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Background: IsoLGs (isolevuglandins) are electrophilic products of lipid peroxidation formed in the presence of reactive oxygen species. IsoLGs contribute to hypertension by an unknown mechanism. Studies have shown that reactive oxygen species production drives the formation of neutrophil extracellular traps (NETs) and that NETs accumulate within the aorta and kidneys of patients with hypertension. The purpose of this study was to determine the role of isoLGs in neutrophil migration and NET formation (NETosis) in hypertension. Methods: Mice were treated with Ang II (angiotensin II) and the specific isoLG scavenger 2-hydroxybenzylamine and examined for tissue neutrophil and NET accumulation by single-cell sequencing and flow cytometry. Isolated human neutrophils were studied to determine the role of isoLGs in NETosis and neutrophil chromatin expansion by immunofluorescence and live cell confocal microscopy. Results: Single-cell sequencing performed on sham, Ang II, and Ang II+2-hydroxybenzylamine treated mice revealed neutrophils as a primary target of 2-hydroxybenzylamine. Peripheral neutrophil migration, aortic NET accumulation, and renal NET accumulation is blocked with 2-hydroxybenzylamine treatment. In isolated human neutrophils, isoLGs accumulate during NETosis and scavenging of isoLGs prevents NETosis. IsoLGs drive neutrophil chromatin expansion during NETosis and disrupt nucleosome structure. Conclusions: These observations identified a critical role of isoLGs in neutrophil migration and NETosis in hypertension and provide a potential therapy for NET-associated diseases including hypertension and associated end organ damage.

Background: Aortic remodeling enhances arterial pulsatility in the microcirculation and we have shown that excessive endothelial stretch increases release of Growth Arrest Specific 6 (GAS6), which activates Axl on monocytes causing immune activation and inflammation. Hypothesis: GAS6/Axl blockade reduces renal and vascular inflammation and lessens renal dysfunction caused by aortic remodeling. Methods and Results: Three month old male C57Bl/6 mice received osmotic minipumps containing vehicle or ang II for 2 weeks and the pumps removed. Aortas demonstrated increased collagen and an 80% loss of Windkessel function that persisted for 6 months following ang II. Renal function studies showed reduced ability to excrete a volume load, a progressive increase in albuminuria and tubular damage as estimated by Periodic Acid Schiff staining. Treatment with the Axl inhibitor R428 beginning 2 months after ang II had minimal effect on aortic remodeling 2 months later, but reduced aortic and renal infiltration of T cells, gamma/delta T cells and macrophages as measured by flow cytometry (Figure). R428 also improved renal excretory capacity, decreased urinary NGAL, reduced albuminuria, and attenuated renal tubular damage. Interestingly, macrophage/T cell complexes were present in aortas of mice previously exposed to hypertension, and were reduced by R428. Conclusion: Brief episodes of hypertension induce chronic aortic remodeling which increases Axl signaling and transformation of monocytes to inflammatory antigen presenting cells that activate T cells. Axl blockade might serve as a therapeutic option to improve end-organ damage after an episode of hypertension.

Autoimmune and inflammatory diseases such as obesity and cancer are highly linked to altered bone growth and osteoporosis. Hypertension (HTN) is an inflammatory condition associated with reduced bone mineral density and increased risk for fragility fractures in adults. As cases of pediatric HTN rise in the United States, HTN may increase bone loss in children and future risk for osteoporosis. We hypothesized that HTN-induced osteoporosis in young mice reduces bone quality like that of aging. To assess this, 3.5- or 16-month-old male C57BL/6J mice received Angiotensin (Ang) II (490 ng/kg/day) or vehicle infusion by subcutaneous osmotic mini-pumps. After 6-weeks of infusion, the lumbar vertebral bodies (VB) were harvested, and analyzed by high-resolution micro-computed tomography and finite element analysis. In young mice, Ang-II induced HTN caused reductions in bone volume fraction (33.4 ± 0.8 vs 25.4 ± 1.1 %, p+ T cells producing IL-17A was increased in hypertensive vs normotensive young mice (230.0 ± 8.9 vs 167.3 ± 16.6 cells per 1 million events, p=0.0035), as were dendritic cells (408.3 ± 29.1 vs. 229.1 ± 20.5 cells per 1 million events, p+ T cells producing IFN-γ also rose (2845 ± 129 vs 2437 ± 174 cells per 1 million events, p=0.07) in young hypertensive mice. Both normotensive and hypertensive old mice experienced overactive inflammatory responses compared to the young group. Thus, HTN mimics aspects of aging on bone health, causing inflammation in the bone marrow and osteoporosis. Efforts to detect and treat HTN in children may deter untoward events related to bone loss in adulthood.

Introduction: Neutrophil extracellular traps (NETs) consist of decondensed chromatin and proteins including citrullinated histone 3, myeloperoxidase, and neutrophil elastase . Previous studies describe a role of neutrophils and NETs in hypertension and associated end-organ damage. Isolevuglandins (IsoLGs) are products of lipid peroxidation that play a role in immune activation in hypertension. We hypothesized that isoLGs are necessary for the process of NETosis and neutrophil migration in hypertension. Methods: C57BL/6 mice were treated with 490 ng/kg/min angiotensin (Ang) II and vehicle or the isoLG scavenger, 2-hydroxybenzylamine (2HOBA). Single cell sequencing was performed on spleens. NETs were quantified by flow cytometry and immunofluorescence. Human neutrophils were isolated and treated with ionomycin and vehicle or ethyl-2HOBA (Et2HOBA). Neutrophil chromatin expansion was determined by live cell confocal microscopy. Results: Single cell sequencing revealed increased splenic neutrophil accumulation following Ang II treatment. This was attenuated with 2HOBA co-treatment (Ang II 7.78%, Ang II + 2HOBA 2.46%, n=3). Flow cytometry revealed a marked increase in aortic NET accumulation that was reduced with 2HOBA (Sham 304 ± 110.6, Ang II 3,522 ± 928.6, Ang II + 2HOBA 1,007 ± 340.9 NETs, n=6-7). Immunofluorescence staining of kidneys revealed an increase in NET area in Ang II treated mice and a reduction with 2HOBA co-treatment (Sham 75.64 ± 27.2, Ang II 636.9 ± 105.0, Ang II + 2HOBA 14.98 ± 7.5 μm 2 , n=4-6). Ionomycin induced NETosis of isolated human neutrophils was associated with isoLG adduct accumulation that was attenuated with Et2HOBA co-treatment (54.24 ± 11.77% vs 1.78 ± 0.41%, n=5). Live cell confocal microscopy revealed NET area was increased 9.2-fold with ionomycin treatment compared to control. Pretreatment with Et2HOBA attenuated this response. Finally, isoLGs directly disrupt nucleosome assembly in vitro . Conclusion: These findings demonstrate an essential role of isoLGs in NETosis and neutrophil migration in hypertension. Moreover, they suggest a role of isoLG scavengers in the treatment of hypertension and additional NET-associated diseases including atherosclerosis and lupus.

