Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs. The peptide engages the AT1R through an extensive interface spanning from the receptor core to its extracellular face and N terminus, remodeling the ligand-binding cavity. Remarkably, the mechanism used to propagate conformational changes through the receptor diverges from other GPCRs at several key sites, highlighting the diversity of allosteric mechanisms among GPCRs. Our structure provides insight into how AngII and its analogs stimulate full or biased signaling, respectively.

Beta-Arrestin1 Prevents Preeclampsia by Downregulation of Mechanosensitive AT1-B2 Receptor Heteromers.

Preeclampsia is the most frequent pregnancy-related complication worldwide with no cure. While a number of molecular features have emerged, the underlying causal mechanisms behind the disorder remain obscure. Here, we find that increased complex formation between angiotensin II AT1 and bradykinin B2, two G protein-coupled receptors with opposing effects on blood vessel constriction, triggers symptoms of preeclampsia in pregnant mice. Aberrant heteromerization of AT1-B2 led to exaggerated calcium signaling and high vascular smooth muscle mechanosensitivity, which could explain the onset of preeclampsia symptoms at late-stage pregnancy as mechanical forces increase with fetal mass. AT1-B2 receptor aggregation was inhibited by beta-arrestin-mediated downregulation. Importantly, symptoms of preeclampsia were prevented by transgenic ARRB1 expression or a small-molecule drug. Because AT1-B2 heteromerization was found to occur in human placental biopsies from pregnancies complicated by preeclampsia, specifically targeting AT1-B2 heteromerization and its downstream consequences represents a promising therapeutic approach.

The Renal Physiology of Pendrin-Positive Intercalated Cells.

Intercalated cells (ICs) are found in the connecting tubule and the collecting duct. Of the three IC subtypes identified, type B intercalated cells are one of the best characterized and known to mediate Cl- absorption and HCO3- secretion, largely through the anion exchanger pendrin. This exchanger is thought to act in tandem with the Na+-dependent Cl-/HCO3- exchanger, NDCBE, to mediate net NaCl absorption. Pendrin is stimulated by angiotensin II and aldosterone administration via the angiotensin type 1a and the mineralocorticoid receptors, respectively. It is also stimulated in models of metabolic alkalosis, such as with NaHCO3 administration. In some rodent models, pendrin-mediated HCO3- secretion modulates acid-base balance. However, of probably more physiological or clinical significance is the role of these pendrin-positive ICs in blood pressure regulation, which occurs, at least in part, through pendrin-mediated renal Cl- absorption, as well as their effect on the epithelial Na+ channel, ENaC. Aldosterone stimulates ENaC directly through principal cell mineralocorticoid hormone receptor (ligand) binding and also indirectly through its effect on pendrin expression and function. In so doing, pendrin contributes to the aldosterone pressor response. Pendrin may also modulate blood pressure in part through its action in the adrenal medulla, where it modulates the release of catecholamines, or through an indirect effect on vascular contractile force. In addition to its role in Na+ and Cl- balance, pendrin affects the balance of other ions, such as K+ and I-. This review describes how aldosterone and angiotensin II-induced signaling regulate pendrin and the contribution of pendrin-positive ICs in the kidney to distal nephron function and blood pressure.

Structural insights into angiotensin receptor signaling modulation by balanced and biased agonists.

The peptide hormone angiotensin II regulates blood pressure mainly through the type 1 angiotensin II receptor AT1 R and its downstream signaling proteins Gq and ß-arrestin. AT1 R blockers, clinically used as antihypertensive drugs, inhibit both signaling pathways, whereas AT1 R ß-arrestin-biased agonists have shown great potential for the treatment of acute heart failure. Here, we present a cryo-electron microscopy (cryo-EM) structure of the human AT1 R in complex with a balanced agonist, Sar1 -AngII, and Gq protein at 2.9 Å resolution. This structure, together with extensive functional assays and computational modeling, reveals the molecular mechanisms for AT1 R signaling modulation and suggests that a major hydrogen bond network (MHN) inside the receptor serves as a key regulator of AT1 R signal transduction from the ligand-binding pocket to both Gq and ß-arrestin pathways. Specifically, we found that the MHN mutations N1113.35 A and N2947.45 A induce biased signaling to Gq and ß-arrestin, respectively. These insights should facilitate AT1 R structure-based drug discovery for the treatment of cardiovascular diseases.

