Ang II-infused Wild-type Mice Research Articles

PS-BPB02-4: LEFT VENTRICLE HYPERTROPHY AND DYSFUNCTION WERE EXAGGERATED BY P2RX7 KNOCKOUT BUT UNAFFECTED BY P2RX7 PHARMACOLOGIC BLOCKADE IN ANGIOTENSIN II-INFUSED MICE

Objective: Extracellular adenosine triphosphate released from damaged cells binds to the P2X7 receptor (P2RX7) to activate of immune cells. Our preliminary data demonstrated that P2RX7 plays a role in vascular injury of hypertensive mice. Angiotensin (Ang) II-induced hypertension, vascular injury and aortic perivascular infiltration of activated T cell were attenuated by P2rx7 knockout or P2RX7 pharmacologic blockade. However, whether P2RX7 is involved in Ang II-induced cardiac dysfunction and hypertrophy is unknown. We hypothesized that Ang II-induced left ventricular (LV) dysfunction and hypertrophy would be blunted by P2rx7 knockout or P2RX7 antagonism. Design and method: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7-/- mice were infused or not with Ang II (1000 ng/kg/min) for 14 days. A second set of Ang II-infused WT mice was infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle for 14 days. Cardiac LV function and remodeling were determined by ultrasound. Total RNA was isolated from the heart using the mirVana miRNA isolation kit. Relative expression was calculated using the Pfaffl method with 40S ribosomal protein S16 (Rps16) as a reference gene. Hypertrophic and fibrotic markers were studied by reverse transcription-quantitative PCR (RT-qPCR) in cardiac ventricles. Results: Ang II-induced 1.5-fold increase in LV mass/body weight (P < 0.001) and ∽25% decrease in fractional shortening in WT mice and mice receiving AZ10606120 (P < 0.05). These changes were exaggerated in P2rx7-/- mice (LV mass/body weight: 1.7-fold increase, fractional shortening: 54% decrease, P < 0.05), but not in mice receiving AZ10606120. Ang II-induced atrial natriuretic peptide (Nppa/Rps16: 7-fold, P < 0.001) and α-skeletal actin 1 expression (Acta1/Rps16: 6-fold, P < 0.001) tended to be further increased in P2rx7-/- mice (Nppa/Rps16: 14-fold and Acta1/Rps16: 12-fold), but not in AZ10606120-treated mice. Ang II-induced myosin heavy chain 7B (14-fold, P < 0.05) was unaffected by P2rx7 knockout or P2RX7 antagonism. Ang II caused a 1.5-fold increase in transforming growth factor β; 1 only in P2rx7-/- mice (P < 0.05). Conclusion: Ang II-induced LV hypertrophy and dysfunction were exaggerated by P2rx7 knockout but were not affected by P2RX7 pharmacologic blockade.

PS-BPB04-2: P2RX7 DEFICIENCY OR P2RX7 ANTAGONISM BLUNTS ANGIOTENSIN II-INDUCED HYPERTENSION, VASCULAR INJURY AND CD8+ T CELL ACTIVATION

Objectives: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β; release, which drives further immune activation. Elevated plasma ATP levels was observed in hypertensive patients. We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Design and method: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7-/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: Plasma ATP was 2.2 fold higher in AngII-infused compared to sham-treated mice (P < 0.05). AngII-induced systolic BP elevation was reduced by P2rx7 deficiency (12 mm Hg, P < 0.05) or P2RX7 antagonism (27 mm Hg, P < 0.01). AngII induced a 26% decrease in LV fractional shortening (FS, P < 0.05) and a 1.5 fold increase in LV mass/body weight (BW) in WT mice (P < 0.001), which were exaggerated in P2rx7-/- mice (FS: 38% lower and LVmass/BW: 1.14 fold higher, P < 0.05), but not in mice receiving AZ10606120. AngII treatment caused a 26% reduction in the dilatation response of mesenteric arteries to acetylcholine in WT mice (P < 0.05), but not in P2rx7-/- or AZ10606120-treated mice. AngII promoted a 3.8 fold increase in CD69+CD8+ T cell infiltration in aortic PVAT of WT mice (P < 0.001), but not in P2rx7-/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8+ T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.