Introduction: Isolevuglandins (isoLGs) are peroxidized lipids that covalently bond lysine residues to modify self-antigens in hypertension. We hypothesized that isoLG-adduction restricts peptide presentation to specific class 1 major histocompatibility complexes (MHC) in mice and humans. Methods and Results: We developed 2 mouse strains expressing either his-tagged class 1 MHC-I H-2D b or H-2K b with truncated membrane binding domains. Shed MHC-I from cultured splenoctyes was adsorbed onto nickel-agarose beads and used to stimulate T cells. We found that CD8+ T cell proliferation is restricted to H-2D b and not H-2K b (Fig 1A) and occurs only if both T-cells and soluble H-2D b are from hypertensive (angiotensin II-treated) mice. Proliferation was blocked if donors received an isoLG-scavenger or by an isoLG specific antibody added during the proliferation assay (Fig 1B). Fluorescence resonance energy transfer (FRET) demonstrated that isoLG-adducts associate with MHC-1 H-2D b and not H-2K b . We transfected HLA-null K562 cells with 18 common human HLA subtypes, and induced isoLG formation with tert-butyl hydroperoxide. Using FRET we identified human HLAs that are high, intermediate and low presenters of isoLG adducts (Fig 1C). Unique modeling software identified candidate peptides and showed that structural characteristics of H-2D b cause lysines at positions 4-7 to project from the MHC groove and present isoLGs (Fig 1D-E). Conclusion: IsoLG adduct presentation is MHC-1 restricted. This may explain differences in the degree of inflammation and end-organ damage observed between populations with hypertension and provides a possible new approach to personalized care.

The National Heart, Lung, and Blood Institute convened a multidisciplinary working group of hypertension researchers on December 6 to 7, 2018, in Bethesda, MD, to share current scientific knowledge in hypertension and to identify barriers to translation of basic into clinical science/trials and implementation of clinical science into clinical care of patients with hypertension. The goals of the working group were (1) to provide an overview of recent discoveries that may be ready for testing in preclinical and clinical studies; (2) to identify gaps in knowledge that impede translation; (3) to highlight the most promising scientific areas in which to pursue translation; (4) to identify key challenges and barriers for moving basic science discoveries into translation, clinical studies, and trials; and (5) to identify roadblocks for effective dissemination and implementation of basic and clinical science in real-world settings. The working group addressed issues that were responsive to many of the objectives of the National Heart, Lung, and Blood Institute Strategic Vision. The working group identified major barriers and opportunities for translating research to improved control of hypertension. This review summarizes the discussion and recommendations of the working group.

Background: The Los Angeles Barbershop Blood Pressure Study (LABBPS) demonstrated that collaboration between barbers and pharmacists delivering hypertension management could significantly improve blood pressure as well as access to care. It is unknown if this model can be translated to other locations. This single-arm, proof-of-concept pilot study tested the development and implementation of a similar protocol in Nashville, TN (NCT04232124). Methods: Between 2019 and 2021, community stakeholders, clinicians, investigators, and the LABBPS team convened to adapt the study design and protocol. Established barbershops with barbers willing to be trained on study procedures were recruited as study sites. Non-Hispanic Black male clients, aged 35-79 years with systolic blood pressure (SBP) > 140 mmHg on two screening days were eligible for participation. Enrolled participants met with a pharmacist for lifestyle and medication management at least once monthly for a six-month period. Barbers measured blood pressure during haircuts for additional monitoring. Results: Eight barbershops in business for an average of 20 (± 5) years participated in the trial. Barbers from each shop (range: 1-4) were trained. A total of 419 clients completed screening visit 1, 82 were eligible and 52 (12%) completed visit 2. We enrolled 36, with 30 completing the initial clinical visit and 27 had complete data at 6-months. Participants were on average age 50 (± 10) years, had a body mass index 33 (± 6), 44% were currently smoking, 52% with high school or less education, and 56% reported current primary care. Baseline BP 157.7±17.1/ 95.1±13.9 mmHg improved to 125.7±11.9/75.6±9.5, a change of -32.1±21.6/ -19.5±14.1, respectively. At 6-months 85% of the group had a BP Conclusion: Implementation of the barber-pharmacist model of hypertension management and care delivery improved BP control among hypertensive Black men in Nashville. Lessons learned regarding adaptation overcoming unforeseen barriers will inform expansion into additional naïve locations for future research.

We have previously reported that depletion Cyclophilin D (CypD), a regulatory subunit of mitochondrial permeability transition pore, improves vascular function and attenuates hypertension, however, specific regulation of CypD in hypertension is not clear. Analysis of human arterioles from hypertensive patients did not reveal alterations in CypD levels but showed 3-fold increase in CypD acetylation. We hypothesized that CypD-K166 acetylation promotes vascular oxidative stress and hypertension, and measures to reduce CypD acetylation can improve vascular function and reduce hypertension. Essential hypertension and animal models of hypertension are linked to inactivation of mitochondrial deacetylase Sirt3 by highly reactive lipid oxidation products, isolevuglandins (isoLGs), and supplementation of mice with mitochondria targeted scavenger of isoLGs, mito2HOBA, improves CypD deacetylation. To test the specific role of CypD-K166 acetylation, we developed CypD-K166R deacetylation mimic mutant mice. Mitochondrial respiration, vascular function and systolic blood pressure in CypD-K166R mice was similar to wild-type C57Bl/6J mice. Meanwhile, angiotensin II-induced hypertension was substantially attenuated in CypD-K166R mice (144 mmHg) compared with wild-type mice (161 mmHg). Angiotensin II infusion in wild-type mice significantly increased mitochondrial superoxide, impaired endothelial dependent relaxation, and reduced the level of endothelial nitric oxide which was prevented in angiotensin II-infused CypD-K166R mice. Hypertension is linked to increased levels of inflammatory cytokines TNFα and IL-17A promoting vascular oxidative stress and end-organ damage. We have tested if CypD-K166R mice are protected from cytokine-induced oxidative stress. Indeed, ex vivo incubation of aorta with the mixture of angiotensin II, TNFα and IL-17A (24 hours) increased mitochondrial superoxide by 2-fold in wild-type aortas which was abrogated in CypD-K166R mice. These data support the pathophysiological role of CypD acetylation in inflammation, oxidative stress and hypertensive end-organ damage. We propose that targeting CypD acetylation may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Isolevuglandins (isoLG) are products of lipid oxidation that covalently ligate self-proteins and contribute to immune activation in hypertension. We hypothesized that these are recognized as “non-self” elicit an antibody response. To determine the presence of anti-isoLG adduct antibodies in humans, immunoblots were performed using endogenous antibodies present in plasma of healthy and hypertensive subjects against human proteins artificially adducted with isoLG. Protein-G HRP was used to visualize the binding of IgG antibodies. We found that humans with hypertension possess IgG antibodies that bind isoLG protein adducts to a greater extent compared to unadducted protein. To identify unique monoclonal antibodies to isoLG adducts C57Bl/6 mice were made hypertensive via angiotensin II infusion for two weeks and then boosted with kidney protein that was adducted with isoLG. Boosted mice were then sacrificed and splenic B-cells were fused with a myeloma cell line. Individual colonies were screened for reactivity with isoLG adducts identifying 11 monoclonal antibody clones with a high specificity for isoLG adducts. Four clones were chosen for further analysis that exhibited 1.5 to 6-fold affinity to adducted vs unadducted mouse proteins. Western blots of normotensive and hypertensive kidney, bone marrow, and heart protein lysate using one of our monoclonal antibodies (clone 2B11) revealed a 90 kDa protein that is increased in bone marrow of hypertensive mice. We conclude that hypertension results in the production of anti-isoLG adduct antibodies in mice and humans. Identification of unique monoclonal antibodies that react with isoLG adducts suggests clonal expansion of anti-isoLG antibody producing cells. Finally, a hypertensive antigen is recognized by the 2B11 clone. Future studies will utilize these antibodies to identify peptide antigens that drive immune activation in hypertension.

