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Myocardial infarction (MI) is the leading cause of mortality worldwide. Interleukin (IL)-33 (IL-33) is a cytokine present in most cardiac cells and is secreted on necrosis where it acts as a functional ligand for the ST2 receptor. Although IL-33/ST2 axis is protective against various forms of cardiovascular diseases, some studies suggest potential detrimental roles for IL-33 signaling. The aim of the present study was to examine the effect of IL-33 administration on cardiac function post-MI in mice. MI was induced by coronary artery ligation. Mice were treated with IL-33 (1 μg/day) or vehicle for 4 and 7 days. Functional and molecular changes of the left ventricle (LV) were assessed. Single cell suspensions were obtained from bone marrow, heart, spleen, and peripheral blood to assess the immune cells using flow cytometry at 1, 3, and 7 days post-MI in IL-33 or vehicle-treated animals. The results of the present study suggest that IL-33 is effective in activating a type 2 cytokine milieu in the damaged heart, consistent with reduced early inflammatory and pro-fibrotic response. However, IL-33 administration was associated with worsened cardiac function and adverse cardiac remodeling in the MI mouse model. IL-33 administration increased infarct size, LV hypertrophy, cardiomyocyte death, and overall mortality rate due to cardiac rupture. Moreover, IL-33-treated MI mice displayed a significant myocardial eosinophil infiltration at 7 days post-MI when compared with vehicle-treated MI mice. The present study reveals that although IL-33 administration is associated with a reparative phenotype following MI, it worsens cardiac remodeling and promotes heart failure.

We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive isoLG (isolevuglandin) accumulate in dendritic cells (DCs). PGE 2 (Prostaglandin E2) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE 2 via its EP (E prostanoid) 3 receptor contributes to DC activation in hypertension. EP3 −/− mice and wild-type littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the nitric oxide synthase inhibitor N ω -nitro-L-arginine methyl ester hydrochloride in drinking water, followed by a 2-week washout period, and a subsequent 4% high-salt diet for 3 weeks. In wild-type mice, this protocol increased systolic pressure from 123±2 to 148±8 mm Hg ( P −/− mice. Similar protective effects were also observed in wild-type mice that received intracerebroventricular injection of a lentiviral vector encoding shRNA targeting the EP3 receptor. Further, in vitro experiments indicated that PGE 2 also acts directly on DCs via its EP1 receptors to stimulate intracellular isoLG formation. Together, these findings provide new insight into how EP receptors in both the central nervous system and peripherally on DCs promote inflammation in salt-induced hypertension.

Background: Early metabolic impairment is associated with significant cardiorenal risk. Specifically, subtle serum insulin and lipids changes in prediabetes trigger cardioautonomic and renovascular deterioration. Interestingly, data from our laboratory suggest that these insidious alterations are triggered by perirenal and perivascular adipose inflammation in response to hypoxia evoked by a localized increase in uncoupling protein1 (UCP1) expression and activity. This affects the adjacent organs in a paracrine manner. Hypothesis: Increased UCP1 expression is triggered by sympathetic stimulation. The sympathovagal imbalance in early metabolic dysfunction underlies the observed localized adipose pool involvement. Pharmacological interventions to offset the sympathetic predominance should prevent increased UCP1 expression and inflammation, and reverse cardiorenal manifestations. Methods: A non-obese prediabetic rat model was induced by twelve weeks of hypercaloric feeding. Moxonidine and galantamine were administered orally at daily doses of 2.4 mg/Kg and 4 mg/Kg for the last two weeks of feeding to reduce and facilitate sympathetic and parasympathetic discharge, respectively. Baroreflex sensitivity and renovascular reactivity were assessed by invasive hemodynamics and in isolated perfused kidneys, respectively. Adipose tissue T Cell distribution and macrophage polarization were examined by flow cytometry. Results: Correcting the autonomic imbalance in prediabetic rats with either moxonidine or galantamine restored cardioautonomic and renovascular activity to those observed in control rats. Although prediabetic rats showed an increased oxidative stress and proinflammatory cytokine production in both adipose pools, no change in T Cell population distribution was observed. However, the increased M 1 /M 2 macrophage polarization profile observed in prediabetic rats was reversed by either drug treatment. This was associated with a normalization of UCP1 expression and hypoxic status. Conclusions: Pharmacological interventions to reverse sympathetic predominance could be of therapeutic value treating perivascular and perirenal adipose inflammation that cause early cardiorenal manifestations of prediabetes.

According to the World Health Organization (WHO), an estimated 1.28 billion adults aged 30–79 years worldwide have hypertension; and every year, hypertension takes 7.6 million lives. High intakes of salt and sugar (mainly fructose from added sugars) have been linked to the etiology of hypertension, and this may be particularly true for countries undergoing the nutrition transition, such as Lebanon. Salt-induced hypertension and fructose-induced hypertension are manifested in different mechanisms, including Inflammation, aldosterone-mineralocorticoid receptor pathway, aldosterone independent mineralocorticoid receptor pathway, renin-angiotensin system (RAS), sympathetic nervous system (SNS) activity, and genetic mechanisms. This review describes the evolution of hypertension and cardiovascular diseases (CVDs) in Lebanon and aims to elucidate potential mechanisms where salt and fructose work together to induce hypertension. These mechanisms increase salt absorption, decrease salt excretion, induce endogenous fructose production, activate fructose-insulin-salt interaction, and trigger oxidative stress, thus leading to hypertension. The review also provides an up-to-date appraisal of current intake levels of salt and fructose in Lebanon and their main food contributors. It identifies ongoing salt and sugar intake reduction strategies in Lebanon while acknowledging the country’s limited scope of regulation and legislation. Finally, the review concludes with proposed public health strategies and suggestions for future research, which can reduce the intake levels of salt and fructose levels and contribute to curbing the CVD epidemic in the country.

