymosin α Gene Transfer Inhibits the Development of Atherosclerosis in Apoe-Deficient Mice

Prothymosin α (ProT) is involved in regulating expression of the oxidative stress-protective genes and it also exerts immunomodulatory activities. In this study, we investigated the therapeutic effects of ProT gene transfer on atherosclerosis in endothelial cells and in ApoE-deficient mice. Adenoviruses encoding mouse ProT (AdProT) were used for the management of atherosclerosis. In vitro, the effects of ProT on antioxidant gene expressions and the protection effect against oxidant-mediated injury in endothelial cells were examined. In vivo, AdProT were administered intraventricularly into the heart of ApoE^-/- mice. Histopathological and immunohistochemical assessments of the aortic tissues were performed. Expressions of HO-1 and antioxidant genes in the aortic tissues were also determined. Our results demonstrated that ProT gene transfer increased antioxidant gene expressions, eNOS expression and NO release, as well as reduced the reactive oxygen species production in endothelial cells. Intraventricular administration of AdProT reduced the lesion formation, increased expressions of HO-1 and SOD genes, and reduced infiltrating macrophages in the aorta of ApoE^-/- mice. This study suggests that ProT gene transfer may have the therapeutic potential for the management of atherosclerosis via inducing antioxidant gene expressions, eNOS expression and NO release, reducing ROS production and macrophage infiltration in endothelium.     

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS^?/?) mice hearts that Na⁺/K⁺- and Ca²⁺-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS^?/? mice. Na⁺/K⁺-ATPase activity from crude preparations of adult male eNOS^?/? mice hearts and kidneys was reduced compared with wild-type animals (32 %, p?<?0.05 and 16 %, p?<?0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na⁺/K⁺-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p?<?0.0001). In addition, although cardiac Ca²⁺-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 protein in eNOS^?/? mice was very high (290 % compared with wild-type animals, p?<?0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p?<?0.01 and 35 %, p?<?0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.     

Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

Specific Activation of Insulin-like Growth Factor-1 Receptor by Ginsenoside Rg5 Promotes Angiogenesis and Vasorelaxation

Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE^−/− mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and G_i-mediated phospholipase C/Ca²⁺/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC₅₀ of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.     

Probiotics (VSL#3) Prevent Endothelial Dysfunction in Rats with Portal Hypertension: Role of the Angiotensin System

Aims

Portal hypertension characterized by generalized vasodilatation with endothelial dysfunction affecting nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) has been suggested to involve bacterial translocation and/or the angiotensin system. The possibility that ingestion of probiotics prevents endothelial dysfunction in rats following common bile duct ligation (CBDL) was evaluated.

Methods

Rats received either control drinking water or the probiotic VSL#3 solution (50 billion bacteria.kg body wt⁻¹.day⁻¹) for 7 weeks. After 3 weeks, rats underwent surgery with either resection of the common bile duct or sham surgery. The reactivity of mesenteric artery rings was assessed in organ chambers, expression of proteins by immunofluorescence and Western blot analysis, oxidative stress using dihydroethidium, and plasma pro-inflammatory cytokine levels by flow cytometry.

Results

Both NO- and EDH-mediated relaxations to acetylcholine were reduced in the CBDL group compared to the sham group, and associated with a reduced expression of Cx37, Cx40, Cx43, IK_Ca and SK_Ca and an increased expression of endothelial NO synthase (eNOS). In aortic sections, increased expression of NADPH oxidase subunits, angiotensin converting enzyme, AT1 receptors and angiotensin II, and formation of ROS and peroxynitrite were observed. VSL#3 prevented the deleterious effect of CBDL on EDH-mediated relaxations, vascular expression of connexins, IK_Ca, SK_Ca and eNOS, oxidative stress, and the angiotensin system. VSL#3 prevented the CBDL-induced increased plasma TNF-α, IL-1α and MCP-1 levels.

Conclusions

These findings indicate that VSL#3 ingestion prevents endothelial dysfunction in the mesenteric artery of CBDL rats, and this effect is associated with an improved vascular oxidative stress most likely by reducing bacterial translocation and the local angiotensin system.     

Angiotensin II modulates interleukin-1β-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-κB crosstalk

Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1β-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-κB, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1β-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1β, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE^−/−) mice. VCAM-1 and iNOS expression were higher in ApoE^−/− than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE^−/− mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-κB crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.     

