AngII Infusion Research Articles

Abstract MP13: Hemolysis-mediated Prolyl Endo-Peptidase plasma release – a natural protective mechanism against extreme blood pressure elevations?

During hypertensive crises (BP >180/120 mm Hg), turbulent passage of red blood cells through severely constricted blood vessels can cause blood cell fragmentation and intravascular hemolysis. Consistent with this, in mice we found that a rapid and large increase in BP (by ~60-70 mm Hg) induced by an Ang II-infusion (0.2 ug/g BW) resulted in hemolysis. Prolyl Endo-Peptidase (PEP) is an intracellular enzyme that converts Ang II to Ang-(1-7), thereby shifting from a powerful vasopressor peptide to a counter regulatory peptide. The Ang II-induced acute BP increase that caused a visible hemolysis was associated with a ~4-fold increase in PEP activity (Figure, Panel A). A massive hemolysis induced by freeze-thawing of blood (cryo-hemolysis), moreover, was associated with a ~20x increase in PEP activity as compared to native plasma (Panel B). Of note, mouse cryo-hemolyzed plasma (HP) ex vivo was able to produce substantially more Ang-(1-7) peptide from spiked Ang II than equivalent amount of non-hemolyzed plasma (P). In addition, when PEP inhibitor, S-17092, was added to cryo-hemolyzed plasma (HP inh), Ang-(1-7) formation from Ang II was greatly diminished (Panel C). This suggests that blood corpuscles acting via intracellular PEP are endowed with a larger capacity than native plasma to catabolize the vasoconstrictor peptide Ang II to the vasodilator peptide Ang-(1-7). In conclusion, a large and rapid increase in BP after Ang II infusion causes hemolysis which is associated with the release of intracellular PEP enzyme from blood cells. We propose that the release of PEP confers a protective mechanism against severe BP elevations during hypertensive crisis by granting higher plasma Ang II-degrading capability thereby lowering Ang II and BP. This occurs at the expense of temporary hemolysis in the setting of hypertensive crisis as seen with malignant hypertension.

Abstract P389: Immunogenic Peptides Responsible for Experimental Hypertension Contribute to Systemic Lupus Erythematosus

Introduction: Hypertension and systemic lupus erythematosus (SLE) lead to modification of self-peptides by isolevuglandin (isoLG) lipid oxidation products. These self-peptides are presented by class 1 major histocompatibility complexes and drive CD8 + T cell activation. We have identified self-peptides from the sodium/glucose cotransporter2 (SGLT2); cadherin 16; Kelch domain-containing protein 7A and solute carrier family 23 that are IsoLG-adducted and cause CD8 + T cell activation in both ang II and L-NAME/high salt models of hypertension. These prime hypertension in response to low dose ang II. Hypothesis: We hypothesized that the same immunogenic peptides responsible for experimental hypertension contribute to systemic lupus erythematosus. Methods: A fluorescent probe made of recombinant IgG and the murine class 1 MHC H2-D b was loaded with either unadducted SGLT2 peptide or an isoLG-adducted SGLT2 peptide. This probe was used in flow cytometry to detect CD8 + T cells specific for this peptide antigen in 12-week-old C57Bl/6 and female B6SLE1.2.3 mice. Results: The unadducted SGLT2 probe recognized < 1% of CD8 + T cells (Figure). In contrast, ang II-infusion caused a striking increase in IsoLG-SGLT2 specific CD8 + T cells in C57Bl/6 aortas (Figure panels A and D). In C57Bl/6 very few CD8 + T cells recognizing IsoLG-SGLT2 were observed in the spleen (Panel B and E). Importantly, from 5 to 20% of CD8 + T cells in spleens of female B6SLE1.2.3 mice recognized the IsoLG-SGLT2 peptide (Figure panels C and E). Image 1. Conclusions: These findings strongly suggest that IsoLG-adducted peptides responsible for ang II-induced hypertension contribute to SLE and suggest that these conditions share similar immunogenicity.

Abstract MP04: Brain miR-410-3p Expression Sex-dependently Reduces Ang-II-induced Hypertension

