Gautam Sikka

Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels

Rationale: Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H2S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine &ggr;-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. Objective: The purpose of this study was to determine if H2S is a major physiological EDHF. Methods and Results: We now show that H2S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H2S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H2S-elicited hyperpolarization. The endothelial intermediate conductance (IKCa) and small conductance (SKCa) potassium channels mediate in part the effects of H2S, as selective IKCa and SKCa channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H2S-induced vasorelaxation. Conclusions: H2S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H2S biosynthesis offer therapeutic potential.

Endothelial Arginase II A Novel Target for the Treatment of Atherosclerosis

Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E–null (ApoE−/−) and wild-type mice fed a high cholesterol diet. In ApoE−/− mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II−/− mice) prevents high-cholesterol diet–dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein–dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.

Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats

There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2(-)) production than young. Acute inhibition of both NOS, with N(G)-nitro-l-arginine methyl ester, and arginase, with 2S-amino- 6-boronohexanoic acid (ABH), significantly reduced O2(-) production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692-702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2(-) production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.

Decreased S-Nitrosylation of Tissue Transglutaminase Contributes to Age-Related Increases in Vascular Stiffness

Rationale: Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S-nitrosylation in young vessels, a process that is reversed in aging. Objective: We sought to determine whether endothelium-dependent NO regulates TG2 activity by S-nitrosylation and whether this contributes to age-related vascular stiffness. Methods and Results: We first demonstrate that NO suppresses activity and increases S-nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix–associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2−/− mice chronically treated with the NOS inhibitor l-NG-nitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S-nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged. Conclusions: Decreased S-nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension.

Serine Racemase Deletion Protects Against Cerebral Ischemia And Excitotoxicity

d-Serine, formed from l-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for d-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.

Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age.

UNLABELLED Cardiovascular dysfunction is a primary independent predictor of age-related morbidity and mortality. Frailty is associated with activation of inflammatory pathways and fatigue that commonly presents and progresses with age. Interleukin 10 (IL-10), the cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine produced by immune and non-immune cells. Homozygous deletion of IL-10 in mice yields a phenotype that is consistent with human frailty, including age-related increases in serum inflammatory mediators, muscular weakness, higher levels of IGF-1 at midlife, and early mortality. While emerging evidence suggests a role for IL-10 in vascular protection, a clear mechanism has not yet been elucidated. METHODS In order to evaluate the role of IL-10 in maintenance of vascular function, force tension myography was utilized to access ex-vivo endothelium dependent vasorelaxation in vessels isolated from IL-10 knockout IL-10(tm/tm) and control mice. Pulse wave velocity ((PWV), index of stiffness) of vasculature was measured using ultrasound and blood pressure was measured using the tail cuff method. Echocardiography was used to elucidated structure and functional changes in the heart. RESULTS Mean arterial pressures were significantly higher in IL-10(tm/tm) mice as compared to C57BL6/wild type (WT) controls. PWV was increased in IL-10(tm/tm) indicating stiffer vasculature. Endothelial intact aortic rings isolated from IL-10(tm/tm) mice demonstrated impaired vasodilation at low acetylcholine doses and vasoconstriction at higher doses whereas vasorelaxation responses were preserved in rings from WT mice. Cyclo-oxygenase (COX-2)/thromboxane A2 inhibitors improved endothelial dependent vasorelaxation and reversed vasoconstriction. Left ventricular end systolic diameter, left ventricular mass, isovolumic relaxation time, fractional shortening and ejection fraction were all significantly different in the aged IL-10(tm/tm) mice compared to WT mice. CONCLUSION Aged IL-10(tm/tm) mice have stiffer vessels and decreased vascular relaxation due to an increase in eicosanoids, specifically COX-2 activity and resultant thromboxane A2 receptor activation. Our results also suggest that aging IL-10(tm/tm) mice have an increased heart size and impaired cardiac function compared to age-matched WT mice. While further studies will be necessary to determine if this age-related phenotype develops as a result of inflammatory pathway activation or lack of IL-10, it is essential for maintaining the vascular compliance and endothelial function during the aging process. Given that a similar cardiovascular phenotype is present in frail, older adults, these findings further support the utility of the IL-10(tm/tm) mouse as a model of frailty.

Melanopsin mediates light-dependent relaxation in blood vessels.

