Ang II-treated Mice Research Articles

Angiotensin-Converting Enzyme-Dependent Intrarenal Angiotensin II Contributes to CTP: Phosphoethanolamine Cytidylyltransferase Downregulation, Mitochondrial Membranous Disruption, and Reactive Oxygen Species Overgeneration in Diabetic Tubulopathy.

Aims: The limited therapeutic options for diabetic tubulopathy (DT) in early diabetic kidney disease (DKD) reflect the difficulty of targeting renal tubular compartment. While renin-angiotensin-aldosterone system (RAS) inhibitors are commonly utilized in the management of DKD, how intrarenal RAS contributes to diabetic tubular injury is not fully understood. Mitochondrial disruption and reactive oxygen species (ROS) overgeneration have been involved in diabetic tubular injury. Herein, we aim to test the hypothesis that angiotensin-converting enzyme (ACE)-dependent intrarenal angiotensin II (AngII) disrupts tubular mitochondrial membranous homeostasis and causes excessive ROS generation in DT. Results: Mice suffered from renal tubular mitochondrial disruption and ROS overgeneration following high-fat diet/streptozocin-type 2 diabetic induction. Intrarenal AngII generation is ACE-dependent in DT. Local AngII accumulation in renal tissues was achieved by intrarenal artery injection. ACE-dependent intrarenal AngII-treated mice exhibit markedly elevated levels of makers of tubular injury. CTP: Phosphoethanolamine cytidylyltransferase (PCYT2), the primary regulatory enzyme for the biosynthesis of phosphatidylethanolamine, was enriched in renal tubules according to single-cell RNA sequencing. ACE-dependent intrarenal AngII-induced tubular membranous disruption, ROS overgeneration, and PCYT2 downregulation. The diabetic ambiance deteriorated the detrimental effect of ACE-dependent intrarenal AngII on renal tubules. Captopril, the ACE inhibitor (ACEI), showed efficiency in partially ameliorating ACE-dependent intrarenal AngII-induced tubular deterioration pre- and post-diabetic induction. Innovation and Conclusion: This study uncovers a critical role of ACE-dependent intrarenal AngII in mitochondrial membranous disruption, ROS overgeneration, and PCYT2 deficiency in diabetic renal tubules, providing novel insight into DT pathogenesis and ACEI-combined therapeutic targets. Antioxid. Redox Signal. 00, 000-000.

Hypertension-induced heart failure disrupts cardiac sympathetic innervation.

About 26 million people worldwide live with heart failure (HF), and hypertension is the primary cause in 25% of these cases. Autonomic dysfunction and sympathetic hyperactivity accompany cardiovascular diseases, including HF. However, changes in cardiac sympathetic innervation in HF are not well understood. We hypothesized that cardiac sympathetic innervation is disrupted in hypertension-induced HF. Male and female C57BL6/J mice were infused with Angiotensin II (AngII) for 4 weeks to generate hypertension leading to HF; controls were infused with saline. AngII-treated mice displayed HF phenotype including reduced cardiac function, hypertrophy, and fibrosis. AngII-treated mice also had significantly reduced sympathetic nerve density in the left ventricle, intraventricular septum, and right ventricle. In the left ventricle, the subepicardium remained normally innervated, while the subendocardium was almost devoid of sympathetic nerves. Loss of sympathetic fibers led to loss of norepinephrine content in the left ventricle. Several potential triggers for axon degeneration were tested and ruled out. AngII-treated mice had increased premature ventricular contractions after isoproterenol and caffeine injection. Although HF can induce a cholinergic phenotype and neuronal hypertrophy in stellate ganglia, AngII treatment did not induce a cholinergic phenotype or activation of trophic factors in this study. Cardiac neurons in the left stellate ganglion were significantly smaller in AngII-treated mice, while neurons in the right stellate were unchanged. Our findings show that AngII-induced HF disrupts sympathetic innervation, particularly in the left ventricle. Further investigations are imperative to unveil the mechanisms of denervation in HF and to develop neuromodulatory therapies for patients with autonomic imbalance.

Trifolin Attenuates Hypertension-Mediated Cardiac Injury by Inhibiting Cardiomyocyte Apoptosis: Mechanistic Insights and Therapeutic Potential

BackgroundHypertension-induced cardiac disease is a common complication and a significant contributor to mortality in hypertensive patients, largely due to cardiomyocyte apoptosis. Although Trifolin has been identified as a potential antihypertensive compound, its therapeutic role in hypertension-induced cardiac injury remains uncertain. PurposeThis study aims to evaluate the protective effects of Trifolin and explore the underlying mechanisms of its action against hypertension-induced cardiac injury. MethodsIn vivo, mice were infused with Angiotensin II (AngII, 500 ng/kg/min) or saline via osmotic pumps and treated with Trifolin (0.1, 1.0, or 10.0 mg/kg/day) or Valsartan (10 mg/kg/day) for four weeks. In vitro, H9C2 cells were stimulated with AngII (1 μM) and treated with Trifolin (25, 50, or 100 μM). Various assays, including echocardiography, hematoxylin and eosin staining, TUNEL assay, Annexin-V/propidium iodide staining, and JC-1 staining, were used to assess Trifolin's therapeutic effects on hypertension-related cardiac injury and cardiomyocyte apoptosis. Potential pharmacological mechanisms were analyzed through network pharmacology and confirmed via Western blotting. ResultsTrifolin treatment improved cardiac function by increasing left ventricular ejection fraction and fractional shortening while reducing tissue disorganization in AngII-treated mice. It also reduced cardiomyocyte apoptosis, reversing the upregulation of Bax and cleaved caspase-3 and the downregulation of Bcl-2. Network pharmacology identified 314 common targets of Trifolin linked to hypertensive heart disease, with involvement in apoptosis, MAPK, PI3K/AKT, and HIF-1 signaling pathways. Trifolin treatment increased p-PI3K/PI3K and p-AKT/AKT ratios while decreasing p-ERK/ERK, p-p38 MAPK/p38 MAPK, and p-JNK/JNK ratios in both mouse and cell models. ConclusionTrifolin alleviates AngII-induced cardiac injury and cardiomyocyte apoptosis, potentially through the regulation of MAPK, PI3K/AKT, and HIF-1 signaling pathways.