The central renin-angiotensin system: A genetic pathway, functional decoding, and selective target engagement characterization in humans.

Accumulating evidence suggests that the brain renin angiotensin system (RAS) plays a pivotal role in the regulation of cognition and behavior as well as in the neuropathology of neurological and mental disorders. The angiotensin II type 1 receptor (AT1R) mediates most functional and neuropathology-relevant actions associated with the central RAS. However, an overarching comprehension to guide translation and utilize the therapeutic potential of the central RAS in humans is currently lacking. We conducted a comprehensive characterization of the RAS using an innovative combination of transcriptomic gene expression mapping, image-based behavioral decoding, and pre-registered randomized controlled discovery-replication pharmacological resting-state functional magnetic resonance imaging (fMRI) trials (N = 132) with a selective AT1R antagonist. The AT1R exhibited a particular dense expression in a subcortical network encompassing the thalamus, striatum, and amygdalo-hippocampal formation. Behavioral decoding of the AT1R gene expression brain map showed an association with memory, stress, reward, and motivational processes. Transient pharmacological blockade of the AT1R further decreased neural activity in subcortical systems characterized by a high AT1R expression, while increasing functional connectivity in the cortico-basal ganglia-thalamo-cortical circuitry. Effects of AT1R blockade on the network level were specifically associated with the transcriptomic signatures of the dopaminergic, opioid, acetylcholine, and corticotropin-releasing hormone signaling systems. The robustness of the results was supported in an independent pharmacological fMRI trial. These findings present a biologically informed comprehensive characterization of the central AT1R pathways and their functional relevance on the neural and behavioral level in humans.

SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

BACKGROUND: T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. METHODS: Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. RESULTS: LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. CONCLUSIONS: LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Mitochondrial CypD Acetylation Promotes Endothelial Dysfunction and Hypertension.

BACKGROUND: Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial CypD (cyclophilin D) at K166 contributes to endothelial dysfunction and hypertension. METHODS: To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial-specific GCN5L1 (general control of amino acid synthesis 5 like 1)-deficient mice using an Ang II (angiotensin II) model of hypertension. RESULTS: Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 (sirtuin 3) and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation, which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells, GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension. Ang II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in a 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins (mito2HOBA) normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in the endothelial function was tested in endothelial-specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented Ang II-induced mitochondrial oxidative stress, reduced the maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. CONCLUSIONS: These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation, which may be beneficial in cardiovascular disease.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. METHODS: Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. RESULTS: In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor ß) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. CONCLUSIONS: Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

NETosis Drives Blood Pressure Elevation and Vascular Dysfunction in Hypertension.

BACKGROUND: Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction. METHODS: NETosis-deficient Padi4-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. RESULTS: Padi4-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. CONCLUSIONS: These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

Experimental TET2 Clonal Hematopoiesis Predisposes to Renal Hypertension Through an Inflammasome-Mediated Mechanism.

