Ang II-infused Mice Research Articles

α-Mangostin Attenuates Blood Pressure and Reverses Vascular Remodeling by Balancing ACE/AT1R and ACE2/Ang-(1-7)/MasR Axes in Ang II-Infused Hypertensive Mice.

Hyperuricemia is a common comorbidity of hypertension and probably has a causal relationship with hypertension. Alpha-mangostin (α-MG) has been reported to have uric acid lowering effect. This study aimed to investigate the dual effects of α-MG on blood pressure (BP) and uric acid levels in angiotensin II (Ang II)-infused hypertensive mice. Male C57BL/6 mice were randomized into five groups: control, Ang II infusion (500 ng/kg/min for 2 weeks), Ang II infusion with gavage administration of α-MG 4.0 and 8.0 mg/kg and benzbromarone (25 mg/kg) respectively. BP, uric acid levels, vascular structure and function, and renin-Ang II system expressions in the aorta were assessed. Treatment with α-MG reduced BP, improved endothelial relaxation, and reversed aortic wall thickening and collagen deposition in Ang II-induced hypertensive mice. It also downregulated Ang II receptor 1 (AT1R) and angiotensin converting enzyme (ACE) expression, while upregulating ACE2, Mas receptor (MasR), and angiotensin (1-7) in the aorta. Moreover, α-MG demonstrated a significant enhancement in uric acid clearance and reduction in serum uric acid levels. Conversely, benzbromarone did not result in a decrease in BP, indicating that the hypotensive effect of α-MG may not be necessarily dependent on its urate-lowering properties. α-MG can attenuate Ang II-induced hypertension and reverse vascular remodeling, potentially by balancing the ACE/Ang II/AT1R axis and the ACE2/Ang-(1-7)/MasR axis. Our findings provide insights into α-MG as a novel anti-hypertensive drug especially in patients with hyperuricemia.

Paeoniflorin Inhibits Atrial Fibrosis and Atrial Fibrillation in Angiotensin II-Infused Mice Through the PI3K-Akt Pathway.

Objective: The investigation aimed to analyze the effect of Paeoniflorin (PF) on the initiation of atrial fibrosis and atrial fibrillation (AF) induced by angiotensin II (Ang II) and explore its associated underlying mechanism. Introduction: PF has anti-inflammatory, immunomodulatory, antioxidant, hepatoprotective, and hypolipidemic properties. However, the protective effect of PF against atrial fibrosis and AF remains unclear. Methods: Male C57BL/6 mice aged 8-10 weeks, with 40 individuals, were subjected to subcutaneous infusion of either saline or Ang II at a dosage of 2.0mg/kg/day. Furthermore, PF at a dosage of 100mg/kg/day was administered through gavage once daily for 28 days. Morphological, histological, and biochemical examinations were undertaken. AF was elicited through in vivo transesophageal burst pacing. Results: PF treatment significantly improved AF in Ang II-infused mice. In addition, PF attenuated cardiac hypertrophy, atrial fibrotic area, atrial apoptosis and oxidative stress in Ang II-induced mice. The effect of PF on the PI3K-Akt pathway reduced the expression of phosphoinositide 3-kinase (p-PI3K) and Phosphorylated Akt (p-Akt) in Ang II-induced mice. Conclusion: PF may, therefore, avert Ang II-induced atrial fibrosis and AF by inhibiting the PI3K-Akt pathway.

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.

Gastrodin Alleviates Angiotensin II-Induced Hypertension and Myocardial Apoptosis via Inhibition of the PRDX2/p53 Pathway In Vivo and In Vitro.

Gastrodin, a highly potent compound found in the traditional Chinese medicine Gastrodia elata Blume, exhibits significant antihypertensive properties. However, its role and the mechanism behind its protective effects on hypertensive cardiac conditions are not well understood. This study aims to investigate the cardiac protective effects and underlying mechanisms of gastrodin in angiotensin II (Ang II)-induced hypertensive models, both in vivo and in vitro. Treatment with gastrodin significantly decreased blood pressure and the heart weight/tibial length (HW/TL) ratio and attenuated cardiac dysfunction and pathological damage in Ang II-infused C57BL/6 mice. RNA sequencing analysis (RNA-seq) revealed 697 up-regulated and 714 down-regulated transcripts, along with 1105 signaling pathways, in Ang II-infused C57BL/6 mice following gastrodin treatment, compared to Ang II-induced hypertensive mice. Furthermore, the analyses of the top 30 Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway indicated significant enrichment in apoptosis and the peroxiredoxin 2 (PRDX2)/p53 pathway. Consistently, gastrodin treatment significantly reduced myocardial apoptosis in both the cardiac tissues of Ang II-induced hypertensive mice and Ang II-stimulated H9c2 cells. Additionally, gastrodin treatment significantly decreased the protein levels of PRDX2, p53, cleaved caspase-3, cleaved caspase-9, and Bax/Bcl-2 ratio in the cardiac tissues of Ang II-infused mice and H9c2 cells stimulated with Ang II. In conclusion, gastrodin treatment can mitigate hypertension-induced myocardial apoptosis in hypertensive mice by inhibiting the PRDX2/p53 pathway.

