Ang II-infused Mice Research Articles

Collagen fibril abnormalities in human and mice abdominal aortic aneurysm

Vascular diseases like abdominal aortic aneurysms (AAA) are characterized by a drastic remodeling of the vessel wall, accompanied with changes in the elastin and collagen content. At the macromolecular level, the elastin fibers in AAA have been reported to undergo significant structural alterations. While the undulations (waviness) of the collagen fibers is also reduced in AAA, very little is understood about changes in the collagen fibril at the sub-fiber level in AAA as well as in other vascular pathologies. In this study we investigated structural changes in collagen fibrils in human AAA tissue extracted at the time of vascular surgery and in aorta extracted from angiotensin II (AngII) infused ApoE-/- mouse model of AAA. Collagen fibril structure was examined using transmission electron microscopy and atomic force microscopy. Images were analyzed to ascertain length and depth of D-periodicity, fibril diameter and fibril curvature. Abnormal collagen fibrils with compromised D-periodic banding were observed in the excised human tissue and in remodeled regions of AAA in AngII infused mice. These abnormal fibrils were characterized by statistically significant reduction in depths of D-periods and an increased curvature of collagen fibrils. These features were more pronounced in human AAA as compared to murine samples. Thoracic aorta from Ang II-infused mice, abdominal aorta from saline-infused mice, and abdominal aorta from non-AAA human controls did not contain abnormal collagen fibrils. The structural alterations in abnormal collagen fibrils appear similar to those reported for collagen fibrils subjected to mechanical overload or chronic inflammation in other tissues. Detection of abnormal collagen could be utilized to better understand the functional properties of the underlying extracellular matrix in vascular as well as other pathologies. STATEMENT OF SIGNIFICANCE: Several vascular diseases including abdominal aortic aneurysm (AAA) are characterized by extensive remodeling in the vessel wall. Although structural alterations in elastin fibers are well characterized in vascular diseases, very little is known about the collagen fibril structure in these diseases. We report here a comprehensive ultrastructural evaluation of the collagen fibrils in AAA, using high-resolution microscopy techniques like transmission electron microscopy (TEM) and atomic force microscopy (AFM). We elucidate how abnormal collagen fibrils with compromised D-periodicity and increased fibril curvature are present in the vascular tissue in both clinical AAA as well as in murine models. We discuss how these abnormal collagen fibrils are likely a consequence of mechanical overload accompanying AAA and could impact the functional properties of the underlying tissue.

Erythrocyte Metabolic Reprogramming by Sphingosine 1-Phosphate in Chronic Kidney Disease and Therapies.

Hypoxia promotes renal damage and progression of chronic kidney disease (CKD). The erythrocyte is the only cell type for oxygen (O2) delivery. Sphingosine 1-phosphate (S1P)-a highly enriched biolipid in erythrocytes-is recently reported to be induced under high altitude in normal humans to enhance O2 delivery. However, nothing is known about erythrocyte S1P in CKD. To investigate the function and metabolic basis of erythrocyte S1P in CKD with a goal to explore potential therapeutics. Using erythrocyte-specific SphK1 (sphingosine kinase 1; the only enzyme to produce S1P in erythrocytes) knockout mice (eSphK1-/-) in an experimental model of hypertensive CKD with Ang II (angiotensin II) infusion, we found severe renal hypoxia, hypertension, proteinuria, and fibrosis in Ang II-infused eSphk1-/- mice compared with controls. Untargeted metabolomics profiling and in vivo U-13C6 isotopically labeled glucose flux analysis revealed that SphK1 is required for channeling glucose metabolism toward glycolysis versus pentose phosphate pathway, resulting in enhanced erythroid-specific Rapoport-Luebering shunt in Ang II-infused mice. Mechanistically, increased erythrocyte S1P functioning intracellularly activates AMPK (AMP-activated protein kinase) 1α and BPGM (bisphosphoglycerate mutase) by reducing ceramide/S1P ratio and inhibiting PP2A (protein phosphatase 2A), leading to increased 2,3-bisphosphoglycerate (an erythrocyte-specific metabolite negatively regulating Hb [hemoglobin]-O2-binding affinity) production and thus more O2 delivery to counteract kidney hypoxia and progression to CKD. Preclinical studies revealed that an AMPK agonist or a PP2A inhibitor rescued the severe CKD phenotype in Ang II-infused eSphK1-/- mice and prevented development of CKD in the control mice by inducing 2,3-bisphosphoglycerate production and thus enhancing renal oxygenation. Translational research validated mouse findings in erythrocytes of hypertensive CKD patients and cultured human erythrocytes. Our study elucidates the beneficial role of eSphk1-S1P in hypertensive CKD by channeling glucose metabolism toward Rapoport-Luebering shunt and inducing 2,3-bisphosphoglycerate production and O2 delivery via a PP2A-AMPK1α signaling pathway. These findings reveal the metabolic and molecular basis of erythrocyte S1P in CKD and new therapeutic avenues.