Valproic acid increases NO production via the SH-PTP1–CDK5–eNOS-Ser116 signaling cascade in endothelial cells and mice

Valproic acid (VPA) with its inhibitory activity of histone deacetylase has been used in the treatment of epilepsy and bipolar disorder associated with cerebrovascular dysfunction. Because nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a role in the maintenance of vascular function, NO is likely to mediate VPA׳s drug effect, but its effect on NO production remains controversial. We investigated whether and how VPA regulates NO production in bovine aortic endothelial cells (BAECs) and mice. VPA increased NO production in BAECs, which was accompanied by a decrease in phosphorylation of eNOS at serine 116 (eNOS-Ser¹¹⁶) and cyclin-dependent kinase 5 at tyrosine 15 (CDK5-Tyr¹⁵). Ectopic expression of p25, a CDK5 activator, restored the VPA-inhibited eNOS-Ser¹¹⁶ phosphorylation. In silico analysis revealed that the CDK5-Tyr¹⁵ residue might be a substrate for SH2 domain-containing protein tyrosine phosphatase 1 (SH-PTP1), and CDK5 actually interacted with SH-PTP1. VPA increased SH-PTP1 expression and its activity. Stibogluconate, a specific SH-PTP1 inhibitor, reversed the VPA-inhibited phosphorylation of CDK5-Tyr¹⁵ and eNOS-Ser¹¹⁶. Knockdown of SH-PTP1 using small interfering RNA also reversed all the observed effects of VPA. Finally, both serum NO level and acetylcholine-induced aortic relaxation increased in VPA-medicated male mice. These increases were accompanied by increased SH-PTP1 expression and decreased phosphorylation of CDK5-Tyr¹⁵ and eNOS-Ser¹¹⁶ in mouse aortas. In conclusion, VPA increases NO production by inhibiting the CDK5-Tyr¹⁵–eNOS-Ser¹¹⁶ phosphorylation axis; this process is mediated by SH-PTP1. VPA may be useful in the treatment of NO-related cerebrocardiovascular diseases.     

Inflammatory Monocytes Determine Endothelial Nitric-oxide Synthase Uncoupling and Nitro-oxidative Stress Induced by Angiotensin II

Endothelial nitric-oxide synthase (eNOS) uncoupling and increased inducible NOS (iNOS) activity amplify vascular oxidative stress. The role of inflammatory myelomonocytic cells as mediators of these processes and their impact on tetrahydrobiopterin availability and function have not yet been defined. Angiotensin II (ATII, 1 mg/kg/day for 7 days) increased Ly6C^high and CD11b⁺/iNOS^high leukocytes and up-regulated levels of eNOS glutathionylation in aortas of C57BL/6 mice. Vascular iNOS-dependent NO formation was increased, whereas eNOS-dependent NO formation was decreased in aortas of ATII-infused mice as assessed by electron paramagnetic resonance (EPR) spectroscopy. Diphtheria toxin-mediated ablation of lysozyme M-positive (LysM⁺) monocytes in ATII-infused LysM^iDTR transgenic mice prevented eNOS glutathionylation and eNOS-derived N^ω-nitro-l-arginine methyl ester-sensitive superoxide formation in the endothelial layer. ATII increased vascular guanosine triphosphate cyclohydrolase I expression and biopterin synthesis in parallel, which was reduced in monocyte-depleted LysM^iDTR mice. Vascular tetrahydrobiopterin was increased by ATII infusion but was even higher in monocyte-depleted ATII-infused mice, which was paralleled by a strong up-regulation of dihydrofolate reductase expression. EPR spectroscopy revealed that both vascular iNOS- and eNOS-dependent NO formation were normalized in ATII-infused mice following monocyte depletion. Additionally, deletion as well as pharmacologic inhibition of iNOS prevented ATII-induced endothelial dysfunction. In summary, ATII induces an inflammatory cell-dependent increase of iNOS, guanosine triphosphate cyclohydrolase I, tetrahydrobiopterin, NO formation, and nitro-oxidative stress as well as eNOS uncoupling in the vessel wall, which can be prevented by ablation of LysM⁺ monocytes.     

Loss of CD226 protects apolipoprotein E-deficient mice from diet-induced atherosclerosis

CD226 is a costimulatory molecule that regulates immune cell functions in T cells, natural killer cells, and macrophages. Because macrophage-derived foam cell formation is a crucial factor contributing to the development of atherosclerosis, we aimed to evaluate the potential roles of CD226 in the pathogenesis of atherosclerosis. The effects of CD226 on atherosclerosis were investigated in CD226 and apolipoprotein E double-knockout (CD226^?/? ApoE^?/?) mice fed with a high-cholesterol atherogenic diet. CD226 expression in macrophages was evaluated using flow cytometry. Histopathological analysis was performed to evaluate the atherosclerotic lesions. Inflammatory cell infiltration was detected using immunofluorescence staining. Bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PEMs) were isolated from the mice and used to explore the mechanism in vitro. The in vivo results indicated that CD226 knockdown protected against atherosclerosis in ApoE^?/? mice, evidenced by reduced plaque accumulation in the brachiocephalic artery, aortic roots, and main aortic tree. CD226 gene-deficient macrophages showed reduced foam cell formation under ox-low density lipoprotein stimulation compared with wild-type (WT) cells. CD226 deficiency also decreased the expression of CD36 and scavenger receptor (SR)-A (responsible for lipoprotein uptake) but increased the expression of ATP-binding cassette transporter A1 and G1 (two transporters for cholesterol efflux). Therefore, loss of CD226 hinders foam cell formation and atherosclerosis progression, suggesting that CD226 is a promising new therapeutic target for atherosclerosis.     