Abstract 118: P2X7 Receptor Contributes To Angiotensin II-induced Hypertension, Vascular Injury And Cd8 + T Cell Activation

Introduction: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β release, which drives further immune activation. Hypothesis: We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: AngII increased plasma ATP in WT mice (4.4±1.2 vs 2.0±0.9 μM, P <0.05). AngII-induced systolic BP elevation was reduced by P2rx7 deficiency (164±3 vs 176±2 mm Hg, P <0.05) or P2RX7 antagonism (143±5 vs 170±5 mm Hg, P <0.01). AngII decreased LV fractional shortening (FS, 32.5±3.1% vs 43.8±2.4%, P <0.05) and increased LV mass/body weight (BW) in WT mice (LVmass/BW, 6.3±0.2 vs 4.2±0.2 mg/g, P <0.001), which were exaggerated in P2rx7 -/- (FS: 20.2±3.1% and LVmass/BW: 7.2±0.5 mg/g, P <0.05), but not in mice receiving AZ10606120. AngII reduced the dilatation response of mesenteric arteries to acetylcholine in WT (61±7 vs 83±4%, P <0.05), but not in P2rx7 -/- or AZ10606120-treated mice. AngII increased CD69 + CD8 + T cell infiltration in aortic PVAT of WT (60±16 vs 16±3 cells/aortic PVAT, P <0.001), but not in P2rx7 -/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.

Blocking of interleukin-1 suppresses both angiotensin II-induced renal inflammation and hypertension

Abstract Background Clinical hypertension is associated with renal inflammation and elevated circulating levels of proinflammatory cytokines. IL-1 receptor antagonist (IL-1Ra) is one of the most important anti-inflammatory cytokines and plays a crucial role in inflammation. Inhibition of IL-1 may contribute to modulation of the Angiotensin II (AngII)-induced hypertension response. This study aimed to elucidate the effects of IL-1Ra and anti-IL-1beta antibody (01BSUR) on AngII-induced hypertension and renal inflammation. Methods and results To determine the contribution of IL-1Ra to AngII-induced renal inflammation, male wild-type (WT) and IL-1Ra-deficient (IL-1Ra−/−) mice were infused with AngII (1000ng/kg/min) using subcutaneous osmotic pumps for 14 days. We checked blood pressure, histological change, and several mRNA expressions 14 days after infusion. Fourteen days after infusion, systolic blood pressure (197±5 vs 169±9 mmHg, p<0.05) in IL-1Ra−/− mice significantly increased compared with WT mice. Furthermore, on day 14 of AngII infusion, plasma IL-6 was 5.9-fold higher in IL-1Ra−/− versus WT mice (p<0.001); renal preproendothelin-1 mRNA expression was also significantly higher in IL-1Ra−/− mice (p<0.05). To examine renal function, we analyzed 24-hour urinary protein excretion and serum levels of blood urea nitrogen, creatinine, and uric acid in IL-1Ra−/− and WT mice. On day 14 of Ang II infusion, all levels increased significantly in IL-1Ra−/− mice compared with WT mice, suggesting that IL-1Ra deficiency reduced renal function following Ang II infusion. In addition, renal histology revealed that glomerular injury (Figure upper panels: PAS staining) and tubulointerstitial fibrosis (Figure lower panels: Elastica Masson staining) increased significantly in Ang II-infused IL-1Ra−/− versus Ang II-infused WT mice. Finally, we administrated 01BSUR to both IL-1Ra−/− and WT mice, and 01BSUR treatment decreased AngII-induced hypertension (162±17 vs 204±6 mmHg, p<0.05) and renal damage (glomerular injury and fibrosis of the tubulointerstitial area) in both IL-1Ra−/− and WT mice compared with IgG2a treatment. These findings suggest that 01BSUR suppresses Ang II-induced inflammation and renal injury. Conclusions Inhibition of interleukin-1 by both endogenous IL-1Ra and exogenous 01BSUR decreased AngII-induced hypertension and renal damage in mice, suggesting suppression of IL-1 may provide an additional strategy to protect against renal damage in hypertensive patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

Chemokine Receptor CXCR-2 Initiates Atrial Fibrillation by Triggering Monocyte Mobilization in Mice.