Biogenesis of circRBM33 mediated by N6-methyladenosine and its function in abdominal aortic aneurysm

ABSTRACT This study aimed to explore whether m6A modification affects the biogenesis of circRBM33, which is involved in the progression of abdominal aortic aneurysm (AAA). For in vitro experiments, vascular smooth muscle cells (VSMCs) were treated with Ang II. MeRIP‒PCR was used to assess m6A modification of circRBM33. Gene expression was measured using RT‒qPCR and Western blotting. For in vivo experiments, a mouse model of AAA was established via Ang II infusion. HE, Sirius Red and TUNEL staining was performed to evaluate pathological changes and cell apoptosis in aortic vessels. The results showed that the m6A level of circRBM33 was abnormally increased in Ang II-induced VSMCs. In addition, METTL3 positively regulated circRBM33 expression. YTHDC1 deficiency decreased circRBM33 expression but had no effect on RBM33 mRNA expression. Notably, neither METTL3 nor YTHDC1 influenced the stability of circRBM33 or RBM33 mRNA. The interaction between circRBM33 and METTL3/YTHDC1 was verified by RIP analysis. Moreover, the Ang II-induced increase in circRBM33 expression was reversed by cycloleucine (an inhibitor of m6A methylation). Importantly, the m6A modification and expression of circRBM33 in the circRBM33-m6A-mut2-expressing VSMCs were not altered by METTL3 silencing. Mechanistically, METTL3/YTHDC1 modulates the biogenesis of circRBM33 in an m6A-dependent manner. In addition, circRBM33 knockdown alleviated AAA by reducing ECM degradation in the Ang II-infused mice. In conclusion, this study demonstrated that METTL3/YTHDC1-mediated m6A modification modulates the biogenesis of circRBM33 from exons of the RBM33 gene. Moreover, knockdown of circRBM33 alleviated AAA by reducing ECM degradation, which may provide a novel therapeutic strategy for treating AAA.

Vγ6/Vδ1+ γδ T cells protect from angiotensin II effects on blood pressure and endothelial function in mice.

γδ T cells mediate angiotensin II (AngII)-induced hypertension and vascular injury. γδ T cells expressing specific T-cell receptor (TCR) variable (V) γ chains develop in several waves in the thymus and migrate to specific or diverse tissues. We hypothesized that γδ T cells expressing specific Vγ subtypes in perivascular tissue mediate AngII hypertensive effects. C57BL/6J male mice were infused or not with AngII (490 ng/kg/min, subcutaneously) for 14 days. γδ T-cell Vγ subtypes were profiled by flow cytometry in the spleen, descending thoracic aorta with adherent perivascular adipose tissue (DTAo/PVAT) and mesenteric vessels (MV)/PVAT. Other sets of AngII-infused mice were injected with control or specific anti-Vγ6 or Vγ4 antibodies. Blood pressure (BP) was determined by telemetry, and mesenteric artery function and remodeling by pressurized myography. Vγ6/Vδ1+ γδ T cells represented more than 50% of the γδ T-cell Vγ subtypes in DTAo/PVAT and MV/PVAT, whereas Vγ1/2+, Vγ4+ and Vγ6/Vδ1+ γδ T cells were the most abundant Vγ subtypes in the spleen. The frequency of Vγ6/Vδ1+ γδ T cells was increased at least 1.5-fold in the spleen and DTAo/PVAT, and tended to increase in MV/PVAT by AngII. A majority of Vγ6/Vδ1+ γδ T cells were activated in perivascular tissues. Vγ6/Vδ1+ γδ T-cell neutralization caused a steeper BP elevation and greater mesenteric artery endothelial dysfunction in mice infused with AngII. This was associated with more than three-fold increase in activated Vγ6/Vδ1- γδ T cells in perivascular tissues. Depletion of Vγ4+ γδ T cells did not alter AngII detrimental effects. Vγ6/Vδ1+ γδ T cells reduce the BP elevation and endothelial dysfunction induced by AngII infusion.