Dysregulation of microRNA (miRNA) is associated with hypertension and other diseases. We previously observed direct interaction of miR-410-3p with AT 1 R and sex-dependent downregulation of miR-410-3p in the hypothalamus of a mouse cardiometabolic disease (CMD) model, suggesting a possible involvement in the development of CMD through modulation of the renin-angiotensin system. To further investigate the role of hypothalamic miR-410-3p expression as a therapeutic target for hypertension, acute Ang-II injection and chronic infusion experiments were performed. In acute experiments, C57BL/6J mice (males and females) were injected with Lenti-miR-410-3p or a control virus (icv, 2 µl, n=5-6). After 3 weeks, mice were anesthetized and a catheter was inserted to the carotid artery for blood pressure (BP) monitoring, followed by a bolus icv injection of Ang-II (200 ng/200 nl). Changes in systolic BP (ΔSBP) were recorded. In chronic experiments, mice were fed with a high calorie diet for 2 months before icv virus injection (2 µl). Telemetry probes (DSI) were implanted for conscious BP monitoring. Three weeks following virus injection, all mice were infused with Ang-II using osmotic minipumps (sc, 600 ng/kg/min, 14 days) to induce CMD. BP was recorded weekly and 24 h average of mean arterial pressure (MAP) was calculated. Acute Ang-II injection-induced pressor responses were similar in males (ΔSBP; 22.2±4.7 mmHg) and females (20.6±6.5 mmHg) transfected with the control virus. In lenti-miR-410-3p transfected mice, the pressor responses were blunted in both males (6.7±4.8 mmHg, P <0.001 vs. control) and females (10.9±4.7 mmHg, P<0.05 vs. control), with a greater reduction observed in males (69.8% vs. 47%). In the HCD model, baseline MAP was not different between male control and Lenti-miR-410, or female control and Lenti-miR-410 group. Following Ang-II infusion, MAP was increased in both sexes treated with control virus. Interestingly, this increase was blunted by Lenti-miR-410-3p expression in males (137.3 ± 9.4 vs. 160.7 ± 4.3 mmHg, P =0.05) but not in females (140.4±4.6 vs. 140.5 ± 4.2 mmHg). These data are aligned with our previous findings that male mice had higher expression of hypothalamic AT 1 R than in females in CMD. In summary, miR-410-3p inhibits both acute and chronic Ang-II induced increases in BP in a sex-dependent manner, with more profound effects in males. miR-410-3p could be a new strategy for the treatment of hypertension in CMD.

O100 IL15RA MEDIATES BLOOD PRESSURE ELEVATION, INFLAMMATION AND VASCULAR DAMAGE IN ANGIOTENSIN II-INDUCED HYPERTENSION

Cytokines are key mediators regulating blood pressure (BP) and target organ damage in hypertension. IL15 levels increase in cardiovascular diseases, but the role of IL15 and its receptor alpha (IL15R) in hypertension and hypertension-mediated inflammation and vascular damage is still unknown. Wild-type and IL15Rα-/-mice (n=5-9) were infused with vehicle or angiotensin II (AngII, 490ng/min/kg). Recombinant IL15 was administered to WT mice (1ug, daily, 14 days) during AngII infusion. Telemetry, wire myography, luminometry, PSR staining, qPCR, immunoblotting, flow cytometry, and primary cell lines were used. Hypertensive patients have increased levels of IL15Rα in arteries, which significantly correlated with systolic (r=0.33, p=0.002), diastolic (r=0.27, p=0.02) pressure and maximal constriction to phenylephrine ex vivo (r=0.27, p=0.03). Similarly, AngII infusion significantly increased serum IL15/IL15Rα (8.8±0.7pg/ml vs 12.1±0.7pg/ml, p<0.01), IL15Rα mRNA (1±0.1 vs 1.8±0.1, p<0.01) and protein (1±0.03 vs 1.8±0.28, p<0.01) levels in the aortas. BP lowering using hydralazine and hydrochlorothiazide treatment prevented these changes in vasculature. IL15Rα was induced by pathophysiological stretch (15%) or interferon-gamma in adventitial fibroblasts, vascular smooth muscles, and endothelial cells in vitro. In contrast, AngII, other inflammatory cytokines or oxidative stress mediators have no such direct effect. Il15Rα-/- blunted AngII-induced hypertension (199±5.6mmHg vs 148±7.3mmHg, p<0.001), protected against endothelial dysfunction (p<0.01) and aortic superoxide production (59.4±7.8 vs 36.4±7.6 RLU/sec/mg) compared to wild-type mice. This was accompanied by reduced perivascular inflammation, lack of immune cell recruitment and effector and memory T cell formation. Similarly, T cell interferon-gamma and interleukin-17 production was attenuated in Il15Rα-/- hypertensive mice. In contrast, simultaneous administration of AngII and rIL15 into WT mice enhanced these outcomes. Perivascular fibrosis was observed in WT, but not Il15Rα-/- hypertensive animals (111036±19117 μm2 vs 48689±5515 μm2, p<0.01) and was in line with significantly lower mRNA of collagen and fibronectin protein levels in Il15Rα-/- aortas. IL15Rα plays a crucial role in immune activation, vascular damage, and AngII-induced hypertension. Targeting this pathway may offer potential therapeutic benefits for controlling hypertension and preventing associated vascular complications.