Endothelial cells (ECs) activated by hypertensive (10%) cyclical stretch releases factors including IL-6 and hydrogen peroxide that stimulate the conversion of human monocytes to an intermediate inflammatory phenotype. A novel subset of DCs in humans has been identified that express Axl and Sigelc-6 + (AS DCs) which drive T cells proliferation and produce inflammatory cytokines. The interplay between ECs and AS DCs in hypertension is unkown. We assessed AS DCs by flow cytometry in normotensive (n=23) and hypertensive (n=11) subjects and found a significant increase in AS DCs in hypertensive compared to normotensive subjects (297 ± 73 vs. 108 ± 26/ml; p =0.0304). When moncoytes were exposed to human aortic endothelial cells (HAECs) undergoing 10% stretch, the formation of AS DCs was markedly enhanced compared to 5%. The ligand for Axl is growth arrest specific 6 (GAS6), and we found that 10% HAEC stretch caused a 50% increase in the release of GAS6 by ECs comapred to 5%. We knocked down either EC GAS6 or Axl using siRNA and either of these abrogated the ability of ECs to promote AS DC formation. Using flow cytometry to analyze venous ECs that had been harvested from 23 volunteers to quantify EC activation and GAS6 secretion in vivo, we found a positive association between GAS6 and ICAM-1 (R 2 =0.39, p =0.0012). We found a positive association between pulse pressure and plasma GAS6 (R 2 =0.25, p =0.0079) ands systolic blood pressure and GAS6 (R 2 =0.19, p=0.0025) in volunteers. We found that plasma GAS6 is increased in Ang II hypertension and that either genetic deletion or pharmacological inhibition of Axl lowered blood pressure in reposne to Ang II and reduced renal inflammation. To investigate the role of immunological vs. stromal Axl in vivo, we perfomed bone marrow transplant studies and found that both Axl WT/WT ->Axl -/- and Axl -/- ->Axl W/WT had a significant reduction in blood pressure by 20 mmHg compared to the Axl WT/WT -> Axl WT/WT control. These data show that both immunological and stromal Axl contribute to hypertension and inflammation and GAS6/Axl signlaing may be a novel therapeutic target in this disease.

Justification: Hypertension and systemic lupus erythematosus (SLE) share similarities including elevations of blood pressure, proteinuria, inflammation and renal dysfunction and both involve formation of isoLG adducts. We hypothesized that isoLG scavenging would modulate overlapping gene pathways in inflammatory cells in these two conditions. Methods: We performed 10 X genomics single cell sequencing on splenocytes of C57Bl/6 mice with angiotensin II (Ang II)-induced hypertension, Ang II co-treatment with the isoLG scavenger 2HOBA, and 24-week-old B6.SLE123 mice with or without 6-weeks of 2HOBA. Matched C57Bl/6 females were used as controls for the B6.SLE123 mice. Results: Both models exhibited myeloid expansion and genes associated with neutrophil infiltration compared to their respective controls and 2HOBA attenuated this ( Table 1 ). Hypertension was associated with neutrophil expansion whereas SLE was associated an expansion of neutrophils, monocytes, and dendritic cells. In SLE, 2HOBA predominantly modulated gene expression in dendritic cells. Gene ontology revealed 2HOBA downregulated genes governing inflammation including Il1 β (Avglog2(fold change) = -1.6, P adj = 0.002) in both hypertension and SLE. Conclusions: In a mouse model of SLE, scavenging of isoLGs with 2HOBA downregulates inflammatory genes specifically in DCs. In models of both hypertension and SLE, scavenging of isoLG prevents neutrophil expansion. Combined these data describe a shared role of isoLGs in hypertension and SLE and suggest a specific role of DCs and neutrophil activation in the pathogenesis of both conditions. Table 1. Percentage of myeloid derived cells in all groups.

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.

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.

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products isolevuglandins (isoLGs) and that scavenging of mitochondrial isoLG improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isoLG-protein adducts in human patients with essential hypertension and angiotensin II mouse model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isoLG was tested by the novel mitochondria-targeted isoLG scavenger, mito2HOBA. Mitochondrial isoLG in arterioles isolated from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects improved deacetylation of a key mitochondrial antioxidant, superoxide dismutase 2 (SOD2). In human aortic endothelial cells, mito2HOBA diminished mitochondrial superoxide and inhibited cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In angiotensin II-infused mice, mito2HOBA prevented accumulation of mitochondrial isoLG-protein adducts, improved Sirt3 mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in angiotensin II-infused mice. These data support the role of mitochondrial isoLGs in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoLGs may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Hypertension affects approximately one-half of the adult population. Another common disease is osteoporosis, which affects 53 million people in the United States. Clinical studies have shown an association between hypertension and bone loss. We hypothesized that experimental hypertension would be associated with osteoporosis. In Angiotensin (Ang) II-induced hypertension, there is an increase in hematopoietic stem cells (HSCs) expressing the master myeloid transcription factor PU.1 (2.99 ± 0.92) vs. sham (0.17 ± 0.04,p=0.007). Here we reported an increase an osteoclasts number per mm 2 by Tartrate-resistant acid phosphatase staining in DOCA-salt hypertension (27.4 ± 2.3) vs. sham (12.09 ± 1.7, p=0.0002). Using microCT analysis of femoral bone, we found that Ang II-induced hypertension caused striking bone loss as reflected by decreases in cortical bone area, cortical thickness and trabecular number (see Table). We also examined bone strength using a three-point biomechanical testing. In keeping with the micro-CT data, both forms of hypertension were associated with increased bone fragility, reflected by several parameters including maximum force to failure and rigidity (see Table). Recent studies have implicated T cells in the genesis of osteoporosis, and in keeping with this, we found almost two-fold increase in both CD4 + and CD8 + T cells in the marrow of mice with DOCA-salt hypertension. Thus, in two models of experimental hypertension there is marked bone loss and the development of bone fragility, potentially related to T cell accumulation and cytokine release. These findings might explain the association between osteoporosis and hypertension.

