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Evidence from nonhuman animal models demonstrates an important role for immune cells in hypertension, but immune cell changes in human hypertension are less clear. Using mass cytometry, we demonstrate novel and selective reductions in CCR10(+) regulatory T cells (Tregs) and PD-1(+)CD57(−)CD8(+) memory T cells. RNA sequencing reveals that CCR10(+) Tregs exhibit gene expression changes consistent with enhanced immunosuppressive function. In addition, CITE-Seq demonstrates that PD-1(+)CD57(−)CD8(+) memory T cells exhibit features of T-cell exhaustion. Taken together, these results provide novel evidence for decreases in anti-inflammatory and/or hypofunctional T-cell populations that may contribute to enhanced inflammation in human hypertension.

SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism inWe used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of thisTrp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Background: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. Methods: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. Results: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8 + T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8 + T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8 + T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. Conclusions: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

OBJECTIVE: Chronic inflammation contributes to the high burden of cardiovascular disease (CVD) in persons with HIV (PWH). HIV has broad effects on innate and adaptive immune cells, including innate lymphoid cells (ILCs) and CD4(+) T-helper cells. At present, the relationship between CVD and plasma cytokines reflecting ILC/T-helper responses in PWH is not well defined. We investigated relationships between plasma cytokines and subclinical atherosclerosis. DESIGN: Cross-sectional study. METHODS: We recruited 70 PWH on a single antiretroviral regimen (efavirenz, tenofovir, and emtricitabine) with at least 12 months of suppressed viremia and 30 HIV-negative controls. We quantified plasma cytokines and chemokines including interferon-γ, interleukin (IL)-4, IL-13, and IL-17A, markers of macrophage activation, and endothelial activation using multiplex assays and ELISA. Cytokines were grouped using Ward’s hierarchical clustering. Brachial artery flow-mediated dilation (FMD) and carotid plaque burden were determined using ultrasound. Multivariable linear regression and negative binomial regression analyses were used to assess the relationships of plasma biomarkers and endpoints adjusted for CVD risk factors. RESULTS: We identified three distinct clusters in PWH, one containing Th1/Th2/ILC1/ILC2 type cytokines, one with Th17/ILC3/macrophage-related cytokines, and a less specific third cluster. Lower FMD was associated with higher plasma IL-17A and macrophage inflammatory protein-1α. In contrast, IL-4, a Th2/ILC2 type cytokine, was associated with carotid plaque. When HIV-negative controls were added to the models clustering was more diffuse, and these associations were attenuated or absent. CONCLUSIONS: Th17/ILC3 and Th2/ILC2-mediated immune mechanisms may have distinct roles in endothelial dysfunction and atherosclerotic plaque formation, respectively, in PWH.

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome where patients, despite having a normal left ventricular systolic function, present with symptoms of volume overload. Recent data have shown that HFpEF is associated with elevation of inflammatory biomarkers. In a well-known rodent hypertensive model (DOCA-salt), we isolated CD45+ leukocytes from the heart and then performed CITE-Seq, a novel technique to obtain transcriptomic and surface marker expression on single cells, on a total of 4,359 and 7,600 cells from four sham and four DOCA-salt left ventricles, respectively. Analysis showed significant differential gene expression in the myeloid (macrophage/monocytic) population. We then took the top 20 genes that were differentially expressed between DOCA-salt vs sham treated myeloid cells and performed a genetic analysis called PrediXcan in Vanderbilt’s DNA databank, BioVU. Our analysis used gene expression prediction models built from the GTEx Project and tested its association with the HFpEF phenotype, which was derived on ICD-9 and 10 codes and natural language programming. From BioVU, 88,660 subjects were included in the association analysis. Of the 20 genes, 8 did not meet prediction model criteria for PrediXcan. In the remaining 12 genes, genetically predicted expression of only 2 ( Lgals1 and Ctsl ) are associated with a HFpEF phenotype. Of these, the gene encoding galectin 1, Lgals1 , had the lowest p-value (0.02) and highest beta coefficient (0.32) corresponding to an odds ratio for HFpEF of 1.38. Galectin-1 is a well known mediator of inflammation resolution in infection and tumor biology, however its role in heart failure is unknown. We are currently exploring the role of galectin-1 in the pathophysiology of HFpEF through Lgals1 -/- and LysM-Cre x Lgals1 fl/fl mice. In conclusion, using two different approaches in mice and humans, we identified galectin-1 as a new potential mediator in HFpEF development.

Objective: Persons with HIV have double the risk of developing cardiovascular disease compared with the general population. A persistent and heightened immune response to cytomegalovirus coinfection may be one contributing factor, but the relationship between cytomegalovirus replication, virus-specific immune cells, and plaque burden is unclear. Approach and Results: We assessed the relationship between CD4 + T-cell subsets and carotid plaque burden in a cohort of 70 HIV-positive participants with sustained viral suppression on a single antiretroviral regimen and without known cardiovascular disease. We evaluated relationships between immune parameters, carotid plaque burden, and brachial artery flow-mediated vasodilation using multivariable linear and logistic regression models. We found that participants with carotid plaque had increased circulating CX3CR1 + ~GPR56 + ~CD57 + (ie, C~G~C) + CD4 + T cells ( P =0.03), which is a marker combination associated with antiviral and cytotoxic responses. In addition, a median of 14.4% (IQR, 4.7%–32.7%) of the C~G~C + CD4 + T-cells expressed antigen receptors that recognized a single cytomegalovirus glycoprotein-B epitope. Using immunofluorescence staining, we found that CX3CR1 + CD4 + T cells were present in coronary plaque from deceased HIV-positive persons. C~G~C + CD4 + T cells were also present in cells isolated from the aorta of HIV-negative donors. Conclusions: HIV-positive persons with carotid atheroma have a higher proportion of circulating CD4 + T-cells expressing the C~G~C surface marker combination associated with antiviral and cytotoxic responses. These cells can be cytomegalovirus-specific and are also present in the aorta.