The role of IL15 in angiotensin II-mediated cardiac remodelling

Abstract Hypertension (HTN) mediated target organ damage, such as cardiac remodelling, is a major cause of death and disability. Cytokines are important regulators of cardiac function, inflammation, and remodelling in cardiovascular disease. Increased levels of interleukin 15 (IL15) have been noted in HTN, but its role in HTN heart disease remains unknown. This study investigates the role of the IL15 axis in the pathogenesis of cardiac remodelling in HTN. Male 12-week-old IL15 receptor α knock-out (IL15RAKO), IL15 knock-out (IL15KO) and matching wild type (WT) mice were administered vehicle or angiotensin II (AngII) (490 ng/min/kg) for 2 weeks by osmotic minipump. Blood pressure was measured by tail cuff (n=5-8). Cardiac function was analysed by echocardiography (n=3-4). Cardiac infiltrating immune cells were analysed by flow cytometry (n=2-3 for IL15RAKO, n=6-10 for IL15KO). Cardiac remodelling was assessed by picrosirius red, WGA staining (n=5-8) and expression of markers of remodelling using RT-qPCR and western blots (n=5-8). Data are presented as mean±SEM and analysed using two-way ANOVA. AngII infusion caused a significant increase in systolic blood pressure in WT and IL15KO animals. In contrast, in IL15RAKO, the response to AngII was blunted. This was in line with a decrease in ejection fraction, however only WT mice had an increase in global longitudinal strain (-20.3±2.0 vs -10.9±1.2, p=0.0039 in WT and 19.9±0.8 vs -16.0±2.1, p=0.1878 in IL15RAKO). HTN was associated with increased cardiac IL15 protein levels in mice. The heart-to-body ratio, along with cardiomyocyte cross-sectional, were significantly increased in WT, but not in IL15RAKO HTN mice. Furthermore, histological analysis showed significantly higher cardiac collagen accumulation in WT but not IL15RAKO upon AngII infusion (5.8±1.0% in WTvvs. 2.1±0.5% in IL15RAKO, p=0.0021). This was in line with a higher expression of col1a1 in WT but not IL15RAKO HTN hearts (20.0±6.0 vs 4.5±1.7, p=0.0173). Similarly, the FN1 protein level was significantly increased in WT but not IL15RAKO AngII-treated mice. These changes were associated with a higher number of Cd11b+ macrophages accumulated in WT hypertensive hearts only. In contrast to IL15RAKO, mice lacking IL15 showed similar increases in global longitudinal strain, heart hypertrophy, and cardiomyocyte cross-sectional area as WT mice upon AngII infusion. Similarly, IL15KO hypertensive hearts showed a significant increase of col1a1 and FN1 in comparison to vehicle-treated mice. A similar level of Cd11b+ macrophages was found in hypertensive IL15KO and WT mice's hearts. In conclusion, IL15RA, but not IL15, plays a critical role in cardiac remodelling mediated by AngII infusion. This suggests that the IL15-IL15RA axis may play an important yet complex role in hypertensive heart disease.

Transient High Salt Intake Promotes T-Cell-Mediated Hypertensive Vascular Injury.

Dietary high salt (HS) intake has a strong impact on cardiovascular diseases. Here, we investigated the link between HS-aggravated immune responses and the development of hypertensive vascular disease. ApolipoproteinE-deficient mice were transiently treated with HS (1% NaCl) via drinking water for 2 weeks, followed by a washout period, and subsequent Ang II (angiotensin II) infusion (1000 ng/kg per min for 10 days) to induce abdominal aortic aneurysms/dissections and inflammation. While transient HS intake alone triggered nonpathologic infiltration of activated T cells into the aorta, subsequent Ang II infusion increased mortality and the incidence of abdominal aortic aneurysms/dissections and atherosclerosis compared with hypertensive control mice. There were no differences in blood pressure between both groups. In transient HS-treated hypertensive mice, the aortic injury was associated with increased inflammation, accumulation of neutrophils, monocytes, CD69+CD4+ T cells, as well as CD4+ and CD8+ memory T cells. Mechanistically, transient HS intake increased expression levels of aortic RORγt as well as splenic CD4+TH17 and CD8+TC1 T cells in Ang II-treated mice. Isolated aortas of untreated mice were incubated with supernatants of TH17, TH1, or TC1 cells polarized in vitro under HS or normal conditions which revealed that secreted factors of HS-differentiated TH17 and TC1 cells, but not TH1 cells accelerated endothelial dysfunction. Our data suggest that transient HS intake induces a subclinical T-cell-mediated aortic immune response, which is enhanced by Ang II. We propose a 2-hit model, in which HS acts as a predisposing factor to enhance hypertension-induced TH17 and TC1 polarization and aortic disease.