Essential hypertension and systemic lupus erythematosus (SLE) are devastating conditions that disproportionately affect women. SLE has heterogeneous manifestations and treatment is limited to the use of non-specific global immunosuppression. Importantly, there is an increased prevalence of hypertension in women with SLE compared to healthy controls. Isolevuglandins (IsoLGs) are oxidation products of fatty acids that form as a result of reactive oxygen species. These molecules adduct covalently to lysine residues of proteins. Adducted proteins are then presented as autoantigens to T-cells resulting in immune cell activation. Previous studies have shown an essential role of IsoLGs in immune cell activation and the development of hypertension in animal models. We hypothesize that isoLGs are important for the development of hypertension and systemic immune activation in SLE. We first examined isoLG adduct accumulation within monocytes of human subjects with SLE compared to healthy controls. By flow cytometry, we found marked accumulation of isoLG adducts within CD14 + monocytes (34.2% ± 12.4% vs 3.81% ± 2.1% of CD14 + , N = 10-11, P B6.SLE123 and NZBWF1 mouse models of SLE. Animals were treated with the isoLG scavenger 2-hydroxybenzylamine (2-HOBA) or vehicle beginning at 7 weeks and were sacrificed at 32 weeks of age. C57BL/6 and NZW were used as controls. Importantly, treatment with 2-HOBA attenuated blood pressure in both mouse models (systolic BP 136.2 ± 5.6 mmHg for B6.SLE123 vs 120.9 ± 4.46 mmHg for B6.SLE123 +2HOBA; 164.7 ± 24.4 mmHg for NZBWF1 vs 136.9 ± 14.9 mmHg for NZBWF1 +2HOBA, N = 6-8, P < 0.05). Moreover, treatment with 2-HOBA reduced albuminuria and renal injury in the B6.SLE123 model (albumin/creatinine ratio 33.8 ± 2.0 x 10 -2 μg/mg for B6.SLE123 vs 5.5 ± 0.9 x 10 -2 μg/mg for B6.SLE123 +2HOBA, N = 7-9, P < 0.05). Finally, immune cell accumulation in primary and secondary lymphoid organs is significantly attenuated by 2-HOBA. These studies suggest a critical role of isoLG adduct accumulation in both systemic immune activation and hypertension in SLE.

We have shown that dendritic cells (DCs) from hypertensive mice convey hypertension when adoptively transferred to recipients. Recently a novel subset of DCs in humans that express Axl and Sigelc-6 + (AS DCs) have been identified which drive T cell proliferation and produce IL-1β, IL-6 and IL-23, consistent with DCs we have observed in hypertension. We hypothesized that AS cells are increased in hypertension and contribute to immune activation in this disease. We quantified circulating AS DCs by flow cytometry in normotensive (n=23) and hypertensive (n=11) subjects and found a more than 2-fold increase in circulating AS DCs in hypertensive compared to normotensive subjects (297 ± 73 vs. 108 ± 26/ml; p =0.0304). To investigate the mechanism by which AS DCs are formed in hypertension, we co-cultured human aortic endothelial cells (HAECs) undergoing either normotensive (5%) or hypertensive (10%) cyclical stretch for 48 hours with CD14 + monocytes from normotensive donors. Co-culture of monocytes with HAECs exposed to 10% stretch significantly increased AS DCs and AS DC IL-1β production when compared to 5% stretch alone as assessed by flow cytometry (21 ± 5 vs. 131 ± 32 IL-1β + AS DCs). Moreover, inhibition of Axl signaling with R248, completely abolished the production of IL-1β in AS DCs (34 ± 8 IL-1β + AS DCs). In additional experiments we found that 10% stretch caused a 50% increase in release of growth arrest 6 (GAS6), the ligand for Axl, from HAECs compared to 5% stretch. Treatment of human monocytes with GAS6 mimicked the effect of 10% stretch in promoting AS cell formation and IL-1β production. Based on the increased secretion of GAS6 from HAECs, we used a J-wire to harvest human endothelial cells from 23 additional volunteers to assess endothelial cell activation and GAS6 secretion in vivo. We found a positive association between pulse pressure and plasma GAS6 (R 2 =0.25, p =0.0079) and a striking positive association between GAS6 and ICAM-1 (R 2 =0.39, p =0.0012). These data show that secretion of GAS6 by an activated endothelial seems to promote the formation and activation of AS DCs. Thus, the interplay between endothelial-derived GAS6 and AS DCs seem to be an important mechanism in human hypertension and might be a novel therapeutic target for this disease.

Apparent treatment-resistant hypertension (ATRH) is highly prevalent and associated with cardiovascular disease (CVD) risk in patients with chronic kidney disease (CKD). We analyzed the association of inflammatory biomarkers with ATRH and its complications in CKD patients. ATRH was defined as blood pressure (BP) ≥140/90 mm Hg while taking ≥3 antihypertensive medications or BP

Hypertension represents a major risk factor for stroke, myocardial infarction, and heart failure and affects 30% of adult population. Vascular dysfunction is crucial in hypertension pathophysiology and exhibits bidirectional relationship. Metabolic disorders and oxidative stress contribute to vascular dysfunction, and mitochondrial deacetylase Sirt3 is critical in the regulation of metabolic and antioxidant functions. We hypothesized that reduced Sirt3 expression contributes to vascular dysfunction and essential hypertension. We studied Sirt3 in arterioles from human mediastinal fat in patients with essential hypertension compared with normotensive subjects. Pathophysiological role of vascular Sirt3 depletion was studied in mice with tamoxifen-inducible endothelial specific Sirt3 depletion (Ec Sirt3KO ). For the first time, we showed a 40% decrease in vascular Sirt3 level and 400% increase in SOD2 acetylation in hypertensive subjects which was linked to increased oxidative stress measured by superoxide overproduction and vascular inflammation due to elevated NfKB activity, VCAM, ICAM and MCP-1 levels. Interestingly, global Sirt3 depletion in mice increases blood pressure, inactivates key mitochondrial antioxidant SOD2 due to acetylation, increases vascular p65, VCAM, ICAM and MCP1, and increased T cell infiltration in kidney, induces cell-senescence and promotes vascular aging while global Sirt3 overexpression prevents these deleterious effects. We tested if Ec Sirt3KO mice have vascular dysfunction and hypertension. Both basal and angiotensin II-induced blood pressure was increased in Ec Sirt3KO mice accompanied by endothelial dysfunction and vascular O 2 • overproduction. Treatment of Ec Sirt3KO mice with SOD2-mimetic mitoTEMPO reduced vascular inflammation, markers of cell-senescence and vascular aging to the wild-type control levels while treatment of Ang II-infused Ec Sirt3KO mice partially rescues from vascular inflammation and attenuates accelerated vascular aging. We suggest that Sirt3 depletion in hypertension promotes both endothelial oxidative stress and metabolic dysfunction. Our data support a therapeutic potential of targeting Sirt3 expression in vascular dysfunction and hypertension.