Salt-sensing mechanisms in hypertension involving the kidney, vasculature, and central nervous system have been well studied; however, recent studies suggest that immune cells can sense sodium (Na + ). Antigen-presenting cells (APCs) including dendritic cells critically modulate inflammation by activating T cells and producing cytokines. We recently found that Na + enters dendritic cells through amiloride-sensitive channels including the α and γ subunits of the epithelial sodium channel (ENaC) and mediates nicotinamide adenine dinucleotide phosphate oxidase-dependent formation of immunogenic IsoLG (isolevuglandin)-protein adducts leading to inflammation and hypertension. Here, we describe a novel pathway in which the salt-sensing kinase SGK1 (serum/glucocorticoid kinase 1) in APCs mediates salt-induced expression and assembly of ENaC-α and ENaC-γ and promotes salt-sensitive hypertension by activation of the nicotinamide adenine dinucleotide phosphate oxidase and formation of IsoLG-protein adducts. Mice lacking SGK1 in CD11c + cells were protected from renal inflammation, endothelial dysfunction, and developed blunted hypertension during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride/high salt model of salt-sensitive hypertension. CD11c + APCs treated with high salt exhibited increased expression of ENaC-γ which coimmunoprecipitated with ENaC-α. This was associated with increased activation and expression of various nicotinamide adenine dinucleotide phosphate oxidase subunits. Genetic deletion or pharmacological inhibition of SGK1 in CD11c + cells prevented the high salt-induced expression of ENaC and nicotinamide adenine dinucleotide phosphate oxidase. These studies indicate that expression of SGK1 in CD11c + APCs contributes to the pathogenesis of salt-sensitive hypertension.

Tobacco smoking is a major risk factor for cardiovascular disease and hypertension. It is associated with the oxidative stress and induces metabolic reprogramming, altering mitochondrial function. We hypothesized that cigarette smoke induces cardiovascular mitochondrial oxidative stress, which contributes to endothelial dysfunction and hypertension. To test this hypothesis, we studied whether the scavenging of mitochondrial H2O2 in transgenic mice expressing mitochondria-targeted catalase (mCAT) attenuates the development of cigarette smoke/angiotensin II-induced mitochondrial oxidative stress and hypertension compared with wild-type mice. Two weeks of exposure of wild-type mice with cigarette smoke increased systolic blood pressure by 17 mmHg, which was similar to the effect of a subpresssor dose of angiotensin II (0.2 mg·kg−1·day−1), leading to a moderate increase to the prehypertensive level. Cigarette smoke exposure and a low dose of angiotensin II cooperatively induced severe hypertension in wild-type mice, but the scavenging of mitochondrial H2O2 in mCAT mice completely prevented the development of hypertension. Cigarette smoke and angiotensin II cooperatively induced oxidation of cardiolipin (a specific biomarker of mitochondrial oxidative stress) in wild-type mice, which was abolished in mCAT mice. Cigarette smoke and angiotensin II impaired endothelium-dependent relaxation and induced superoxide overproduction, which was diminished in mCAT mice. To mimic the tobacco smoke exposure, we used cigarette smoke condensate, which induced mitochondrial superoxide overproduction and reduced endothelial nitric oxide (a hallmark of endothelial dysfunction in hypertension). Western blot experiments indicated that tobacco smoke and angiotensin II reduce the mitochondrial deacetylase sirtuin-3 level and cause hyperacetylation of a key mitochondrial antioxidant, SOD2, which promotes mitochondrial oxidative stress. NEW & NOTEWORTHY This work demonstrates tobacco smoking-induced mitochondrial oxidative stress, which contributes to endothelial dysfunction and development of hypertension. We suggest that the targeting of mitochondrial oxidative stress can be beneficial for treatment of pathological conditions associated with tobacco smoking, such as endothelial dysfunction, hypertension, and cardiovascular diseases. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/mitochondrial-oxidative-stress-in-smoking-and-hypertension/ .

Traumatic brain injury (TBI) remains a major cause of morbidity and disability worldwide and a healthcare burden. TBI is an important risk factor for neurodegenerative diseases hallmarked by exacerbated neuroinflammation. Neuroinflammation in the cerebral cortex plays a critical role in secondary injury progression following TBI. The NOD-like receptors (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is a key player in initiating the inflammatory response in various central nervous system disorders entailing TBI. This current study aims to investigate the role of NLRP3 in repetitive mild traumatic brain injury (rmTBI) and identify the potential neuroprotective effect of saffron extract in regulating the NLRP3 inflammasome. 24 hours following the final injury, rmTBI causes an upregulation in mRNA levels of NLRP3, caspase-1, the apoptosis-associated speck-like protein containing a CARD (ASC), nuclear factor kappa B (NF-κB), interleukin-1Beta (IL-1β), interleukin 18 (IL-18), nuclear factor erythroid 2–related factor 2 (NRF2) and heme oxygenase 1 (HMOX1). Protein levels of NLRP3, sirtuin 1 (SIRT1), glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba1), and neuronal nuclei (Neu N) also increased after rmTBI. Administration of saffron alleviated the degree of TBI, as evidenced by reducing the neuronal damage, astrocyte, and microglial activation. Pretreatment with saffron inhibited the activation of NLRP3, caspase-1, and ASC concurrent to reduced production of the inflammatory cytokines IL-1β and IL-18. Additionally, saffron extract enhanced SIRT1 expression, NRF2, and HMOX1 upregulation. These results suggest that NLRP3 inflammasome activation and the subsequent inflammatory response in the mice cortex are involved in the process of rmTBI. Saffron blocked the inflammatory response and relieved TBI by activating detoxifying genes and inhibiting NLRP3 activation. The effect of saffron on the NLRP3 inflammasome may be SIRT1 and NF-κB dependent in the rmTBI model. Thus, brain injury biomarkers will help in identifying a potential therapeutic target in treating TBI-induced neurodegenerative diseases.