Abstract 32: Vγ6 + γδ T cells protect against angiotensin II-induced hypertension and vascular injury in male mice

Background: γδ T cells mediate angiotensin II (AngII)-induced blood pressure (BP) elevation, vascular injury and activated T cell infiltration in mesenteric vessels with adherent perivascular adipose tissue (MV/PVAT). γδ T cells expressing specific T cell receptor (TCR) variable (V) chain γ develop in several waves in the thymus and migrate to diverse tissues. We hypothesized that γδ T cells expressing specific Vγ subtypes in perivascular tissue are implicated in AngII detrimental hypertensive effects. Methods: C57BL/6J male mice were infused or not with AngII (490 ng/kg/min, SC) for 14 days. Vγ1 and Vγ2 (Vγ1/2) + , Vγ4 + , Vγ5 + , Vγ6 + , and Vγ7 + γδ T cell frequencies and T cell phenotypes were determined by flow cytometry in the spleen, descending thoracic aorta (DTAo)/PVAT, and MV/PVAT (n=7-13). Other sets of AngII-infused mice were injected with control or anti-Vγ6 (n=7-12) or Vγ4 antibodies (n=5-8). BP was determined by telemetry, mesenteric artery (MA) function and remodeling by pressurized myography, and T cell phenotypes by flow cytometry. Results: In DTAo/PVAT and MV/PVAT, respectively, 43±5% and 51%±6% of γδ T cells were Vγ6 + , and 17±2% and 23±4% Vγ4 + , whereas in the spleen, 36±2% of the γδ T cells were Vγ1/2 + , 32±1% Vγ4 + and 24±1% Vγ6 + . AngII increased the frequency of Vγ6 + γδ T cells in the spleen (0.8±0.1 vs 0.5±0.0% of CD3, P &lt;0.001) and DTAo/PVAT (5.9±0.8 vs 3.6±0.5% of CD3, P &lt;0.05), and tended to increase their frequency in MV/PVAT. The majority of Vγ6 + γδ T cells were activated (CD69 + ) in DTAo/PVAT (75±5%) and MV/PVAT (84±2%). Vγ6 + γδ T cell depletion caused a steeper BP elevation ( P &lt;0.05) and worsened MA endothelial dysfunction (maximal acetylcholine relaxation response: 28±3 vs 55±8%, P &lt;0.05) in mice infused with AngII. This was associated with increased CD69 + non-Vγ6 + γδ T cells in DTAo/PVAT (3.0±0.7 vs 0.8±0.4% of CD3, P &lt;0.01) and MV/PVAT (15.2±2.6 vs 4.9±1.0% of CD3, P &lt;0.01). Depletion of Vγ4 + γδ T cells in perivascular tissues, did not alter the AngII detrimental effects. Conclusions: Vγ6 + γδ T cells are the most abundant γδ T cell Vγ subtype in perivascular tissues. AngII increased Vγ6 + γδ T cells in the spleen and perivascular tissue. Vγ6 + γδ T cell depletion in AngII-infused mice induced a steeper BP elevation, worsened MA endothelial dysfunction, and activated non-Vγ6 + γδ T cells. Vg6 + γδ T cells may play a protective role in AngII-induced hypertension and vascular injury.

Quercetin Protects Against Hypertensive Renal Injury by Attenuating Apoptosis: An Integrated Approach Using Network Pharmacology and RNA Sequencing.

Quercetin is known for its antihypertensive effects. However, its role on hypertensive renal injury has not been fully elucidated. In this study, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and Annexin V staining were used to assess the pathological changes and cell apoptosis in the renal tissues of angiotensin II (Ang II)-infused mice and Ang II-stimulated renal tubular epithelial cell line (NRK-52E). A variety of technologies, including network pharmacology, RNA-sequencing, immunohistochemistry, and Western blotting, were performed to investigate its underlying mechanisms. Network pharmacology analysis identified multiple potential candidate targets (including TP53, Bcl-2, and Bax) and enriched signaling pathways (including apoptosis and p53 signaling pathway). Quercetin treatment significantly alleviated the pathological changes in renal tissues of Ang II-infused mice and reversed 464 differentially expressed transcripts, as well as enriched several signaling pathways, including those related apoptosis and p53 pathway. Furthermore, quercetin treatment significantly inhibited the cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells. In addition, quercetin treatment inhibited the upregulation of p53, Bax, cleaved-caspase-9, and cleaved-caspase-3 protein expression and the downregulation of Bcl-2 protein expression in both renal tissue of Ang II-infused mice and Ang II-stimulated NRK-52E cells. Moreover, the molecular docking results indicated a potential binding interaction between quercetin and TP53. Quercetin treatment significantly attenuated hypertensive renal injury and cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells and by targeting p53 may be one of the potential underlying mechanisms.

Baicalin ameliorates angiotensin II-induced cardiac hypertrophy and mitogen-activated protein kinase signaling pathway activation: A target-based network pharmacology approach