Salidroside Pre-Treatment Inhibits Hypertensive Renal Injury and Fibrosis Through Inhibiting Wnt/β-Catenin Pathway.

This study aimed to explore the protective effects and underlying mechanisms of salidroside (SAL) in angiotensin II (Ang II)-induced hypertensive renal injury and fibrosis, using in vivo and in vitro models. In this study, we generated Ang II-induced hypertensive renal injury and fibrosis in mice and the recombinant interferon-gamma (IFN-γ)-stimulated murine podocyte clone 5 (MPC5) model in vitro. Histological and oxidative stress analyses were performed to evaluate the renal injury. SAL pre-treatment reduced systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MAP), and attenuated serum creatinine (Scr), blood urea nitrogen (BUN), and serum cystatin C (Cys-C) levels in Ang II-infused mice (all, P < 0.001). SAL reduced renal fibrosis and related molecules expression, including Collagen I, Collagen III, and α-smooth muscle actin (α-SMA) (all, P < 0.001). SAL decreased the content of malondialdehyde (MDA) while increasing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in Ang II-treated mice (all, P < 0.001). In addition, SAL pre-treatment inhibited AT1R, Wnt1, Wnt3a, and β-catenin expressions (all, P < 0.001), both in vivo and in vitro. Our experimental data demonstrate that SAL pre-treatment protects against Ang II-induced hypertensive renal injury and fibrosis by suppressing the Wnt/β-catenin pathway in vivo and in vitro.

Abstract MP35: Angiotensin II-induced vascular response is enhanced by Extracellular Vesicles

Intro: The exact effects of extracellular vesicles (EVs) on renal hemodynamics and blood pressure remain to be determined. In this study, we examined the effect of EVs from hypertensive mice and humans on Angiotensin II (ANG II)-induced vascular response in an ex vivo model of isolated and perfused renal afferent arterioles (Af-Arts) and also assessed their effect on renal blood flow (RBF) and systolic blood pressure (SBP) in normotensive mice in vivo. Method: Circulating EVs were collected from mice with ANG II-induced hypertension (n=5) and hypertensive humans (n=2). Differential centrifugation (DCF) (5000G x 30min, followed by 17.000G for 30 minutes) was applied to generate an EV pellet (p20) which was subsequently run through size exclusion chromatography (SEC). EV size and concentration was determined by nanoparticle tracking analysis and spectral flow cytometry. Basic EV characterization was performed with cryo electron microscopy and western blotting of EV and non EV proteins. Following a 30-minute perfusion with EVs (p20 EV pellet (10 10 /ml) or fraction 1-3 or 4-6 of SEC (doses 1-9 7/ml )), dose-response curves to ANG II (10 -12 to 10 -6 mol/l) were measured in Af-Arts of C57BL/6 mice. Similar doses of EVs were infused into the penile vein of C57BL/6 mice and RBF and SBP were measured. As EVs might deliver AT1R we explored the final common pathway in vasoconstriction in vessels treated with EVs from hypertensive animals vs controls by performing immunoblotting of myosin light chain II S19 phosphorylation. Results: EVs deriving from ANG II-treated mice and hypertensive humans, but not those from normotensive control mice and humans, significantly enhanced the vasoconstrictor response to ANG II in Af-Arts of C57BL/6 mice. The same vasoconstrictor response was observed when Af-Arts were perfused with combined EV preps from DCF and SEC (fraction 1-3 vs fraction 1-6) from hypertensive animals. In vivo injection of EVs from ANG II-treated hypertensive mice reduced RBF and increased SBP in C57BL/6 mice compared to EVs from normotensive control mice. Immunoblotting of myosin light chain II S19 phosphorylation was inconclusive. Conclusion: These data suggest that EVs from hypertensive animals and humans directly affect ANG II response, renal hemodynamic and blood pressure regulation. Further work is needed to identify the EV effector candidate for enhanced ANG II response and possible effect on ART1 activation.

USP11 promotes renal tubular cell pyroptosis and fibrosis in UUO mice via inhibiting KLF4 ubiquitin degradation.

The pyroptosis of renal tubular epithelial cells leads to tubular loss and inflammation and then promotes renal fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) can bidirectionally regulate the transcription of target genes. Our previous study revealed that sustained elevation of KLF4 is responsible for the transition of acute kidney injury (AKI) into chronic kidney disease (CKD) and renal fibrosis. In this study, we explored the upstream mechanisms of renal tubular epithelial cell pyroptosis from the perspective of posttranslational regulation and focused on the transcription factor KLF4. Mice were subjected to unilateral ureteral obstruction (UUO) surgery and euthanized on D7 or D14 for renal tissue harvesting. We showed that the pyroptosis of renal tubular epithelial cells mediated by both the Caspase-1/GSDMD and Caspase-3/GSDME pathways was time-dependently increased in UUO mouse kidneys. Furthermore, we found that the expression of the transcription factor KLF4 was also upregulated in a time-dependent manner in UUO mouse kidneys. Tubular epithelial cell-specific Klf4 knockout alleviated UUO-induced pyroptosis and renal fibrosis. In Ang II-treated mouse renal proximal tubular epithelial cells (MTECs), we demonstrated that KLF4 bound to the promoter regions of Caspase-3 and Caspase-1 and directly increased their transcription. In addition, we found that ubiquitin-specific protease 11 (USP11) was increased in UUO mouse kidneys. USP11 deubiquitinated KLF4. Knockout of Usp11 or pretreatment with the USP11 inhibitor mitoxantrone (3 mg/kg, i.p., twice a week for two weeks before UUO surgery) significantly alleviated the increases in KLF4 expression, pyroptosis and renal fibrosis. These results demonstrated that the increased expression of USP11 in renal tubular cells prevents the ubiquitin degradation of KLF4 and that elevated KLF4 promotes inflammation and renal fibrosis by initiating tubular cell pyroptosis.

