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.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

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.

PIEZO Ion Channels in Cardiovascular Functions and Diseases.

The cardiovascular system provides blood supply throughout the body and as such is perpetually applying mechanical forces to cells and tissues. Thus, this system is primed with mechanosensory structures that respond and adapt to changes in mechanical stimuli. Since their discovery in 2010, PIEZO ion channels have dominated the field of mechanobiology. These have been proposed as the long-sought-after mechanosensitive excitatory channels involved in touch and proprioception in mammals. However, more and more pieces of evidence point to the importance of PIEZO channels in cardiovascular activities and disease development. PIEZO channel-related cardiac functions include transducing hemodynamic forces in endothelial and vascular cells, red blood cell homeostasis, platelet aggregation, and arterial blood pressure regulation, among others. PIEZO channels contribute to pathological conditions including cardiac hypertrophy and pulmonary hypertension and congenital syndromes such as generalized lymphatic dysplasia and xerocytosis. In this review, we highlight recent advances in understanding the role of PIEZO channels in cardiovascular functions and diseases. Achievements in this quickly expanding field should open a new road for efficient control of PIEZO-related diseases in cardiovascular functions.

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.

G Protein-Coupled Receptors: A Century of Research and Discovery.

GPCRs (G protein-coupled receptors), also known as 7 transmembrane domain receptors, are the largest receptor family in the human genome, with ≈800 members. GPCRs regulate nearly every aspect of human physiology and disease, thus serving as important drug targets in cardiovascular disease. Sharing a conserved structure comprised of 7 transmembrane α-helices, GPCRs couple to heterotrimeric G-proteins, GPCR kinases, and ß-arrestins, promoting downstream signaling through second messengers and other intracellular signaling pathways. GPCR drug development has led to important cardiovascular therapies, such as antagonists of ß-adrenergic and angiotensin II receptors for heart failure and hypertension, and agonists of the glucagon-like peptide-1 receptor for reducing adverse cardiovascular events and other emerging indications. There continues to be a major interest in GPCR drug development in cardiovascular and cardiometabolic disease, driven by advances in GPCR mechanistic studies and structure-based drug design. This review recounts the rich history of GPCR research, including the current state of clinically used GPCR drugs, and highlights newly discovered aspects of GPCR biology and promising directions for future investigation. As additional mechanisms for regulating GPCR signaling are uncovered, new strategies for targeting these ubiquitous receptors hold tremendous promise for the field of cardiovascular medicine.

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.

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.

Dectin-1 Acts as a Non-Classical Receptor of Ang II to Induce Cardiac Remodeling.

BACKGROUND: Cardiac remodeling in heart failure involves macrophage-mediated immune responses. Recent studies have shown that a PRR (pattern recognition receptor) called dectin-1, expressed on macrophages, mediates proinflammatory responses. Whether dectin-1 plays a role in pathological cardiac remodeling is unknown. Here, we identified a potential role of dectin-1 in this disease. METHODS: To model aberrant cardiac remodeling, we utilized mouse models of chronic Ang II (angiotensin II) infusion. In this model, we assessed the potential role of dectin-1 through using D1KO (dectin-1 knockout) mice and bone marrow transplantation chimeric mice. We then used cellular and molecular assays to discover the underlying mechanisms of dectin-1 function. RESULTS: We found that macrophage dectin-1 is elevated in mouse heart tissues following chronic Ang II administration. D1KO mice were significantly protected against Ang II-induced cardiac dysfunction, hypertrophy, fibrosis, inflammatory responses, and macrophage infiltration. Further bone marrow transplantation studies showed that dectin-1 deficiency in bone marrow-derived cells prevented Ang II-induced cardiac inflammation and dysfunction. Through detailed molecular studies, we show that Ang II binds directly to dectin-1, causing dectin-1 homodimerization and activating the downstream Syk (spleen tyrosine kinase)/NF-κB (nuclear factor kappa B) signaling pathway to induce expression of inflammatory and chemoattractant factors. Mutagenesis studies identified R184 in the C-type lectin domain to interact with Ang II. Blocking dectin-1 in macrophages suppresses Ang II-induced inflammatory mediators and subsequent intercellular cross talk with cardiomyocytes and fibroblasts. CONCLUSIONS: Our study has discovered dectin-1 as a new nonclassical receptor of Ang II and a key player in cardiac remolding and dysfunction. These studies suggest that dectin-1 may be a new target for treating hypertension-related heart failure.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. METHODS: Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. RESULTS: Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. CONCLUSIONS: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Smooth Muscle Mineralocorticoid Receptor Promotes Hypertension After Preeclampsia.

BACKGROUND: Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. METHODS: Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. RESULTS: Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. CONCLUSIONS: These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3.