Treatments for metabolic diseases, such as diet and therapeutics, often provide short-term therapy for metabolic stressors, but relapse is common. Repeated bouts of exposure to, and relief from, metabolic stimuli results in a phenomenon we call “metabolic cycling.” Recent human and rodent data suggest metabolic cycling promotes an exaggerated response and ultimately worsened metabolic health. This is particularly evident with cycling of body weight and hypertension. The innate and adaptive immune systems have a profound impact on development of metabolic disease, and current data suggest that immunologic memory may partially explain this association, especially in the context of metabolic cycling. In this Brief Review, we highlight recent work in this field and discuss potential immunologic mechanisms for worsened disease prognosis in individuals who experience metabolic cycling.

Salt-sensing mechanisms in hypertension involving the kidney, vasculature and central nervous system have been well studied; however, recent studies suggest that immune cells can sense sodium (Na(+)). Antigen presenting cells (APCs) including dendritic cells (DCs) critically modulate inflammation by activating T cells and producing cytokines. We recently found that Na(+) enters DCs through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel (ENaC) and mediates NADPH oxidase-dependent formation of immunogenic isolevuglandin (IsoLG)-protein adducts leading to inflammation and hypertension. Here, we describe a novel pathway in which the salt-sensing kinase serum/glucocorticoid kinase 1 (SGK1) in APCs mediates salt-induced expression and assembly of ENaCα and ENaCγ and promotes salt-sensitive hypertension by activation of the NADPH oxidase and formation of IsoLG-protein adducts. Mice lacking SGK1 in CD11c(+) cells were protected from renal inflammation, endothelial dysfunction and developed blunted hypertension during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride (L-NAME)/high salt model of salt-sensitive hypertension. CD11c(+) APCs treated with high salt exhibited increased expression of ENaCγ which co-immunoprecipitated with ENaCα. This was associated with increased activation and expression of various NADPH oxidase subunits. Genetic deletion or pharmacological inhibition of SGK1 in CD11c(+) cells prevented the high salt induced expression of ENaC and NADPH oxidase. These studies indicate that expression of SGK1 in CD11c(+) APCs contributes to the pathogenesis of salt-sensitive hypertension.

T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.

Vascular superoxide (O˙ 2 - ) and inflammation contribute to hypertension. The mitochondria are an important source of O˙ 2 - ; however, the regulation of mitochondrial O˙ 2 - and the antihypertensive potential of targeting the mitochondria remain poorly defined. Angiotensin II and inflammatory cytokines, such as interleukin 17A and tumor necrosis factor-α (TNFα) significantly contribute to hypertension. We hypothesized that angiotensin II and cytokines co-operatively induce cyclophilin D (CypD)–dependent mitochondrial O˙ 2 - production in hypertension. We tested whether CypD inhibition attenuates endothelial oxidative stress and reduces hypertension. CypD depletion in CypD −/− mice prevents overproduction of mitochondrial O˙ 2 - in angiotensin II–infused mice, attenuates hypertension by 20 mm Hg, and improves vascular relaxation compared with wild-type C57Bl/6J mice. Treatment of hypertensive mice with the specific CypD inhibitor Sanglifehrin A reduces blood pressure by 28 mm Hg, inhibits production of mitochondrial O˙ 2 - by 40%, and improves vascular relaxation. Angiotensin II–induced hypertension was associated with CypD redox activation by S-glutathionylation, and expression of the mitochondria-targeted H 2 O 2 scavenger, catalase, abolished CypD S-glutathionylation, prevented stimulation mitochondrial O˙ 2 - , and attenuated hypertension. The functional role of cytokine–angiotensin II interplay was confirmed by co-operative stimulation of mitochondrial O˙ 2 - by 3-fold in cultured endothelial cells and impairment of aortic relaxation incubated with combination of angiotensin II, interleukin 17A, and tumor necrosis factor-α which was prevented by CypD depletion or expression of mitochondria-targeted SOD2 and catalase. These data support a novel role of CypD in hypertension and demonstrate that targeting CypD decreases mitochondrial O˙ 2 - , improves vascular relaxation, and reduces hypertension.

Vascular dysfunction plays a key role in the development of hypertension and heart disease which causes one-third of deaths worldwide. Cardiovascular disease risk factors reduce mitochondrial deacetylase Sirt3 and Sirt3 declines with age paralleling the increased incidence of cardiovascular disease and hypertension; however, the role of Sirt3 was largely ignored. We hypothesized that Sirt3 plays critical role in vascular dysfunction and hypertension. To test this hypothesis we have developed novel tamoxifen-inducible endothelium specific Sirt3 knockout (Ec Sirt3KO ), endothelial Sirt3 overexpressing (Ec Sirt3OX ), smooth muscle Sirt3 knockout (Smc Sirt3KO ), smooth muscle Sirt3 overexpressing (Smc Sirt3OX ) and global Sirt3 overexpressing (Sirt3OX) mice on C57Bl/6J background, and examined the effect of Sirt3 expression on vascular dysfunction and angiotensin II-induced hypertension. It was found that cell-specific Sirt3 depletion in endothelial and smooth muscle in Ec Sirt3KO and Smc Sirt3KO mice increases vascular permeability by 2-fold, raises T cell and monocyte vascular accumulation, diminishes endothelial NO and increases blood pressure by 10 mm Hg compared with wild-type Sham mice. Angiotensin II infusion in Ec Sirt3KO and Smc Sirt3KO mice caused vascular hyperpermeability, increased vascular hypertrophy, exacerbated endothelial dysfunction and hypertension. Meanwhile, Sirt3 overexpression reduced vascular permeability and diminished vascular inflammation compared to Sham wild-type mice. Sirt3 overexpression in endothelial and smooth muscle prevents angiotensin II-induced vascular hyperpermeability, inhibits vascular oxidative stress, preserves endothelial-dependent relaxation, diminishes vascular hypertrophy and attenuates hypertension by 20 mm Hg in angiotensin II-infused Ec Sirt3OX and Smc Sirt3OX mice compared with the wild-type littermates. Interestingly, Sirt3 depletion in human aortic endothelial cells and human vascular smooth muscle cells reduces expression of differentiation markers. We suggest that Sirt3 is central in redox and metabolic regulations of smooth muscle and endothelial cells. Our data support a therapeutic potential of targeting Sirt3 in vascular dysfunction and hypertension.