Altered heart rate variability in angiotensin II–mediated hypertension is associated with impaired autonomic nervous system signaling and intrinsic sinoatrial node dysfunction

Hypertensive heart disease is associated with sinoatrial node (SAN) dysfunction and reductions in heart rate variability (HRV). Alterations in HRV could occur in association with changes in autonomic nervous system (ANS) activity, changes in SAN function and responsiveness to ANS agonists, or both. These relationships are unclear. The purpose of this study was to investigate the roles of ANS signaling, intrinsic SAN function, and changes in HRV in a mouse model of angiotensin II (AngII)-mediated hypertensive heart disease. Mice were treated with saline or AngII (2.5 mg/(kg⋅d)) for 3 weeks. ANS activity was assessed through HRV analysis of electrocardiograms collected invivo by telemetry as well as direct recordings of vagal nerve activity and renal sympathetic nerve activity from anesthetized mice. The effects of the ANS agonists isoproterenol and carbachol on SAN function and beating interval variability were assessed from electrogram recordings in intact isolated atrial preparations and from spontaneous action potential recordings in isolated SAN myocytes. Time and frequency domain analysis demonstrates that mice infused with AngII had reduced HRV. AngII-infused mice had elevated renal sympathetic nerve activity while resting vagal nerve activity was unchanged. AngII caused an increase in SAN beating interval variability in isolated atrial preparations and isolated SAN myocytes. Furthermore, isolated atrial preparations and SAN myocytes from AngII-infused mice had impaired responses to both isoproterenol and carbachol. Reduced HRV in hypertension occurs in association with altered sympathovagal balance as well as intrinsic SAN dysfunction and reduced responsiveness of SAN myocytes to ANS agonists.

Runx2 (Runt-Related Transcription Factor 2)-Mediated Microcalcification Is a Novel Pathological Characteristic and Potential Mediator of Abdominal Aortic Aneurysm

Abdominal aortic aneurysms (AAAs) are highly lethal diseases without effective clinical predictors and therapeutic targets. Vascular microcalcification, as detected by fluorine-18-sodium fluoride, has recently been recognized as a valuable indicator in predicting atherosclerotic plaque rupture and AAA expansion. However, whether vascular microcalcification involved in the pathogenesis of AAA remains elusive. Approach and Results: Microcalcification was analyzed in human aneurysmal aortas histologically and in AngII (angiotensin II)-infused ApoE-/- mouse aortas by fluorine-18-sodium fluoride positron emission tomography and X-ray computed tomography scanning in chronological order in live animals. AAA patients' aortic tissue showed markedly enhanced microcalcification in the aortic media within the area proximal to elastic fiber degradation, compared with non-AAA patients. Enhanced fluorine-18-sodium fluoride uptake preceded significant aortic expansion in mice. Microcalcification-positive mice on day 7 of AngII infusion showed dramatic aortic expansion on subsequent days 14 to 28, whereas microcalcification-negative AngII-infused mice and saline-induced mice did not develop AAA. The application of hydroxyapatite, the main component of microcalcification, aggravated AngII-induced AAA formation in vivo. RNA-sequencing analysis of the suprarenal aortas of 4-day-AngII-infused ApoE-/- mice and bioinformatics analysis with ChIP-Atlas database identified the potential involvement of the osteogenic transcriptional factor Runx2 (runt-related transcription factor 2) in AAA. Consistently, vascular smooth muscle cell-specific Runx2 deficiency markedly repressed AngII-induced AAA formation in the ApoE-/- mice compared with the control littermates. Our studies have revealed microcalcification as a novel pathological characteristic and potential mediator of AAA, and targeting microcalcification may represent a promising strategy for AAA prevention and treatment.

Interleukin-9 Deletion Relieves Vascular Dysfunction and Decreases Blood Pressure via the STAT3 Pathway in Angiotensin II-Treated Mice.

Background Multiple interleukin (IL) family members were reported to be closely related to hypertension. We aimed to investigate whether IL-9 affects angiotensin II- (Ang II-) induced hypertension in mice. Methods Mice were treated with Ang II, and IL-9 expression was determined. In addition, effects of IL-9 knockout (KO) on blood pressure were observed in Ang II-infused mice. To determine whether the effects of IL-9 on blood pressure was mediated by the signal transducer and activator of the transcription 3 (STAT3) pathway, Ang II-treated mice were given S31-201. Furthermore, circulating IL-9 levels in patients with hypertension were measured. Results Ang II treatment increased serum and aortic IL-9 expression in a dose-dependent manner; IL-9 levels were the highest in the second week and continued to remain high into the fourth week after the treatment. IL-9 KO downregulated proinflammatory cytokine expression, whereas it upregulated anti-inflammatory cytokine levels, relieved vascular dysfunction, and decreased blood pressure in Ang II-infused mice. IL-9 also reduced smooth muscle 22α (SM22α (SM22Conclusions IL-9 KO alleviates inflammatory response, prevents phenotypic transformation of smooth muscle, reduces vascular dysfunction, and lowers blood pressure via the STAT3 pathway in Ang II-infused mice. IL-9 might be a novel target for the treatment and prevention of clinical hypertension.