Baicalin, a flavonoid glycoside from Scutellaria baicalensis Georgi., exerts anti-hypertensive effects. The present study aimed to assess the cardioprotective role of baicalin and explore its potential mechanisms. Network pharmacology analysis pointed out a total of 477 potential targets of baicalin were obtained from the PharmMapper and SwissTargetPrediction databases, while 11,280 targets were identified associating with hypertensive heart disease from GeneCards database. Based on the above 382 common targets, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed enrichment in the regulation of cardiac hypertrophy, cardiac contraction, cardiac relaxation, as well as the mitogen-activated protein kinase (MAPK) and other signaling pathways. Moreover, baicalin treatment exhibited the amelioration of increased cardiac index and pathological alterations in angiotensin II (Ang II)-infused C57BL/6 mice. Furthermore, baicalin treatment demonstrated a reduction in cell surface area and a down-regulation of hypertrophy markers (including atrial natriuretic peptide and brain natriuretic peptide) in vivo and in vitro. In addition, baicalin treatment led to a decrease in the expression of phosphorylated c-Jun N-terminal kinase (p-JNK)/JNK, phosphorylated p38 (p-p38)/p38, and phosphorylated extracellular signal-regulated kinase (p-ERK)/ERK in the cardiac tissues of Ang II-infused mice and Ang II-stimulated H9c2 cells. These findings highlight the cardioprotective effects of baicalin, as it alleviates hypertensive cardiac injury, cardiac hypertrophy, and the activation of the MAPK pathway.

Interleukin-5 alleviates cardiac remodelling via the STAT3 pathway in angiotensin II-infused mice.

Interleukin-5 (IL-5) has been reported to be involved in cardiovascular diseases, such as atherosclerosis and cardiac injury. This study aimed to investigate the effects of IL-5 on cardiac remodelling. Mice were infused with angiotensin II (Ang II), and the expression and source of cardiac IL-5 were analysed. The results showed that cardiac IL-5 expression was time- and dose-dependently decreased after Ang II infusion, and was mainly derived from cardiac macrophages. Additionally, IL-5-knockout (IL-5-/-) mice were used to observe the effects of IL-5 knockout on Ang II-induced cardiac remodelling. We found knockout of IL-5 significantly increased the expression of cardiac hypertrophy markers, elevated myocardial cell cross-sectional areas and worsened cardiac dysfunction in Ang II-infused mice. IL-5 deletion also promoted M2 macrophage differentiation and exacerbated cardiac fibrosis. Furthermore, the effects of IL-5 deletion on cardiac remodelling was detected after the STAT3 pathway was inhibited by S31-201. The effects of IL-5 on cardiac remodelling and M2 macrophage differentiation were reversed by S31-201. Finally, the effects of IL-5 on macrophage differentiation and macrophage-related cardiac hypertrophy and fibrosis were analysed invitro. IL-5 knockout significantly increased the Ang II-induced mRNA expression of cardiac hypertrophy markers in myocardial cells that were co-cultured with macrophages, and this effect was reversed by S31-201. Similar trends in the mRNA levels of fibrosis markers were observed when cardiac fibroblasts and macrophages were co-cultured. In conclusions, IL-5 deficiency promote the differentiation of M2 macrophages by activating the STAT3 pathway, thereby exacerbating cardiac remodelling in Ang II-infused mice. IL-5 may be a potential target for the clinical prevention of cardiac remodelling.

2-Methoxyestradiol protects from angiotensin II-induced renal CD3+/FOXP3+ T cell-dysregulation via 12/15 lipoxygenase inhibition

We previously showed that 2-methoxyestradiol (2-ME), an active metabolite of 17β-estradiol (E2) and lox (12/15-lipoxygenase) gene disruption, protects against angiotensin (Ang) II-induced hypertension. Moreover, 2-ME reduces Ang II-induced increases in plasma levels of 12(S)-hydroxyeicosatetraenoic acid (HETE), a major arachidonic acid-LOX-generated metabolite in ovariectomized (OVX, endogenous E2 depleted) wild-type (WT) mice. 2-ME also exerts anti-inflammatory effects, and regulatory T cells (Tregs) have been reported to impact Ang II-induced hypertension in rodent models, with premenopausal females being protected relative to males. This study tested the hypothesis that 2-ME protects against the inflammatory effects of Ang II by upregulating Tregs in the kidney via LOX inhibition in female mice. Intact (ovaries present) or OVX WT and OVX-LOX knock-out (KO) mice (8-10 weeks, n=3-4/group) were infused with Ang II (700 ng/kg/min) or saline (vehicle) via osmotic minipumps (S.C.) with or without 2-ME (15 μg/g, I.P., every third day) treatment for 2 weeks. Renal protein expressions of CD3+ T cells and FOXP3+ Tregs were evaluated by immunofluorescence staining and mRNA expressions of tnf-α and il-10 by RT-PCR using TaqMan assay (relative to saline-infused intact WT) in the whole kidney. Results represented as mean ± SEM were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The CD3+, tnf-α and il-10 expressions were comparable between saline-infused intact WT, OVX-WT, and OVX-KO (CD3+: 8.3±0.3, 7.0±0.5, 6.3±0.3; tnf-α: 1.0±0.0, 0.9±0.0, 0.8±0.2; il-10: 1.1±0.1, 0.9±0.1, 1.0±0.0). However, saline-infused intact WT and OVX-KO expressed higher Tregs (p&lt;0.05) as compared to OVX-WT (7.6±0.3, 7.3±0.3 vs 4.0±0.5). Ang II increased CD3+, Tregs, and il-10 (p&lt;0.05) but not tnf-α expressions as compared to its vehicle in intact WT (12.6±0.3, 15.3±0.3, 3.7±0.3,0.8±0.1). Ang II also increased CD3+ (11.0±0.6) and Tregs (11.0±0.6) in OVX-KO (p&lt;0.05), without altering mRNA expressions of tnf-α (0.9±0.3) and il-10 (1.5±0.1) . In contrast, Ang II resulted in a greater increase in CD3+ (25.0±1.1) in OVX-WT than OVX-KO and intact WT, besides increasing tnf-α expression (2.5±0.4) (p&lt;0.05). 2-ME reduced CD3+ (16.7±0.9) and tnf-α (1.4±0.4) expressions, and increased Tregs (10.0±1.0) and il-10 (2.0±0.0) expressions in Ang II-infused OVX-WT mice (p&lt;0.05). 2-ME also reduced CD3+ (6.7±0.3, p&lt;0.05) and showed a trend to increase Tregs (14.0±1.1, P=0.056), and il-10 (2.3±0.2, P=0.058) expressions without altering tnf-α (0.7±0.1) expression in Ang II-infused OVX-KO mice. In conclusion, Ang II produces a specific effect on the renal T cell profile, greater increases in proinflammatory T cells in OVX mice, and anti-inflammatory Tregs in intact female mice, most likely mediated by E2 through metabolite 2-ME via inhibition of LOX. R01HL19134-47, AHA Award#940538. 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 149: The Protective Effect Of Vamp8 Deficiency In Aortopathies: Potential Impact Of Impaired Platelet Cargo Release