O47 THE ROLE OF IL-33 AND ST2 IN THE MODULATION OF TARGET ORGAN DAMAGE IN ANGIOTENSIN II-DEPENDENT HYPERTENSION

Introduction: Hypertension is associated with a chronic inflammatory state and aberrant immune system activity. Interleukin-33/suppression of tumorigenicity 2 (IL-33/ST2) signalling axis is important in the modulation of atherosclerosis and cardiovascular fibrosis. We aimed to evaluate the modulation of IL-33 and ST2 in hypertensive mouse models and patients. Methods and Results: Hypertension was induced by a 14-day infusion of Ang-II (490ng/min/kg) in 12-week-old WT C57BL/6, ST2KO, IL33KO mice. IL-33 and ST2 mRNA and protein were evaluated using qPCR and Western blot in WT mice. Ang II-infusion significantly increased IL-33 and ST2 protein expression in target organs related to hypertension, particularly the aorta (1.0±0.1 vs 2.8±0.2, p<0.0001, and 0.963±0.068 vs 2.094±0.231, p=0.002, respectively). Further, mRNA expression of IL-33 and ST2 was elevated in hypertensive aortas when compared with control (1.0±0.053 vs 1.76±0.271, p=0.0185 and 1.0±0.039 vs 1.978±0.221, p=0.0039, respectively). IHC from WT mice illustrated the localisation of IL-33 and ST2 in Ang-II aortas in ECs and SMCs. Further in human hypertensive mammary arteries, the localisation was. In-vivo, IL33KO and ST2KO mice showed no altered BP at baseline or after 14 days of Ang-II compared to WT littermates. ST2KO Ang-II mice showed protection against endothelial dysfunction compared to WT Ang-II mice through isometric tension studies (p=0.0317). Additionally, Ang-II-induced IL33KO and ST2KO mice worsen cardiac fibrosis compared to WT littermates (p=0.0005,p<0.0001, respectively). Conclusion: Both IL-33 and its receptor, ST2 are increased in the vascular and perivascular tissues in hypertension. Additionally, the IL-33/ST2 axis has a role in regulating cardiac fibrosis.

P109 AMINOPEPTIDASE A: A POTENTIAL NOVEL THERAPY FOR ANGIOTENSIN-DEPENDENT HYPERTENSION?

Background: We have shown that mice with a genetic deficiency in Aminopeptidase A (APA) have elevatedblood pressure. APA is an enzyme that removes a single aspartate residue from the amino-terminus of several peptides in the renin-angiotensin systems (RAS), including angiotensin (Ang) II. In this study, we investigated the impact of a recombinant APA on the cleavage of various RAS peptides and its effect on blood pressure. Methods: An in vitro assay was used to identify Ang peptides as substrates for APA. Ang peptides identified as APA substrates using this assay were then injected to anesthetized WT and APA-deficient mice to assess their pressor effect by measuring systolic blood pressure (SBP). Subsequently, murine recombinant (r)APA was used to evaluate its action on the acute SBP elevation elicited by Ang peptides that were identified to trigger pressor response. Results: In the in vitro assay, Ang II (22427±3353, n=12), Ang I (21883±1692, n=3), Ang1-7 (15833±3395,n=3), Ang1-9 (15773±3395, n=3) and Ang1-12 (13170±220, n=3), were identified as a substrate for rAPA as indicated by a strong aspartate-driven fluorescence formation. When these five Ang peptides were administered to WT mice, only Ang I, Ang II and Ang 1-12 caused a significant increase in SBP. This effect was exaggerated in APA deficient mice. The SBP increase elicited by Ang II in WT mice was markedly reduced by the pre-administration of rAPA (Fig). Conclusion: Aminopeptidase A can cleave the N-terminal aspartate from Ang II as well as from other RASpeptides that elicit (Ang 1-12, Ang I) and do not elicit (Ang 1-9, Ang 1-7) pressor response in mice. APA deficiency is associated with an exaggerated SBP response to Ang I, Ang II and Ang 1-12 while the recombinant enzyme mitigates the acute hypertensive effect. rAPA markedly reduced the increase in SBP caused by AngII infusion suggesting that it may be used to treat hypertension caused by excess of AngII.

Abstract Tu027: Impact of Atherosclerosis-Related Small Cerebral Vessel Damages on Tau Pathology in Atherosclerosis Mouse Model

Background: The linkage between atherosclerosis and vascular dementia, including its ties to Alzheimer’s disease (AD), is well-established. This research explores how small cerebral vessel damages, a less recognized consequence of atherosclerosis, contribute to the development of tau pathology, a key AD hallmark. Methods and Results: Through the ApoE-/- mouse model, we evaluated the impact of hypercholesterolemia and AngII infusion on tau pathology. The regimen included an atherogenic diet for three months and subsequent AngII infusion (1mg/kg/min) for 28 days in three-month-old mice. Key outcomes measured were phosphorylated tau (AT8), cerebral microbleeds, blood component deposition, and oxidative stress markers. Results indicated a synergistic effect of diet and AngII on increasing microbleeds, vascular leakage, and oxidative stress, which coincided with heightened p-tau in affected brain regions. A specific focus on endothelial glutaredoxin-1 (Glrx) deficiency revealed its critical role in moderating these pathological changes, linking endothelial oxidative stress directly with tau pathology. Conclusion: Our research highlights the significant role of small cerebral vessel damage in advancing tau pathology in atherosclerosis, with endothelial oxidative stress as a key mediator. This underscores novel pathways through which vascular health influences AD pathology.

WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin β1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin β1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Abstract 3038: Platelet-specific Deletion Of Transforming Growth Factor-β1 Augments Angiotensin Ii-induced Abdominal Aortic Aneurysm In Mice

Background: Transforming growth factor beta (TGFβ) blockade via systemic antibody targeting has been shown to augment murine AAA progression in multiple independent studies. Despite these findings, the primary source of protective TGFβ within AAA remains unclear. Previous research conducted in our laboratory demonstrates deletion of platelet-specific TGFβ1 exacerbates aneurysms in the elastase mouse model of AAA. Our present study extends these results by showing a comparable phenotype in the angiotensin II (Ang II) murine model of AAA. Methods and Results: Low density lipoprotein receptor deficient ( Ldlr -/- ) mice with platelet-specific deletion of Tgfβ1 were created by breeding male mice expressing Cre recombinase under the control of a Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Male mice were fed high fat diet 1 week prior to and throughout subcutaneous infusion of AngII (1,000 ng/kg/min). After day 28 of infusion, ex vivo aortic diameter was significantly increased in Cre+ (n = 17) vs Cre- (n = 18) mice ( Cre- : 1.75 ± 0.17 mm; Cre+ : 2.43 ± 0.21 mm; P < 0.05). AAA incidence, defined as aortic diameter ≥ 1.2mm, was 56% for Cre- mice vs. 93% in the Cre+ group. Histological analysis of picrosirius red staining revealed a marked reduction in type 1 abdominal collagen deposition as a proportion of total cellular area in aortas from Cre+ mice versus Cre- controls. OCT embedded sections of aneurysms from both cohorts were also probed for macrophage marker CD68 and analyzed using immunofluorescent microscopy. We found Cre+ aortas had significantly increased macrophage infiltration compared to Cre- aortas. Conclusions: This study of platelet-specific deletion of TGFβ1 in a secondary model of murine AAA supports the conclusion that TGFβ1 sourced form platelets plays a critically important role in the protection of AAA.

Investigating mechanisms of diminished GABAergic inhibitory effcacy in the hypothalamic paraventricular nucleus (PVN) of AngII-salt hypertensive mice

The PVN receives glutamatergic excitation from sodium and AngII sensing regions of the forebrain along with dense GABAergic inhibition from the surrounding peri-nuclear zone (PNZ). Studies in various models of hypertension and heart failure report that GABAergic inhibitory tonus is diminished, contributing to the net increase in PVN-driven sympathetic nerve activity that contributes to the pathogenesis of these chronic and prevalent cardiovascular diseases. Here we compared GABAergic inhibition of the PVN in normotensive mice consuming a normal salt (0.4% NaCl) diet (NSD) and hypertensive mice consuming a high salt (4% NaCl) diet (HSD) with systemic infusion of AngII (600 ng/kg/min, s.c.) (AngII-salt HTN). Using gramicidin perforated patch recordings in brain slices, we determined that the GABA equilibrium potential (EGABA) of PVN neurons was significantly depolarized in the AngII-salt HTN group (Δ+34.3 ± 7.5 mV) compared to the NSD group (n=3 cells) (P=0.003), consistent with diminished inhibitory effcacy of GABA and reflecting increased intracellular [Cl−]. Western blot analysis of PVN punches yielded perplexing results, showing increased expression of the Cl− efflux transporter KCC2 (+12%) and diminished expression of the Cl− influx transporter NKCC1 (-38%) — suggestive of compensation for a separate cause of elevated intracellular [Cl−]. Previously, we determined that EGABA can be rapidly and reversibly depolarized (+9 mV) by perfusing slices with high K+ (+10 mM) aCSF in the presence of ionotropic glutamate receptor blockade with CNQX (10 μM) and APV (50 μM). Hence it seems that heightened synaptic GABA release is capable of increased intracellular [Cl−]. Given that the magnitude of the latter effect is small relative to the measured depolarization of EGABA in AngII-salt HTN neurons, increased synaptic GABA release appears inadequate to explain the measured increase of EGABA in AngII-salt HTN. We posit that glial buffering of extracellular Cl− might contribute to the unexpectedly large EGABA depolarization in AngII-salt HTN. Notably, EGABA among PNZ GABAergic neurons was also significantly depolarized in the AngII-salt HTN group (Δ+22.4 ± 8.8 mV, n=3 cells) compared to NSD controls (n=3 cells) (P=0.037). Mechanism(s) supporting this are presently unknown. Of special interest, preliminary data indicate that most GABAergic PNZ neurons have recurrent GABAergic responses that are predicted (based on having depolarized EGABA) to have diminished feedback inhibition in the AngII-salt group. If true, PNZ neurons would be expected to increase synaptic GABA release in the PVN of mice with AngII-salt HTN. What contribution the latter effect makes to the net depolarization of EGABA in PVN neurons is under investigation. NS115072 (GMT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2020: Vascular Glucose Transporter 1 Is Dispensable For Abdominal Aortic Aneurysm Induced By Angiotensin II