AVE0991, a nonpeptide angiotensin-(1-7) mimic, inhibits angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E knockout mice.

AVE0991, a nonpeptide angiotensin-(1-7) mimic, has similar protective effects for cardiovascular system to Ang-(1-7). In this article, we aimed to explore the effects of AVE0991 and Ang-(1-7) on abdominal aortic aneurysm (AAA) induced by Ang II in apolipoprotein E knockout mice. The mice AAA model was established by Ang II infusion, and then mice received different treatment with saline, Ang II (1.44mg/kg/day), different dose AVE0991 (0.58 or 1.16μmol/kg/day), or Ang-(1-7) (400ng/kg/min). The incidence of AAA was 76%, 48%, 28%, and 24% in the vehicle, the low-dose AVE0991, high-dose AVE0991, and the Ang-(1-7) group, respectively. In comparison with control group, AVE0991 and Ang-(1-7) treatment significantly increased smooth muscle cells and decreased macrophage accumulation, the expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α), and the expression and activity of metalloproteinases 2 and 9 in mice AAA model or in human smooth muscle cells (hVSMCs). The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation, whereas the positive impacts were reversed by co-administration with the Mas antagonist A779 (400ng/kg/min) and AVE0991 in Ang II-infused mice or in hVSMCs. Therefore, AVE0991 and Ang-(1-7) might be novel and promising interventions in the prevention and treatment of AAA. KEY MESSAGES: • AVE0991 dose-dependently inhibited Ang II-induced AAA formation in Apoe-/- mice. • Ang-(1-7) played the same protective role as high-dose AVE0991. • Inhibition of Mas receptor with A779 could reverse the protective effect of AVE0991. • The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation.

6\u03b2-Hydroxytestosterone, a metabolite of testosterone generated by CYP1B1, contributes to vascular changes in angiotensin II-induced hypertension in male mice

BackgroundPreviously, we showed that 6β-hydroxytestosterone (6β-OHT), a cytochrome P450 1B1 (CYP1B1)-derived metabolite of testosterone, contributes to angiotensin II (Ang II)-induced hypertension in male mice. This study was conducted to test the hypothesis that 6β-OHT contributes to increased vascular reactivity, endothelial dysfunction, vascular hypertrophy, and reactive oxygen species production associated with Ang II-induced hypertension.MethodsEight- to 10-week-old intact or castrated C57BL/6 J (Cyp1b1+/+ and Cyp1b1−/−) mice were anesthetized for implantation of a micro-osmotic pump which delivered Ang II (700 ng/kg/day) or saline for 14 days. Mice were injected with 6β-OHT (15 μg/g b.w every third day), flutamide (8 mg/kg every day), or its vehicle. Blood pressure was measured via tail-cuff. Vascular reactivity, endothelial-dependent and endothelial-independent vasodilation, media to lumen ratio, fibrosis by collagen deposition, and reactive oxygen species production by dihydroethidium staining were determined in the isolated thoracic aorta.ResultsThe response of thoracic aorta to phenylephrine and endothelin-1 was increased in Ang II-infused Cyp1b1+/+ mice compared to intact Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice; these effects of Ang II were restored by treatment with 6β-OHT. Ang II infusion caused endothelial dysfunction, as indicated by decreased relaxation of the aorta to acetylcholine in Cyp1b1+/+ but not Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice. 6β-OHT did not alter Ang II-induced endothelial dysfunction in Cyp1b1+/+ mice but restored it in Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice. Ang II infusion increased media to lumen ratio and caused fibrosis and reactive oxygen species production in the aorta of Cyp1b1+/+ mice. These effects were minimized in the aorta of Cyp1b1−/− or castrated Cyp1b1+/+ and Cyp1b1−/− mice and restored by treatment with 6β-OHT. Treatment with the androgen receptor antagonist flutamide reduced blood pressure and vascular hypertrophy in castrated Ang II-infused mice injected with 6β-OHT.Conclusions6β-OHT is required for the action of Ang II to increase vascular reactivity and cause endothelial dysfunction, hypertrophy, and increase in oxygen radical production. The effect of 6β-OHT in mediating Ang II-induced hypertension and associated hypertrophy is dependent on the androgen receptor. Therefore, CYP1B1 could serve as a novel target for the development of therapeutics to treat vascular changes in hypertensive males.

Sympathetic Enhancement of Memory T-Cell Homing and Hypertension Sensitization.