Introduction and Objective: Platelet activation and subsequent cargo secretion play a critical role in thrombus formation and aortic remodeling, factors potentially influencing the growth and rupture of abdominal aortic aneurysms (AAA). Despite the compelling data that platelet activation is associated with AAA and rupture, the underlying mechanisms remain poorly understood. This study hypothesizes that VAMP8, a key element of platelets’ secretory machinery, significantly contributes to AAA formation. Approach and Results: A retrospective, single-center multiple comparisons study of adult aneurysmal patients admitted to UKHealthCare between 2004 and 2023 revealed a significantly lower platelet count in AAA patients (n=3131, 213.76 ± 3.46) vs. healthy individuals (n= 6309, 243.18 ± 4.09) suggesting an increase in platelets consumption, potentially in the aneurysmal sack. Thoracic aortic aneurysm and aortic dissection patients also indicated lower platelet count. In vivo , platelets were shown to accumulate at elastin break sites and false lumens of abdominal aortas from 28-day AngII-infused hypercholesteremic mice. To assess the role of platelets, specifically platelet secretion, we utilized VAMP8 null mice. We first validated the platelet secretion defect in these mice and then used RNASeq and proteomics array to define the role of VAMP8 in platelet cargo packaging. Next, VAMP8 -/- or WT hypercholesterolemic male mice fed a Western diet were infused with AngII (1,000 ng/kg/min) for 4 weeks. Our results revealed that VAMP8 -/- mice are protected against AngII-driven aortic rupture. Additionally, aortic ex vivo analysis indicated that the VAMP8 deficiency (n=15, aortic diameter: 0.9 mm ± 0.03) profoundly attenuated AngII-induced AAA compared to control (n=9, aortic diameter: 2.0 mm ± 0.2). Further analysis also indicated a marked reduction in thoracic aortic aneurysm and atherosclerosis development in VAMP8 -\- vs. Control group. Conclusion: Our results elucidate that a deficiency in VAMP8 results in the profound attenuation of aortic aneurysms and atherosclerosis development, introducing a novel paradigm for understanding the impact of platelet cargo secretion in the development of aortopathies.

Abstract 1005: Hypertriglyceridemia Promotes Aortic Aneurysm Formation And Rupture In Angiotensin Ii Infused Mice