Atrial fibrillation (AF) is frequently associated with increased inflammatory response characterized by infiltration of monocytes/macrophages. The chemokine receptor CXCR-2 is a critical regulator of monocyte mobilization in hypertension and cardiac remodeling, but it is not known whether CXCR-2 is involved in the development of hypertensive AF. AF was induced by infusion of Ang II (angiotensin II; 2000 ng/kg per minute) for 3 weeks in male C57BL/6 wild-type mice, CXCR-2 knockout mice, bone marrow-reconstituted chimeric mice, and mice treated with the CXCR-2 inhibitor SB225002. Microarray analysis revealed that 4 chemokine ligands of CXCR-2 were significantly upregulated in the atria during 3 weeks of Ang II infusion. CXCR-2 expression and the number of CXCR2+ immune cells markedly increased in Ang II-infused atria in a time-dependent manner. Moreover, Ang II-infused wild-type mice had increased blood pressure, AF inducibility, atrial diameter, fibrosis, infiltration of macrophages, and superoxide production compared with saline-treated wild-type mice, whereas these effects were significantly attenuated in CXCR-2 knockout mice and wild-type mice transplanted with CXCR-2-deficient bone marrow cells or treated with SB225002. Moreover, circulating blood CXCL-1 levels and CXCR2+ monocyte counts were higher and associated with AF in human patients (n=31) compared with sinus rhythm controls (n=31). In summary, this study identified a novel role for CXCR-2 in driving monocyte infiltration of the atria, which accelerates atrial remodeling and AF after hypertension. Blocking CXCR-2 activation may serve as a new therapeutic strategy for AF.

Abstract 710: Deficiency of IL12p40 Promotes Angiotensin II-Induced Abdominal Aortic Aneurysm

Introduction: Abdominal aortic aneurysm (AAA) is caused by the immune and inflammatory cell accumulation of the aortic wall, leading to enhanced inflammation and aneurysm formation. Our recent studies demonstrate that inhibition of Notch signaling attenuates AAA formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40 knockout (KO) mice, we investigated the direct effects of M2-like macrophages in the development of AAA. Methods and Results: Male (8-10 week-old) wild-type (WT) and IL12p40-KO mice (n=10) on C57BL/6 background were infused with angiotensin-II (AngII, 1,000 ng/kg/min) by implanting osmotic pumps subcutaneously for 28 days. Comparable increase in mean, systolic and diastolic arterial blood pressure was observed between WT and IL12p40-KO mice in response to AngII. Unexpectedly, AngII significantly increased the AAA-associated mortality (2/10) and luminal expansion (7/10) in IL12p40-KO mice as determined by transabdominal ultrasound. In addition, IL12p40-deficiency significantly increased aortic stiffness in the abdominal aortas in response to AngII than WT as measured by pulse wave velocity (PWV) and atomic force microscopy (AFM). Macroscopic and histological examination also showed that IL12p40-KO mice exhibited severe abdominal aortic lesion formation and increased maximal aortic width compared to WT mice. Increased elastin fragmentation and collagen deposition was detected in aorta of AngII-infused IL12p40-KO mice compared to AngII-infused WT mice. Further, there was no significant change in the total cholesterol, triglycerides and HDL-cholesterol levels in the serum of WT and IL12p40-KO mice treated with AngII. Importantly, bone marrow derived macrophages (BMDM) from IL12p40-KO mice showed increased mRNA expression of M2-markers (TGF-β1 and IL10) and decreased expression of M1-markers (iNOS and TNF-α) at basal level compared to WT BMDM. Conclusions: IL12p40 deficiency may promote the development of AAA, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic-stiffness and fibrosis mediated by TGF-β1 and IL10. In summary, optimal M1/M2 balance rather than predominance of M2-phenotype may be prerequisite to prevent AAA formation.

The immunoproteasome subunit LMP10 mediates angiotensin II-induced retinopathy in mice