Humans have had a long and complex relationship with salt. Although highly valued in many societies, dietary salt has long been associated with high blood pressure1–3 and, more recently, with other diseases.4–6 Some individuals with hypertension often display salt-sensitive blood pressure changes, which is a condition more prevalent among blacks, older people, and individuals with renal insufficiency or diabetes mellitus.7–9 In general, for those with salt-sensitive hypertension, excess sodium intake is associated with higher blood pressure, whereas a low-salt diet decreases blood pressure.3 In spite of this well-known association, the basic molecular and cellular mechanisms underlying the effects of salt on blood pressure regulation are still not well understood. Furthermore, individuals with high blood pressure are at increased risk for multiple diseases (ie, coronary artery disease, heart failure, stroke, and renal disease) although at present whether or not a high dietary salt intake can directly lead to these diseases (ie, in the absence of hypertension) is not known. Our understanding of the effect of salt on health has grown even more complex recently. Researchers have reported a new connection between salt and autoimmunity: a high-salt diet was shown to accelerate autoimmune activity in a mouse model of multiple sclerosis.10,11 In addition, a close connection between hypertension and the immune system has been revealed.12–16 However, the causal relationships between salt, immunity, and hypertension (eg, how salt could mediate interactions between the immune system and the vasculature, brain, or kidney to increase blood pressure) are not well understood. The National Heart, Lung, and Blood Institute convened a Working Group (WG) in 2014 to discuss this new emerging scientific area in hypertension research. The WG brought together experts from diverse backgrounds including hypertension, epidemiology, preeclampsia, cardiovascular disease, …

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.

Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1 + ) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1 + progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1 + . Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I + cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1 + progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix.

We have shown that monocyte-derived dendritic cells (DCs) are activated in hypertension to produce large amounts of cytokines and to activate T cells. DCs from hypertensive mice can convey hypertension when adoptively transferred to recipients. Single cell sequencing has recently identified a novel subset of DCs in humans that express Axl and Sigelc6 + (AS DCs). These cells have been reported to potently drive T cell proliferation and to produce large amounts of IL-8 and IL12. The role of AS DCs in hypertension remains unknown. We isolated total peripheral blood mononuclear cells (PBMCs) from normotensive (n=23) and hypertensive (n=12) patients and assessed DC populations, including AS DCs, using flow cytometry. We found a significant increase in the AS DCs in hypertensive compared to normotensive patients (297 ± 73 vs. 108 ± 26/ml; P=0.0304). In contrast, there were no differences in CD1c + DCs (3398 ± 776 vs. 5245 ± 122/ml) or CD141 + DCs (164 ± 20 vs. 218 ± 49/ml) between normotensive and hypertensive subjects. To investigate the mechanism by which AS DCs are formed in hypertension, we used two in vitro hypertensive stimuli: exposure to salt and hypertensive stretch of adjacent human endothelial cells. Human PBMCs were cultured in either normal NaCl (NS, 150 mM) or high NaCl (HS, 190 mM) for 48 hours. Flow cytometry indicated a striking increase in AS DCs by exposure to HS compared to NS (516 ± 181 vs 201 ± 57/ml) and this was prevented by co-treatment of cells with the salt-sensing Serum Glucocorticoid Kinase 1 inhibitor GSK650394. As a second approach, we co-cultured human aortic endothelial cells (HAECs) with PBMCs from normotensive donors and exposed the HAEC monolayer to either normal (5%) or hypertensive cyclical stretch (10%) for 24 hours. Co-culture of PBMCs with HAECs exposed to 10% stretch doubled AS DCs as compared to PBMCs cultured with HAECs undergoing 5% stretch (1.4 ± 0.5 vs 0.7 ± 0.3%; P=0.0217). These data show that AS DC population are increased in hypertensive patients and that known hypertensive stimuli in vitro promote formation of AS DCs. Thus, AS DCs seem to be an important immune cell subset in human hypertension and might be a novel therapeutic target for this disease.

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.

Monocytes play a crucial role in immune activation and the development of hypertension. Deletion of monocytes prevents experimental hypertension, and monocyte derived dendritic cells (DCs) and macrophages promote T cell activation and tissue damage. Our group recently found that monocytes exposed to hypertensive mechanical stretch convert to a pro-inflammatory phenotype denoted by expression CD14 + CD16 + , also known as intermediate monocytes. We aimed to determine whether hypertension has a direct effect on the bone marrow to activate the production and mobilization of monocytes. To study this, we initially analyzed the circulating monocytes from 15 normotensive and 12 hypertensive subjects using flow cytometry and found that hypertensive subjects have an absolute monocytosis compared to normotensives (1014 ± 113 vs. 567.5 ± 97 per uL, p < 0.05). This is associated with high numbers in both the classical CD14 + CD16 - monocytes and the non-classical CD14 low CD16 + monocytes. To further study mechanisms involved, we analyzed monocytes in the blood and bone marrow from C57BL/6 mice following two weeks of sham or Ang II infusion (490 ng/kg/min, s.c.). Similar to the findings in humans, more total monocytes were found in mice with Ang II-induced hypertension (46.7±10.1 vs 18.7±4.7 per μL, p

Salt-sensitive hypertension affects nearly 50% of the population and reducing salt intake decreases blood pressure and cardiovascular events in the general population. The precise mechanism of how dietary salt contributes to blood pressure (BP) elevation, renal injury, and cardiovascular disease remains unclear. Substantial evidence supports a role of increased sympathetic output in salt-dependent hypertension and this promotes renal inflammation and dysfunction. It has been shown that salt accumulates in the interstitium of hypertensive humans and animals and drives immune cells toward a proinflammatory phenotype through the salt sensing kinase serum/glucocorticoid kinase 1 (SGK1). We have also shown an important role of monocytes and monocyte-derived dendritic cells in hypertension. In this study, we tested the hypothesis that SGK1 in myeloid CD11c + cells promote salt-sensitive hypertension by increasing sympathetic outflow. To test this hypothesis, we created mice lacking SGK1 in CD11c + cells (SGK1 CreCD11c mice) and used SGK1 fl/fl mice as controls. To induce salt-sensitivity, mice received 0.5 mg/ml of N-Nitro-L-arginine methyl ester hydrochloride (L-NAME) in the drinking water for 2 weeks. This was followed by a 2-week washout period and then a 4% high salt diet for 3 weeks. BP was monitored using telemetry. We found that BP elevation during high salt feeding was significantly attenuated in SGK1 CreCD11c mice compared to SGK1 fl/fl mice (125 ± 1 vs. 141 ± 1 mmHg; p < 0.01). SGK1 CreCD11c mice had a significant reduction in CD45 + , CD3 + , CD4 + CD8 + and CD19 + cells in the kidney compared to SGK1 fl/fl mice as assessed by flow cytometry (p < 0.05). Interestingly, on high salt, SGK1 CreCD11c mice had a significant reduction in heart rate (HR) compared to SGK1 fl/fl mice (528 ± 2 vs. 592 ± 7 bpm; p = 0.006). Microglial cells of the brain express CD11c, and we found that the reduction in HR was associated with a marked reduction in HR variability in SGK1 CreCD11c mice compared to SGK1 fl/fl mice (1.65 ± 0.38 vs. 3.00 ± 0.57; p = 0.0018), indicating a reduction in sympathetic outflow. Our data indicate that SGK1 in CD11c + myeloid cells and likely those of the central nervous system, modulate BP and sympathetic outflow promoting the pathogenesis of salt-sensitive hypertension.