Chronic Ingestion of H1-Antihistamines Increase Progression of Atherosclerosis in Apolipoprotein E-/- Mice

Although increased serum histamine levels and H1R expression in the plaque are seen in atherosclerosis, it is not known whether H1R activation is a causative factor in the development of the disease, or is a host defense response to atherogenic signals. In order to elucidate how pharmacological inhibition of histamine receptor 1 (H1R) signaling affects atherogenesis, we administered either cetirizine (1 and 4 mg/kg. b.w) or fexofenadine (10 and 40 mg/kg. b.w) to ApoE^−/− mice maintained on a high fat diet for three months. Mice ingesting a low dose of cetirizine or fexofenadine had significantly higher plaque coverage in the aorta and cross-sectional lesion area at the aortic root. Surprisingly, the higher doses of cetirizine or fexofenadine did not enhance atherosclerotic lesion coverage over the controls. The low dose of fexofenadine, but not cetirizine, increased serum LDL cholesterol. Interestingly, the expression of iNOS and eNOS mRNA was increased in aortas of mice on high doses of cetirizine or fexofenadine. This may be a compensatory nitric oxide (NO)-mediated vasodilatory mechanism that accounts for the lack of increase in the progression of atherosclerosis. Although the administration of cetirizine did not alter blood pressure between the groups, there was a positive correlation between blood pressure and lesion/media ratio at the aortic root in mice receiving the low dose of cetirizine. However, this association was not observed in mice treated with the high dose of cetirizine or either doses of fexofenadine. The macrophages or T lymphocytes densities were not altered by low doses of H1-antihistamines, whereas, high doses decreased the number of macrophages but not T lymphocytes. The number of mast cells was decreased only in mice treated with low dose of fexofenadine. These results demonstrate that chronic ingestion of low therapeutic doses of cetirizine or fexofenadine enhance progression of atherosclerosis.     

SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol

Endothelial nitric oxide synthase (eNOS) plays a crucial role in endothelial cell functions. SIRT1, a NAD⁺-dependent deacetylase, is shown to regulate endothelial function and hence any alteration in endothelial SIRT1 will affect normal vascular physiology. Cigarette smoke (CS)-mediated oxidative stress is implicated in endothelial dysfunction. However, the role of SIRT1 in regulation of eNOS by CS and oxidants are not known. We hypothesized that CS-mediated oxidative stress downregulates SIRT1 leading to acetylation of eNOS which results in reduced nitric oxide (NO)-mediated signaling and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) and H₂O₂ showed decreased SIRT1 levels, activity, but increased phosphorylation concomitant with increased eNOS acetylation. Pre-treatment of endothelial cells with resveratrol significantly attenuated the CSE- and oxidant-mediated SIRT1 levels and eNOS acetylation. These findings suggest that CS- and oxidant-mediated reduction of SIRT1 is associated with acetylation of eNOS which have implications in endothelial dysfunction.     

Endothelial nitric oxide deficiency promotes Alzheimer's disease pathology

Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer's disease. A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14–15 month) endothelial nitric oxide synthase deficient (eNOS^?/?) mice. APP, β‐site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS^?/? mice as compared with LMA wild‐type controls. APP and Aβ_1‐40 were increased in hippocampal tissue of eNOS^?/? mice as compared with wild‐type mice. LMA eNOS^?/? mice displayed an increased inflammatory phenotype as compared with LMA wild‐type mice. Importantly, LMA eNOS^?/? mice performed worse in a radial arm maze test of spatial learning and memory as compared with LMA wild‐type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline.

     

KCa3.1 upregulation preserves endothelium‐dependent vasorelaxation during aging and oxidative stress

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium‐dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial K_Ca3.1, which contributes to EDR, is upregulated by H₂O₂. We investigated whether K_Ca3.1 upregulation compensates for diminished EDR to NO during aging‐related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1‐phosphate were increased in aged wild‐type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild‐type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age‐matched wild‐type mice. Increased H₂O₂ levels induced Fyn and extracellular signal‐regulated kinases (ERKs) phosphorylation and K_Ca3.1 upregulation. Catalase/GPX1 double knockout (catalase^?/?/GPX1^?/?) upregulated K_Ca3.1 in MAECs. NO production was decreased in aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? MAECs. However, K_Ca3.1 activation‐induced, N^G‐nitro‐l ‐arginine‐, and indomethacin‐resistant EDR was increased without a change in acetylcholine‐induced EDR in aortic rings from aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1‐phosphate induced similar changes in levels of the antioxidant enzymes and upregulated K_Ca3.1. Our findings suggest that, during aging‐related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H₂O₂ and thereby upregulate K_Ca3.1 expression and function via a H₂O₂/Fyn‐mediated pathway. Altogether, enhanced K_Ca3.1 activity may compensate for decreased NO signaling during vascular aging.     

Arginase inhibition by rhaponticin increases L-arginine concentration that contributes to Ca2+-dependent eNOS activation

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca²⁺-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca²⁺ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca²⁺ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca²⁺ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1^−/−) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca²⁺ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation.     

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO₂) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO₂ via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO₂-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO₂-FAs stimulated the phosphorylation of eNOS at Ser¹¹⁷⁹. These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO₂-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.