Plasma total cholesterol (TC) and triglyceride (TG) are reported to be positively associated with the risk of abdominal aortic aneurysm (AAA). Our group discovered that hypercholesterolemia augments angiotensin II-induced AAA in mice. We also found that remnants of triglyceride-rich lipoproteins (TRLs) may be crucial for AngII-induced AAA. Recently, it was reported that hypertriglyceridemia (HTG) caused by inducible lipoprotein lipase deficiency (iLpl-/-) results in uptake and accumulation of TRL lipid in aortic endothelial cells in vivo. Based upon these findings, we hypothesized that HTG stimulates AAA formation. Methods: Adult male and female Lplf/f.beta-actin-Mer/Cre/Mer 1/0 and Lplf/f mice were administered 75 mg/kg/day tamoxifen for 5 consecutive days. Mice were fed either a standard diet (SD) throughout the study or a Western-type diet (WD) starting 1 wk after the completion of tamoxifen administration and continuing for a total of 5 wks. Mini osmotic pumps were implanted in mice 2 wks after completion of tamoxifen administration and delivered saline or AngII at 1,000 ng/kg/min for 4 wks. Results: AngII infused and SD fed iLpl-/- versus Lplf/f mice had elevated plasma TG and TC levels but similar abdominal aortic external diameter and AAA incidence. Since the SD was low in fat, a high fat WD was then fed to AngII-infused mice. Plasma TG and TC were increased in female iLpl-/- versus Lplf/f mice. However, plasma lipid concentrations could not be measured in male iLpl-/- because 10 of 11 animals had died from aortic rupture. In contrast, none of the male Lplf/f mice died and only 4 of 11 had abdominal aortic dilation. Female compared to male iLpl-/- mice were protected from AngII-induced aortic rupture (1/11 died) and only 1 of 9 females had abdominal aortic dilation. To eliminate the possibility that HTG alone caused aortic rupture in male mice, the study was repeated with the addition of saline infusion. In agreement with the first study, AngII plus WD lead to death by aortic rupture in all male iLpl-/- mice (9/9). In contrast, iLpl-/- male mice infused with saline had markedly greater survival (1/5 died of unknown cause). Conclusions: HTG causes aortic aneurysm development in AngII-infused male iLpl-/- mice. Thus, treating HTG could reduce AAA risk.

Inhibition of smooth muscle cell death by Angiotensin 1-7 protects against abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) represents a debilitating vascular disease characterized by aortic dilatation and wall rupture if it remains untreated. We aimed to determine the effects of Ang 1-7 in a murine model of AAA and to investigate the molecular mechanisms involved. Eight- to 10-week-old apolipoprotein E-deficient mice (ApoEKO) were infused with Ang II (1.44 mg/kg/day, s.c.) and treated with Ang 1-7 (0.576 mg/kg/day, i.p.). Echocardiographic and histological analyses showed abdominal aortic dilatation and extracellular matrix remodeling in Ang II-infused mice. Treatment with Ang 1-7 led to suppression of Ang II-induced aortic dilatation in the abdominal aorta. The immunofluorescence imaging exhibited reduced smooth muscle cell (SMC) density in the abdominal aorta. The abdominal aortic SMCs from ApoEKO mice exhibited markedly increased apoptosis in response to Ang II. Ang 1-7 attenuated cell death, as evident by increased SMC density in the aorta and reduced annexin V/propidium iodide-positive cells in flow cytometric analysis. Gene expression analysis for contractile and synthetic phenotypes of abdominal SMCs showed preservation of contractile phenotype by Ang 1-7 treatment. Molecular analyses identified increased mitochondrial fission, elevated cellular and mitochondrial reactive oxygen species (ROS) levels, and apoptosis-associated proteins, including cytochrome c, in Ang II-treated aortic SMCs. Ang 1-7 mitigated Ang II-induced mitochondrial fission, ROS generation, and levels of pro-apoptotic proteins, resulting in decreased cell death of aortic SMCs. These results highlight a critical vasculo-protective role of Ang 1-7 in a degenerative aortic disease; increased Ang 1-7 activity may provide a promising therapeutic strategy against the progression of AAA.

Abstract 16975: Wheel-Running Exercise Abrogates Enhanced Sympathetic Neural-Immune Interface in Aortic Adventitia of Angiotensin II-Infused Hypertensive Mice

Background: Artery stiffness causes cardiovascular pathology. Although mechanisms underlying endothelial dysfunction and arterial stiffness are well characterized, it is unknown if neural-immune interaction in aortic adventitia (AA) influences arterial stiffness. We hypothesized that sympathetic innervation and CD68 + macrophages are increased in AA of Angiotensin II (Ang II)-infused mice leading to increased arterial fibrosis and stiffness, and that voluntary wheel-running exercise (VWR) mitigates these effects. Methods and Results: In eight-week-old C57BL/6 male mice, Ang II was infused at 1.5 μg/g/day for 4 weeks with and without VWR. Mean arterial blood pressure [MAP] measured by tail-cuff averaged 135±11 mmHg in Ang II-infused mice (n=5) and 90±10 mmHg in vehicle-infused mice (n=4). Staining for markers of nerve fibers/bundles with Neurofilament A (NF200), sympathetic nerves with tyrosine hydroxylase (TH), nerve endings in AA with synapsin, and CD68 + macrophages were all increased in AA removed from Ang II-infused mice compared with vehicle-infused mice. The increases in CD68 + macrophages and extracellular matrix (ECM) were associated with elevated pulse wave velocity (PWV), an index for arterial stiffness (Ang II: p &lt; 0.05 vs. vehicle). scRNA-seq analyses further corroborated that Ang II infusion promoted fibrosis and macrophage-associated genes in AA. VWR significantly attenuated the Ang II-induced increases in MAP, sympathetic innervation, CD68 + expression, ECM, and PWV (Ang II+VWR: all p&lt;0.05 vs. Ang II). To chemically “denervate” the sympathetic nervous system, we injected 6-hydroxydopamine (6-OHDA, 100 mg/kg on day 0 and 250 mg/kg on day 2, then weekly for 4 weeks) or vehicle in Ang II-infused mice. 6-OHDA decreased MAP, sympathetic innervation, macrophage expression, ECM accumulation, and PWV (Ang II+6-OHDA: p&lt;0.05 vs. Ang II+vehicle, n=3). Conclusion: Ang II-induced hypertensive mice develop increases in sympathetic innervation and macrophages in close proximity within AA that define an enhanced sympathetic neural-immune interface in association with vascular fibrosis, arterial stiffness, and hypertension. VWR significantly mitigates these pathophysiological responses.