Understanding the molecular mechanisms of pathological vascular remodeling is important for treating cardiovascular diseases and complications. Recent studies have highlighted a role of deubiquitinases in vascular pathophysiology. Here, we investigate the role of a deubiquitinase, OTUD1, in angiotensin II (Ang II)-induced vascular remodeling. We detect upregulated OTUD1 in the vascular endothelium of Ang II-challenged mice and show that OTUD1 deletion attenuates vascular remodeling, collagen deposition, and EndMT. Conversely, OTUD1 overexpression aggravates these pathological changes both in vivo and in vitro. Mechanistically, SMAD3 is identified as a substrate of OTUD1 using co-immunoprecipitation followed by LC-MS/MS. We find that OTUD1 stabilizes SMAD3 and facilitates SMAD3/SMAD4 complex formation and subsequent nuclear translocation through both K48- and K63-linked deubiquitination. OTUD1-mediated SMAD3 activation regulates transcription of genes involved in vascular EndMT and remodeling in HUVECs. Finally, SMAD3 inhibition reverses OTUD1-promoted vascular remodeling. Our findings demonstrate that endothelial OTUD1 promotes Ang II-induced vascular remodeling by deubiquitinating SMAD3. We identify SMAD3 as a target of OTUD1 and propose OTUD1 as a potential therapeutic target for diseases related to vascular remodeling.

Small AAAs: Recommendations for Rodent Model Research for the Identification of Novel Therapeutics.

CLINICAL PROBLEM: Most abdominal aortic aneurysms (AAAs) are small with low rupture risk (<1%/y) when diagnosed but slowly expand to ≥55 mm and undergo surgical repair. Patients and clinicians require medications to limit AAA growth and rupture, but drugs effective in animal models have not translated to patients. RECOMMENDATIONS FOR INCREASING TRANSLATION FROM MOUSE MODELS: Use models that simulate human AAA tissue pathology, growth patterns, and rupture; focus on the clinically relevant outcomes of growth and rupture; design studies with the rigor required of human clinical trials; monitor AAA growth using reproducible ultrasound; and perform studies in both males and females. SUMMARY OF STRENGTHS AND WEAKNESSES OF MOUSE MODELS: The aortic adventitial elastase oral ß-aminopropionitrile model has many strengths including simulating human AAA pathology and modeling prolonged aneurysm growth. The Ang II (angiotensin II) model performed less well as it better simulates acute aortic syndrome than AAA. The elastase plus TGFß (transforming growth factor-ß) blocking antibody model displays a high rupture rate, making prolonged monitoring of AAA growth not feasible. The elastase perfusion and calcium chloride models both display limited AAA growth.

Targeting of G protein-coupled receptor 39 alleviates angiotensin II-induced renal damage by reducing ribonucleotide reductase M2.

Renal fibrosis, apoptosis and autophagy are the main pathological manifestations of angiotensin II (Ang II)-induced renal injury. G protein-coupled receptor 39 (GPR39) is highly expressed in various tissues including the kidney, but its role in the kidney is entirely unclear. This study was performed to investigate the underlying mechanism by which knockdown of GPR39 alleviated Ang II-induced renal injury. In vivo, GPR39 knockout (KO) mice were constructed and infused with Ang II for 4 weeks, followed by renal function tests. In vitro, Ang II-induced cells were treated with si-GPR39 for 48 h. Fibrosis, apoptosis and autophagy were detected in both cells and mice. The underlying mechanism was sought by mRNA transcriptome sequencing and validated in vitro. GPR39 was upregulated in renal tissues of mice with Ang II-mediated renal injury. Knockdown of GPR39 ameliorated renal fibrosis, apoptosis, and autophagy, and decreased the expression of ribonucleotide reductase M2 (RRM2). In vitro, knockdown of GPR39 was also identified to improve the Ang II-induced cell fibrosis, apoptosis, and autophagy. mRNA transcriptome results showed that knockout of GPR39 reduced the expression of RRM2 in Ang II-induced kidney tissue. Activation of RRM2 could reverse the therapeutic effect of GPR39 knockout, and the inhibitor of RRM2 could improve the cell fibrosis, apoptosis and autophagy caused by GPR39 agonist. These results indicated that targeting of GPR39 could alleviate Ang II-induced renal fibrosis, apoptosis, and autophagy via reduction of RRM2 expression, and GPR39 may serve as a potential target for Ang II-induced renal injury.

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

Inhibition of the angiotensin II type 2 receptor AT2R is a novel therapeutic strategy for glioblastoma.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

Angiotensin II enhances bacterial clearance via myeloid signaling in a murine sepsis model.