Inflammation has been implicated in a variety of retinal diseases. The immunoproteasome plays a critical role in controlling inflammatory responses, but whether activation of immunoproteasome contributes to angiotensin II (Ang II)-induced retinopathy remains unclear. Hypertensive retinopathy (HR) was induced by infusion of Ang II (3000 ng/kg/min) in wild-type (WT) and immunoproteasome subunit LMP10 knockout (KO) mice for 3 weeks. Changes in retinal morphology, vascular permeability, superoxide production and inflammation were examined by pathological staining. Our results showed that immunoproteasome subunit LMP10 expression and its trypsin-like activity were significantly upregulated in the retinas and serum of Ang II-infused mice and in the serum from patients with hypertensive retinopathy. Moreover, Ang II-infused WT mice showed an increase in the central retinal thickness, vascular permeability, reactive oxygen species (ROS) production and inflammation compared with saline controls, and these effects were significantly attenuated in LMP10 KO mice, but were aggravated in mice intravitreally injected with rAAV2-LMP10. Interestingly, administration of IKKβ specific inhibitor IMD-0354 remarkably blocked an Ang II-induced increase in vascular permeability, oxidative stress and inflammation during retinopathy. Mechanistically, Ang II-induced upregulation of LMP10 promoted PTEN degradation and activation of AKT/IKK signaling, which induced IkBα phosphorylation and subsequent degradation ultimately leading to activation of NF-kB target genes in retinopathy. Therefore, this study provided novel evidence demonstrating that LMP10 is a positive regulator of NF-kB signaling, which contributes to Ang II-induced retinopathy. Strategies for inhibiting LMP10 or IKKβ activity in the eye could serve as a novel therapeutic target for treating hypertensive retinopathy.

Activation of AMP-activated protein kinase by metformin ablates angiotensin II-induced endoplasmic reticulum stress and hypertension in mice in vivo.

Metformin, one of the most frequently prescribed medications for type 2 diabetes, reportedly exerts BP-lowering effects in patients with diabetes. However, the effects and underlying mechanisms of metformin on BP in non-diabetic conditions remain to be determined. The aim of the present study was to determine the effects of metformin on angiotensin II (Ang II) infusion-induced hypertension in vivo. The effects of metformin on BP were investigated in wild-type (WT) C57BL/6J mice and in mice lacking AMP-activated protein kinase α2 (AMPKα2) mice with or without Ang II infusion. Also, the effect of metformin on Ang II-induced endoplasmic reticulum (ER) stress was explored in cultured human vascular smooth muscle cells (hVSMCs). Metformin markedly reduced BP in Ang II-infused WT mice but not in AMPKα2-deficient mice. In cultured hVSMCs, Ang II treatment resulted in inactivation of AMPK, as well as the subsequent induction of spliced X-box binding protein-1, phosphorylation of eukaryotic translation initiation factor 2α and expression of glucose-regulated protein 78kDa, representing three well-characterized ER stress biomarkers. Moreover, AMPK activation by metformin ablated Ang II-induced ER stress in hVSMCs. Mechanistically, metformin-activated AMPKα2 suppressed ER stress by increasing phospholamban phosphorylation. Metformin alleviates Ang II-triggered hypertension in mice by activating AMPKα2, which mediates phospholamban phosphorylation and inhibits Ang II-induced ER stress in vascular smooth muscle cells.

Abstract 072: TRIF-Pathway of Innate Immune Responses Mediates Angiotensin II Hypertension

We tested the role of two distinct adaptors of toll-like receptor (TLR) signaling on Ang II-induced hypertension and cardiac hypertrophy. These TLR adaptors, myeloid differentiation protein 88 (MyD88) and TIR domain-containing adaptor inducing interferon β (TRIF) facilitate distinct inflammatory signaling pathways. In an earlier study, we reported that MyD88-/- mice are protected from cardiac hypertrophy and pro-inflammatory gene expression after myocardial infarction. Our current results with 3 weeks infusion of Ang II (3000 ng/kg/min) vs. saline indicate that in MyD88-/- mice, the pressor response to Ang II and cardiac hypertrophy were increased more than in wild type (WT) mice. In Ang II-infused WT, systolic blood pressure (SBP) peaked at 147 ± 4 mmHg whereas in Ang II-infused MyD88-/- mice SBP reached a peak value of 163 ± 6 mmHg. However, in mice with non-functional TRIF adaptor mutant (Trifmut), SBP did not increase during Ang II infusion and remained similar to the SBP in saline-infused mice (115 ± 3 mmHg). Baseline SBP was not different among WT, MyD88-/- and Trifmut mice. The increase in heart weight to body weight ratio (HW/BW) between saline and Ang II-infused mice was greater in MyD88-/- mice than WT mice (60% increase in MyD88-/- vs. 40% increase in WT), whereas it was less in Trifmut mice (22% increase). Accordingly, expression of several inflammatory genes (Tnfa, Nox4 and Agtr1a) was significantly greater (P< 0.05) in the heart and kidney of Ang II-infused MyD88-/- mice compared with Ang II-infused WT mice, whereas expression of these genes in Trifmut mice was either unchanged or reduced. We conclude that- (1) Ang II-induced hypertension, cardiac hypertrophy and inflammatory gene expression are mediated by activation of a TRIF-dependent pathway, but not by the MyD88-dependent pathways, and (2) Enhanced Ang II effects on SBP and hypertrophy in MyD88-/- mice suggest that MyD88 may serve as a negative regulator of the TRIF pathway in Ang II-induced hypertension. Selective targeting of these adaptor proteins may have significant therapeutic implications.