Visual Abstract, Highlights • Mentees are more satisfied with their mentorship experience when they have had more than 3 mentors or a mentor from outside of their practice/institution. • Satisfaction with the mentoring relationship is significantly associated with perceived satisfaction in achieving professional goals. • Sex and race/ethnicity concordance in mentoring relationships is associated with positive outcomes. • Characteristics that mentees desire in a mentor tend to change with time/career stage., Summary The effects of mentorship on measurable outcomes of success and the aspects of mentorship that are most valuable in promoting the careers of cardiologists are unclear. To address this, we conducted a large-scale survey of cardiologists in a real-world setting. We identified factors that enhance the mentorship experience, and found that mentee needs change with career stage. Importantly, satisfaction with the mentoring relationship is significantly associated with perceived satisfaction in achieving professional goals. Furthermore, we found that gender and race concordance in mentoring relationships is an important variable with the potential to increase diversity in the field of cardiology.

Extracellular fluid (ECF) potassium concentration ([K+]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K+] in male C57BL/6J mice fed a K+-deficient diet (0K) versus 1% K+ diet (1K) for 10 days ( n = 5–6/group). During 0K feeding, plasma [K+] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K+ (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na+ (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na+] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-β2 subunits, decreased K+-Cl− cotransporter isoform 3, and increased phosphorylated (p) Na+,K+,2Cl− cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K+ loss and Na+ gain. Renal transporters’ adaptations, effecting a 98% reduction in K+ excretion, included two- to threefold increased phosphorylated Na+-Cl− cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na+ delivery to epithelial Na+ channels where Na+ reabsorption drives K+ secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 μmol K+ into the urine and muscle shifts ~47 μmol K+ from ICF to ECF, illustrating the importance of the concerted responses during K+ deficiency.

Aims Elevated serum immunoglobulins have been associated with experimental and human hypertension for decades but whether immunoglobulins and B cells play a causal role in hypertension pathology is unclear. In this study, we sought to determine the role of B cells and high-affinity class-switched immunoglobulins on hypertension and hypertensive end-organ damage to determine if they might represent viable therapeutic targets for this disease. Methods and results We purified serum IgG from mice exposed to vehicle or angiotensin (Ang) II to induce hypertension and adoptively transferred these to wild type (WT) recipient mice receiving a subpressor dose of Ang II. We found that transfer of IgG from hypertensive animals leads does not affect blood pressure, endothelial function, renal inflammation, albuminuria, or T cell derived cytokine production compared with transfer of IgG from vehicle infused animals. As an alternative approach to investigate the role of high affinity, class-switched immunoglobulins, we studied mice with genetic deletion of activation-induced deaminase (Aicda-/-). These mice have elevated levels of IgM but virtual absence of class-switched immunoglobulins such as IgG subclasses and IgA. Neither male nor female Aicda-/- mice were protected from Ang II-induced hypertension and renal/vascular damage. To determine if IgM or non-immunoglobulin dependent innate functions of B cells play a role in hypertension, we studied mice with severe global B cell deficiency due to deletion of the membrane exon of the IgM heavy chain (μMT-/-). μMT-/- mice were also not protected from hypertension or end-organ damage induced by Ang II infusion or deoxycorticosterone acetate (DOCA)-salt treatment. Conclusions These results suggest that B cells and serum immunoglobulins do not play a causal role in hypertension pathology. Translational perspective Our results suggest that in most cases of essential hypertension, B cells are not a causal factor in the pathophysiology of disease. Thus, elevated serum immunoglobulins seen in hypertensive animals and humans may reflect a biomarker of aberrant immune activation in hypertension and not a therapeutic target. However, autoantibodies may cause hypertension in special cases, and more work is needed to determine whether specific B cell subsets might play an important role that is masked by global B cell deficiency.

Diet-induced obesity is associated with an increased risk of developing cardiovascular disease (CVD) . Aortic damage and stiffness are critical risk factors for CVD progression, especially with chronic high fat diet (HFD). Mice fed HFD ad libitum have increased food intake during the inactive period. We previously showed that HFD leads to significantly reduced blood pressure (BP) dipping and that time restricted food availability to the active period (TRF) in the final 2 weeks of HFD restores BP dipping. Thus, we hypothesized that TRF will reduce aortic damage and stiffness in chronic HFD mice. Utilizing this same mouse model of ad libitum chronic HFD (20 weeks 45% fat diet, male C57Bl6/J mice) compared to normal fat diet (NFD, 10% fat) and TRF intervention (food during lights off active period only from weeks 18-20), we determined aortic fibrosis and stiffness. By histological staining and quantitative analysis of the fibrosis area, HFD+TRF mice had significantly reduced fibrosis compared to HFD mice (% fibrosis per area, NFD: 143, NFD+TRF: 132, HFD: 175, HFD+TRF: 105, 2-way ANOVA, TRF effect: p=0.02). Aortic stiffness was measured by pulse wave velocity (PWV, Vevo 3100). We found that HFD fed mice have a significant increase in PWV compared to NFD group with no significant difference in the HFD+TRF and NFD+TRF groups compared to the NFD group (PWV m/sec, NFD: 1.60, NFD+TRF: 1.5, HFD: 2.30, HFD+TRF: 1.70, 2-way ANOVA, diet effect: p=0.02). We further analyzed plasma sE-selectin (endothelial-specific damage marker) and 8-isoprostane (oxidative stress marker) in HFD and HFD+TRF mice. Plasma from HFD+TRF mice showed significantly reduced sE-selectin and 8-isoprostane compared to HFD mice (sE-selectin ng/ml, HFD: 3.50, HFD+TRF: 1.85, p=0.027; 8-isoprostane ng/ml, HFD: 159.1, HFD+TRF: 59.6, p=0.008). Thus, TRF in chronic HFD mice leads to reduced endothelial injury and oxidative stress that is correlated with reduced aortic fibrosis and stiffness compared to ad libitum HFD mice. This study highlights that restricting the time of food intake in a model of diet-induced obesity minimizes CVD risk by reducing aortic damage and oxidative stress.