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.

Abstract P237: Bainiku-ekisu Attenuates Cardiac Remodeling In Angiotensin Ii-infused Mice

A fruit of the Prunus mume is a traditional food in Japan. Recently, bainiku-ekisu, an infused juice concentrate of Japanse Prunus mume , is attracting attention as a health promoting supplement. Angiotensin II (AngII) plays a central role in development of hypertension. It has been reported that bainiku-ekisu treatment attenuates the growth-promoting signaling induced by AngII in vascular smooth muscle cells. However, whether bainiku-ekisu has any effect on an animal model of hypertension remains unknown. Therefore, this study was designed to explore the potential anti-hypertensive benefit of bainiku-ekisu utilizing a mouse model of hypertension with AngII infusion. 8- to 10-week-old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump or sham-operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement and at 2 weeks by telemetry. Cardiac hypertrophy was assessed by heart-to-body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular remodeling. Increase in heart-to-body weight ratios by Ang II were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 μm vs. 71 μm, respectively, p&lt;0.01). Control mice with angiotensin II infusion showed cardiac matrix deposition, which was attenuated in BEW group. In addition, attenuation of hypertension was observed in Ang II-infused mice with BEW treatment. We also observed attenuation of protein synthesis induced by AngII with BE incubation in cultured aortic VSMC. In conclusion, BEW prevented Ang II-induced vascular remodeling and hypertension. Potential cardiovascular health benefit to taking BE as a functional food should be further studied.

Abstract P432: CTRP1 Contributes To Angiotensin II-Induced Hypertension Via Activation Of Intrarenal Renin

C1q/TNF-related protein 1(CTRP1) is generally regarded as an adipokine and its circulating level is elevated in patients with hypertension. However, the functional role and mechanism of CTRP1 in blood pressure (BP) regulation largely remains elusive. In the present study, we investigated the BP phenotype of global CTRP1 knockout mice (termed CTRP1 KO) during angiotensin II (AngII) infusion and further explored the underlying mechanism involving the intrarenal renin. At baseline, 4-mo-old CTRP1 KO mice exhibited no developmental abnormalities or had normal BPs as compared with wild-type controls (WT). AngII infusion (300 ng/min/kg) induced a 3-fold increase in plasma and urinary CTRP1 levels as assessed by ELISA, but it was undetectable in CTRP1 KO mice. By radiotelemetry, AngII-infused CTRP1 KO mice exhibited reduced MAP as compared with AngII-infused WT mice (MAP [mmHg] in Day 7: 124.1 ± 2.4 in CTRP1 KO mice vs. 135 ± 3.1 in WT mice, n = 4 for each group, p &lt; 0.05 by Student’s t test). This reduction was accompanied by a diminished response in intrarenal renin expression, as evidenced by urinary total prorenin/renin excretion ([ng/24h]: 0.51 ± 0.08 in WT/Vehicle vs. 1.22 ± 0.12 in WT/AngII; 0.32 ± 0.11 in CTRP1 KO/Vehicle vs. 0.54 ± 0.09 in CTRP1 KO/AngII; p &lt; 0.05 by Two-way ANOVA analysis) and renal medullary mRNA levels (qRT-PCR: 1.00 ± 0.18 in WT/Vehicle vs. 3.73 ± 0.59 in WT/AngII; 0.52 ± 0.21 in CTRP1 KO/Vehicle vs. 1.31 ± 0.37 in CTRP1 KO/AngII, p &lt; 0.05 by Two-way ANOVA analysis). In contrast, renal cortical renin mRNA was suppressed by AngII infusion more significantly in WT than in CTRP1 KO mice. In cultured mouse collecting duct-derived M-1 cells, treatment with recombinant human CTRP1-His protein (hCTRP1-His) at 20 nM for 24 hours consistently upregulated renin expression at both protein (Densitometry: 1.00 ± 0.23 in Vehicle group vs. 2.58 ± 0.42 in hCTRP1-His group, p &lt; 0.05) and mRNA levels (qRT-PCR: 1.00 ± 0.12 in Vehicle group vs. 11.60 ± 1.21 in hCTRP1-His group, p &lt; 0.05) levels. Collectively, these findings provide evidence favoring CTRP1 as a regulator of intrarenal renin mediating AngII-induced hypertension and further suggest CTRP1 as a potential mediator of adipose-renal crosstalk.