Background: Pharmacological inhibition of glycolysis attenuated abdominal aortic aneurysm (AAA) development in mice. Glucose transporters (GLUTs) incorporate glucose as a first step of glycolysis. GLUT1 over-expression in vascular smooth muscle cells (VSMC) leads to enhancement of atherosclerosis. However, the role of VSMC GLUT1 in AAA remains unclear. Hypothesis: VSMC GLUT1 deletion attenuates AAA development and rupture in mice. Goals/Aims: Our goal was to establish contribution of VSMC GLUT1 in AAA. Methods: The glucose consumption of cultured VSMCs was evaluated in the presence or absence of AngII stimulation and GLUT1 inhibitor BAY-876. GLUT1 floxed mice were crossed with tamoxifen-inducible SMMHC-Cre mice. At 6 weeks of age, mice were injected with tamoxifen. As a control, tamoxifen-treated SMMHC-Cre male mice were utilized. At 8 weeks, angiotensin II (AngII, 1 μg/kg/min) was delivered for 4 weeks. In addition, mice received 1 mg/mL BAPN during the first 2 weeks of AngII infusion. After 4weeks, all animals were euthanized and maximal external diameter of the abdominal aorta was measured. Results: In cultured VSMC, the glucose consumption increased by AngII was attenuated by pretreatment with GLUT1 inhibitor (media glucose 257.9±4.5 vs 238.9±6.2 mg/dL, p<0.05). AngII plus BAPN significantly developed AAA in control mice (diameter 1.05±0.07 vs 2.37±0.31mm, p<0.05). However, silencing of VSMC GLUT1 did not affect AAA development by AngII plus BAPN (2.41±0.46mm, p=0.66). The survival rate due to aortic rupture in VSMC GLUT1 silenced mice and control mice treated with BAPN and AngII were 60.0% and 57.1%, respectively (p=0.820). AngII induced hypertension in control mice (102±3.8 vs 133±7.1mmHg, p<0.05) and VSMC GLUT1 silenced mice (149±11.4mmHg, p=0.99). Discussion Although GLUT1 may be a primary VSMC GLUT to enhance glucose utilization in response to AngII stimulation, VSMC GLUT1 silencing did not alter AngII-dependent AAA development or rupture in a mouse model. Since pharmacological inhibition of GLUT attenuated AAA and associated macrophage infiltration, it is possible that GLUT1 expressed in macrophages may contribute to development of AAA. Conclusion: VSMC GLUT1 silencing did not alter AngII-dependent AAA development in a mouse model.

Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway.

Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. We generated a microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t-test were used to analyze the data. Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.

TEA domain transcription factor 1 (TEAD1) induces cardiac fibroblasts cells remodeling through BRD4/Wnt4 pathway

Cardiac fibroblasts (CFs) are the primary cells tasked with depositing and remodeling collagen and significantly associated with heart failure (HF). TEAD1 has been shown to be essential for heart development and homeostasis. However, fibroblast endogenous TEAD1 in cardiac remodeling remains incompletely understood. Transcriptomic analyses revealed consistently upregulated cardiac TEAD1 expression in mice 4 weeks after transverse aortic constriction (TAC) and Ang-II infusion. Further investigation revealed that CFs were the primary cell type expressing elevated TEAD1 levels in response to pressure overload. Conditional TEAD1 knockout was achieved by crossing TEAD1-floxed mice with CFs- and myofibroblasts-specific Cre mice. Echocardiographic and histological analyses demonstrated that CFs- and myofibroblasts-specific TEAD1 deficiency and treatment with TEAD1 inhibitor, VT103, ameliorated TAC-induced cardiac remodeling. Mechanistically, RNA-seq and ChIP-seq analysis identified Wnt4 as a novel TEAD1 target. TEAD1 has been shown to promote the fibroblast-to-myofibroblast transition through the Wnt signalling pathway, and genetic Wnt4 knockdown inhibited the pro-transformation phenotype in CFs with TEAD1 overexpression. Furthermore, co-immunoprecipitation combined with mass spectrometry, chromatin immunoprecipitation, and luciferase assays demonstrated interaction between TEAD1 and BET protein BRD4, leading to the binding and activation of the Wnt4 promoter. In conclusion, TEAD1 is an essential regulator of the pro-fibrotic CFs phenotype associated with pathological cardiac remodeling via the BRD4/Wnt4 signalling pathway.

Abstract TP36: Angiotensin II Induced Hypertension Increases the Brain Expression and Enzymatic Activity of CD38