Circadian rhythms govern our daily physiological processes. However, disruption of circadian rhythms, as can occur with ad libitum Western diets, disrupt these processes leading to cardiometabolic diseases. Our lab and others have shown that Th17 cells, which produce interleukin 17A (IL-17A), are implicated in the development of cardiovascular and renal end-organ damage associated with high fat and/or high salt diets. Th17 cell differentiation and trafficking is regulated by the circadian clock and influenced by light-dark cycles. However, whether feeding-fasting rhythms influence Th17 cell responses is poorly understood. We tested the hypothesis that limiting food intake to the 12-hr active period (time-restricted feeding, TRF) mitigates high fat and high salt (HF/HS) diet induced T cell IL-17A production and target organ damage. Beginning at 8 weeks of age, male C57Bl/6J mice were placed on either a normal chow/normal salt (NC/NS) or a HF/HS diet for 20 weeks, with TRF intervention occurring during the last two weeks in the HF/HS + TRF group. Body weight was similarly significantly increased in the HF/HS and HF/HS + TRF groups in comparison to the NC/NS group. Th17 cells were significantly increased (2.6-fold increase, p = 0.02) in the Peyer’s patches (lymphoid aggregates found in the small intestines) of mice on HF/HS diet in comparison to those on NC/NS. Importantly, TRF abolished this increase. Renal CD4 + T cell IL-17A production, as measured by flow cytometry, was increased by HF/HS diet compared to NC/NS (3-fold increase, p = 0.02). Similarly, TRF abolished this increase. This study highlights how Western diets exacerbate intestinal and renal IL-17A production and the potential beneficial impact of a behavioral intervention, TRF, to mitigate the Th17 mediated inflammation associated with diet-induced obesity.

Hypertension, or an elevated blood pressure, is the primary modifiable risk factor for cardiovascular disease, the number one cause of mortality worldwide. We previously demonstrated that Th17 activation and interleukin 17A (IL-17A)/IL-21 production is integral for the full development of a hypertensive phenotype as well as the renal and vascular damage associated with hypertension. Rho-associated coiled-coil containing protein Kinase 2 (ROCK2) serves as a molecular switch upregulating Th17 and inhibiting regulatory T cell (Treg) differentiation. We hypothesize that hypertension is characterized by excessive T cell ROCK2 activation leading to increased Th17/Treg ratios and ultimately end-organ damage. We first showed in vitro that KD025, an experimental orally bioavailable ROCK2 inhibitor inhibits Th17 cell proliferation and IL-17A/IL-21 production. To determine if hypertensive stimuli such as endothelial stretch increases T cell ROCK2 expression, we cultured human aortic endothelial cells exposed to 5% (normotensive) or 10% (hypertensive) stretch with circulating human T cells and HLA-DR+ antigen presenting cells. Hypertensive stretch increased T cell ROCK2 expression 2-fold. We then tested the effect of ROCK2 inhibition with KD025 (50mg/kg i.p. daily) in vivo on angiotensin II (Ang II)-induced hypertension. Treatment with KD025 significantly attenuated the hypertensive response within 1 week of Ang II treatment (systolic blood pressure: 139± 8 vs 108±7mmHg) and this persisted for the duration of the 4 week study reaching blood pressures 20 mmHg lower (135±13mmHg) than vehicle treated mice (158±4mmHg p

Interleukin-33 (IL-33) is an alarmin critical for alternative macrophage activation and has been linked to hypertension in humans. The goal of this study was to evaluate whether IL-33 contributes to (deoxycorticosterone acetate) DOCA-salt induced cardiac remodeling and macrophage polarization. C57Bl/6J (WT, n=8) and IL33-deficient (IL-33 KO, n=8) mice underwent uninephrectomy, implantation of a DOCA pellet, and 1% NaCl supplementation of drinking water for three weeks. A control group of WT mice (n=10) underwent a sham procedure and were maintained on normal water. Systolic blood pressure measured by tail cuff showed a significant increase in WT DOCA-salt mice (128±9 mmHg; mean±SD) and IL-33 KO DOCA-salt mice (129±11) compared to WT sham (103±11). Hypertension was accompanied by significantly increased heart to body weight ratios in WT DOCA-salt (7.0±1.0 mg/g) and IL-33 KO DOCA-salt (6.8±0.6) compared to WT sham (5.3±0.3) mice. Glomerular injury was assessed by urine albumin-to-creatinine ratios, which were similarly increased in WT DOCA-salt (0.92±0.4) and IL-33 KO DOCA-salt (0.42±0.09) mice compared to WT controls (0.03±0.003). Echocardiography was performed to assess left ventricle (LV) dimensions and systolic function. IL-33 KO DOCA-salt mice exhibited a significantly higher cardiac output (22±2 mL/min) compared to WT sham (16±3) and WT DOCA-salt (15±3) mice, driven by increased stroke volume. No significant changes in heart rate or ejection fraction were observed between groups. Additionally, IL33-KO DOCA-salt mice had an increased diastolic LV internal dimension (3.2±0.2 mm) compared to WT sham (2.7±0.3) and WT DOCA-salt (2.7±0.3). DOCA-salt induced increased mRNA expression of alternative macrophage activation markers in both LV ( Chil3, Il10) and isolated peritoneal macrophages ( Arg1, Retnla, Tgfb1 ). Peritoneal macrophages deficient in IL-33 expressed less Il10 compared to WT. Cardiac fibrosis evaluated by Masson’s trichrome showed a significant increase in LV collagen deposition in WT DOCA-salt mice (1.09±0.8% trichrome positive) compared to WT sham (0.43±0.3) but no increase in IL-33 KO DOCA-salt mice (0.54±0.3). In conclusion, IL-33 deficiency alters macrophage and cardiac function in the DOCA-salt model of hypertension.

Emerging evidence from animal models has demonstrated the importance of multiple innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets are altered in human hypertension. To test this, we performed high dimensional single cell profiling of human peripheral blood mononuclear cells using mass cytometry. Unsupervised computational analysis revealed a 40% decrease in CD8 + memory T cells in hypertensive individuals. Using Phenograph to identify subsets of these cells revealed a selective 60% decrease in PD-1 + CD8 + memory T cells in hypertension. This observation was confirmed in a validation cohort using flow cytometry in which PD-1 + CD8 + memory T cells were significantly decreased 44% in hypertensive compared to control individuals. To determine the phenotype of these PD-1 + CD8 + memory T cells, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) on four control and four hypertensive individuals. Using antibodies to identify PD-1 + and PD-1 - CD8 + memory T cells, gene set enrichment analysis of the coordinate single cell transcriptomic data revealed that PD-1 + cells exhibit over-representation of features of both immunologically active effector T cells and hypofunctional exhausted T cells. Thus, clustering analysis of PD-1 + CD8 + memory T cells was performed which demonstrated 4 distinct subclusters. One of these subclusters was decreased in hypertension and exhibited selective expression of multiple inhibitory receptors characteristic of exhausted T cells. At the protein level, this subcluster was marked by expression of the inhibitory receptor LAG3 and low levels of CD57. Combining these markers to identify PD-1 + LAG3 + CD57 - CD8 + memory T cells permitted identification of exhausted cells which demonstrated a significant 35% decrease in hypertensive compared to control individuals using flow cytometry. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1 + CD8 + memory T cells with features of exhaustion in human hypertension. These findings provide new insights into the pathogenesis of human hypertension including loss and/or re-invigoration of exhausted T cells.