BACKGROUND: Hypertension incidence increases with age and represents one of the most prevalent risk factors for cardiovascular disease. Clonal events in the hematopoietic system resulting from somatic mutations in driver genes are prevalent in elderly individuals who lack overt hematologic disorders. This condition is referred to as age-related clonal hematopoiesis (CH), and it is a newly recognized risk factor for cardiovascular disease. It is not known whether CH and hypertension in the elderly are causally related and, if so, what are the mechanistic features. METHODS: A murine model of adoptive bone marrow transplantation was employed to examine the interplay between Tet2 (ten-eleven translocation methylcytosine dioxygenase 2) clonal hematopoiesis and hypertension. RESULTS: In this model, a subpressor dose of Ang II (angiotensin II) resulted in elevated systolic and diastolic blood pressure as early as 1 day after challenge. These conditions led to the expansion of Tet2-deficient proinflammatory monocytes and bone marrow progenitor populations. Tet2 deficiency promoted renal CCL5 (C-C motif ligand 5) chemokine expression and macrophage infiltration into the kidney. Consistent with macrophage involvement, Tet2 deficiency in myeloid cells promoted hypertension when mice were treated with a subpressor dose of Ang II. The hematopoietic Tet2-/- condition led to sodium retention, renal inflammasome activation, and elevated levels of IL (interleukin)-1ß and IL-18. Analysis of the sodium transporters indicated NCC (sodium-chloride symporter) and NKCC2 (Na+-K+-Cl- cotransporter 2) activation at residues Thr53 and Ser105, respectively. Administration of the NLRP3 (NLR family pyrin domain containing 3) inflammasome inhibitor MCC950 reversed the hypertensive state, sodium retention, and renal transporter activation. CONCLUSIONS: Tet2-mediated CH sensitizes mice to a hypertensive stimulus. Mechanistically, the expansion of hematopoietic Tet2-deficient cells promotes hypertension due to elevated renal immune cell infiltration and activation of the NLRP3 inflammasome, with consequences on sodium retention. These data indicate that carriers of TET2 CH could be at elevated risk for the development of hypertension and that immune modulators could be useful in treating hypertension in this patient population.

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

BACKGROUND: Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. METHODS: IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. RESULTS: We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. CONCLUSIONS: These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.

L-Wnk1 Deletion in Smooth Muscle Cells Causes Aortitis and Inflammatory Shift.

BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-ß (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-ß blockade using neutralizing anti-TGF-ß antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.

Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

KLF15 maintains contractile phenotype of vascular smooth muscle cells and prevents thoracic aortic dissection by interacting with MRTFB.

Thoracic aortic dissection (TAD) is a highly dangerous cardiovascular disorder caused by weakening of the aortic wall, resulting in a sudden tear of the internal face. Progressive loss of the contractile apparatus in vascular smooth muscle cells (VSMCs) is a major event in TAD. Exploring the endogenous regulators essential for the contractile phenotype of VSMCs may aid the development of strategies to prevent TAD. Krüppel-like factor 15 (KLF15) overexpression was reported to inhibit TAD formation; however, the mechanisms by which KLF15 prevents TAD formation and whether KLF15 regulates the contractile phenotype of VSMCs in TAD are not well understood. Therefore, we investigated these unknown aspects of KLF15 function. We found that KLF15 expression was reduced in human TAD samples and ß-aminopropionitrile monofumarate-induced TAD mouse model. Klf15KO mice are susceptible to both ß-aminopropionitrile monofumarate- and angiotensin II-induced TAD. KLF15 deficiency results in reduced VSMC contractility and exacerbated vascular inflammation and extracellular matrix degradation. Mechanistically, KLF15 interacts with myocardin-related transcription factor B (MRTFB), a potent serum response factor coactivator that drives contractile gene expression. KLF15 silencing represses the MRTFB-induced activation of contractile genes in VSMCs. Thus, KLF15 cooperates with MRTFB to promote the expression of contractile genes in VSMCs, and its dysfunction may exacerbate TAD. These findings indicate that KLF15 may be a novel therapeutic target for the treatment of TAD.

Classical and nonclassical effects of angiotensin-converting enzyme: How increased ACE enhances myeloid immune function.

As part of the classical renin-angiotensin system, the peptidase angiotensin-converting enzyme (ACE) makes angiotensin II which has myriad effects on systemic cardiovascular function, inflammation, and cellular proliferation. Less well known is that macrophages and neutrophils make ACE in response to immune activation which has marked effects on myeloid cell function independent of angiotensin II. Here, we discuss both classical (angiotensin) and nonclassical functions of ACE and highlight mice called ACE 10/10 in which genetic manipulation increases ACE expression by macrophages and makes these mice much more resistant to models of tumors, infection, atherosclerosis, and Alzheimer's disease. In another model called NeuACE mice, neutrophils make increased ACE and these mice are much more resistant to infection. In contrast, ACE inhibitors reduce neutrophil killing of bacteria in mice and humans. Increased expression of ACE induces a marked increase in macrophage oxidative metabolism, particularly mitochondrial oxidation of lipids, secondary to increased peroxisome proliferator-activated receptor α expression, and results in increased myeloid cell ATP. ACE present in sperm has a similar metabolic effect, and the lack of ACE activity in these cells reduces both sperm motility and fertilization capacity. These nonclassical effects of ACE are not due to the actions of angiotensin II but to an unknown molecule, probably a peptide, that triggers a profound change in myeloid cell metabolism and function. Purifying and characterizing this peptide could offer a new treatment for several diseases and prove potentially lucrative.