Introduction: CD38 is an ectoenzyme that was shown to mediate the age-dependent decrease in NAD + levels. We have shown that CD38 expression and enzymatic activity are increased in the brain of spontaneously hypertensive stroke-prone rats, a model of human cerebral small vessel disease. Therefore, we hypothesized that hypertension (HTN) induced by angiotensin II (ANG-II) infusion leads to an increase in CD38 expression and enzymatic activity. Methods: Male C57BL/6J mice (10-13) weeks of age underwent subcutaneous infusion of ANG-II (600 ng/kg/min) or saline for a 2-week duration through implanted osmotic pumps. CD38 expression was measured by immunofluorescence and Western Blotting. CD38 enzymatic activity was assessed in the brain homogenate by measuring the NAD + analogue conversion to the fluorescent product (ε-ADPR). Dihydroethidium, diaminofluorescein, JZL1707 sensor, and immunohistochemistry were used to measure the levels of superoxide, nitric oxide (NO), NAD(P)H, and endothelial nitric oxide synthase (eNOS) in the brain, respectively. Results: Ang-II infusion caused a slow rise of blood pressure leading to HTN. The brain of ANG-II infused mice showed 48% increase in the expression (P=0.013) and 73% increase in enzymatic activity (P=0.044) of CD38 compared to control mice. The expression of CD38 localized mainly to the endothelial cells in the ANG-II infused mice brains. The brains of ANG-II infused mice also had 26% lower levels of NAD(P)H (P<0.0001), 226% higher superoxide (P<0.0001), 32% lower NO (P<0.0001) and 30% lower eNOS levels (P=0.003) compared to control mice. Conclusions: ANG-II induced HTN increases CD38 expression and enzymatic activity with oxidative stress and lower levels of NAD(P)H. These results suggest a potential role of elevated CD38 enzymatic activity in the pathogenesis of cerebrovascular dysfunction induced by ANG-II as well as that seen in other models of HTN.

Histamine H2-receptor antagonism improves conduit artery endothelial function and reduces plasma aldosterone level without lowering arterial blood pressure in angiotensin II–hypertensive mice

Aldosterone through the mineralocorticoid receptor MR has detrimental effects on cardiovascular disease. It reduces the bioavailability of nitric oxide and impairs endothelium-dependent vasodilatation. In resistance arteries, aldosterone impairs the sensitivity of vascular smooth muscle cells to nitric oxide by promoting the local secretion of histamine which activates H2 receptors. The present experiments tested in vivo and ex vivo the hypothesis that systemic H2-receptor antagonism reduces arterial blood pressure and improves vasodilatation in angiotensin II–induced chronic hypertension. Hypertension was induced by intravenous infusion of angiotensin II (60 ng kg−1 min−1) in conscious, unrestrained mice infused concomitantly with the H2-receptor antagonist ranitidine (27.8 µg kg−1 min−1) or vehicle for 24 days. Heart rate and arterial blood pressure were recorded by indwelling arterial catheter. Resistance (mesenteric) and conductance (aortae) arteries were harvested for perfusion myography and isometric tension recordings by wire myography, respectively. Plasma was analyzed for aldosterone concentration. ANGII infusion resulted in elevated arterial blood pressure and while in vivo treatment with ranitidine reduced plasma aldosterone concentration, it did not reduce blood pressure. Ranitidine improved ex vivo endothelial function (acetylcholine 10−9 to 10−6 mol L−1) in mesenteric resistance arteries. This was abolished by ex vivo treatment with aldosterone (10−9 mol L−1, 1 h). In aortic segments, in vivo ranitidine treatment impaired relaxation. Activation of histamine H2 receptors promotes aldosterone secretion, does not affect arterial blood pressure, and protects endothelial function in conduit arteries but promotes endothelial dysfunction in resistance arteries during angiotensin II–mediated hypertension. Aldosterone contributes little to angiotensin II–induced hypertension in mice.

Abstract 11310: T Helper 1 Lymphocytes Promote Diastolic Dysfunction and Fibrosis in a Non-Hypertensive Angiotensin II Mouse Model of Heart Failure

Introduction: Angiotensin (Ang) II induces cardiac fibrosis and diastolic dysfunction, also in absence of hypertension (HTN). We found that a non-HTN AngII infusion fails to induce dysfunction and fibrosis in T cell receptor α deficient mice ( Tcrα -/- , bereft of T cells). Hypothesis: Since T helper 1 (Th1) lymphocytes promote adverse remodeling in models of heart failure (HF), we hypothesized that Th1 cells are sufficient to induce diastolic dysfunction and fibrosis in a non-HTN AngII model. Methods: Naïve CD4+ cells were isolated from wild type (WT) mice lymph nodes (LNs) and spleens (MACS columns) and differentiated into Th1 cells over 7 days using plates coated with anti-CD3 antibody (5 μg/ml) plus IL-12 (10 ng/ml) and anti-IL-4 (1 μg/ml) and from day 4, IL-2 (20 ng/ml). Adoptive transfer (AT) of 10 7 Th1 cells/500 μl PBS, naïve CD4+ cells, or PBS alone was performed into Tcrα -/- mice (3-11 months old, N=8-16/group) implanted with AngII (0.2 μg/kg/day) osmotic pumps for 4 wks. AT was repeated two weeks later. LV dimensions, ejection fraction (EF), and diastolic function (isovolumetric relaxation time - IRT) were measured with Doppler echocardiography. LV end-diastolic pressure (EDP) and peak systolic pressure (PSP) were measured with a Millar catheter. Heart slides were stained with picrosirius red to measure interstitial fibrosis. Results: All groups had similar LV dimensions, EF, and PSP (not shown). AT of naïve CD4+ cells or PBS did not increase the IRT vs baseline (7.3±0.9 vs 7.6±0.6 and 9.9±0.7 vs 8.8±0.7 ms). AT of Th1 cells significantly increased the IRT (15.6±0.9 vs 9.0±0.7 ms, all p<0.0001). EDP was significantly higher in Th1 mice (11.5±0.8 mmHg) vs naïve CD4+ mice (4.6±0.8 mmHg) or PBS (5.2±0.6 mmHg) (all p<0.0001). Interstitial fibrosis in Th1 mice (1.4±0.2%) was significantly higher than in naïve CD4+ mice (0.7±0.1%) or PBS (0.8±0.1%) (all p<0.001). Conclusions: Th1 cells are sufficient to induce diastolic dysfunction and fibrosis in a non-HTN AngII HF mouse model.