Calcific aortic valve disease (CAVD) is highly prevalent and has no pharmaceutical treatment. Surgical replacement of the aortic valve has proved effective in advanced disease but is costly, time limited, and in many cases not optimal for elderly patients. This has driven an increasing interest in noninvasive therapies for patients with CAVD. Adaptive immune cell signaling in the aortic valve has shown potential as a target for such a therapy. Up to 15% of cells in the healthy aortic valve are hematopoietic in origin, and these cells, which include macrophages, T lymphocytes, and B lymphocytes, are increased further in calcified specimens. Additionally, cytokine signaling has been shown to play a causative role in aortic valve calcification both in vitro and in vivo. This review summarizes the physiological presence of hematopoietic cells in the valve, innate and adaptive immune cell infiltration in disease states, and the cytokine signaling pathways that play a significant role in CAVD pathophysiology and may prove to be pharmaceutical targets for this disease in the near future.

Genetic variants at SH2B3 are associated with blood pressure and circulating β 2 M (β-2 microglobulin), a well-characterized kidney filtration biomarker. We hypothesize that circulating β 2 M is an independent risk predictor of hypertension and may causally contribute to its development. The study sample consisted of 7 065 Framingham Heart Study participants with measurements of plasma β 2 M. Generalized estimating equations were used to test the association of β 2 M with prevalent and new-onset hypertension. There were 2 145 (30%) cases of prevalent hypertension at baseline and 886 (21%) cases of incident hypertension during 6 years of follow-up. A 1-SD increase in baseline plasma β 2 M was associated with a greater risk of prevalent (odds ratio 1.14, 95% CI 1.05–1.24) and new-onset (odds ratio 1.18, 95% CI 1.07–1.32) hypertension. Individuals within the top β 2 M quartile had a greater risk than the bottom quartile for prevalent (odds ratio 1.29, 95% CI 1.05−1.57) and new-onset (odds ratio 1.59, 95% CI 1.20–2.11) hypertension. These associations remained essentially unchanged in analyses restricted to participants free of albuminuria and chronic kidney disease. Mendelian randomization demonstrated that lower SH2B3 expression is causal for increased circulating β 2 M levels, and in a hypertensive mouse model, knockout of Sh2b3 increased β 2 M gene expression. In a community-based study of healthy individuals, higher plasma β 2 M levels are associated with increased risk of prevalent and incident hypertension independent of chronic kidney disease status. Overlapping genetic signals for hypertension and β 2 M, in conjunction with mouse knockout experiments, suggest that the SH2B3 -β 2 M axis plays a causal role in hypertension.

Elevated cardiovascular risk including stroke, heart failure, and heart attack is present even after normalization of blood pressure in patients with hypertension. Underlying immune cell activation is a likely culprit. Although immune cells are important for protection against invading pathogens, their chronic overactivation may lead to tissue damage and high blood pressure. Triggers that may initiate immune activation include viral infections, autoimmunity, and lifestyle factors such as excess dietary salt. These conditions activate the immune system either directly or through their impact on the gut microbiome, which ultimately produces chronic inflammation and hypertension. T cells are central to the immune responses contributing to hypertension. They are activated in part by binding specific antigens that are presented in major histocompatibility complex molecules on professional antigen-presenting cells, and they generate repertoires of rearranged T-cell receptors. Activated T cells infiltrate tissues and produce cytokines including interleukin 17A, which promote renal and vascular dysfunction and end-organ damage leading to hypertension. In this comprehensive review, we highlight environmental, genetic, and microbial associated mechanisms contributing to both innate and adaptive immune cell activation leading to hypertension. Targeting the underlying chronic immune cell activation in hypertension has the potential to mitigate the excess cardiovascular risk associated with this common and deadly disease.

OBJECTIVE: Persons with human immunodeficiency virus (HIV) have double the risk of developing cardiovascular disease (CVD) compared to the general population. A persistent and heightened immune response to cytomegalovirus (CMV) co-infection may be one contributing factor, but the relationship between CMV replication, virus-specific immune cells and plaque burden is unclear. APPROACH AND RESULTS: We assessed the relationship between CD4(+) T-cell subsets and carotid plaque burden in a cohort of 70 HIV-positive participants with sustained viral suppression on a single antiretroviral regimen and without known CVD. We evaluated relationships between immune parameters, carotid plaque burden, and brachial artery flow-mediated vasodilation (FMD) using multivariable linear and logistic regression models. We found that participants with carotid plaque had increased circulating CX3CR1(+)~GPR56(+)~CD57(+) (i.e., C~G~C)(+) CD4(+) T cells (p=0.03), which is a marker combination associated with anti-viral and cytotoxic responses. In addition, a median of 14.4% [IQR 4.7, 32.7%] of the C~G~C(+) CD4(+) T-cells expressed antigen receptors that recognized a single CMV glycoprotein-B epitope. Notably, using immunofluorescence staining we found that CX3CR1(+) CD4(+) T-cells were present in coronary plaque from deceased HIV-positive persons. C-G-C(+) CD4(+) T cells were also present in cells isolated from the aorta of HIV-negative donors. CONCLUSIONS: HIV-positive persons with carotid atheroma have a higher proportion of circulating CD4(+) T-cells expressing the C~G~C surface marker combination associated with antiviral and cytotoxic responses. These cells can be CMV-specific and are also present in the aorta.

PURPOSE OF REVIEW: To summarize key advances in our understanding of the role of interleukin 17A (IL-17A) in the pathogenesis of hypertension and highlight important areas for future research and clinical translation. RECENT FINDINGS: While T helper 17 (Th17) cells are major producers of IL-17A, there are several additional innate and adaptive immune cell sources including gamma-delta T cells, innate lymphoid cells, and natural killer cells. IL-17A promotes an increase in blood pressure through multiple mechanisms including inhibiting endothelial nitric oxide production, increasing reactive oxygen species formation, promoting vascular fibrosis, and enhancing renal sodium retention and glomerular injury. IL-17A production from Th17 cells is increased by high salt conditions in vitro and in vivo. There is also emerging data linking salt, the gut microbiome, and intestinal T cell IL-17A production. Novel therapeutics targeting IL-17A signaling are approved for the treatment of autoimmune diseases and show promise in both animal models of hypertension and human studies. SUMMARY: Hypertensive stimuli enhance IL-17A production. IL-17A is a key mediator of renal and vascular dysfunction in hypertensive mouse models and correlates with hypertension in humans. Large randomized clinical trials are needed to determine whether targeting IL-17A might be an effective adjunct treatment for hypertension and its associated end-organ dysfunction.