Effector memory T lymphocytes (TEM cells) exacerbate hypertension in response to repeated hypertensive stimuli. These cells reside in the bone marrow for prolonged periods and can be reactivated on reexposure to the hypertensive stimulus. Because hypertension is associated with increased sympathetic outflow to the bone marrow, we hypothesized that sympathetic nerves regulate accumulation and reactivation of bone marrow-residing hypertension-specific TEM cells. Using unilateral superior cervical ganglionectomy in wild-type C57BL/6 mice, we showed that sympathetic nerves create a bone marrow environment that supports residence of hypertension-specific CD8+ T cells. These cells, defined by their proliferative response on coculture with dendritic cells from Ang (angiotensin) II-infused mice, were reduced in denervated compared with innervated bone of Ang II-infused mice. Adoptively transferred CD8+ T cells from Ang II-infused mice preferentially homed to innervated compared with denervated bone. In contrast, ovalbumin responsive T cells from OT-I mice did not exhibit this preferential homing. Increasing superior cervical ganglion activity by activating Gq-coupled designer receptor exclusively activated by designer drug augmented CD8+ TEM bone marrow accumulation. Adoptive transfer studies using mice lacking β2AR (β2 adrenergic receptors) indicate that β2AR in the bone marrow niche, rather than T-cell β2AR is critical for TEM cell homing. Inhibition of global sympathetic outflow using Gi-coupled DREADD (designer receptor exclusively activated by designer drug) injected into the rostral ventrolateral medulla or treatment with a β2AR antagonist reduced hypertension-specific CD8+ TEM cells in the bone marrow and reduced the hypertensive response to a subsequent response to low dose Ang II. Sympathetic nerves contribute to the homing and survival of hypertension-specific TEM cells in the bone marrow after they are formed in hypertension. Inhibition of sympathetic nerve activity and β2AR blockade reduces these cells and prevents the blood pressure elevation and renal inflammation on reexposure to hypertension stimuli.

Sirt6 deficiency aggravates angiotensin II-induced cholesterol accumulation and injury in podocytes.

Disturbed renal lipid metabolism, especially cholesterol dysregulation plays a crucial role in the pathogenesis of chronic kidney disease (CKD). We recently reported that angiotensin (Ang) II could induce cholesterol accumulation and injury in podocytes. However, the underlying mechanisms for these alterations remain unknown.Methods: Bioinformatics analysis of renal biopsy specimens from patients with hypertensive nephropathy (HN) suggests the involvement of Sirtuin 6 (Sirt6) in Ang II-induced dysregulation of glomerular cholesterol. Using a podocyte-specific Sirt6 knockout mouse model, the effects of Sirt6 on Ang II-induced cholesterol accumulation in podocytes and the therapeutic efficacies of cholesterol-lowering agents were evaluated.Results: Cholesterol accumulation was detected in the podocytes of Ang II-infused mice, whereas selective deletion of Sirt6 in podocytes not only increased cholesterol accumulation in these cells but also exacerbated Ang II-induced kidney injury. Deletion of Sirt6 also attenuated the protective effect of cyclodextrin (CD) on Ang II-induced urinary albumin excretion, glomerulosclerosis and podocyte injury. In addition, we demonstrated that Sirt6 affected cholesterol efflux in podocytes by regulating the expression of ATP-binding cassette transporter G1 (ABCG1).Conclusions: These findings provide evidence that Sirt6 is a potential target for renin-angiotensin system (RAS)-associated podocyte injury and provide a rationale for the application of cholesterol-lowering agents in patients with CKD.

GPR174 suppression attenuates retinopathy in angiotensin II (Ang II)-treated mice by reducing inflammation via PI3K/AKT signaling

Hypertension is closely associated with eye diseases, including hypertensive retinopathy. However, the molecular mechanism controlling the progression of hypertensive retinopathy remains poorly understood. G-Protein Coupled Receptor 174 (GPR174) is expressed in various tissues, and plays a critical role in regulating immune and inflammatory responses. However, if GPR174 is involved in angiotensin II (Ang II)-induced retinopathy is still unclear. In this study, hypertensive retinopathy was induced by Ang II infusion in the wild type (GPR174+/+) and GPR174 knockout (GPR174−/−) mice. Clinical data indicated that GPR174 levels were markedly up-regulated in serum of patients with hypertension (HP) or hypertensive retinopathy (HR), along with increased trypsin-like activity. Similar changes of GRP174 and trypsin-like activity were observed in Ang II-infused mice. Furthermore, Ang IIsignificantly increased the central retinal thickness, vascular permeability and inflammatory response in GPR174+/+ micewhen compared with the saline GPR174+/+ mice. Of note, these effects were markedly alleviated by the knockout ofGPR174 in Ang II-treated mice. Consistently, vascular endothelial growth factor (VEGF) expression levels in retinal tissue were also stimulated by Ang II, which were clearly attenuated by GPR174−/−. In addition, phosphatidylinositol 3-kinase (PI3 K)/AKT and nuclear factor-κB (NF-κB) signaling pathways were markedly activated in retinas of Ang II-infused GPR174+/+ mice, whereas being greatly ameliorated by GPR174−/−. The in vitro data showed that pre-treatment of PI3 K/AKT specific inhibitor LY294002 remarkably abrogated GPR174 over-expression-accelerated expression levels of Iba-1, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in lipopolysaccharide (LPS)-incubated retinal microglial cells.Furthermore, in LPS-exposed retinal microglial cells, PI3 K/AKT and NF-κB pathways were further promoted by GPR174 over-expression, which were significantlyabolished by LY294002. Thus, GPR174 might be a positive meditator of inflammation, contributing to Ang II-induced retinopathy by activating PI3 K/AKT signaling, and could be considered as a novel therapeutic target for the treatment of hypertensive retinopathy.