Significance Non–image-forming opsins such as Opn4 regulate important physiological functions such as circadian photo-entrainment and affect. The recent discovery that melanopsin (Opn4) functions outside the central nervous system prompted us to explore a potential role for this receptor in blood vessel regulation. We hypothesized that Opn4-mediated signaling might explain the phenomenon of photorelaxation, for which a mechanism has remained elusive. We report the presence in blood vessels of Opn4 and demonstrate that it mediates wavelength-specific, light-dependent vascular relaxation. This photorelaxation signal transduction involves cGMP and phosphodiesterase 6, but not protein kinase G. Furthermore it is regulated by G protein-coupled receptor kinase 2 and involves vascular hyperpolarization. This receptor pathway can be harnessed for wavelength-specific light-based therapy in the treatment of diseases that involve altered vasoreactivity. Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4−/− mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430–460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.

Restoring leptin signaling reduces hyperlipidemia and improves vascular stiffness induced by chronic intermittent hypoxia

Chronic intermittent hypoxia (IH) during sleep can result from obstructive sleep apnea (OSA), a disorder that is particularly prevalent in obesity. OSA is associated with high levels of circulating leptin, cardiovascular dysfunction, and dyslipidemia. Relationships between leptin and cardiovascular function in OSA and chronic IH are poorly understood. We exposed lean wild-type (WT) and obese leptin-deficient ob/ob mice to IH for 4 wk, with and without leptin infusion, and measured cardiovascular indices including aortic vascular stiffness, endothelial function, cardiac myocyte morphology, and contractile properties. At baseline, ob/ob mice had decreased vascular compliance and endothelial function vs. WT mice. We found that 4 wk of IH decreased vascular compliance and endothelial relaxation responses to acetylcholine in both WT and leptin-deficient ob/ob animals. Recombinant leptin infusion in both strains restored IH-induced vascular abnormalities toward normoxic WT levels. Cardiac myocyte morphology and function were unaltered by IH. Serum cholesterol and triglyceride levels were significantly decreased by leptin treatment in IH mice, as was hepatic stearoyl-Coenzyme A desaturase 1 expression. Taken together, these data suggest that restoring normal leptin signaling can reduce vascular stiffness, increase endothelial relaxation, and correct dyslipidemia associated with IH.

Transcriptional Regulation of Endothelial Arginase 2 by Histone Deacetylase 2

Objective—Arginase 2 (Arg2) is a critical target in atherosclerosis because it controls endothelial nitric oxide, proliferation, fibrosis, and inflammation. Regulators of Arg2 transcription in the endothelium have not been characterized. The goal of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial Arg2 transcription and endothelial function. Approach and Results—The HDAC inhibitor trichostatin A increased levels of Arg2 mRNA, protein, and activity in both human aortic endothelial cells and mouse aortic rings. These changes occurred in both time- and dose-dependent patterns and resulted in Arg2-dependent endothelial dysfunction. Trichostatin A and the atherogenic stimulus oxidized low-density lipoprotein enhanced the activity of common promoter regions of Arg2. HDAC inhibition with trichostatin A also decreased endothelial nitric oxide, and these effects were blunted by arginase inhibition. Nonselective class I HDAC inhibitors enhanced Arg2 expression, whereas the only selective inhibitor that increased Arg2 expression was mocetinostat, a selective inhibitor of HDACs 1 and 2. Additionally, mouse aortic rings preincubated with mocetinostat exhibited dysfunctional relaxation. Overexpression of HDAC2 (but not HDAC 1, 3, or 8) cDNA in human aortic endothelial cells suppressed Arg2 expression in a concentration-dependent manner, and siRNA knockdown of HDAC2 enhanced Arg2 expression. Chromatin immunoprecipitation indicated direct binding of HDAC2 to the Arg2 promoter, and HDAC2 overexpression in human aortic endothelial cells blocked oxidized low-density lipoprotein–mediated activation of the Arg2 promoter. Finally, overexpression of HDAC2 blocked oxidized low-density lipoprotein–mediated vascular dysfunction. Conclusions—HDAC2 is a critical regulator of Arg2 expression and thereby endothelial nitric oxide and endothelial function. Overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis.

Nitroprusside inhibits calcium-induced impairment of red blood cell deformability

Red blood cell (RBC) deformation is critical for microvascular perfusion and oxygen delivery to tissues. Abnormalities in RBC deformability have been observed in aging, sickle cell disease, diabetes, and preeclampsia. Although nitric oxide (NO) prevents decreases in RBC deformability, the underlying mechanism is unknown.