Qianyang yuyin granule ameliorates mitochondrial dysfunction of hypertensive myocardial remodeling

Ethnopharmacological relevanceClinical studies have found that Qianyang Yuyin granule (QYYYG), a kind of oral Chinese patent medicine, had definite clinical effect for hypertensive myocardial remodeling. However, the potential mechanism is not entirely clear. Aim of the studyThe purpose of this research was to explore the underlying mechanism QYYYG on the treatment of hypertensive myocardial remodeling. Materials and methodsAnalysis the transcriptome data from the NCBI public platform GEO database and our study to explore the key pathological change of myocardial tissues in hypertensive mice and the main pathway of QYYYG in treating hypertensive myocardial remodeling. Network pharmacological analysis was used to predict the potential target of QYYYG. The molecular docking and molecular dynamics simulation was used for molecular binding analysis of specific compounds and target proteins. In the experiment in vivo, the effect of QYYYG on hypertensive myocardial remodeling and myocardial mitochondrial dysfunction in hypertensive mice caused by Ang Ⅱ was estimated. In the experiment in vitro, the Ang Ⅱ-induced myocardial remodeling model in H9c2 cells was constructed, and the effect of QYYYG on ameliorating myocardial remodeling and mitochondrial dysfunction was evaluated. ResultsTranscriptome analysis suggested that mitochondrial dysfunction was a key pathological change of myocardial tissues in hypertensive mice, and QYYYG could improve hypertensive myocardial remodeling through enhancing mitochondrial biogenesis to repair myocardial mitochondrial dysfunction. Network pharmacological analysis predicted that SIRT1 was an important potential target of QYYYG in treating hypertensive myocardial remodeling, and basically all the active components, especially quercetin, had a great binding affinity with SIRT1. Experiments in vivo proved that QYYYG had great efficacy hypertensive myocardial remodeling in Ang Ⅱ-treated mice. It was found that QYYYG improved the quality and quantity of mitochondria, and increased SIRT1 levels in myocardial tissue of Ang Ⅱ-treated mice. In Ang Ⅱ-treated H9c2 cells, with intervention of QYYYG, myocardial remodeling and myocardial mitochondrial dysfunction was ameliorated. In addition, QYYYG up-regulated SIRT1 expression and enhanced mitochondrial biogenesis in Ang Ⅱ-treated H9c2 cells. ConclusionThis study suggested that mitochondrial dysfunction was an important pathological change of myocardial tissues in hypertensive mice. QYYYG might ameliorate the mitochondrial dysfunction of hypertensive myocardial remodeling through up-regulating SIRT1 expression to enhance the mitochondrial biogenesis.