Ginsenoside Rg3 Attenuates Angiotensin II-Mediated Renal Injury in Rats and Mice by Upregulating Angiotensin-Converting Enzyme 2 in the Renal Tissue.

Angiotensin II- (Ang II-) mediated renal injury represents a major pathogenetic mechanism in most chronic kidney diseases. Our previous research demonstrated that ginsenoside Rg3 (Rg3) attenuates Ang II elevation in the myocardium in spontaneously hypertensive rats (SHR). It is possible that Rg3 has similar effects in the renal tissue. In this research, we first demonstrated that Rg3 could attenuate Ang II increase in the kidney of SHR and reduce hypertensive nephropathy progression. Then, we found that Rg3 attenuated Ang II increase by upregulating angiotensin-converting enzyme 2 (ACE2) in the renal tissue. We confirmed this finding in an exogenous Ang II-infused mice model of renal injury, and two models showed consistent results. In conclusion, Rg3 attenuates Ang II-mediated renal injury in rats and mice by upregulating ACE2 in the renal tissue. This research is the first to demonstrate that Rg3 increases tissue ACE2 levels in vivo.

Abstract P120: Leukocyte and Lymphocyte-Derived Extracellular Vesicles are Elevated in Angiotensin-Induced Hypertension

Infiltration of macrophages and T cells in kidneys and vasculature may play a role in the development of hypertension (HTN) and extracellular vesicles (EVs) have emerged as novel cell-to-cell communicators in the pathogenesis HTN. Immune cell-derived EVs can reflect the pathophysiological activity of the parental cell and mediate immune stimulation or inhibition in paracrine fashion. We investigated subgroups of circulating leukocyte-derived EVs (LEVs) in angiotensin II (Ang II)-induced HTN with or without candesartan (C) or hydralazine, hydrochlorothiazide, and reserpine (HHR). Ang II was delivered via osmotic mini pumps (500 ng/kg/min; n=15) and with C (10 mg/kg/day, n=5) or HHR (30 mg/kg/day, n=5) in drinking water for 4 weeks to 129S6 mice. Systolic blood pressure (SBP) was measured daily by tail-cuff manometry. After 4 weeks, mice were euthanized and citrated blood collected. EV isolation was performed by differential centrifugation, followed by EV phenotyping with imaging flow cytometry. We used EV surface markers for leukocyte (CD45), T- and B-cells (CD3,CD19), neutrophils (Ly6g+/CD11b-) and monocytes/macrophages (Ly6g-/CD11b+). Ang II infused mice had significantly higher SBP compared to normotensive controls (NC) (n=5) (173.79 ± 4.9mmHg vs 129.8 ± 6.2mmHg, p <0.0001) and C/HHR treated mice (134.42 ± 7.5mmHg (C) and 138.21 ± 5.2mmHg (HHR), p = NS). Leukocyte-derived EV counts (CD45+ EVs for Ang II: 2.16E+06 particles/ml; NC: 6.15E+05 particles/mL; p=0.0001) and T cell-derived EV counts (CD3+) (Ang II: 2.14E+06 particles/ml, NC: 4.46E+05 particles/mL; p=0.0001] increased after 4 weeks in Ang II-infused mice compared to NC. In C and HHR treated groups, leukocyte- and T cell-derived EV concentrations were similar to NC, suggesting a BP effect rather than drug class effect. CD45+ and CD3+ EV concentrations correlated significantly with SBP [(r2=0.9343; p= 0.0004) and (r2= 0.8982; p= 0.0012), respectively]. In conclusion, leukocyte- and T cell-derived EVs were increased in Ang II-induced HTN and correlated significantly with SBP. Therefore, leukocyte- and T cell-derived EVs are potential bio-markers in HTN. Further studies are required to understand the functional role of leukocyte- and T cell-derived EVs in the pathogenesis of HTN.