Reactive oxygen species (ROS) have been shown to be important mediators of hypertension. Isolevuglandins (isoLG) are oxidation products of fatty acids that adduct to lysine residues of proteins. Our prior data suggest that isoLG-adducted proteins are presented by dendritic cells (DCs) and activate T-cells resulting in hypertension. Identification of the source and mechanism of presentation of isoLG adducts is critical for the development of therapies targeting this process. Antigens formed intracellularly are processed by the proteasome and presented to CD8 + T cells in MHC class I (MHC-I). We hypothesize that within DCs, isoLG-adducted peptides are processed by the proteasome and presented in MHC-I. Treatment of mouse DCs with the oxidant tert-butyl hydroperoxide (TBHP) increased levels of isoLG-adducts on the DC surface, as detected by flow cytometry, from 1.3 x 10 3 to 11.4 x 10 3 /10 6 DCs (N = 9, P < 0.01). Co-treatment of mouse DCs with TBHP and proteasome inhibitors (PIs) bortezomib (BTZ) or MG132 attenuated surface isoLG-adduct levels when compared to TBHP alone (2.7 x 10 3 or 3.5 x 10 3 vs 11.4 x 10 3 /10 6 DCs, N = 9, P < 0.001). Moreover, using flow cytometry-based fluorescence resonance energy transfer, we found that isoLG-adducted peptides and MHC-I interact at the intermolecular level on mouse DCs, suggesting that IsoLG-modified peptides are presented within MHC-I. Co-treatment of these cells with TBHP and BTZ attenuated this interaction compared with TBHP treatment alone (2.9 x 10 3 vs 32.7 x 10 3 /10 6 DCs, N = 9, P < 0.001). Next, mice were treated with angiotensin II (ang II) with or without co-treatment with BTZ. Co-treated animals exhibited reduced mean systolic blood pressure (161.7 ± 7.2 vs 122.9 ± 17.9 mmHg, N = 4-5, P < 0.05) and a reduction in renal macrophages (13.4 x 10 3 vs 6.57 x 10 3 DCs/Kidney, N = 4-5, P < 0.001) and DCs (129.4 x 10 3 vs 79.9 x 10 3 DCs/Kidney, N = 4-5, P < 0.05). These studies provide evidence that the proteasome processes isoLG adducts into peptides that are presented within MHC -I, and that proteasome inhibitors can not only block this process, but in doing so, can reduce renal inflammation and lower blood pressure. Efforts to inhibit processing and presentation of isoLG-modified peptides may therefore have therapeutic benefit in hypertension.

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

We and others have shown an important role CD8+ cells in both experimental and human hypertension. CD8+ T cells are activated by antigens presented by major histocompatibility complex 1 (MHC1). C57BL/6 mice express two MHC1, referred to as H2-Kb and H2-Db. To identify antigenic peptides responsible for hypertension, we made two transgenic mice lacking the transmembrane domains of MHC1 and an added His-tag to the modified MHC1. These transgenes are driven by CD11c promoter, allowing expression in antigen presenting cells. The soluble Kb and Db (sKb and sDb) mice received 2 week infusions of sham or angiotensin II and their splenocytes placed in culture for two days. Ni-NTA beads were then used to bind the shed MHC-1 and these beads mixed with 10 6 T cells from other ang II infused mice (Figure panel A). CFSE dilution was used to monitor T cell proliferation. We found that sDb from ang II infused male mice, but not sham infused mice, potently stimulated proliferation of CD8+ T cells from ang II infused male mice. This degree of stimulation was significantly greater than that observed by sKb (Figure panel B). The shed MHC1 from female ang II-treated mice caused significantly less stimulation of female CD8+ T cells compared to male MHC1, in keeping with prior observations that female mice have less immune activation in hypertension. In summary, these data strongly suggest that unique antigens, presented in the context of H2-Db, are generated in hypertension. These data also suggest that there may be unique human lymphocyte antigens (HLAs), the analog of mouse MHC, that predispose to hypertension and related end-organ damage.

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.

Salt-sensitivity is present in 50% of all hypertensive individuals. Prior studies have focused on the roles of kidney, vasculature and sympathetic activity in salt-sensitivity but the contribution of immune cells is poorly understood. We recently found that in murine dendritic cells amiloride sensitive channels sense salt and trigger NADPH oxidase-dependent formation of isolevuglandin-(IsolG)-adducts. We tested the hypothesis that human monocytes exhibit salt-sensitivity leading to activation via IsoLG-adduct formation and this is associated with cardiovascular risk factors. In a cohort of 18 subjects, we found that the sodium intake, measured by 24 hours urine excretion (UNa) was positively correlated with plasma levels of IsolGs. We also measured accumulation of interstitial sodium in 70 subjects by Magnetic Sodium Resonance and evaluated their circulating monocytes by flow cytometry. Subjects with high skin sodium had higher levels of IsoLGs in their monocytes (24±6 vs 38±6 %, pp =0.04). To investigate the ability of monocytes to respond to salt, we cultured monocytes from 17 subjects in high salt environment (HS:190 mM NaCl) or normal media (NS:150mM NaCl) for 48 hours. In culture, 47% of the subjects respond to salt, denoted by an increase of at least 20% in IsoLG formation (NS: 1327±240 vs. HS: 2217±653, p =0.009) as well as increased expression of the activation markers CD83 and CD86. The subjects’ cardiovascular risk factors including pulse pressure, BMI, glucose and total cholesterol positively correlated with the amount of IsolGs produced (ΔHS-NS) in response to salt (r=0.51 p

Several studies have established a relationship between hypertension and salt intake; however the mechanisms by which salt causes hypertension are poorly understood. There is also evidence that sodium (Na+) accumulates in the interstitium with aging and hypertension in concentrations exceeding the plasma. We tested the hypothesis that increased NaCl would convert human monocytes to an inflammatory phenotype and to define mechanisms involved. We exposed monocytes from 17 human volunteers to normal physiological NaCl (NS: 150 mM/L), elevated NaCl (HS: 190 mM/L), or an equiosmoloar concentration of mannitol. Exposure of human monocytes to high salt, but not mannitol, increased formation of immunogenic isolevuglandins (isoLG) (NS: 1688±384 vs Mann:1762±429 vs HS: 2381± 635 MFI p

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.