Angiotensin II-Induced Heart Failure Disrupts Cardiac Sympathetic Innervation

About 26 million people worldwide live with heart failure (HF), and 25% have hypertension (HTN) as the primary cause. These patients have a high risk for cardiac arrhythmias and sudden cardiac death. Autonomic dysfunction and sympathetic hyperactivity accompany these diseases, and sympathetic activation is crucial for triggering fatal arrhythmias. However, changes in cardiac sympathetic innervation in HF are not well understood. We hypothesized that cardiac sympathetic innervation is disrupted in a mouse model of Angiotensin II (AngII)-induced HF. To test this hypothesis, C57BL6 male (N=6, 10) and female (N=6, 10) mice 12-20 weeks old were implanted with minipumps releasing either AngII (712ng/min/Kg, N=6-10) or saline (N=6-10) for 4 weeks. HTN and cardiac function were measured with tail cuff quantification and echocardiography, respectively. Fibrosis was assessed using Trichrome staining. Cardiac sympathetic innervation was quantified using immunohistochemistry for Tyrosine Hydroxylase (TH). Cardiac norepinephrine (NE) was measured by High-Performance Liquid Chromatography with electrochemical detection. Values were compared with a Student’s unpaired t-test unless noted, data were represented as mean ± SD, and significance was defined as p&lt;0.05. AngII-treated mice had significantly increased mean arterial pressure (Saline 91.0±15.6 vs. AngII 152.9±22.3 mmHg, p&lt;0.05, N=6-7) 7 days after minipump implantation. Four weeks of AngII treatment led to HF, as indicated by reduced ejection fraction (Saline 54.8±3.8 vs. AngII 43.1±8.2 %, p&lt;0.05, N=10) and fractional shortening (Saline 14.3±2.6 vs. AngII 10.6±3.3 %, p&lt;0.05, N=10). Cardiac hypertrophy was also observed in AngII-treated mice and was reflected by increased heart weight to body weight ratio (Saline 5.2±0.2 vs. AngII 7.7±1.0, p&lt;0.05, N=6). Fibrosis was detected in the intraventricular septum (IVS) and left ventricle (LV) of AngII-treated mice. TH+ fibers in the LV and IVS were decreased significantly (LV: Saline 0.6±0.2 vs. AngII 0.4±0.1 %; IVS: Saline 0.3±0.1 vs. AngII 0.09±0.04 %, p&lt;0.05, N=6), while no differences were detected in the right ventricle (Saline 1.1±0.2 vs. AngII 0.8±0.3 %, P&gt;0.05, N=6). In addition, the subepicardium-subendocardium (EPI-ENDO) gradient was altered (EPI: Saline 0.9±0.2 vs. AngII 0.8±0.3 %, P&gt;0.05; ENDO: Saline 0.3±0.05 vs. AngII 0.1±0.1 %, p&lt;0.05; EPI:ENDO ratio: Saline 2.7±0.7 vs. AngII 15.7±16.8, p&lt;0.05, Mann-Whitney test, N=6). A significant decrease in NE content accompanied the loss of TH+ fibers in the LV (LV: Saline 3.9±0.4 vs. AngII 1.6±0.2 pmol/mg, p&lt;0.05; RV: Saline 4.4±1.0 vs. AngII 3.8±0.8 pmol/mg, P&gt;0.05, N=4-6). Our data demonstrate that AngII-induced HF disrupts cardiac sympathetic nerve density, distribution, and NE content, confirming our initial hypothesis. This evidence contrasts with sympathetic hyperactivity seen in cardiac diseases. Future studies will assess the overall cardiac innervation and, determine whether the loss of TH+ fibers is due to a reduced sympathetic innervation after cardiac remodeling or the cholinergic transdifferentiation of TH+ fibers. A better understanding of the cardiac sympathetic nervous system in HF is imperative to deciphering the mechanistic changes to cardiac innervation in disease, and may help to develop and implement novel and precise neuromodulatory therapies for patients with autonomic imbalance. Supported by NIH R01 HL146833 to Dr. Habecker. 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.

Galantamine Reduces Blood Pressure and Renal Injury in Female Angiotensin II-induced Hypertensive Mice

Inflammation is a known culprit in the development and progression of hypertension. The autoimmune disease, systemic lupus erythematosus (SLE), is an important model of hypertension in the setting of chronic kidney inflammation. We have shown that administration of an acetylcholinesterase inhibitor, galantamine, attenuates the hypertension, kidney inflammation, and kidney injury in female SLE mice by activating the vagally-mediated cholinergic anti-inflammatory pathway. Although SLE hypertension represents a specific population of patients, we hypothesized that galantamine would have similar anti-inflammatory effects in the angiotensin II (AngII)-induced hypertensive mouse, which more commonly models essential hypertension. Female C57BL/6 mice were implanted with minipumps at 9 weeks of age for subcutaneous delivery of either AngII (1000 ng/kg/min) or saline (controls), and galantamine hydrobromide (4 mg/kg/day) or vehicle, for 21 days. At 12 weeks, carotid catheters were implanted and mean arterial pressure (MAP) was measured in conscious mice for two consecutive days. Mice were then euthanized, and kidneys collected. Markers of kidney inflammation and injury, including interleukin (IL)-6 and kidney injury molecule (KIM)-1, were measured by ELISA and normalized to total kidney protein. MAP (mmHg) was higher in AngII-treated mice compared to controls (174±4 vs. 133±3; n=4-5, p=0.0023). Galantamine lowered MAP in AngII mice (157±8; n=7, p=0.2567) without altering MAP in controls (133±4; n=5, p=0.9999). Renal IL-6 (pg/μg) was not different between AngII and control mice (0.004±0.0001 vs. 0.005±0.0006; p=0.6685). Although galantamine lowered IL-6 in both groups, it was only significant in controls (0.003±0.0002; p=0.0134) and not AngII mice (0.003±0.0003; p=0.4677). Renal cortical KIM-1 (pg/μg) trended higher in AngII mice compared to controls (2.53±0.50 vs 0.63±0.16; p=0.0741). Galantamine lowered KIM-1 in AngII mice (1.18±0.39, p=0.2064), but not controls (1.45 ± 0.59; p=0.6285). In summary, galantamine protected female mice from hypertension and renal injury in a model of AngII hypertension. This suggests that acetylcholinesterase inhibitor therapy may be effective in patients with hypertension. Studies funded by NIH R01HL153703 and K01HL139859 for KWM. 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.

Endothelial deubiquinatase YOD1 mediates Ang II-induced vascular endothelial-mesenchymal transition and remodeling by regulating β-catenin.