Klotho inhibits angiotensin II-induced cardiac hypertrophy, fibrosis, and dysfunction in mice through suppression of transforming growth factor-β1 signaling pathway

Recent studies have revealed critical roles of transforming growth factor-β1 (TGF-β1) and microRNA-132 (miR-132), a downstream mediator of TGF-β1, in the pathogenesis of cardiac remodeling. In this study, we tested whether the antiaging protein klotho modifies angiotensin II (Ang II)-induced cardiac remodeling through regulating TGF-β1-miR-132 axis. We found that both klotho and the TGF-β1 inhibitor LY364947 significantly inhibited cardiac hypertrophy, fibrosis, and dysfunction in Ang II-infused mice, as evidenced by the ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL), cardiomyocyte cross-sectional area, fibrotic area, and expression of prohypertrophic genes (ANP, β-MHC) and fibrotic marker genes (α-SMA, collagen I), echocardiographic parameters. Meanwhile, klotho also significantly inhibited Ang II-induced protein expression of TGF-β1 and phosphorylated Smad2/3 in the heart tissues and cultured cardiomyocytes and cardiac fibroblasts. In vitro experiments demonstrated that Ang II-induced cardiomyocyte hypertrophy and proliferation and activation of cardiac fibroblasts were markedly inhibited by klotho, LY364947 or the miR-132 inhibitor anti-miR-132. Both klotho and the TGF-β1 inhibitor LY364947 downregulated the miR-132 expression. Additionally, klotho decreased Ang II-induced protein expressions of cardiac fibroblast growth factor (FGF)23 in vivo and in vitro. The decreased protein levels of klotho in serum and renal tissues of Ang II-infused mice were elevated by klotho. Klotho downregulated the protein levels of TGF-β1 in renal tissues of Ang II-infused mice. In conclusion, our results suggest that klotho prevents Ang II-induced cardiac remodeling and dysfunction through modifying the TGF-β1-miR-132 axis, providing an experimental basis for clinical treatment on cardiac remodeling.

Circulating miR-103a-3p contributes to angiotensin II-induced renal inflammation and fibrosis via a SNRK/NF-\u03baB/p65 regulatory axis

Although angiotensin II (AngII) is known to cause renal injury and fibrosis, the underlying mechanisms remain poorly characterized. Here we show that hypertensive nephropathy (HN) patients and AngII-infused mice exhibit elevated levels of circulating miR103a-3p. We observe a positive correlation between miR-103a-3p levels and AngII-induced renal dysfunction. miR-103a-3p suppresses expression of the sucrose non-fermentable-related serine/threonine-protein kinase SNRK in glomerular endothelial cells, and glomeruli of HN patients and AngII-infused mice show reduced endothelial expression of SNRK. We find that SNRK exerts anti-inflammatory effects by interacting with activated nuclear factor-κB (NF-κB)/p65. Overall, we demonstrate that AngII increases circulating miR-103a-3p levels, which reduces SNRK levels in glomerular endothelial cells, resulting in the over-activation of NF-κB/p65 and, consequently, renal inflammation and fibrosis. Together, our work identifies miR-103a-3p/SNRK/NF-κB/p65 as a regulatory axis of AngII-induced renal inflammation and fibrosis.

Effects of rikkunshito on renal fibrosis and inflammation in angiotensin II-infused mice

The underlying pathogenesis of chronic kidney disease involves an activated renin-angiotensin system and systemic inflammation which ultimately develop renal injury. Rikkunshito (RKT) has been reported to exert anti-fibrotic and anti-inflammatory effects through enhancement of ghrelin signaling pathway. In this study, we investigated the effects of RKT on renal fibrosis and inflammation in angiotensin II (Ang II)-induced renal injury model. Ang II-infused mice exhibited hypertension, cardiac hypertrophy, increases in blood urea nitrogen and serum creatinine, moderate albuminuria and renal pathological changes such as mild urinary cast, interstitial macrophage infiltration and modest interstitial fibrosis. RKT had no evident effects on the Ang II-induced renal functional insufficiency and fibrosis, but attenuated renal interstitial macrophage infiltration. In addition, RKT significantly restored the Ang II-induced alteration in the expression of renal fibrosis- and inflammation-related genes such as type 3 collagen, transforming growth factor-β, monocyte chemoattractant protein-1 and interleukin-6. Furthermore, although RKT did not affect the expression of renal ghrelin receptor, an Ang II-induced decrease in renal sirtuin 1 expression, a critical down-stream pathway of the ghrelin receptor, was restored by RKT. These findings suggest that RKT potentially has a renal anti-inflammatory effect in the development of renal injury, and this effect could be mediated by the ghrelin signaling pathway.

Exogenous Vasohibin-2 Exacerbates Angiotensin II-Induced Ascending Aortic Dilation in Mice.