Sepsis, defined as organ dysfunction caused by a dysregulated host-response to infection, is characterized by immunosuppression. The vasopressor norepinephrine is widely used to treat low blood pressure in sepsis but exacerbates immunosuppression. An alternative vasopressor is angiotensin-II, a peptide hormone of the renin-angiotensin system (RAS), which displays complex immunomodulatory properties that remain unexplored in severe infection. In a murine cecal ligation and puncture (CLP) model of sepsis, we found alterations in the surface levels of RAS proteins on innate leukocytes in peritoneum and spleen. Angiotensin-II treatment induced biphasic, angiotensin-II type 1 receptor (AT1R)-dependent modulation of the systemic inflammatory response and decreased bacterial counts in both the blood and peritoneal compartments, which did not occur with norepinephrine treatment. The effect of angiotensin-II was preserved when treatment was delivered remote from the primary site of infection. At an independent laboratory, angiotensin-II treatment was compared in LysM-Cre AT1aR-/- (Myeloid-AT1a-) mice, which selectively do not express AT1R on myeloid-derived leukocytes, and littermate controls (Myeloid-AT1a+). Angiotensin-II treatment significantly reduced post-CLP bacteremia in Myeloid-AT1a+ mice but not in Myeloid-AT1a- mice, indicating that the AT1R-dependent effect of angiotensin-II on bacterial clearance was mediated through myeloid-lineage cells. Ex vivo, angiotensin-II increased post-CLP monocyte phagocytosis and ROS production after lipopolysaccharide stimulation. These data identify a mechanism by which angiotensin-II enhances the myeloid innate immune response during severe systemic infection and highlight a potential role for angiotensin-II to augment immune responses in sepsis.

Reduced cardiac antioxidant defenses mediate increased susceptibility to workload-induced myocardial injury in males with genetic cardiomyopathy.

Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking ß-sarcoglycan (ß-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this ß-SG-/- model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in ß-SG-/- mice. Male ß-SG-/- hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female ß-SG-/- hearts. This protection of females was independent of ovarian hormones. Male ß-SG-/- hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT1R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of ß-SG-/- male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT1R-mediated amplification of workload-induced myocardial injury in male ß-SG-/- mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male ß-SG-/- heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury.

Bidirectional relation between dipeptidyl peptidase 4 and angiotensin II type I receptor signaling.

Cardiometabolic diseases are often associated with heightened levels of angiotensin II (Ang II), which accounts for the observed oxidative stress, inflammation, and fibrosis. Accumulating evidence indicates a parallel upregulation of dipeptidyl dipeptidase 4 (DPP4) activity in cardiometabolic diseases, with its inhibition shown to mitigate oxidative stress, inflammation, and fibrosis. These findings highlight an overlap between the pathophysiological mechanisms used by Ang II and DPP4. Recent evidence demonstrates that targeted inhibition of DPP4 prevents the rise in Ang II and its associated molecules in experimental models of cardiometabolic diseases. Similarly, inhibitors of the angiotensin I-converting enzyme (ACE) or Ang II type 1 receptor (AT1R) blockers downregulate DPP4 activity, establishing a bidirectional relationship between DPP4 and Ang II. Here, we discuss the current evidence supporting the cross talk between Ang II and DPP4, along with the potential mechanisms promoting this cross regulation. A comprehensive analysis of this bidirectional relationship across tissues will advance our understanding of how DPP4 and Ang II collectively promote the development and progression of cardiometabolic diseases.

Honokiol ameliorates angiotensin II-induced cardiac hypertrophy by promoting dissociation of the Nur77-LKB1 complex and activating the AMPK pathway.

Pathological cardiac hypertrophy is a key contributor to heart failure, and the molecular mechanisms underlying honokiol (HNK)-mediated cardioprotection against this condition remain worth further exploring. This study aims to investigate the effect of HNK on angiotensin II (Ang II)-induced myocardial hypertrophy and elucidate the underlying mechanisms. Sprague-Dawley rats were exposed to Ang II infusion, followed by HNK or vehicle treatment for 4 weeks. Our results showed that HNK treatment protected against Ang II-induced myocardial hypertrophy, fibrosis and dysfunction in vivo and inhibited Ang II-induced hypertrophy in neonatal rat ventricular myocytes in vitro. Mechanistically, HNK suppressed the Ang II-induced Nur77 expression at the transcriptional level and promoted ubiquitination-mediated degradation of Nur77, leading to dissociation of the Nur77-LKB1 complex. This facilitated the translocation of LKB1 into the cytoplasm and activated the LKB1-AMPK pathway. Our findings suggest that HNK attenuates pathological remodelling and cardiac dysfunction induced by Ang II by promoting dissociation of the Nur77-LKB1 complex and subsequent activation of AMPK signalling. This study uncovers a novel role of HNK on the LKB1-AMPK pathway to protect against cardiac hypertrophy.