Introduction: Hypertension, a common condition that affects 33% of the US population, is a major risk factor for heart disease and stroke. Treatments for hypertension are limited and there is a critical need to develop new therapies. The Renin Angiotensin Aldosterone System (RAAS) plays a key role in regulating blood pressure, and renin controls its rate-limiting step. In adults, renin is produced and stored by renal Juxtaglomerular (JG) cells. During sodium restriction and other pathophysiological stresses that require an increase in renin expression and release, the adult kidney increases the number of cells expressing renin, in a process known as JG cell expansion. JG cells formation mechanisms remain unclear. Our aim is to determine new regulators of renin and blood pressure control. Methods: In vivo: JG cell expansion was induced by 10 days of low sodium diet (0.01% Na) and furosemide (0.1 mg/g body weight). In vitro: primary renal Mesenchymal Stromal Cells (MSC) were isolated from wild type mice and used until passage 5. MSC were differentiated into renin expressing cells by 7 days I&F (IBMX-100 uM and Forskolin-10 uM) treatment. Sox6 was down regulated in MSCs with lentivirus carrying vectors for Sox6 shRNA or controls scramble shRNA. Results: In vitro, I&F induced renin expression in MSCs (Renin relative expression to gapdh 0.0153±0.005, n=3, P= 0.05, no renin expression in MSCs). Gene arrays comparing renal MSCs and JG cells identified Sox6 as a potential gene that controlled MSC differentiation. In vitro silencing of Sox6 by shRNA inhibited the differentiation of renal MSCs into renin producing cells (3.5 fold decrease compared to control shRNA, n= 4, P= 0.01). In a new transgenic mouse model, in which Sox6 is deleted specifically in renin expressing cells (Ren1d Cre /Sox6 fl/fl ), plasma renin concentration was not affected at baseline (0.3 vs W.T. 0.5 ug Ang I/ml/h, n=3, P>0.05). However, under conditions that promote JG cell expansion, Sox6 ablation halted the increase in the number of JG cells (8.75 fold decrease, n= 6 to 9, P= 0.001). Conclusion: Our data indicates that Sox6 plays a novel role in renal physiology; modulating renin expression during pathological conditions. These findings suggest that Sox6 may become a new target for blood pressure control.

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.

Reactive oxygen species and the NADPH oxidases contribute to hypertension via mechanisms that remain undefined. Reactive oxygen species produced in the central nervous system have been proposed to promote sympathetic outflow, inflammation, and hypertension, but the contribution of the NADPH oxidases to these processes in chronic hypertension is uncertain. We therefore sought to identify how NADPH oxidases in the subfornical organ (SFO) of the brain regulate blood pressure and vascular inflammation during sustained hypertension. We produced mice with loxP sites flanking the coding region of the NADPH oxidase docking subunit p22 phox . SFO-targeted injections of an adenovirus encoding cre-recombinase markedly diminished p22 phox , Nox2, and Nox4 mRNA in the SFO, as compared with a control adenovirus encoding red-fluorescent protein injection. Increased superoxide production in the SFO by chronic angiotensin II infusion (490 ng/kg min –1 ×2 weeks) was blunted in adenovirus encoding cre-recombinase–treated mice, as detected by dihydroethidium fluorescence. Deletion of p22 phox in the SFO eliminated the hypertensive response observed at 2 weeks of angiotensin II infusion compared with control adenovirus encoding red-fluorescent protein-treated mice (mean arterial pressures=97±15 versus 154±6 mm Hg, respectively; P =0.0001). Angiotensin II infusion also promoted marked vascular inflammation, as characterized by accumulation of activated T-cells and other leukocytes, and this was prevented by deletion of the SFO p22 phox . These experiments definitively identify the NADPH oxidases in the SFO as a critical determinant of the blood pressure and vascular inflammatory responses to chronic angiotensin II, and further support a role of reactive oxygen species in central nervous system signaling 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.

High salt intake and inflammation are implicated in the genesis of hypertension. Recently it has become clear that sodium can accumulate in the interstitial space in concentrations exceeding that of the plasma, and that these high salt (HS) concentrations can be pro-inflammatory. Our laboratory recently published a new pathway in which increased oxidative stress in dendritic cells (DCs) leads to formation of isoketal-modified proteins which act as neo-antigens to activate T cells. We hypothesized that increasing sodium chloride (NaCl) in excess activates antigen presenting cells via formation of immunogenic isoketals. We exposed monocytes from human volunteers to normal physiological NaCl (NS: 150 mM/L), elevated NaCl concentrations (HS: 190 mM/L), or an equiosmoloar concentration of mannitol. We found that exposure of human monocytes to high salt, but not mannitol, caused a 2-fold increase in formation of isoketal-modified proteins. This was associated with an increase in activation marker CD86 (NS: 466 ± 192 vs HS: 596 ± 324 MFI p

We have shown that dendritic cells (DCs) from hypertensive mice and monocytes in humans accumulate highly reactive isolevuloglandins (isoLGs) or isoketals that adduct to protein lysines and promote T cell activation, specifically. Monocytes that traverse the endothelium have three different fates: reemerge into circulation as an activated monocyte; differentiate into macrophages or into monocyte-derived DCs. We hypothesized that human endothelial cells exposed to hypertensive mechanical stretch will promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive uniaxial cyclical stretch (10%) for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and the p22 phox subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also accumulated isoLG-modified proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively, p = 0.0001) as evidenced by intracellular staining and flow cytometry. We also co-cultured these monocytes with T cells from the same patient and examined proliferation of the latter using CFSE. Monocytes co-cultured with 10% stretched HAECs induced a 1,500-fold increase in CD4 + T cell proliferation and a 1,300-fold increase in CD8 + T cells proliferation. In addition, monocytes co-cultured with 10% stretched HAECs had a 2-fold increase in phosphorylated STAT3 expression compared to 5% stretch cultures. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, reduced their differentiation into DCs and prevented both CD4 + and CD8 + T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote differentiation into activated DCs potentially via the STAT3 pathway.

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.