Background: Chronic angiotensin II (AngII) infusion promotes ascending aortic dilation in C57BL/6J mice. Meanwhile, vasohibin-2 (VASH2) is an angiogenesis promoter in neovascularization under various pathologic conditions. The aim of this study was to investigate whether exogenous VASH2 influences chronic AngII-induced ascending aortic dilation. Methods and Results: Eight–ten-week-old male C57BL/6J mice were injected with adenovirus (Ad) expressing either VASH2 or LacZ. One week after the injection, mice were infused with either AngII or saline s.c. for 3 weeks. Mice were divided into 4 groups: AngII+VASH2, AngII+LacZ, saline+VASH2, and saline+LacZ. Overexpression of VASH2 significantly increased AngII-induced intimal areas as well as the external diameter of the ascending aorta. In addition, VASH2 overexpression promoted ascending aortic medial elastin fragmentation in AngII-infused mice, which was associated with increased matrix metalloproteinase activity and medial smooth muscle cell (SMC) apoptosis. On western blot analysis, accumulation of apoptotic signaling proteins, p21 and p53 was increased in the AngII+VASH2 group. Furthermore, transfection of human aortic SMC with Ad VASH2 increased p21 and p53 protein abundance upon AngII stimulation. Positive TUNEL staining was also detected in the same group of the human aortic SMC. Conclusions: Exogenous VASH2 exacerbates AngII-induced ascending aortic dilation in vivo, which is associated with increased medial apoptosis and elastin fragmentation.

Fra-1 plays a critical role in angiotensin II-induced vascular senescence.

Vascular aging has a strong relationship with cardiovascular disease. Fos-related antigen 1 (Fra-1), also referred to as Fos-like antigen 1, is a transcription factor and has been reported to be involved in many pathologic processes. Here, we demonstrate that Fra-1 plays a critical role in angiotensin II (Ang II)-induced vascular senescence. Fra-1 expression is increased significantly in Ang II-induced rat aortic endothelial cell (RAEC) senescence and the arteries from Ang II-infused mice. Interestingly, silencing Fra-1 blocks Ang II-induced senescence phenotypes in RAECs, including decreased senescence-associated β-galactosidase staining, and mitigated proliferation suppression and senescence-associated secretory phenotype. Further, knocking down Fra-1 inhibits vascular aging phenotypes in an Ang II-infused mice model. The up-regulated Fra-1 also exists in human atherosclerotic plaques and Ang II-induced vascular smooth muscle cells as well as in replicated senescence RAECs. Mechanistic studies reveal that Fra-1 preferentially associates with c-Jun and binds to the cyclin-dependent kinase inhibitor 1a (p21) and cyclin-dependent kinase inhibitor 2a (p16) promoter region, leading to elevated gene expression, which causes senescence-related phenotypes. In conclusion, our results identify that Fra-1 plays a novel and key role in promoting vascular aging by directly binding and transcriptionally activating p21 and p16 signaling, suggesting intervention of Fra-1 is a potential strategy for preventing aging-associated cardiovascular disorders.-Yang, D., Xiao, C., Long, F., Wu, W., Huang, M., Qu, L., Liu, X., Zhu, Y. Fra-1 plays a critical role in angiotensin II-induced vascular senescence.

Mas receptor deficiency augments angiotensin II-induced atherosclerosis and aortic aneurysm ruptures in hypercholesterolemic male mice

ObjectivePrevious studies demonstrated that deficiency of angiotensin-converting enzyme 2 (ACE2) augmented angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. Effects of ACE2 deficiency could arise from increased concentrations of its substrate, AngII, or decreased concentrations of its product, angiotensin-(1-7) [Ang-(1-7)]. Infusion of Ang-(1-7), a Mas receptor (MasR) ligand, to hypercholesterolemic male mice reduced AngII-induced atherosclerosis, suggesting a protective role of the Ang-(1-7)/MasR axis. However, it is unclear whether endogenous Ang-(1-7) acts at MasR to influence AngII-induced vascular diseases. The purpose of this study was to define the role of MasR deficiency in AngII-induced atherosclerosis and AAA formation and severity in hypercholesterolemic male mice. MethodsMasR+/+ and MasR−/− male mice on a low-density lipoprotein receptor-deficient (Ldlr−/−) or apolipoprotein E-deficient (Apoe−/−) background were infused with AngII at either 600 or 1000 ng/kg/min by osmotic minipump for 28 days. Atherosclerosis was quantified at study end point as percentage lesion surface area of the aortic arch in Ldlr−/− mice. Abdominal aortic internal diameters were quantified by ultrasound, and maximal external AAA diameters were quantified at study end point. Blood pressure was quantified by radiotelemetry and a tail cuff-based technique. Serum cholesterol concentrations and vascular tissue characterization were examined at study end point. ResultsMasR deficiency did not influence body weight, systolic blood pressure at baseline and during AngII infusion, or serum cholesterol concentrations in either Apoe−/− or Ldlr−/− mice. MasR deficiency increased AngII-induced atherosclerosis in aortic arches of Ldlr−/− mice (P < .05), associated with increased oxidative stress and apoptosis in aortic root sections (P < .05). MasR deficiency also augmented internal and external AAA diameters and increased aortic ruptures of both Ldlr−/− and Apoe−/− mice (P < .05). These effects were associated with increased elastin breaks and T-lymphocyte and macrophage accumulation into abdominal aortas of AngII-infused MasR-deficient mice (P < .05). ConclusionsThese results demonstrate that MasR deficiency augmented AngII-induced atherosclerosis and AAA rupture through mechanisms involving increased oxidative stress, inflammation, and apoptosis, suggesting that MasR activation may provide therapeutic efficacy against vascular diseases.

Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Krüppel-like factor (KLF) 15 has emerged as a critical regulator of fibrosis in cardiovascular diseases. However, the precise role that KLF15 and its functional domain played in adventitial inflammation and fibrosis remains unclear. This study aims to investigate the role of the transactivation domain (TAD) of KLF15 in angiotensin II (Ang II)-induced adventitial pathologic changes. KLF15 expression was decreased in the vascular adventitia of Ang II-infused mice (1000 ng/kg/min, 14 d) and in adventitial fibroblasts (AFs) stimulated by Ang II (10-7 M). Adenovirus-mediated KLF15 overexpression normalized Ang II-induced vascular hypertrophy, increased collagen deposition, macrophage infiltration, and CCL2 and VCAM-1 expression. Interestingly, KLF15-ΔTAD (KLF15 with deletion of TAD at amino acids 132-152) overexpression showed no effect on the above pathologic changes. Similarly, perivascularly overexpression of KLF15 but not KLF15-ΔTAD in carotid arteries also attenuated Ang II-induced vascular inflammation and fibrosis. Furthermore, KLF15 overexpression after Ang II infusion rescued Ang II-induced vascular remodeling. CCL2 or VCAM-1-mediated monocyte and macrophage migration or adhesion to AFs in response to Ang II was negatively regulated by KLF15 through TAD. Ang II-enhanced Smad2/3 activation and adventitial migration, proliferation, and differentiation of AFs were suppressed by KLF15 but not KLF15-ΔTAD overexpression. Conversely, small interfering RNA knockdown of KLF15 aggravated Ang II-induced Smad2/3 activation and dysfunction of AFs. Luciferase, coimmunoprecipitation, and chromatin immunoprecipitation assay were used to demonstrate that interaction of KLF15 with Smad2/3 suppressed CCL2 expression through TAD. Mechanistically, activation of Ang II type 1 receptor/phospholipase Cγ 1/ERK1/2 signaling resulted in a decrease of KLF15 expression. In conclusion, these results demonstrate that KLF15 negatively regulates activation of AFs through TAD, which plays an important role in Ang II-induced adventitial inflammation and fibrosis.-Lu, Y.-Y., Li, X.-D., Zhou, H.-D., Shao, S., He, S., Hong, M.-N., Liu, J.-C., Xu, Y.-L., Wu, Y.-J., Zhu, D.-L., Wang, J.-G., Gao, P.-J. Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Endothelial α1AMPK modulates angiotensin II-mediated vascular inflammation and dysfunction.

Mice with a global deletion of α1AMPK are characterized by endothelial dysfunction and NADPH oxidase subunit 2 (NOX-2)-mediated vascular oxidative stress. However, the underlying mechanisms are incompletely understood and may involve endothelial NOX-2 upregulation or facilitated vascular infiltration of phagocytic cells. Therefore, the current study was designed to investigate the vascular effects of chronic angiotensin II (AngII) infusion in mice with an endothelial-specific α1AMPK deletion. A mouse strain with endothelial-specific α1AMPK deletion was generated by breeding α1AMPKflox/flox mice with TekCre+ or Cadh5Cre+ mice. Chronic AngII infusion (0.5mg/kg/day for 7day) caused mild endothelial dysfunction in wild-type mice that was significantly aggravated in endothelial α1AMPK knockout mice. Aortic NOX-2 and CD68 expression wereincreased, indicating that infiltrating leukocytes may significantly contribute to enhanced vascular oxidative stress. Flow cytometry revealed a higher abundance of aortic CD90.2+ T-cells, CD11b+F4/80+ macrophages and Ly6G-Ly6C+ monocytes. Vascular mRNA expression of monocyte chemoattractant protein 1, CCL5 and vascular cell adhesion molecule 1 was enhanced in AngII-infused mice lacking endothelial α1AMPK, facilitating the recruitment of inflammatory cells to the vessel wall. In addition, AngII-induced upregulation of cytoprotective heme oxygenase 1 (HO-1) was blunted in mice with endothelial α1AMPK deletion, compatible with an impaired antioxidant defense in these animals. In summary,endothelial expressed α1AMPK limits the recruitment of inflammatory cells to the vessel wall and maintains HO-1 mediated antioxidant defense. Both mechanisms reduce vascular oxidative damage and preserve endothelial function during chronic AngII treatment.