T cell derived cytokines, interleukin 17A (IL17A) and interferon gamma (IFNg), promote angiotensin II (Ang II)-induced hypertension and end-organ damage. Interleukin 21 (IL21), produced primarily by T follicular helper (Tfh) cells, has been shown to induce IL17A and IFNγ production from T effector cells. IL21 is also a potent activator of germinal center (GC) B cells and immunoglobulin (Ig) class switching. We hypothesized that IL21 plays a fundamental role in hypertension and hypertensive end-organ damage through its effects on pro-inflammatory T cell polarization, vascular dysfunction, and enhanced Ig secretion. CD4+ T cells from Ang II infused WT mice exhibit 1.5-fold increased IL21 mRNA expression and 2-fold increased IL-21 production compared to vehicle. We found IL21 -/- mice exhibit a 30mmHg reduction in systolic blood pressure (SBP) in response to 4 weeks of Ang II infusion compared to age-matched wild type (WT) mice. Further, IFNg and IL17A production from CD8+ or CD4+ T cells, respectively, was abrogated in IL21 -/- mice vs. WT mice infused with Ang II. We also found Tfh cells and GC B cells significantly increased in the aortas and mesenteric vessels of Ang II WT mice but not IL21 -/- mice. Ang II induced an increase in percent of lymph node GC B cells and IgG/IgM ratio, consistent with Ig class switching. Ang II-induced increase in renal injury was also blunted in IL21 -/- mice vs. WT mice. Mesenteric vessels from IL21 -/- mice were protected from Ang II-induced endothelial dysfunction. IL21 -/- mice also developed blunted aortic fibrosis and reduced smooth muscle cell hypertrophy after 4 weeks of Ang II vs. WT mice. IL-21 neutralization beginning 2 weeks after Ang II resulted in a 15 mmHg reduction in SBP and reversal of aortic inflammation and vascular endothelial dysfunction compared to isotype control treated animals. Lastly, CD4+ T cell production of IL21 positively correlated with SBP in human volunteers, and in fact, IL-21 production was significantly higher in hypertensive compared to normotensive subjects. Taken together, these studies provide compelling evidence that IL21 may function as a master cytokine in the pathogenesis of hypertension and suggest that therapies targeting IL21 may reduce hypertension and the associated end-organ damage.

Aims: \ud Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function.\ud \ud Methods and Results: \ud Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1β, IL-23, chemokine (C-C motif) ligand 4 and tumor necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells and macrophages with activated STAT3.\ud \ud Conclusions: \ud These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of nitric oxide signaling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable nitric oxide, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions.

Effector memory T cells (T EM cells) play a crucial role in hypertension. Formed during an initial blood pressure surge, T EM cells can reside in bone marrow (BM) in a quiescent state for prolonged periods. Upon re-exposure to a hypertensive stimulus, these cells can be reactivated and aggravate hypertension and renal damage. Hypertension is also associated with elevated sympathetic outflow. We hypothesized that sympathetic nerves promote accumulation and reactivation of hypertension-specific T EM cells in the BM. We performed unilateral superior cervical ganglionectomy (SCGx) in C57BL/6 mice, causing sympathectomy of the forelimb on the surgical side. After 2-week Ang II infusion (490 ng/kg/min s.c.), 5х10 6 BM cells from both the SCGx and control limbs were isolated and co-cultured with 0.5х10 6 dendritic cells from other hypertensive mice. We found 30% fewer CD8 + T cell proliferation in the SCGx BM than intact side (1.9±0.2 vs. 2.8±0.1х10 4 , p+ T cells. To study mechanisms involved in T cell homing, 10 7 pan T cells were isolated from CD45.2 mice after Ang II infusion and adoptively transferred to CD45.1 mice with unilateral SCGx. Flow cytometry indicated that 35% fewer donor CD8 + T EM cells homed to the SCGx than the intact BM 7 days after transfer (5.3±0.8 vs. 8.1±1.5 per 10 4 total T cells, p

One third of adult population has hypertension and third of hypertensive patients remain hypertensive despite treatment with multiple drugs. There have been no mechanistically novel treatments for hypertension in the past 30 years. We propose that new agents targeting CypD activation, a regulatory subunit of mitochondrial permeability transition pore, could add to the currently available therapeutic armamentarium to improve treatment of hypertension. To test this hypothesis we have studied CypD acetylation which was associated with CypD activation. We found that angiotensin II-induced hypertension is linked to CypD-K166 hyperacetylation, mPTP opening, impaired mitochondrial respiration and reduced kidney ATP. CypD hyperacetylation may result from imbalance between GCN5L1 acetyltransferase and reduced Sirt3 deacetylase activity. Indeed, aortic mitochondrial level of GCN5L1 increased by 50% while Sirt3 was reduced by 40% in angiotensin II infused mice. Sirt3 can be inactivated by cytotoxic reactive lipid dicarbonyls derived from arachidonic acid, isoketals, leading to mitochondrial hyperacetylation. To test the role of mitochondrial isoketals we synthesized a novel mitochondria-targeted isoketal scavenger mito2HOBA. mito2HOBA supplementation in drinking water (0.1g/Liter) attenuates angiotensin II-induced hypertension, prevents accumulation of mitochondrial isoketal protein adducts in the aorta and kidney measured by LC/MS and D11 antibody, improves Sirt3/GCNL1 ratio, normalizes the CypD acetylation, attenuates mPTP opening, protects mitochondrial respiration and preserves normal kidney ATP level. Decreased NO bioavailability is a hallmark of endothelial oxidative stress in hypertension due to superoxide overproduction and NO oxidation. Interestingly, angiotensin II increases vascular superoxide by 2-fold and reduces endothelial NO level to 50% while mito2HOBA diminishes vascular O 2 • and preserves NO bioavailability in angiotensin II-infused mice. These data support the role of isoketals-mediated CypD hyperacetylation in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoketals may have therapeutic potential in treatment of hypertension and target-organ-damage.