Objective: Chronic inflammation contributes to the high burden of cardiovascular disease (CVD) in persons with HIV (PWH). HIV has broad effects on immune function, including CD4+T-helper cells which secrete cytokines that regulate innate and adaptive immune pathways implicated in CVD progression. At present, the relationship between T-helper cell responses and CVD in PWH is not well defined. Methods: We recruited 70 PWH on a single antiretroviral regimen (efavirenz, tenofovir, and emtricitabine) with at least 12 months of suppressed viremia. We quantified 19 plasma cytokines and chemokines including interferon (IFN)-γ, interleukin (IL)-2, 4, 13, and 17A, markers of macrophage activation, and markers of endothelial activation using multiplex assays and ELISA. Cytokines were grouped using Ward's hierarchical clustering. Brachial artery flow-mediated dilation (FMD), as a marker of endothelial function, and carotid plaque burden were measured using ultrasound. Multivariable linear regression was used to assess the relationships of biomarkers and endpoints with adjustment for CVD risk factors. Results: We identified three distinct cytokine clusters, one containing Th1/Th2 cytokines, one with Th17-type and macrophage-related cytokines, and a third less specific. Lower brachial artery FMD was associated with higher plasma IL-17A and macrophage inflammatory protein 1α, while higher soluble vascular cell adhesion molecule-1 and intercellular cell adhesion molecule 1 were associated with higher IL-17A, and IL-5. In contrast, IL-10 and the Th2-type cytokine IL-4 were associated with greater plaque burden. Conclusions: Distinct Th2 and Th17-mediated immune mechanisms may have a role at differing stages of atherosclerotic vascular disease in PWH.

Introduction: Inflammation and oxidative stress are linked to multiple risk factors for atrial fibrillation (AF), and to AF itself in the setting of sterile injury (e.g. after catheter ablation or cardiac surgery). However, anti-inflammatory therapies and conventional antioxidants cause adverse effects or are ineffective to prevent AF. Highly reactive mediators of lipid peroxidation such as isolevuglandins (IsoLGs) have been identified as a major component of oxidative stress-related injury. We hypothesized that during AF promoted by cardiac inflammation, a scavenger of IsoLG will decrease AF susceptibility. Methods: We studied mice with a systemic inflammatory phenotype due to deficiency in the lymphocyte adaptor protein ( Lnk -/- ), a negative regulator of cytokine signaling. At weaning, Lnk -/- mice and their wild-type (WT) littermates received either vehicle or a potent IsoLG scavenger, 2-hydroxybenzylamine (2-HOBA), by oral administration. At age 14 weeks, animals underwent transesophageal burst pacing, echocardiography, and tissue harvest or flow cytometry to measure atrial inflammation and IsoLG-adducts. Results: Cardiac histology and echocardiography revealed no major histologic or structural abnormalities in Lnk -/- mice. Nevertheless, Lnk -/- mice demonstrated a significant increase in AF burden compared to WT controls (124.8±43.3 vs 6.8±3 sec, respectively [mean±SEM, n=28, 12; P 30 sec; 48.1% vs 0%; P-/- mice, with a significant increase in CD3, CD19, NK1.1, and CD11b/MHCII positive cells, compared to atria from WT control mice. Furthermore, there was a 2 to 4-fold increase in IsoLG-adducts for Lnk -/- atrial immune cells positive for CD3, CD19, NK1.1 and CD11b/MHCII, compared to cells from WT atria. Lnk -/- mice treated with 2-HOBA had significantly reduced AF burden (4.7±4.5 sec, n=7; P Conclusions: IsoLGs play a critical role in the pathogenesis of inflammation-mediated AF, and 2-HOBA, a scavenger of IsoLGs, represents a potentially novel therapeutic strategy for AF in this clinical setting.

Deoxycorticosterone acetate (DOCA)-salt is a common hypertension model in mice and has recently been used to study heart failure with preserved ejection fraction (HFpEF) in rats. Our goal was to validate DOCA-salt as a mouse model of HFpEF and determine how DOCA-salt alters the cardiac immunological landscape to identify novel therapeutic targets for this disease. DOCA-salt mice underwent uninephrectomy, implantion of a DOCA pellet, and supplementation of the drinking water with 1% NaCl water for three weeks. Control mice underwent a sham procedure and received normal water. Compared to control mice, DOCA-salt mice exhibited elevated systolic BP, increased heart weight to body weight ratios (5.6 vs 7.1), increased lung wet to dry weight ratios (4.4 vs 4.8) indicative of pulmonary congestion, and decreased time to exhaustion upon treadmill exercise testing (23.0 vs. 18.5 seconds). On conscious echocardiography, DOCA-salt mice exhibited a preserved ejection fraction. Invasive hemodynamic studies revealed an increased tau constant (5.7 vs 8.2) and increased end-diastolic pressures in DOCA-salt mice (1.7 vs 2.6), consistent with diastolic dysfunction. CITE-seq, a novel technique to obtain transcriptomic and surface marker expression on single cells, was performed on a total of 4,359 and 7,600 cells sorted live CD45+ leukocytes from four sham and four DOCA-salt left ventricles, respectively. Unsupervised computational analysis revealed 29 clusters of immune cells. Six clusters containing natural killer, T lymphocyte and myeloid cell populations were overrepresented and five B cell clusters were underrepresented in DOCA-salt samples. Differential expression analysis of CD11b+CD64+ cardiac macrophages revealed transcriptional changes between groups with 146 significantly upregulated and 111 downregulated genes. Gene set enrichment analysis showed upregulation of leukocyte migration, response to type I interferon, and cytokine-mediated signaling pathways in DOCA-salt macrophages. In conclusion, the DOCA-salt mouse model recapitulates key features of HFpEF including diastolic dysfunction with preserved ejection fraction, cardiac hypertrophy, and pulmonary congestion and is associated with an altered cardiac immune cell profile.