Abstract 101: Similar Cd28 + Delta T Effector Memory Cell Subpopulations Are Present In Murine Perivascular Tissues Of Angiotensin II-infused Male Mice And Subcutaneous White Adipose Tissue Of Obese Women

Introduction: The innate-like γδ T cells play a role in angiotensin II (AngII)-induced hypertension, mesenteric artery injury and T cell activation in mesenteric vessels/perivascular adipose tissue (MV/PVAT) in mice. Blood γδ T cells frequency is associated with systolic blood pressure in humans. Obesity was associated with small artery injury in subcutaneous white adipose tissue (WAT) and is a risk factor for hypertension in humans. We hypothesized that single cell RNA sequencing (scRNA-seq) in MV/PVAT of AngII-infused vs control mice and in subcutaneous WAT of obese vs lean humans will reveal γδ T cell subpopulations involved in hypertension, vascular injury and T cell activation. Methods: Male C57BL/6J mice were infused SC or not with 490 ng/kg/min AngII for 14 days (n=3). Hypertension was confirmed by tail cuff blood pressure measurement. MV/PVAT were collected, and T cells were isolated and used for scRNA-seq. γδ T cell subpopulations were studied by subclustering T cells expressing the T cell receptor δ constant chain. Cell subpopulations were validated by flow cytometry. Subcutaneous WAT CD45 + cell ScRNA-seq data of 3 lean and 3 obese women were obtained from GEO NCBI database (GSE155960) and γδ T cell subpopulations analyzed as above. Results: ScRNA-seq identified 6 murine and 9 human δ T cell subclusters. AngII increased Cd28 high (% of T cells: 3.6±0.9 vs 1.4±0.2) and Trdv4 + δ T effector memory (EM) cells (7.3±1.3 vs 3.4±1.7) and lowered resting δ T cells (1.0±0.2 vs 4.2±1.5) in mice. Cd28 high δ T EM and resting δ T cell study by flow cytometry using unique marker pairs, CD28 + SELL - and CD28 - SELL + , showed that AngII increased CD28 + SELL - (cell number: 376±48 vs 220±40) and decreased CD28 - SELL + δ T cells (23±4 vs 45±4) in MV/PVAT. Cd28 / CD28 subclustering of δ T cells identified 4 human and murine cell populations and showed that AngII increased Cd28 + γδ natural killer T EM (% of T cells: 1.9±0.3 vs 0.5±0.2) and Cd28 high δ T EM cell subpopulations (1.6±0.5 vs 0.2±0.1) in mice, and that CD28 + δ T EM-1 cell subpopulation tended to be increased in obese vs lean women. Conclusion: ScRNA-seq revealed similar δ T EM cell subpopulations expressing CD28 in murine MV/PVAT and human subcutaneous WAT that could play a role in hypertension, vascular injury and T cell activation.

PDE4D mediates impaired β-adrenergic receptor signalling in the sinoatrial node in mice with hypertensive heart disease.

The sympathetic nervous system increases HR by activating β-adrenergic receptors (β-ARs) and increasing cAMP in sinoatrial node (SAN) myocytes while phosphodiesterases (PDEs) degrade cAMP. Chronotropic incompetence, the inability to regulate heart rate (HR) in response to sympathetic nervous system activation, is common in hypertensive heart disease; however, the basis for this is poorly understood. The objective of this study was to determine the mechanisms leading to chronotropic incompetence in mice with angiotensin II (AngII)-induced hypertensive heart disease. C57BL/6 mice were infused with saline or AngII (2.5 mg/kg/day for 3 weeks) to induce hypertensive heart disease. HR and SAN function in response to the β-AR agonist isoproterenol (ISO) were studied in vivo using telemetry and electrocardiography, in isolated atrial preparations using optical mapping, in isolated SAN myocytes using patch-clamping, and using molecular biology. AngII-infused mice had smaller increases in HR in response to physical activity and during acute ISO injection. Optical mapping of the SAN in AngII-infused mice demonstrated impaired increases in conduction velocity and altered conduction patterns in response to ISO. Spontaneous AP firing responses to ISO in isolated SAN myocytes from AngII-infused mice were impaired due to smaller increases in diastolic depolarization (DD) slope, hyperpolarization-activated current (If), and L-type Ca2+ current (ICa,L). These changes were due to increased localization of PDE4D surrounding β1- and β2-ARs in the SAN, increased SAN PDE4 activity, and reduced cAMP generation in response to ISO. Knockdown of PDE4D using a virus-delivered shRNA or inhibition of PDE4 with rolipram normalized SAN sensitivity to β-AR stimulation in AngII-infused mice. AngII-induced hypertensive heart disease results in impaired HR responses to β-AR stimulation due to up-regulation of PDE4D and reduced effects of cAMP on spontaneous AP firing in SAN myocytes.