Hypertension is a prominent contributor to vascular injury. Deubiquinatase has been implicated in the regulation of hypertension-induced vascular injury. In the present study we investigated the specific role of deubiquinatase YOD1 in hypertension-induced vascular injury. Vascular endothelial endothelial-mesenchymal transition (EndMT) was induced in male WT and YOD1-/- mice by administration of Ang II (1 μg/kg per minute) via osmotic pump for four weeks. We showed a significantly increased expression of YOD1 in mouse vascular endothelial cells upon Ang II stimulation. Knockout of YOD1 resulted in a notable reduction in EndMT in vascular endothelial cells of Ang II-treated mouse; a similar result was observed in Ang II-treated human umbilical vein endothelial cells (HUVECs). We then conducted LC-MS/MS and co-immunoprecipitation (Co-IP) analyses to verify the binding between YOD1 and EndMT-related proteins, and found that YOD1 directly bound to β-catenin in HUVECs via its ovarian tumor-associated protease (OTU) domain, and histidine at 262 performing deubiquitination to maintain β-catenin protein stability by removing the K48 ubiquitin chain from β-catenin and preventing its proteasome degradation, thereby promoting EndMT of vascular endothelial cells. Oral administration of β-catenin inhibitor MSAB (20 mg/kg, every other day for four weeks) eliminated the protective effect of YOD1 deletion on vascular endothelial injury. In conclusion, we demonstrate a new YOD1-β-catenin axis in regulating Ang II-induced vascular endothelial injury and reveal YOD1 as a deubiquitinating enzyme for β-catenin, suggesting that targeting YOD1 holds promise as a potential therapeutic strategy for treating β-catenin-mediated vascular diseases.

Activation of Cannabinoid Type 2 Receptor in Microglia Reduces Neuroinflammation through Inhibiting Aerobic Glycolysis to Relieve Hypertension.

Studies have shown that the chronic use of cannabis is associated with a decrease in blood pressure. Our previous studies prove that activating the cannabinoid type 2 (CB2) receptor in the brain can effectively reduce blood pressure in spontaneously hypertensive rats; however, the exact mechanism has not been clarified. The objective of this study is to demonstrate that activation of microglial CB2 receptors can effectively reduce the levels of TNF-α, IL-1β, and IL-6 in the paraventricular nucleus (PVN) through inhibiting aerobic glycolysis, thereby relieving hypertension. AngiotensinII (AngII) was administered to BV2 cells and C57 mice to induce hypertension and the release of proinflammatory cytokines. The mRNA and protein expression of the CB2 receptor, TNF-α, IL-1β, IL-6, and the PFK and LDHa enzymes were detected using RT-qPCR and Western blotting. The Seahorse XF Energy Metabolism Analyzer was used to measure the oxidative phosphorylation and aerobic glycolysis metabolic pathways in BV2 cells. The long-term effects of injecting JWH133, a selective CB2 receptor agonist, intraperitoneally on blood pressure were ascertained. ELISA was used to measure norepinephrine and lactic acid levels while immunofluorescence labeling was used to locate the CB2 receptor and c-Fos. By injecting pAAV-F4/80-GFP-mir30shRNA (AAV2-r-CB2shRNA) into the lateral cerebral ventricle, the CB2 receptor in microglia was specifically knocked down. Activation of CB2 receptors by the agonist JWH133 suppressed TNF-α, IL-1β, and IL-6 by inhibiting PFK and LDHa enzymes involved in glycolysis, as well as lactic acid accumulation, along with a reduction in glycoPER levels (marks of aerobic glycolysis) in AngII-treated BV2 cells. In AngII-treated mice, the administration of JWH133 specifically activated CB2 receptors on microglia, resulting in decreased expression levels of PFK, LDHa, TNF-α, IL-1β, and IL-6, subsequently leading to a decrease in c-Fos protein expression within PVN neurons as well as reduced norepinephrine levels in plasma, ultimately contributing to blood pressure reduction. The results suggest that activation of the microglia CB2 receptor decreases the neuroinflammation to relieve hypertension; the underlying mechanism is related to inhibiting aerobic glycolysis of microglia.

Vascular protein disulfide isomerase A1 mediates endothelial dysfunction induced by angiotensin II in mice.

Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed invivo with magnetic resonance imaging and exvivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine invivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated exvivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.

STING activation in cardiomyocytes drives hypertrophy-associated heart failure via NF-κB-mediated inflammatory response

Accumulating evidence highlights the key importance of innate immunity in heart hypertrophy and failure. Though stimulator of interferon genes (STING) is an integral innate immunity regulator, whether cardiomyocyte-derived STING driving cardiac hypertrophy and failure has rarely been explored, nor has its underlying mechanism been clarified. Herein, we addressed these two questions through several mouse experiments. Our results revealed that cardiac tissues from patients exhibiting cardiac hypertrophy markedly increased STING expression. Myocardial tissues of mice challenged with angiotensin II (Ang II) or transverse aortic constriction (TAC) also showed that STING was consistently upregulated and activated. Activation of STING by cGAMP or DMXAA resulted in cardiomyocyte hypertrophy in vitro, which was abolished by STING knockout. Furthermore, deleting or pharmacologically inhibiting STING attenuated cardiac hypertrophy and dysfunction in TAC or Ang II-treated mice. In contrast, mice with cardiomyocyte-specific STING activation developed cardiac hypertrophy and failure. Mechanistically, NF-κB signaling but not TBK1 or autophagy formation was implicated in STING -induced cardiac hypertrophy and failure. Collectively, we identified that STING-NF-κB axis mediated inflammatory response to drive cardiac hypertrophy-associated heart failure, highlighting its promise as a potential therapeutic target in clinical practice.

OTUD1 promotes hypertensive kidney fibrosis and injury by deubiquitinating CDK9 in renal epithelial cells.