Extracellular fluid (ECF) potassium concentration ([K(+)]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K(+)] in male C57BL/6J mice fed a K(+)-deficient diet (0K) versus 1% K(+) diet (1K) for 10 days (n = 5–6/group). During 0K feeding, plasma [K(+)] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K(+) (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na(+) (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na(+)] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-β2 subunits, decreased K(+)-Cl(−) cotransporter isoform 3, and increased phosphorylated (p) Na(+),K(+),2Cl(−) cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K(+) loss and Na(+) gain. Renal transporters’ adaptations, effecting a 98% reduction in K(+) excretion, included two- to threefold increased phosphorylated Na(+)-Cl(−) cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na(+) delivery to epithelial Na(+) channels where Na(+) reabsorption drives K(+) secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 μmol K(+) into the urine and muscle shifts ~47 μmol K(+) from ICF to ECF, illustrating the importance of the concerted responses during K(+) deficiency.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the global pandemic of coronavirus disease‐2019 (COVID‐19). SARS‐CoV‐2 is a zoonotic disease, but little is known about variations in species susceptibility that could identify potential reservoir species, animal models, and the risk to pets, wildlife, and livestock. Certain species, such as domestic cats and tigers, are susceptible to SARS‐CoV‐2 infection, while other species such as mice and chickens are not. Most animal species, including those in close contact with humans, have unknown susceptibility. Hence, methods to predict the infection risk of animal species are urgently needed. SARS‐CoV‐2 spike protein binding to angiotensin‐converting enzyme 2 (ACE2) is critical for viral cell entry and infection. Here we integrate species differences in susceptibility with multiple in‐depth structural analyses to identify key ACE2 amino acid positions including 30, 83, 90, 322, and 354 that distinguish susceptible from resistant species. Using differences in these residues across species, we developed a susceptibility score that predicts an elevated risk of SARS‐CoV‐2 infection for multiple species including horses and camels. We also demonstrate that SARS‐CoV‐2 is nearly optimal for binding ACE2 of humans compared to other animals, which may underlie the highly contagious transmissibility of this virus among humans. Taken together, our findings define potential ACE2 and SARS‐CoV‐2 residues for therapeutic targeting and identification of animal species on which to focus research and protection measures for environmental and public health.

In only a few months, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, leaving physicians, scientists, and public health officials racing to understand, treat, and contain this zoonotic disease. SARS-CoV-2 has made the leap from animals to humans, but little is known about variations in species susceptibility that could identify potential reservoir species, animal models, and the risk to pets, wildlife, and livestock. While there is evidence that certain species, such as cats, are susceptible, the vast majority of animal species, including those in close contact with humans, have unknown susceptibility. Hence, methods to predict their infection risk are urgently needed. SARS-CoV-2 spike protein binding to angiotensin converting enzyme 2 (ACE2) is critical for viral cell entry and infection. Here we identified key ACE2 residues that distinguish susceptible from resistant species using in-depth sequence and structural analyses of ACE2 and its binding to SARS-CoV-2. Our findings have important implications for identification of ACE2 and SARS-CoV-2 residues for therapeutic targeting and identification of animal species with increased susceptibility for infection on which to focus research and protection measures for environmental and public health.

Objective: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1 +/− model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1 +/− murine aortic valves were characterized by flow cytometry. Macrophages in Notch1 +/− aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1 +/− macrophages drive disease. Notch1 +/− mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1 +/− aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3β isoform. Conclusions: This study reveals that Notch1 +/− aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

ObjectiveMacrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1+/- model of calcific aortic valve disease.Approach and ResultsMacrophages in wild-type and Notch1+/- murine aortic valves were characterized by flow cytometry. Macrophages in Notch1+/- aortic valves had increased expression of MHCII. We then used bone marrow transplants to test if differences in Notch1+/- macrophages drive disease. Notch1+/- mice had increased valve thickness, macrophage infiltration, and M1-like macrophage polarization regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1+/- aortic valve cells promote macrophage invasion as quantified by migration index and M1-like polarization quantified by Ly6C and CCR2 positivity regardless of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification by decreasing abundance of the STAT3β isoform.ConclusionsThis study reveals that Notch1+/- aortic valve disease involves increased macrophage recruitment and polarization driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification through STAT3 splicing changes. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.Graphical Abstract

Visual Abstract, Highlights • IsoLGs are highly reactive lipid dicarbonyl metabolites that constitute a major component of oxidative stress-related injury, and they promote the formation of amyloid. • In a hypertensive murine model, IsoLG adducts and PAOs developed in the atria, along with inducible AF. • IsoLG and PAO accumulation and AF were prevented by the dicarbonyl scavenger 2-HOBA, but not by an inactive analog 4-hydroxybenzylamine. • Mechanically stretched atrial cells generated cytosolic IsoLG adducts and PAOs that were prevented by 2-HOBA. • Natriuretic peptides generated cytotoxic oligomers, a process accelerated by IsoLGs, contributing to atrial PAO formation. • These findings identify a novel pathway during oxidative stress to increase AF susceptibility, and they support the concept of preemptively scavenging reactive downstream mediators as a potential therapeutic approach to prevent AF., Summary Oxidative damage is implicated in atrial fibrillation (AF), but antioxidants are ineffective therapeutically. The authors tested the hypothesis that highly reactive lipid dicarbonyl metabolites, or isolevuglandins (IsoLGs), are principal drivers of AF during hypertension. In a hypertensive murine model and stretched atriomyocytes, the dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) prevented IsoLG adducts and preamyloid oligomers (PAOs), and AF susceptibility, whereas the ineffective analog 4-hydroxybenzylamine (4-HOBA) had minimal effect. Natriuretic peptides generated cytotoxic oligomers, a process accelerated by IsoLGs, contributing to atrial PAO formation. These findings support the concept of pre-emptively scavenging reactive downstream oxidative stress mediators as a potential therapeutic approach to prevent AF.