Sodium can accumulate in the interstitium and promote inflammation through poorly defined mechanisms. Here we describe a novel pathway by which dendritic cells (DCs) are activated by increased extracellular sodium (190 mM). We show that sodium entry through the epithelial sodium channel (ENaC) and the sodium hydrogen exchanger (NHE) leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin 1β (IL-1β) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation and hypertension involving increased oxidative stress and IsoLG production in DCs.

We have recently reported SOD2 hyperacetylation and reduced Sirt3 level in human subjects with essential hypertension. We hypothesized that diminished Sirt3 expression promotes endothelial dysfunction and hypertension while Sirt3 overexpression protects endothelial function and attenuates hypertension. Indeed, hypertension was markedly increased in Sirt3 knockout (Sirt3 -/- ) in response to angiotensin II (0.7 mg/kg/day) compared with wild-type C57Bl/6J mice. Sirt3 depletion caused SOD2 inactivation due to SOD2 hyperacetylation, increased mitochondrial O 2 • and diminished endothelial nitric oxide. Angiotensin II infusion in wild-type mice was associated with Sirt3 inactivation and SOD2 hyperacetylation in aorta and kidney. To test the specific role of Sirt3 in vasculature we have generated tamoxifen-inducible endothelium specific Sirt3 knockout mice (Ec Sirt3 KO ) and tamoxifen-inducible smooth muscle specific Sirt3 knockout mice (Smc Sirt3 KO ). Deletion of Sirt3 in smooth muscle exacerbated hypertension (165 mm Hg vs 155 mm Hg in wild-type) and significantly increased mortality in angiotensin II infused Smc Sirt3 KO mice (30% vs 3% in wild-type) which was associated with higher rate of aortic aneurysm. Ec Sirt3 KO mice had elevated basal blood pressure by 12 mm Hg and hypertension was exacerbated in Ec Sirt3 KO mice accompanied by impaired vascular relaxation and reduced endothelial nitric oxide. Treatment of angiotensin II-infused Sirt3 -/- mice with SOD2 mimetic mitoTEMPO rescued endothelial-dependent relaxation and reduced blood pressure. We tested if Sirt3 overexpression protects endothelial function and attenuates angiotensin II-induced hypertension. These new mice were obtained by crossing the EIIa-cre with Sirt3flox mice resulting in constitutively increased Sirt3 in the whole body. Sirt3 overexpression abolished angiotensin II induced impairment of vasorelaxation and attenuated development of hypertension. Our data suggest that diminished Sirt3 activity leads to SOD2 hyperacetylation and contributes to the pathogenesis of hypertension. It is conceivable that Sirt3 agonists and SOD2 mimetics may have therapeutic potential in cardiovascular disease.

Accumulating evidence indicates that NaCl can be concentrated in tissues with high salt-intake, age and in the setting of hypertension. Elevated NaCl has been shown to promote T H 17 cell formation in an SGK1-dependent fashion. We have previously shown that Memory T cells play a major role in the genesis of hypertension. These long-lived cells remain responsive to repeated hypertensive stimuli, such as salt feeding, and can be mobilized to enter the kidney where they release cytokines that promote renal dysfunction. To examine mechanisms by which T cells sense salt and contribute to salt-sensitivity, we tested the hypothesis that SGK1 in T cells is necessary for formation of memory T cells and their activation in salt-sensitive hypertension. To study the role of SKG1 in hypertension, we produced mice with T cell specific deletion of SGK1, SGK1 fl/fl x tg CD4cre mice and used SGK1 fl/fl mice as controls. To impose repeated episodes of hypertension, we treated these mice with L-NAME (0.5mg/ml) in drinking water for two weeks, allowed a two-week normotensive interval and a then fed high salt (4% NaCl) for three weeks. L-NAME followed by high salt increased memory T cells in the kidney, aorta and bone marrow of SGK1 fl/fl control mice but not in SGK1 fl/fl x tg CD4cre mice, as identified by the surface marker CD44 hi . To assess markers of renal injury, we measured albumin in 24-hour urine samples collected at the end of the L-NAME/high salt. L-NAME/high salt caused striking albuminuria in SGK1 fl/fl mice and was absent in SGK1 fl/fl x tg CD4cre mice. In additional studies, we found that loss of SGK1 in T cells abrogates renal and vascular inflammation and protects against hypertensive renal and vascular injury in the L-NAME/high salt model. Thus, our data provide a potential mechanism by which SGK1 in T cells promotes their development of salt sensitivity and their mediation of renal and vascular dysfunction in hypertension.

We have recently identified a critical role of hypertension-specific effector memory T lymphocytes (T EM cells) in the blood pressure elevation and renal dysfunction caused by repeated hypertensive stimuli. Formed during an initial immune challenge, T EM cells reside in the bone marrow (BM) in a quiescent state for prolonged periods, and can be reactivated upon re-exposure to the hypertensive stimulus. Hypertension is associated with increased sympathetic outflow. We therefore hypothesized that sympathetic nerves regulate accumulation and reactivation of hypertension-specific T EM cells in the BM. We performed unilateral superior cervical ganglionectomy (SCGx) in wild-type C57BL/6 mice, causing selectively sympathectomy of the forelimb on the surgical side. After recovery, mice received Ang II infusion for two weeks. To determine the changes of T cells in the BM that were specific to hypertension, 5х10 6 BM cells were isolated from either the SCGx or control limbs, loaded with proliferation marker CFSE, and co-cultured with 0.5х10 6 splenic dendritic cells isolated from another Ang II-infused mouse. We found 30% less CD8 + T cell proliferation in the SCGx BM compared to control side (1.8±0.1 vs. 2.6±0.3х10 4 ), but no difference in CD4 + T cells. To further study the effect of sympathetic nerves on BM T cell homing, 1х10 7 pan T cells were isolated from CD45.2 + wild-type mice after Ang II infusion and adoptively transferred to CD45.1 mice that had previously undergone unilateral SCGx. Flow cytometry indicated that 7 days after transfer, 25% fewer CD8 + T EM cells from the hypertensive donors homed to the SCGx BM compared to the innervated BM (27.8±2.6 vs. 20.8±2.5 per 10 3 total BM cells). This effect was specific for hypertension as homing of OT-I T EM cells, which are responsive to ovalbumin, was not influenced by sympathectomy. Further adoptive transfer studies using mice lacking beta 2 adrenergic receptors (β2AR) indicate that β2AR in the bone marrow niche, rather than T cell β2AR is critical for T EM cell homing. These data define a novel role of sympathetic nerves in regulation of memory T cell trafficking, and this likely contributes to the predisposition to hypertension and end-organ damage for prolonged periods following an initial episode of hypertension.