Hypertensive renal disease (HRD) contributes to the progression of kidney dysfunction and ultimately leads to end-stage renal disease. Understanding the mechanisms underlying HRD is critical for the development of therapeutic strategies. Deubiquitinating enzymes (DUBs) have been recently highlighted in renal pathophysiology. In this study, we investigated the role of a DUB, OTU Domain-Containing Protein 1 (OTUD1), in HRD models. HRD was induced in wild-type or Otud1 knockout mice by chronic infusion of angiotensin II (Ang II, 1 μg/kg per min) through a micro-osmotic pump for 4 weeks. We found that OTUD1 expression levels were significantly elevated in the kidney tissues of Ang II-treated mice. Otud1 knockout significantly ameliorated Ang II-induced HRD, whereas OTUD1 overexpression exacerbated Ang II-induced kidney damage and fibrosis. Similar results were observed in TCMK-1 cells but not in SV40 MES-13 cells following Ang II (1 μM) treatment. In Ang II-challenged TCMK-1 cells, we demonstrated that OTUD1 bound to CDK9 and induced CDK9 deubiquitination: OTUD1 catalyzed K63 deubiquitination on CDK9 with its Cys320 playing a critical role, promoting CDK9 phosphorylation and activation to induce inflammatory responses and fibrosis in kidney epithelial cells. Administration of a CDK9 inhibitor NVP-2 significantly ameliorated Ang II-induced HRD in mice. This study demonstrates that OTUD1 mediates HRD by targeting CDK9 in kidney epithelial cells, suggesting OTUD1 is a potential target in treating this disease.

Abstract 16902: Molecular Basis of the Vascular Cell Phenotypic Switch and Wall Remodeling in Abdominal Aortic Aneurysm: NOX4 NADPH Oxidase-Derived Mitochondrial Oxidative Stress and DNA Damage

Vascular cells phenotypic changes and wall remodeling are the hallmarks of abdominal aortic aneurysm (AAA) pathology. Mitochondrial oxidative stress (mtOS) is associated with the vascular smooth muscle cells (SMC) and macrophage phenotypic switch. We previously reported that NOX4 NADPH oxidase expression is increased in cardiovascular cells in response to pathological insult inducing mtOS and dysfunction and vascular disease progression. We tested the hypothesis that NOX4-dependent mtOS and DNA damage induce vascular phenotypic changes and remodeling resulting in AAA progression. In angiotensin II (Ang II)-induced AAA model Apoe -/- / Nox4 Tg mice with mitochondria-targeted overexpression of NOX4 had the highest, whereas Apoe -/- / Nox4 -/- displayed the lowest incidence of AAA, abdominal aorta area and diameter as compared with Apoe -/- mice. The aortic wall ROS levels, oxidative DNA damage and DNA damage response markers, elastin degradation index, MMP2 expression, and macrophage infiltration were significantly higher in AngII-treated Apoe -/- / Nox4 TG mice compared with Apoe -/- or Apoe -/- / Nox4 -/- mice. Spectral flow cytometry analysis of AAA cell suspension from Ang II-infused mice revealed a decrease in contractile SMCs but an increase in macrophage-like SMC populations, as well as increased number of infiltrating pro-inflammatory macrophages in Apoe -/- / Nox4 TG mice. As compared with wild-type or Nox4 -/- mice, Ang II-treated SMC from Nox4 TG mice showed significantly elevated mtOS, DNA damage, dysfunction, as well as activation of cGAS-STING pathway and NLRP3/AIM2 inflammasome expression. Flow cytometry analysis of Ang II-treated Nox4 TG mice SMC contained a lower proportion of cells with contractile markers (ACTA2 + , MYH11 + , TAGLN + ) and a higher proportion of cells with macrophage-like markers (CD68 + , CD11b + , LGALS3 + ). These data suggest that NOX4-dependent mitochondrial DNA damage and activation of DNA-sensing pathways have a causal role in AAA development, leading to macrophage-like SMCs, increased inflammation, and vessel wall remodeling. NOX4 inhibition and mitochondrial function augmentation may be beneficial in preserving aortic wall integrity in AAA-susceptible patients.

Insulin-Like Growth Factor 1 Receptor Deficiency Alleviates Angiotensin II-Induced Cardiac Fibrosis Through the Protein Kinase B/Extracellular Signal-Regulated Kinase/Nuclear Factor-κB Pathway.