Hypertension afflicts 33% of the U.S. adult population. Despite current treatments, approximately 50% of people are unresponsive to treatment. There is a critical need to develop new therapies to treat this disease and its complications. The Renin Angiotensin Aldosterone System plays a key role in regulating blood pressure in humans. Renin controls the rate-limiting step in the conversion of angiotensinogen to angiotensin I. In adults, renin is produced and stored by Juxtaglomerular (JG) cells in the kidney. However, the transcriptional mechanisms that govern formation of renin expressing cells under normal or pathophysiological conditions remain poorly understood. During blood pressure changes the number of adult renal cells expressing renin increase through a process known as JG cell expansion. We found that this process involves differentiation of mesenchymal stromal-like cells (MSC) to renin expressing cells among others. We aim to determine new regulators of renin expression and blood pressure control. Renin expression in vitro was induced by treatment of MSCs with 3-isobutyl-1-methylxanthine (IBMX) and Forskolin. MSCs were transduced with lentivirus carrying vectors Sox6 shRNA or control shRNA. A new transgenic mouse, in which Sox6 is deleted specifically in renin expressing cells (Ren1dCre/Sox6 fl/fl ), was used to study the impact of Sox6 in renin expression in vivo . Gene array comparing renal MSC and JG cells identified potential genes that control MSC differentiation, including Sox6. In vitro silencing of Sox6 by shRNA decreased differentiation of renal MSCs into renin producing cells (3.5 fold, n=4, P= 0.01). Preliminary IHC data showed that the transcription factor Sox6 is expressed by renin producing cells in the adult kidney during JG cell expansion. Plasma renin concentration (PRC) increased during JG cell expansion (induced with low sodium diet and furosemide) in wild type mice (27-fold), whereas in mice lacking Sox6 in JG cells PRC was as low as non-treated mice. We conclude that Sox6 has a novel role in modulating renin expression and thereby can contribute to renal physiology, opening new possibilities of addressing questions regarding physiological regulation of renin and hypertension.

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 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.

We have recently shown that CD70 is important in formation of effector memory T cells and that these cells seem to enhance the hypertensive response and end organ damage caused by repeated hypertensive stimuli. CD70 has traditionally been identified on antigen presenting cells and is thought to interact with CD27 on T cells, promoting proliferation and memory cell formation. The precise cell types that express CD70 and the role of CD70 on non-immune cells has not previously been investigated. In our prior study, we showed that hypertension is associated with a marked increase in CD70 surface expression on dendritic cells (DCs) and macrophages. To examine the specific effect of DC CD70, we performed adoptive transfer of DCs from hypertensive WT mice to normotensive CD70 -/- mice and from hypertensive CD70 -/- mice to normotensive WT mice. The recipient mice were treated with a normally subpressor dose of ang II (140 ng/kg/min). Interestingly the CD70 -/- recipients were protected from the development of hypertension, despite receiving WT DCs from hypertensive donors. In contrast, the WT recipeints that received CD70 -/- DCs developed modest hypertension, indicating that non-DC sources of CD70 likely contribute to the hypertensive phenotype. Studies of mesenteric vascular reactivity showed that CD70 -/- mice have markedly impaired endothelium-dependent vasodilatation to acetylcholine compared to WT mice (43±10 vs 25 ± 3 %) at baseline. In contrast, there were no differences in relaxation responses to sodium nitroprusside. In additional experiments, we showed that human umbillical vein endothelial cells express CD70 mRNA and that this is increased by > 30 fold by laminar shear stress (15 dynes/cm2) compared to oscillatory shear. Finally, using immunohistochemistry, we identified CD70 protein localized to resistance vessels of the kidney of ang II-treated mice. These data identify a new role of CD70 in modulating vascular function independent of its role on antigen presenting cells in memory T cell formation.

Adaptive immunity and especially T lymphocytes play a crucial role in the development of hypertension. Proteins that are oxidatively modified by highly reactive isolevuglandins (isoLGs) accumulate in dendritic cells & lead to subsequent activation of T lymphocytes. This process can be prevented by compounds known to scavenge isoLGs, such as 2-hydroxybenzylamine (2-HOBA). The overall goal of current study is to develop novel antihypertensive drugs based on this isoLG scavenging strategy, that will not only prevent but will also reverse hypertension and its associated inflammatory end-organ damage. Initially, the preventative effects of 10 putative isoLG scavengers were tested. C57Bl/6 mice received angiotensin (Ang) II (490 ng/kg/min) infusion for 14 days. Each compound was administered in the drinking water (1mg/ml) at the onset of Ang II infusion for 7 days to a half of the mice & for entire 14 day period to another half of the animals. Blood pressure was measured by tail cuff method. Among 10 compounds, we found that 2-HOBA, methyl 2HOBA (Me2HOBA) & 3-methoxy2-HOBA (3-Mo2HOBA) were most effective in lowering blood pressure. Interestingly, the pyridoxamine analogs were not effectively lowered blood pressure. We further examined the efficacy of our 3 most effective compounds in reversing established hypertension. Mice received Ang II infusion for 6 weeks and received each study drug (2 mg/ml) during the last 4 weeks. We employed radiotelemetry to monitor blood pressure. Compared with untreated mice, Me2-HOBA, 3-Mo2-HOBA & 2-HOBA were equally effective in lowering blood pressure by 20 mmHg (all p < 0.05 vs no drug). Six weeks of Ang II infusion caused 2- to 4-fold increases in renal T cell (CD3 + ) & monocyte/macrophage (F4/80 + ) infiltration as measured by immunohistochemistry & all three compounds markedly reduced these by 50%. These treatments also reduced aortic fibrosis as measured by Masson’s Trichrome blue staining. We conclude that these isoLG scavengers can be potentially used as new effective therapy for lowering blood pressure & attenuating hypertensive renal & vascular damage. Variability of in vivo effectiveness for the different scavengers likely reflects differences in bioavailability due to structure & lypophilicity.

Hypertension, defined as a condition associated with ≥140-mm Hg systolic blood pressure or ≥90-mm Hg diastolic blood pressure, affects >1 billion people worldwide,1 and in 2009 it cost the US healthcare system more than $73 billion.2 Despite the availability of many antihypertensive therapies, individual responses vary, and efficacy remains a concern. Current treatments have yielded only modest reductions in the overall disease risk even in countries where therapeutics are available and affordable. The initiating causes and the pathogenic mechanisms for disease and its comorbidities remain largely unknown, and prognostic markers for adult hypertension that could improve its diagnosis, prevention, and, ultimately, its management are not yet available. As a result, ≈28% of the US population and a similar proportion of the adult Western European and Canadian populations experience what is known as “essential hypertension,”3 which is a primary component of several complex, multifactorial, multigenic conditions that are commonly associated with high levels of morbidity and mortality from diabetes mellitus, cardiovascular disease, and renal disease. If the current rise in the number of hypertension cases is not abated, total annual global healthcare costs resulting from suboptimal blood pressure for those >30 years of age could amount to $3.6 trillion more over the next 10 years.4 The causes of essential hypertension remain poorly understood, although the complex mechanisms for blood pressure (BP) regulation have been extensively characterized in both humans and animal models.5–7 Comprehensive study of >12 inbred rat strains that recapitulate many aspects of hereditary human hypertension has added substantially throughout the years to our understanding of the underlying physiological and molecular pathways, the genetic complexity of risk and treatment responses, and the pathological consequences of hypertension.8 In fact, many interventions that lead to a …