Mechanical stretch activates the endothelium to produce reactive oxygen species and expression of adhesion molecules and cytokines. Human monocytes that traverse the endothelium differentiate into dendritic cells (DCs) upon exposure to a pro-inflammatory state. We hypothesized that human endothelial cells exposed to mechanical stretch will promote conversion of human monocytes into activated DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the endothelial cells to either normal (5%) or hypertensive (10%) uniaxial cyclical stretch for 48 hours. Co-culturing monocytes with HAECs exposed to 10% stretch showed a marked increase in pro-inflammatory cytokines such as IL-6, IL-23A and IL-1β compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs. We have shown that DCs from hypertensive mice accumulate isolevuglandins (IsoLGs) that adduct to proteins and promote T cell activation. Thus, we performed intracellular staining and flow cytometry and found that these monocytes significantly accumulate higher levels of IsoLG-adducted proteins compared to 5% stretch (69.7 ± 5.8 vs 10.8 ± 1.85, respectively). In addition, monocytes co-cultured with endothelial cells exposed to 10% stretch expressed phosphorylated STAT3 (pSTAT3), which was blocked by stattic, a STAT3 inhibitor. Similarly, monocytes co-cultured with HAECs exposed to 10% stretch induced a 1,500-fold and a 1,300-fold increase in CD4 + and CD8 + T cell proliferation; while, inhibition of STAT3 prevented this T cell proliferation. To test if this is due to cell-cell contact, we seeded monocytes in a transwell with endothelial cells exposed to either 5% or 10% stretch. We found that these monocytes expressed DC markers, pSTAT3, and accumulated IsoLG-peptides when exposed to hypertensive mechanical stretch. In addition, angiotensin II (490ng/kg/min) infusion of C57Bl/6 mice increased pSTAT3 in monocytes, macrophages, and DCs in the renal and vascular tissue compared to sham controls. Thus, our data indicate that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote differentiation into DCs known to be immunogenic via STAT3.

We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1–dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II–induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate–salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl– cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.

Rationale: Clinical studies have shown that Sirt3 (Sirtuin 3) expression declines by 40% by 65 years of age paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 because of increased NADH (nicotinamide adenine dinucleotide, reduced form) and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) because of hyperacetylation. Objective: In this study, we examined whether the loss of Sirt3 activity increases vascular oxidative stress because of SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. Methods and Results: Hypertension was markedly increased in Sirt3-knockout (Sirt3 −/ − ) and SOD2-depleted (SOD2 +/ − ) mice in response to low dose of angiotensin II (0.3 mg/kg per day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O 2 · – , and diminished endothelial nitric oxide. Angiotensin II-induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2, and reduced SOD2 activity. Scavenging of mitochondrial H 2 O 2 in mCAT mice expressing mitochondria-targeted catalase prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H 2 O 2 scavenger, mitochondria-targeted hydrogen peroxide scavenger ebselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation, and reduced blood pressure in wild-type but not in Sirt3 −/− mice, whereas an SOD2 mimetic, (2-[2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino]-2-oxoethyl) triphenylphosphonium (mitoTEMPO), reduced blood pressure and improved vasorelaxation both in Sirt3 −/− and wild-type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels, whereas SOD2 expression was not affected. Conclusions: Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.

Recent studies have emphasized a role of adaptive immunity, and particularly T cells, in the genesis of hypertension. We sought to determine the T-cell subtypes that contribute to hypertension and renal inflammation in angiotensin II–induced hypertension. Using T-cell receptor spectratyping to examine T-cell receptor usage, we demonstrated that CD8 + cells, but not CD4 + cells, in the kidney exhibited altered T-cell receptor transcript lengths in Vβ3, 8.1, and 17 families in response to angiotensin II–induced hypertension. Clonality was not observed in other organs. The hypertension caused by angiotensin II in CD4 −/− and MHCII −/− mice was similar to that observed in wild-type mice, whereas CD8 −/− mice and OT1xRAG-1 −/− mice, which have only 1 T-cell receptor, exhibited a blunted hypertensive response to angiotensin II. Adoptive transfer of pan T cells and CD8 + T cells but not CD4 + /CD25 − cells conferred hypertension to RAG-1 −/− mice. In contrast, transfer of CD4 + /CD25 + cells to wild-type mice receiving angiotensin II decreased blood pressure. Mice treated with angiotensin II exhibited increased numbers of kidney CD4 + and CD8 + T cells. In response to a sodium/volume challenge, wild-type and CD4 −/− mice infused with angiotensin II retained water and sodium, whereas CD8 −/− mice did not. CD8 −/− mice were also protected against angiotensin-induced endothelial dysfunction and vascular remodeling in the kidney. These data suggest that in the development of hypertension, an oligoclonal population of CD8 + cells accumulates in the kidney and likely contributes to hypertension by contributing to sodium and volume retention and vascular rarefaction.