Abstract 081: Transactivation Of Brain ADAM17 In Angiotensin II-Induced Hypertension Can Be Prevented By Kinin B1 Receptor Blockade

A disintegrin and metalloprotease 17 (ADAM17) has been implicated in mediating inflammation in several cardiovascular diseases. Targeting brain ADAM17 using siRNA revealed a reduction in blood pressure, confirming its role in hypertension. Previously, we reported that kinin B1 receptor (B1R) activation increases ADAM17 activity in primary hypothalamic neurons, while pretreatment with a B1R antagonist mitigated this effect. However, whether there is a direct interaction between ADAM17 and B1R in hypertension is unknown. In this study, we tested the hypothesis that B1R blockade will prevent the transactivation of ADAM17, thereby, preventing the development of angiotensin II-induced hypertension. Male and female C57BL/6NJ wild-type (WT) and B1R knockout mice (B1RKO) were administered Ang II (600 ng/kg/min; SC, 2 weeks) or saline via osmotic minipumps. Ang II infusion significantly increased mean arterial pressure (radiotelemetry) in WT mice (145 ±13 mmHg vs. 103 ±6, n=6, p<0.01), which was attenuated in B1RKO mice. Both male and female B1RKO mice showed similar attenuation of Ang II-induced hypertension. Ang II-infusion led to a significant upregulation of ADAM17 in the PVN of WT mice and was blunted in B1RKO mice (p<0.05, n=3). Proximity ligation assay (PLA) was used to examine close proximity (<40nm) interactions between two receptors. ADAM17-B1R interactions in the PVN of hypertensive mice were significantly increased (p<0.05, n=3). To further understand this correlation, primary neurons and microglia from the hypothalamus of neonatal WT mice were cultured and treated with Ang II (300 nM) for 24 hours. Ang II increased B1R and ADAM17 protein expression in both cell types and was prevented by a B1R antagonist SSR240612 (10 uM) (p<0.05, n=5). To supplement these findings, PLA was used on primary neurons and microglia and revealed an increase in ADAM17-B1R interactions following Ang II. Furthermore, B1R antagonism attenuated this ADAM17-B1R interaction in both neurons and microglia (p<0.05, n=5). Our data provides novel evidence that B1R blockade can attenuate Ang II-induced hypertension in the brain, possibly through a reduction in B1R-ADAM17 interactions, suggesting that B1R blockade may serve as a potential antihypertensive therapeutic agent.

Abstract P218: Ubr1 And Nedd4-2 Synergistically Modulate Ace2 Ubiquitination In Hypertensive Male Mice

Angiotensin-converting enzyme 2 (ACE2) is a counter-regulatory player in the renin-angiotensin system (RAS). Downregulation of ACE2 in hypertension results in vasoconstriction, sympathetic over-activation, and hypertension. Recently, we identified UBR1, an E3 ubiquitin-protein ligase, as a potential ubiquitinase modulating ACE2 expression, notably in hypertensive male mice where UBR1 is upregulated in the hypothalamus and peripheral organs. Here, we aim to understand the role of UBR1 in hypertension and its crosstalk with Nedd4-2, another E3 ligase regulating ACE2. C57BI/6J male mice (3 month-old, n=18) were implanted with telemetry probes for weekly 24 h BP recording. One week following baseline BP recording, mice were implanted with subcutaneous osmotic pumps containing Ang-II (600 ng/kg/min). After confirming hypertension, mice were divided into 4 treatment groups (n=6/group); 1) peripheral UBR1 siRNA via osmotic pump (50 μg/day/2 weeks), 2) central UBR1 siRNA via intracerebroventricular infusion (ICV) (50 μg/day/2 weeks) 3) scrambled shRNA + Ang-II, and 4) no treatment. After 6 weeks of BP recording, mice were euthanized, and hypothalami were harvested for protein analysis (Capillary Western). BP was transiently reduced during the resting phase after 3 weeks of ICV UBR1 siRNA infusion compared to the Ang-II group (MAP: 114±7 vs . 135±3 mmHg). Ang-II infusion enhanced UBR1 protein levels versus no treatment (Fold Change:1.6±0.1 vs . 1.0±0.04; p<0.05) which was prevented by peripheral and central UBR1 siRNA infusion, respectively (0.7±0.08, 1.3±0.08 vs. 1.6±0.1, p<0.05). Interestingly, while UBR1 siRNA infusion increased ACE2 expression (1.7±0.1, 2±0.2, p<0.01, respectively), it was also associated with increased Nedd4-2 levels ( 2.9±0.1, 3.5±0.2, p<0.0001, respectively), and pNedd4-2 (4.1±0.4, 3.8±0.5, p<0.001, respectively). Together, these data suggest that while prevention of UBR1 upregulation in hypertension is associated with an increase in ACE2 expression, it is not sufficient to permanently reduce BP as other E3 ligases, like Nedd4-2, appear to compensate for the knockdown.