The novel coronavirus disease (COVID-19) outbreak, caused by SARS-CoV-2, represents the greatest medical challenge in decades. We provide a comprehensive review of the clinical course of COVID-19, its comorbidities, and mechanistic considerations for future therapies. While COVID-19 primarily affects the lungs, causing interstitial pneumonitis and severe acute respiratory distress syndrome (ARDS), it also affects multiple organs, particularly the cardiovascular system. Risk of severe infection and mortality increase with advancing age and male sex. Mortality is increased by comorbidities: cardiovascular disease, hypertension, diabetes, chronic pulmonary disease, and cancer. The most common complications include arrhythmia (atrial fibrillation, ventricular tachyarrhythmia, and ventricular fibrillation), cardiac injury [elevated highly sensitive troponin I (hs-cTnI) and creatine kinase (CK) levels], fulminant myocarditis, heart failure, pulmonary embolism, and disseminated intravascular coagulation (DIC). Mechanistically, SARS-CoV-2, following proteolytic cleavage of its S protein by a serine protease, binds to the transmembrane angiotensin-converting enzyme 2 (ACE2) —a homologue of ACE—to enter type 2 pneumocytes, macrophages, perivascular pericytes, and cardiomyocytes. This may lead to myocardial dysfunction and damage, endothelial dysfunction, microvascular dysfunction, plaque instability, and myocardial infarction (MI). While ACE2 is essential for viral invasion, there is no evidence that ACE inhibitors or angiotensin receptor blockers (ARBs) worsen prognosis. Hence, patients should not discontinue their use. Moreover, renin–angiotensin–aldosterone system (RAAS) inhibitors might be beneficial in COVID-19. Initial immune and inflammatory responses induce a severe cytokine storm [interleukin (IL)-6, IL-7, IL-22, IL-17, etc.] during the rapid progression phase of COVID-19. Early evaluation and continued monitoring of cardiac damage (cTnI and NT-proBNP) and coagulation (D-dimer) after hospitalization may identify patients with cardiac injury and predict COVID-19 complications. Preventive measures (social distancing and social isolation) also increase cardiovascular risk. Cardiovascular considerations of therapies currently used, including remdesivir, chloroquine, hydroxychloroquine, tocilizumab, ribavirin, interferons, and lopinavir/ritonavir, as well as experimental therapies, such as human recombinant ACE2 (rhACE2), are discussed.

Early-career academic cardiologists, who many believe are an important component of the future of cardiovascular care, face myriad challenges. The Early Career Section Academic Working Group of the American College of Cardiology, with senior leadership support, assessed the progress of this cohort from 2013 to 2016 with a global perspective. Data consisted of accessing National Heart, Lung, and Blood Institute public information, data from the American Heart Association and international organizations, and a membership-wide survey. Although the National Heart, Lung, and Blood Institute increased funding of career development grants, only a small number of early-career American College of Cardiology members have benefited as funding of the entire cohort has decreased. Personal motivation, institutional support, and collaborators continued to be positive influential factors. Surprisingly, mentoring ceased to correlate positively with obtaining external grants. The totality of findings suggests that the status of early-career academic cardiologists remains challenging; therefore, the authors recommend a set of attainable solutions.

To characterize the T cell subsets producing interleukin 17 (IL-17) isoforms A and F in hypertensive kidneys and vessels and determine whether inhibition of IL-17 signaling lowers blood pressure and end-organ damage in a mouse model of hypertension.T cell derived cytokines play a central role in the pathophysiology of hypertension and contribute to end-organ dysfunction. We previously showed that mice genetically deficient in IL-17A exhibited blunted hypertension and reduced renal and vascular dysfunction in response to angiotensin II (Ang II) infusion. Monoclonal antibodies to IL-17 isoforms or the IL-17 receptor are emerging as novel therapeutics for psoriasis and related autoimmune disorders.Mice were infused with Ang II for 4 weeks to induce hypertension. Using flow cytometry and intracellular staining, we determined that the primary T cell subsets producing IL-17A and IL-17F in the kidney and aorta are gamma delta (γδ) T cells and CD4+ T helper 17 (TMonoclonal antibodies to IL-17A or IL-17RA, but not IL-17F, may be a useful adjunct treatment for hypertension and the associated end-organ dysfunction.

The role of B cells and serum immunoglobulins (Ig) in hypertension is unclear. Elevated serum IgG has been observed in some hypertensive patients and in animal models of hypertension. Mice with a severe B cell deficiency due to lack of B cell activating factor receptor (BAFF-R) and mice lacking the cytokine interleukin 21 (IL21) - which is a potent inducer of B cell Ig class switching and high affinity Ig production - are protected from Ang II-induced hypertension and exhibit reduced serum IgG levels. Thus, we sought to determine whether hypertensive IgGs play a causative role in hypertension and end-organ dysfunction. First, we purified serum IgG from normotensive and hypertensive wild type (WT) mice and adoptively transferred equal amounts of IgG by intraperitoneal injection into normotensive recipient mice followed by infusion of a sub-pressor dose of angiotensin II (Ang II) for 2 weeks. Flow cytometric analysis of aortas revealed a modest increase in F4/80+ macrophages (p=0.04) and CD8+ T cells (p=0.02) in mice that received hypertensive IgG. However, there was no difference in endothelial dependent relaxation, renal inflammation, albuminuria, and blood pressure (BP) between the two groups. As an alternative method to investigate the role of class-switched high affinity Igs, we studied mice with genetic deletion of activation-induced deaminase (AID) which is an enzyme necessary for somatic hypermutation (to generate high affinity Igs) and isotype class switching. AID -/- mice exhibit undetectable IgG and higher levels of IgM in serum compared to WT mice. Ang II infusion for 4 weeks revealed no difference in BP, aortic inflammation, renal inflammation, and albuminuria. We also studied mice with deficiency of the IgM heavy chain (uMT). These mice exhibit undetectable levels of all immunoglobulins and are severely deficient in mature B cells. uMT-/- mice are also not protected from hypertension induced by Ang II infusion or deoxycorticosterone acetate (DOCA-salt) treatment. Together, these results demonstrate that while elevated serum immunoglobulins seen in hypertensive animals and humans may represent a biomarker of aberrant immune activation, they are likely not playing a causal role in hypertension pathophysiology.