Obesity is associated with vascular diseases that are often attributed to vascular oxidative stress. We tested the hypothesis that vascular oxidative stress could induce obesity. We previously developed mice that overexpress p22phox in vascular smooth muscle, tgsm/p22phox, which have increased vascular ROS production. At baseline, tgsm/p22phox mice have a modest increase in body weight. With high-fat feeding, tgsm/p22phox mice developed exaggerated obesity and increased fat mass. Body weight increased from 32.16 ± 2.34 g to 43.03 ± 1.44 g in tgsm/p22phox mice (vs. 30.81 ± 0.71 g to 37.89 ± 1.16 g in the WT mice). This was associated with development of glucose intolerance, reduced HDL cholesterol, and increased levels of leptin and MCP-1. Tgsm/p22phox mice displayed impaired spontaneous activity and increased mitochondrial ROS production and mitochondrial dysfunction in skeletal muscle. In mice with vascular smooth muscle–targeted deletion of p22phox (p22phoxloxp/loxp/tgsmmhc/cre mice), high-fat feeding did not induce weight gain or leptin resistance. These mice also had reduced T-cell infiltration of perivascular fat. In conclusion, these data indicate that vascular oxidative stress induces obesity and metabolic syndrome, accompanied by and likely due to exercise intolerance, vascular inflammation, and augmented adipogenesis. These data indicate that vascular ROS may play a causal role in the development of obesity and metabolic syndrome.

In recent years, it has become clear that the immune system contributes to the genesis of hypertension. Hypertensive stimuli, such as angiotensin II, DOCA-salt, and norepinephrine, cause T cells and monocytes/macrophages to accumulate in the kidney and vasculature. These cells release inflammatory cytokines, such as IL-6, interferon-γ, and IL-17, that promote renal and vascular dysfunction. These cytokines also promote angiotensinogen production in the proximal tubule and Na+ retention in the distal nephron and contribute to renal fibrosis and glomerular damage. For several years, we have observed accumulation of memory T cells in the kidney and vasculature. Given the propensity for memory cells to produce cytokines such as interferon-γ and IL-17, interventions to prevent the formation or renal accumulation of specific memory T cell subsets could prevent end-organ damage and blood pressure elevation in response to hypertensive stimuli.

Blood flow regulation of normal cerebral arteries is a critical and important factor to supply the brain tissue with nutrients and oxygen. Stroke insult results in a disruption or reduction in cerebral arteries' blood flow with subsequent brain tissue damage. Hemorrhagic stroke is one type of stroke and accounts for about 13 % of all of stroke insults. In this type of stroke, the cerebral artery breaks open and causes bleeding in or surrounding the brain. Subsequently, this bleeding causes blood vessels to constrict in a process called vasospasm, in which the vessels narrow and impede the blood flow to brain tissue. Hemorrhagic stroke is the major cause of prolonged constriction of cerebral arteries. This leads to partial brain damage and sometimes death in patients with aneurysmal subarachnoid hemorrhage. Among the key delicate techniques to assess small blood vessel functionality is the wire myograph, which can be utilized in several cerebral injury models including stroke. The wire myograph is a device that provides information about the reactivity, stiffness, and elasticity of small blood vessels under isometric conditions. In this book chapter, we describe the techniques involved in wire myography assessment and the different measures and parameters recorded; we describe the utility of this technique in evaluating the effects of subarachnoid hemorrhage on basilar artery sensitivity to different agonists.

Endothelial dysfunction is associated with aging, diabetes, hyperlipidemia, obesity and these risk factors affect the expression and activity of the mitochondrial deacetylase Sirt3. Sirt3 activates major antioxidant SOD2 by deacetylation of specific lysine residues and Sirt3 depletion increases oxidative stress. We hypothesized that loss of vascular Sirt3 increases endothelial dysfunction, promotes hypertension and end organ damage. The role of vascular Sirt3 was studied in wild-type C57Bl/6J mice and tamoxifen-inducible smooth muscle specific Sirt3 knockout mice (Smc Sirt3 KO ) using angiotensin II model of hypertension (Ang II, 0.7 mg/kg/day). Western blot showed 30% reduction of vascular Sirt3 and 2-fold increase in SOD2 acetylation in Ang II-infused WT mice. We have tested if ex vivo treatment of aorta with Sirt3 activator resveratrol improves endothelial function. Indeed, ex vivo incubation with resveratrol (10 μM) significantly reduced SOD2 acetylation, diminished mitochondrial O 2 and increased endothelial NO to normal level while Sirt3-inactive analog dihydroresveratrol had no effect. Specific role of vascular Sirt3 was studied in Smc Sirt3 KO mice by crossing floxed Sirt3 mice with mice carrying gene for inducible cre in the vascular smooth muscle. Sirt3 deletion exacerbates hypertension (165 mm Hg vs 155 mm Hg in wild-type) and significantly increases mortality in Ang II-infused Smc Sirt3 KO mice (60% vs 10% in wild-type) associated with severe edema and aortic aneurysm (100% vs 20% in wild-type). Decrease of NO is a hallmark of endothelial dysfunction in hypertension due to vascular oxidative stress. Indeed, Ang II infusion increased vascular O 2 by 2-fold and reduced endothelial NO by 2-fold. Interestingly, Ang II infusion in Smc Sirt3 KO mice caused severe vascular oxidative stress (3-fold increase in O 2 ) and exacerbated endothelial dysfunction (4-fold decrease in NO). These data indicate that reduced vascular Sirt3 activity occurs in hypertension and this promotes vascular oxidative stress, increases endothelial dysfunction, exacerbates hypertension, increases end-organ-damage and mortality. It is conceivable that Sirt3 agonists and SOD2 mimetics may have therapeutic potential in cardiovascular disease.