SCD40L ‐CD40/VLA‐ 5 Signaling Contributes to AngII‐induced Thrombosis and Inflammation.

AngII‐induced hypertension is associated with accelerated thrombus formation and leukocyte recruitment. The objective of this study was to evaluate the mechanisms underlying the prothrombogenic‐proinflammatory responses associated with chronic AngII infusion. We assessed the contribution of CD40/CD40L to these AngII‐mediated responses and whether/how the generation of soluble CD40L(sCD40L) contributes to the exacerbated thrombosis and leukocyte recruitment. Thrombus formation in mouse arterioles was induced using the light/dye injury model, while firm adhesion of leukocytes was quantified in adjacent venules. Wild type (WT), CD40‐/‐, CD40L‐/‐ , bone marrow chimeric CD40‐/‐ and CD40L‐/‐ mice (CD40‐/‐→WT or CD40L‐/‐→WT) were infused with AngII (1 µg/kg/min for 14 days) via osmotic pumps. Some WT mice and AngII infused WT mice were treated with either recombinant sCD40L, a P‐selectin blocking antibody, or a VLA5 antagonist (ATN‐161). The studies revealed that both AngII‐infused CD40‐/‐ and CD40L‐/‐ exhibited reduced leukocyte adhesion and oxidative stress in venules, and a blunted thrombosis response in arterioles. WT‐AngII mice treated with ATN‐161 were completely protected against the enhanced thrombosis and leukocyte adhesion responses to AngII, while P‐selectin immunoblockade offered only partial protection. Infusion of recombinant sCD40L in either WT, CD40‐/‐, or CD40L‐/‐ mice restored the accelerated thrombus formation elicited by AngII infusion. These findings implicate sCD40‐CD40L/VLA‐5‐dependent signaling in the prothrombotic‐proinflammatory responses elicited by chronically elevated AngII levels (Postdoctoral Fellowship from Malcolm Feist Endowment).

ASSA14-03-20 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced vascular remodelling via degradation of angiotensin II type 1 receptor

Background Cellular repressor of E1A-stimulated genes (CREG), a new cardiovascular homeostasis regulator, has been proposed to reduce the neointimal formation after vascular injury via the maintenance of the quiescent mature vascular smooth muscle cell (VSMC) phenotype. We hypothesised that CREG is a negative regulator of angiotensin (Ang) II-mediated vascular remodelling. Methods Ten-week-old male heterozygous CREG-deficient (CREG +/- ) mice and their littermate wild-type (WT) mice were used. An osmotic minipump was implanted subcutaneously to infuse a dose of Ang II (1.5 mg/kg · d) or saline (vehicle) for 14 days. Ang II-infused WT mice were then treated with placebo or recombinant human CREG (rhCREG; 300 μg/kg · d). Systolic blood pressure (SBP); extent of vascular remodelling; protein level of collagen type I/III, angiotensin-converting enzyme-2 (ACE2), angiotensin receptor type 1 (AT1R) and Rab7 were evaluated. Primary VSMC culture was performed to assess AT1R and collagen protein expression before and after CREG gene transfection and knockdown. Results CREG levels are high in vascular media under basal conditions but rapidly decrease in response to Ang II. Ang II infusion for 14 days resulted that levels of SBP, intima medial thickness and vascular remodelling of the aorta and mesenteric artery were significantly greater in CREG +/- mice compared with the WT controls. Vascular gene expression level of CREG was lower in Ang II-treated CREG +/- mice than in WT mice. However, daily treatment of Ang II-infused WT mice with rhCREG blunted the increase of SBP. Ang II-mediated vascular remodelling and expression of collagen type I/III were also suppressed by rhCREG. Moreover, rhCREG treatment inhibited Ang II-mediated up-regulation of AT1R expression and down-regulation of ACE2 expression. Ang II-induced vascular remodelling was inhibited by rhCREG in association with reduced plasma Ang II and increased plasma Ang 1–7 levels. In adult VSMCs, CREG over-expression increased the expression of Rab7 by a regulatory mechanism of ubiquitination. Importantly, Ang II-mediated collagen production and AT1R expression was inhibited by CREG over-expression and augmented by CREG knock-down in a rab7-dependent manner. Conclusions Elevated Ang II induced hypertension and vascular remodelling, which were exacerbated by CREG deficiency, whereas rhCREG attenuated Ang II-induced adverse vascular remodelling. Hence, CREG is an important negative regulator of Ang II-induced vascular disease and suppresses adverse vascular remodelling.