Hypertension is the leading risk factor for morbidity and mortality worldwide. Emerging evidence in animal models demonstrates the importance of a variety of innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of specific circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We performed unbiased high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed consistent increases in a novel memory helper T cell subset in hypertension. Manual two dimensional gating revealed that this CD4 + CD45RO + CD62L + CCR7 - CD161 lo memory cell population is nearly 2-fold increased in hypertensive subjects (1.0 vs 1.9%). As an alternative to starting with unsupervised analysis, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals (3.2 vs 1.9% and 1.3 vs 0.93%, respectively). Unsupervised analysis of the cellular subsets of these populations using Phenograph revealed a selective 63% decrease in PD-1 + memory cytotoxic T cells and 49% decrease in CCR10 + regulatory T cells in hypertension (0.67 vs 0.25% and 0.37 vs 0.19%, respectively). Taken together, these results demonstrate that in human hypertension there are increases in a novel memory helper T cell subset and decreases in PD-1 + memory cytotoxic and CCR10 + regulatory T cell populations. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.

We and others have demonstrated that interleukin-17A (IL-17A) plays a critical role in the pathogenesis of cardiovascular disease and hypertension. Patients with psoriasis, an IL-17A mediated autoimmune disease, are at elevated risk for cardiovascular events. New biologic therapies for psoriasis have been developed that target IL-17A (secukinumab) or its receptor (brodalumab), but their impact on incident hypertension and its associated complications is unknown and remain unexplored outside of short-term safety in clinical trials. Our goal is to perform a retrospective case-control study utilizing the Synthetic Derivative (Vanderbilt’s de-identified version of the electronic health record) to determine whether incident hypertension or other major adverse cardiovascular events is reduced in patients with psoriasis receiving IL-17A targeted therapies versus other therapeutics. Patients are defined as cases by 2 or more mentions of a psoriasis ICD9 or ICD10 code and at least one documented prescription for secukinumab or brodalumab. Controls are defined by the presence of 2 or more psoriasis ICD codes, no documented exposure to the drugs of interest, and a mention of “psoriasis” in their Problem List. Using these definitions, we identified 571 cases and 4484 controls in the Synthetic Derivative. Of the 571 potential cases identified, 65 were confirmed by manual review for an initial validation study. Of the manually confirmed cases, 22 (34%) were male and 43 (66%) were female. Manually reviewed cases had a mean record length of 5.0 years with an average of 29.9 discrete ICD mentions. Twenty-three cases (35%) had an ICD code for hypertension, which predated the first documented psoriasis code in eight patients. Thus, we are able to use the Synthetic Derivative to identify patients with psoriasis receiving biologic therapies and assess temporal relationships relevant to hypertension. We plan to expand this study to the larger cohort identified and analyze rates of hypertension and a composite outcome of hypertension complications, including stroke, myocardial infarction, heart failure, and chronic kidney disease, in cases and controls matched for age, gender, race, and BMI.

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

Genetic variants at SH2B3 are associated with blood pressure and circulating beta-2 microglobulin (β(2)M), a well-characterized kidney filtration biomarker. We hypothesize that circulating β(2)M is an independent risk predictor of hypertension and may causally contribute to its development. The study sample consisted of 7065 Framingham Heart Study participants with measurements of plasma β(2)M. Generalized estimating equations were used to test the association of β(2)M with prevalent and new-onset hypertension. There were 2145 (30%) cases of prevalent hypertension at baseline and 886 (21%) cases of incident hypertension during six years of follow-up. A 1-standard deviation increase in baseline plasma β(2)M was associated with a greater risk of prevalent (odds ratio [OR] 1.14, 95% confidence interval [CI] 1.05–1.24) and new-onset (OR 1.18, 95% CI 1.07–1.32) hypertension. Individuals within the top β(2)M quartile had a greater risk than the bottom quartile for prevalent (OR 1.29, 95% CI .05–1.57) and new-onset (OR 1.59, 95% CI 1.20–2.11) hypertension. These associations remained essentially unchanged in analyses restricted to participants free of albuminuria and chronic kidney disease (CKD). Mendelian randomization demonstrated that lower SH2B3 expression is causal for increased circulating β(2)M levels, and in a hypertensive mouse model, knockout of Sh2b3 increased β(2)M gene expression. In a community-based study of healthy individuals, higher plasma β(2)M levels are associated with increased risk of prevalent and incident hypertension independent of CKD status. Overlapping genetic signals for hypertension and β(2)M, in conjunction with mouse knockout experiments, suggest that the SH2B3-β(2)M axis plays a causal role in 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.

Hypertension is a leading risk factor for cardiovascular disease, which is a major cause of death worldwide. T cells are required for the development of hypertension. Specifically, we showed that T helper 17 (Th17) cells produce the pro-inflammatory cytokine, IL-17A, which leads to sustained elevations of blood pressure and renal and vascular dysfunction in experimental hypertension. T cell differentiation and trafficking is regulated by circadian clocks. High fat and high salt intake, typical of Western diets, disrupt circadian rhythms and are associated with increased risk for developing cardiovascular disease. These diets are also associated with an increase in Th17 cell differentiation. Melatonin is a major circadian hormone with anti-inflammatory and anti-oxidant actions. To determine the interplay between circadian rhythms and Th17 cell differentiation, we tested the hypothesis that in vitro treatment of Th17 cells with melatonin would inhibit IL-17A cytokine production. Splenic naïve CD4+ T cells from wild type C57Bl/6J male mice were cultured for 72 hours on anti-CD3/anti-CD28 antibody coated plates in the presence or absence of Th17-polarizing cytokines and treated with melatonin (2ng/ml; Sigma Aldrich) or vehicle (0.02% DMSO). IL-17A cytokine levels were analyzed by ELISA. In cells treated with Th17 polarizing cytokines, IL-17A cytokine production was significantly reduced by melatonin. Thus, this study highlights the involvement of circadian control of Th17 cells via the actions of melatonin to suppress Th17 differentiation and IL-17A production. Future studies will determine the effect of melatonin treatment in vivo on development of hypertension and renal/vascular dysfunction.

Emerging evidence from animal models demonstrates the importance of a variety of innate and adaptive immune cells in the pathogenesis of hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We thus performed high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed an approximately two-fold increase in a novel memory helper CD62L+CCR7−CD161lo T cell subset in hypertensive subjects. In addition, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals. Unsupervised analysis of the cellular subsets of these populations using Phenograph demonstrated a selective 63% decrease in PD-1+ memory cytotoxic T cells and 49% decrease in CCR10+ regulatory T cells in hypertension. In a validation cohort, circulating PD-1+ memory cytotoxic and CCR10+ regulatory T cells were significantly decreased by 30% and 50%, respectively, without a difference in the memory helper T cell subset. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1+ memory cytotoxic and CCR10+ regulatory T cell populations in human hypertension. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.