We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive γ-ketoaldehydes (isoketals) accumulate in dendritic cells (DCs). These are immunogenic and lead to subsequent T lymphocytes activation. The local signals that stimulate DCs to accumulate isoketal adducts remain undefined. Prostaglandin E 2 (PGE 2 ) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE 2 via its EP3 receptor contributes to DC activation in hypertension. EP3 -/- mice and wild type (WT) littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the NOS inhibitor L-NAME (LN) in drinking water, a 2 week washout period, and a subsequent 4% high salt diet (HS) for 3 weeks. In WT mice, this protocol increased systolic pressure from 123±2 to 148±8 mmHg (p+ and CD8 + effector memory T cells by 2 to 3 fold. This was associated with a striking accumulation of isoketal protein adducts in splenic DCs. However, the increases in blood pressure, renal T cell infiltration and DC isoketal formation were completely prevented in EP3 -/- mice. We further hypothesized that EP3 receptors contribute to oxidative stress production in the kidney. As measured by dihydroethidium with confocal microscopy, the LNHS protocol induced marked increases in superoxide production in WT mice, but not in EP3 -/- mice. To examine the direct effects of PGE 2 , splenic DCs were incubated with PGE 2 in vitro for 24 hours. PGE 2 dose-dependently increased isoketal-adduct formation in DCs (vehicle: 8.8±5.1% vs. 50 nM PGE 2 : 41.4±11.7%, p2 in DC activation in hypertension. In vivo, PGE 2 has a predominant effect on EP3 receptors to enhance renal vascular ROS production, which likely leads to isoketal-adduct formation and accumulation in DCs. PGE 2 also acts directly on DCs via its EP1 receptors to stimulate intracellular isoketal formation. Together, these findings provide additional information as to how PGE 2 modulates inflammation in hypertension.

We have previously shown that the T cell-derived pro-inflammatory cytokine, interleukin 17A (IL17A), is upregulated by and promotes angiotensin II (Ang II)-induced hypertension and contributes to renal and vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride enhances IL17A production from CD4+ T cells in an SGK1 dependent manner. Since dietary salt intake is associated with hypertension, we hypothesized that T cell SGK1 promotes hypertension and contributes to end-organ dysfunction. To test this hypothesis, we crossed SGK1 fl/fl mice with Tg CD4cre mice to delete SGK1 in T lymphocytes. Loss of T cell SGK1 resulted in a blunted blood pressure response following 2 weeks of Ang II infusion (24.8 mmHg reduction, p=0.01). Moreover, renal and vascular inflammation in response to Ang II infusion was abrogated in these mice compared to SGK1 fl/fl control mice. Ang II infusion increased total (CD45+) leukocytes in the kidney from 55.4 to 120.4 x10 3 (pfl/fl mice while there was no increase in mice with T cell deletion of SGK1 (48.1 to 47.5x10 3 , p=ns). Similarly, Ang II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4x10 3 (pfl/fl mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1x10 3 , p=ns). Furthermore, relaxation of mesenteric arterioles isolated from Ang II infused SGK1 fl/fl mice in response to acetylcholine was impaired by 22.37% (pfl/fl mice infused with Ang II compared to 3.23-fold (p=ns) in mice without T cell SGK1, demonstrating that these mice are protected from Ang II-induced renal injury. Finally, we found that total numbers of splenic CD4+IL17A+ cells increase from 0.44% to 1.15% (pfl/fl mice infused with Ang II compared to no change (0.45% to 0.47%, p=ns) in mice without T cell SGK1. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and the associated end-organ dysfunction.

We and others have shown that hypertension (HTN) is linked with striking fibrosis in the aortic adventitia. This leads to aortic stiffening, leading to organ damage. Through a screen of microRNAs (miRNAs) in the aorta, we found that miR-762 is the most upregulated miRNA in Ang II hypertensive mice. qRT-PCR confirmed that miR-762 is upregulated 6.35±1.22 (p=0.03) fold in Ang II-infused mice compared to controls. To study the role of miR-762 in HTN, we administered a locked nucleic acid inhibitor of miR-762. MiR-762 inhibition normalized stress-strain relationships and aortic systolic energy storage (ASE) (Table). Moreover, miR-762 inhibition in the last 2 weeks of Ang II infusion reversed aortic stiffness in mice treated with 4 wk of Ang II (ASE, 4 wk Ang II [51±5.18 kPa] vs 4wk Ang II + LNA-762 (last 2 wk) [20±1.76 kPa], p

Oxidative stress is an important contributor to hypertension. Our group has shown that oxidative stress generates isolevuglandins (isoLG) that modify proteins and that these isoLG-modified proteins seem to act as neoantigens to promote hypertension. In addition to proteins, isoLG can also modify phosphatidylethanolamines (PEs). IsoLG-modified PE (IsoLG-PE) can in turn activate immune cells via reactions with toll-like receptors and downstream pathways including NFκB. The role of isoLG-PE in hypertension has not yet been defined. We have previously shown that the enzyme N-acyl-phosphatidylethanolamine-phospholipase D (NAPE-PLD) cleaves isoLG-PE, and mice lacking this enzyme have elevated cellular levels of isoLG-PE. To assess whether loss of NAPE-PLD and resulting increase in isoLG-PE contributes to hypertension, we infused wild type (WT) and NAPE-PLD -/- mice with low dose Ang II. We found baseline blood pressure was elevated in NAPE-PLD -/- mice compared to WT mice (128 ± 3 vs 111 ± 2 mmHg, p = 0.005) and that the increase in BP to low dose Ang II infusion (140 ng/kg/min) was augmented in NAPE-PLD -/- compared to WT mice (146 ± 9 vs 127 ± 4 mmHg, p=0.045). Endothelium-dependent vascular relaxation of the mesenteric arterioles to acetylcholine was impaired in NAPE-PLD -/- compared to WT mice (maximum relaxation of 43% ± 6% vs 53% ± 5%, p= 0.004), but endothelium-independent responses to sodium nitroprusside were similar in both groups (82% ± 2% in NAPE-PLD -/- mice vs 81% ± 4% in WT mice). Aortic adventitial collagen content by planimetry was likewise increased in NAPE-PLD -/- versus WT mice following low-dose Ang II. These studies in mice lacking NAPE-PLD suggest that in addition to exerting its effects via protein modification to form neo-antigens, the reaction of isoLG with PE and related phospholipids can augment hypertension, alter endothelium-dependent vasodilatation and promote vascular fibrosis.