818 ACTIVATION OF ANG II TYPE 1 RECEPTOR-ASSOCIATED PROTEIN SUPPRESSES VASCULAR HYPERTROPHY AND OXIDATIVE STRESS IN ANG II-MEDIATED HYPERTENSION

Objective: We cloned a novel molecule interacting with Ang II type 1 receptor, which we named ATRAP (for Ang II type 1 receptor-associated protein). The present study was designed to investigate the putative functional role of ATRAP in vascular hypertrophy and oxidative stress in Ang II-mediated hypertension in vivo. Methods & Results: We generated transgenic (Tg) mice expressing ATRAP in tissues including aorta under control of the β-actin promoter. The mRNA levels of ATRAP in Tg mice aorta were increased 15-fold compared with C57BL/6J Wild-type (Wt) mice. Adult male Wt mice and Tg mice at from 10 to 12 weeks of ages, were divided into two groups for the subcutaneous infusion of vehicle or Ang II 500ng/kg/min via osmotic mini-pump for 14days. Although systolic blood pressure was significantly elevated in the Ang II-infused mice, there was no significant difference in systolic blood pressure of Ang II-infused Tg mice compared to that of Ang II-infused Wild type mice. In Wt mice, the Ang II treatment promoted vascular hypertrophy in aorta, concomitant with a significant increase in aortic NADPH oxidase component, p22phox, expression. On the other hand, in ATRAP transgenic mice, the development of vascular hypertrophy and activation of p22phox in the context of Ang II treatment were significantly suppressed. Conclusions: These results demonstrate that activation of ATRAP at local tissue sites including aorta efficiently inhibits the vascular hypertrophy and oxidative stress provoked in Ang II-mediated hypertension, thereby suggesting ATRAP to be a novel therapeutic target in vascular disease.

Abstract 8693: Knockout of p47 phox Attenuates Angiotensin II-induced Endothelial Dysfunction and Vessel Damage in Vivo

Increased bioavailability and activity of angiotensin II (AngII) have been found to be associated with vascular oxidative stress derived from a Nox2 enzyme. The Nox2 has several regulatory subunits i.e. p40 phox , p47 phox , p67 phox and rac1. However, it is unclear about the role of p47 phox (a key regulatory subunit of Nox2 activation) in mediating AngII-induced vessel dysfunction. In this study, we examined the effect of p47 phox knockout (KO) on AngII-induced hypertension and aortic dissection using littermates of C57BL/6 wild-type (WT) and p47 phox KO mice (n=7) at the age of 10~12 months. In WT mice, AngII infusion (1.15mg/kg/day delivered by osmotic mini-pump for 14 days) increased significantly the systolic blood pressure (SBP) from 127 + 13 to 172 + 11 mmHg, and this was accompanied with significant indication of cardiac hypertrophy (heart/body weight ratio increased ~17.9 + 0.1%) as compared to vehicle infused controls (P < 0.05), and 3 out of 7 AngII-infused WT mice developed aortic dissection. However, in p47 phox KO mice, AngII infusion caused a mild increase in SBP (from 119 + 9 to 149 + 10 mmHg, P<0.05) without significant increase in heart/body weight ratio, and there was no aortic dissection in AngII-infused KO mice. Aorta organ bath showed that AngII-infusion reduced significantly the endothelium-dependent vessel relaxation to acetylcholine in WT mice (Emax from 82.10 + 8.73% to 54.37 + 6.91%, P < 0.05), which can be returned to the control level by adding a superoxide scavenger, tiron. However, AngII had no significant effect on endothelium-dependent vessel relaxation to acetylcholine in the p47 phox KO mice (Emax from 73.08 + 1.15% to 65.73 + 5.35%). In conclusion, p47 phox plays a critical role in mediating AngII-induced endothelial dysfunction, hypertension and aortic dissection. Knockout or inhibiting p47 phox may protect vessels from these AngII-induced vascular disorders.