Background The renin-angiotensin system plays a crucial role in the development of heart failure, and Ang II (angiotensin II) acts as the critical effector of the renin-angiotensin system in regulating cardiac fibrosis. However, the mechanisms of cardiac fibrosis are complex and still not fully understood. IGF1R (insulin-like growth factor 1 receptor) has multiple functions in maintaining cardiovascular homeostasis, and low-dose IGF1 treatment is effective in relieving Ang II-induced cardiac fibrosis. Here, we aimed to investigate the molecular mechanism of IGF1R in Ang II-induced cardiac fibrosis. Methods and Results Using primary mouse cardiac microvascular endothelial cells and fibroblasts, in vitro experiments were performed. Using C57BL/6J mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated IGF1R heterozygous knockout (Igf1r+/-) mice, cardiac fibrosis mouse models were induced by Ang II for 2 weeks. The expression of IGF1R was examined by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot. Mice heart histologic changes were evaluated using Masson and picro sirius red staining. Fibrotic markers and signal molecules indicating the function of the Akt (protein kinase B)/ERK (extracellular signal-regulated kinase)/nuclear factor-κB pathway were detected using quantitative reverse transcription polymerase chain reaction and Western blot. RNA sequencing was used to explore IGF1R-mediated target genes in the hearts of mice, and the association of IGF1R and G-protein-coupled receptor kinase 5 was identified by coimmunoprecipitation. More important, blocking IGF1R signaling significantly suppressed endothelial-mesenchymal transition in primary mouse cardiac microvascular endothelial cells and mice in response to transforming growth factor-β1 or Ang II, respectively. Deficiency or inhibition of IGF1R signaling remarkably attenuated Ang II-induced cardiac fibrosis in primary mouse cardiac fibroblasts and mice. We further observed that the patients with heart failure exhibited higher blood levels of IGF1 and IGF1R than healthy individuals. Moreover, Ang II treatment significantly increased cardiac IGF1R in wild type mice but led to a slight downregulation in Igf1r+/- mice. Interestingly, IGF1R deficiency significantly alleviated cardiac fibrosis in Ang II-treated mice. Mechanistically, the phosphorylation level of Akt and ERK was upregulated in Ang II-treated mice, whereas blocking IGF1R signaling in mice inhibited these changes of Akt and ERK phosphorylation. Concurrently, phosphorylated p65 of nuclear factor-κB exhibited similar alterations in the corresponding group of mice. Intriguingly, IGF1R directly interacted with G-protein-coupled receptor kinase 5, and this association decreased ≈50% in Igf1r+/- mice. In addition, Grk5 deletion downregulated expression of the Akt/ERK/nuclear factor-κB signaling pathway in primary mouse cardiac fibroblasts. Conclusions IGF1R signaling deficiency alleviates Ang II-induced cardiac fibrosis, at least partially through inhibiting endothelial-mesenchymal transition via the Akt/ERK/nuclear factor-κB pathway. Interestingly, G-protein-coupled receptor kinase 5 associates with IGF1R signaling directly, and it concurrently acts as an IGF1R downstream effector. This study suggests the promising potential of IGF1R as a therapeutic target for cardiac fibrosis.

Costunolide ameliorates angiotensin II-induced atrial inflammation and fibrosis by regulating mitochondrial function and oxidative stress in mice: A possible therapeutic approach for atrial fibrillation

Atrial fibrillation (AF) is a cardiac disease characterized by disordered atrial electrical activity. Atrial inflammation and fibrosis are involved in AF progression. Costunolide (COS) is a sesquiterpene lactone containing anti-inflammatory and anti-fibrotic activities. This study aims to explore the underlying mechanisms by which COS protects against AF. Male C57BL/6 mice (8- to 10-week-old) were infused with angiotensin (Ang) II for 3 weeks. Meanwhile, different doses of COS (COS-L: 10 mg/kg, COS-H: 20 mg/kg) were administered to mice by intragastric treatment. The results showed irregular and rapid heart rates in Ang II-treated mice. Moreover, the levels of inflammatory cytokines and fibrotic factors were elevated in mice. COS triggered a reduction of Ang II-induced inflammation and fibrosis, which conferred a protective effect. Mechanistically, mitochondrial dysfunction with mitochondrial respiration inhibition and aberrant ATP levels were observed after Ang II treatment. Moreover, Ang-II-induced excessive reactive oxygen species caused oxidative stress, which was further aggravated by inhibiting Nrf2 nuclear translocation. Importantly, COS diminished these Ang-II-mediated effects in mice. In conclusion, COS attenuated inflammation and fibrosis in Ang-II-treated mice by alleviating mitochondrial dysfunction and oxidative stress. Our findings represent a potential therapeutic option for AF treatment.

Abstract P308: Leucine Rich Repeat Containing 8A Anion Channels Modulate Vasodilation In Angiotensin II-induced Hypertensive Mice

Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) comprise Volume-regulated anion channels (VRACs) which are activated by tumor necrosis factor alpha (TNFα) and angiotensin II (AngII). We have previously investigated vascular reactivity in mesenteric arteries from male wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to both endothelium-dependent (acetylcholine, ACh), and independent (sodium nitroprusside, SNP) vasodilators. We hypothesized that LRRC8A KO mice would be protected from AngII-induced hypertension. Blood pressure (BP) was measured by radiotelemetry. Resting BP was unaltered compared to WT and AngII infusion by osmotic minipump (alzet, 90 ng/min for 14 days) induced a similar degree of hypertension in awake mice (nighttime) but the difference between day and night pressures was significantly larger in KO mice. Mesenteric arteries were isolated from WT and KO mice after 14 days and vascular reactivity was quantified by wire myograph. Relaxation responses to both ACh and SNP relaxation were impaired in AngII hypertensive compared to normotensive WT mice. However, KO vessels relaxed better to both vasodilators in rings from both vehicle and AngII-treated mice (ACh LogEC 50 , WT+AngII -5.3 ± 0.27 vs. KO+AngII -6.5 ± 0.19, p &lt; 0.05, n=4-6; SNP LogEC 50 , WT+AngII -7.4 ± 0.10 vs. KO+AngII -8.1 ± 0.10, p &lt; 0.05, n=3-6). These results demonstrate a dissociation between elevated blood pressure and impaired vasodilator function. The data suggest that LRRC8A anion channels play a complex role in the development of AngII-induced hypertension.