S-Nitrosylation of Septin2 Exacerbates Aortic Aneurysm and Dissection by Coupling the TIAM1-RAC1 Axis in Macrophages.

BACKGROUND: S-Nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in cardiovascular disease. Aortic aneurysm and dissection are high-risk cardiovascular diseases without an effective cure. The aim of this study was to determine the role of SNO of Septin2 in macrophages in aortic aneurysm and dissection. METHODS: Biotin-switch assay combined with liquid chromatography-tandem mass spectrometry was performed to identify the S-nitrosylated proteins in aortic tissue from both patients undergoing surgery for aortic dissection and Apoe-/- mice infused with angiotensin II. Angiotensin II-induced aortic aneurysm model and ß-aminopropionitrile-induced aortic aneurysm and dissection model were used to determine the role of SNO of Septin2 (SNO-Septin2) in aortic aneurysm and dissection development. RNA-sequencing analysis was performed to recapitulate possible changes in the transcriptome profile of SNO-Septin2 in macrophages in aortic aneurysm and dissection. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation were used to uncover the TIAM1-RAC1 (Ras-related C3 botulinum toxin substrate 1) axis as the downstream target of SNO-Septin2. Both R-Ketorolac and NSC23766 treatments were used to inhibit the TIAM1-RAC1 axis. RESULTS: Septin2 was identified S-nitrosylated at cysteine 111 (Cys111) in both aortic tissue from patients undergoing surgery for aortic dissection and Apoe-/- mice infused with Angiotensin II. SNO-Septin2 was demonstrated driving the development of aortic aneurysm and dissection. By RNA-sequencing, SNO-Septin2 in macrophages was demonstrated to exacerbate vascular inflammation and extracellular matrix degradation in aortic aneurysm. Next, TIAM1 (T lymphoma invasion and metastasis-inducing protein 1) was identified as a SNO-Septin2 target protein. Mechanistically, compared with unmodified Septin2, SNO-Septin2 reduced its interaction with TIAM1 and activated the TIAM1-RAC1 axis and consequent nuclear factor-κB signaling pathway, resulting in stronger inflammation and extracellular matrix degradation mediated by macrophages. Consistently, both R-Ketorolac and NSC23766 treatments protected against aortic aneurysm and dissection by inhibiting the TIAM1-RAC1 axis. CONCLUSIONS: SNO-Septin2 drives aortic aneurysm and dissection through coupling the TIAM1-RAC1 axis in macrophages and activating the nuclear factor-κB signaling pathway-dependent inflammation and extracellular matrix degradation. Pharmacological blockade of RAC1 by R-Ketorolac or NSC23766 may therefore represent a potential treatment against aortic aneurysm and dissection.

LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology.

The renin-angiotensin-aldosterone system plays crucial roles in cardiovascular physiology and pathophysiology. However, many of the signaling mechanisms have been unclear. The angiotensin II (ANG II) type 1 receptor (AT1R) is believed to mediate most functions of ANG II in the system. AT1R utilizes various signal transduction cascades causing hypertension, cardiovascular remodeling, and end organ damage. Moreover, functional cross-talk between AT1R signaling pathways and other signaling pathways have been recognized. Accumulating evidence reveals the complexity of ANG II signal transduction in pathophysiology of the vasculature, heart, kidney, and brain, as well as several pathophysiological features, including inflammation, metabolic dysfunction, and aging. In this review, we provide a comprehensive update of the ANG II receptor signaling events and their functional significances for potential translation into therapeutic strategies. AT1R remains central to the system in mediating physiological and pathophysiological functions of ANG II, and participation of specific signaling pathways becomes much clearer. There are still certain limitations and many controversies, and several noteworthy new concepts require further support. However, it is expected that rigorous translational research of the ANG II signaling pathways including those in large animals and humans will contribute to establishing effective new therapies against various diseases.