Volumetric Ultrasound Analysis of the Abdominal Aorta Enhances Strain Assessment in Hypertensive Mice

Aortic stiffness (AS) is a well-described and underutilized predictor of cardiovascular morbidity and mortality, particularly in hypertension (HTN). Employing noninvasive ultrasound technology to quantify AS metrics could have therapeutic application, but regional variation has compromised integration. We hypothesize that integrating 4-dimensional (4D) ultrasound with volumetric analysis will enhance the AS analysis in hypertensive mice as a foundation for clinical translation. Systolic blood pressure (SBP) was captured by Coda tail-cuff in normotensive C57Bl/6 mice (n = 3) on day 0. Under inhaled anesthetic, the Vevo3100 high-resolution micro-ultrasound was utilized to capture images of the infrarenal aorta which included (1) axial plane at three segments (proximal, middle, and distal); (2) longitudinal plane; and (3) 4D images of volumetric changes across the cardiac cycle. VevoVasc software enabled quantification of distensibility, pulse propagation velocity, global radial strain, and volumetric strain (VS). HTN was then induced by osmotic pump angiotensin-II infusion (1.46 mg/kg/day). SBP and ultrasound measurements were reassessed at 21 days. The t test was utilized to compare strain metrics at day 0 vs 21 with significance at P < .05. With AngII infusion, SBP increased 32 ± 2% (P < .05). Values for distensibility significantly decreased from proximal to distal aortic regions at day 0, and demonstrated a 50 ± 5% reduction with HTN at day 21 (P < .05). As another indicator of aortic stiffness in HTN, pulse propagation velocity increased >2.8-fold. Global radial strain, on the other hand, provided discordant results along the axial aortic regions at day 0 as well as day 21, and no change could be detected in the longitudinal plane. With 4D ultrasound to capture volume and normalize by pulse pressure to create the VS analysis, however, we detected a significant reduction in VS (0.74 ± 0.3 μL/mm Hg to 0.36 ± 0.1 μL/mm Hg; P < .05) to effectively quantify infrarenal aortic strain as an indicator of increased AS. Methodology to ensure sensitive, specific, and reproducible quantification of AS is vital to establishing clinical utilization. Ultrasound-derived VS analysis decreased regional variability and further investigation is warranted to define this technique in the medical management of HTN and other regional aortic pathology.

CMTM3 deficiency induces cardiac hypertrophy by regulating MAPK/ERK signaling

ObjectivesCardiac hypertrophy is the heart's compensatory response stimulated by various pathophysiological factors. However, prolonged cardiac hypertrophy poses a significant risk of progression to heart failure, lethal arrhythmias, and even sudden cardiac death. For this reason, it is crucial to effectively prevent the occurrence and development of cardiac hypertrophy. CMTM is a superfamily of human chemotaxis, which is involved in immune response and tumorigenesis. CMTM3 expressed widely in tissues, including the heart, but its cardiac function remains unclear. This research aims to explore the effect and mechanism of CMTM3 in the development of cardiac hypertrophy. Methods and resultsWe generated a Cmtm3 knockout mouse model (Cmtm3−/−) as the loss-of-function approach. CMTM3 deficiency induced cardiac hypertrophy and further exacerbated hypertrophy and cardiac dysfunction stimulated by Angiotensin Ⅱ infusion. In Ang Ⅱ-infusion stimulated hypertrophic hearts and phenylephrine-induced hypertrophic neonatal cardiomyocytes, CMTM3 expression significantly increased. However, adenovirus-mediated overexpression of CMTM3 inhibited the hypertrophy of rat neonatal cardiomyocytes induced by PE stimulation. In terms of mechanism, RNA-seq data revealed that Cmtm3 knockout-induced cardiac hypertrophy was related to MAPK/ERK activation. In vitro, CMTM3 overexpression significantly inhibited the increased phosphorylation of p38 and ERK induced by PE stimulation. ConclusionsCMTM3 deficiency induces cardiac hypertrophy and aggravates hypertrophy and impaired cardiac function stimulated by angiotensin Ⅱ infusion. The expression of CMTM3 increases during cardiac hypertrophy, and the increased CMTM3 can inhibit further hypertrophy of cardiomyocytes by inhibiting MAPK signaling. Thus, CMTM3 plays a negative regulatory effect in the occurrence and development of cardiac hypertrophy.