Prevention of Angiotensin II–Mediated Renal Oxidative Stress, Inflammation, and Fibrosis by Angiotensin-Converting Enzyme 2

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase capable of metabolizing angiotensin (Ang) II into Ang 1 to 7. We hypothesized that ACE2 is a negative regulator of Ang II signaling and its adverse effects on the kidneys. Ang II infusion (1.5 mg/kg⁻¹/d⁻¹) for 4 days resulted in higher renal Ang II levels and increased nicotinamide adenine dinucleotide phosphate oxidase activity in ACE2 knockout (Ace2(-/y)) mice compared to wild-type mice. Expression of proinflammatory cytokines, interleukin-1β and chemokine (C-C motif) ligand 5, were increased in association with greater activation of extracellular-regulated kinase 1/2 and increase of protein kinase C-α levels. These changes were associated with increased expression of fibrosis-associated genes (α-smooth muscle actin, transforming growth factor-β, procollagen type Iα1) and increased protein levels of collagen I with histological evidence of increased tubulointerstitial fibrosis. Ang II-infused wild-type mice were then treated with recombinant human ACE2 (2 mg/kg⁻¹/d⁻¹, intraperitoneal). Daily treatment with recombinant human ACE2 reduced Ang II-induced pressor response and normalized renal Ang II levels and oxidative stress. These changes were associated with a suppression of Ang II-mediated activation of extracellular-regulated kinase 1/2 and protein kinase C pathway and Ang II-mediated renal fibrosis and T-lymphocyte-mediated inflammation. We conclude that loss of ACE2 enhances renal Ang II levels and Ang II-induced renal oxidative stress, resulting in greater renal injury, whereas recombinant human ACE2 prevents Ang II-induced hypertension, renal oxidative stress, and tubulointerstitial fibrosis. ACE2 is an important negative regulator of Ang II-induced renal disease and enhancing ACE2 action may have therapeutic potential for patients with kidney disease.

Angiotensin Converting Enzyme 2 Suppresses Pathological Hypertrophy, Myocardial Fibrosis and Diastolic Dysfunction

Background: Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Methods and Results: Ang II infusion (1.5 mg.kg−1.d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2-/y) mice compared to wildtype (WT) mice. Daily treatment of Ang II-infused WT mice with recombinant human ACE2 (rhACE2; 2 mg.kg−1.d−1; i.p.) blunted the hypertrophic response, expressions of hypertrophy markers and reduced the Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of pro-collagen type Ia1, pro-collagen type IIIa1, TGFb1 and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was normalized by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1–7 levels. rhACE2 treatment reversed Ang II-mediated activation of protein kinase C (PKC)α and PKCβ1 protein levels and phosphorylation of the ERK1/2, JAK2 and STAT3 signaling pathways in WT mice. A subpressor dose of Ang II (0.15 mg.kg−1.d−1) resulted in a milder phenotype which was also completed suppressed by rhACE2 (2 mg.kg−1.d−1; i.p). In cultured adult murine ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production and ERK1/2 signaling were suppressed by rhACE2 in an Ang 1–7 dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wildtype mice. Conclusions: Elevated Ang II induces hypertension, myocardial hypertrophy, fibrosis and diastolic dysfunction which were exacerbated by ACE2 deficiency while rhACE2 prevents this Ang II-induced and pressure-overload induced myocardial adverse remodeling. Hence, ACE2 as an important negative regulator of Ang II-induced heart disease can have therapeutic potentials for patients with heart disease. Background: Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system. Methods and Results: Ang II infusion (1.5 mg.kg−1.d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2-/y) mice compared to wildtype (WT) mice. Daily treatment of Ang II-infused WT mice with recombinant human ACE2 (rhACE2; 2 mg.kg−1.d−1; i.p.) blunted the hypertrophic response, expressions of hypertrophy markers and reduced the Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of pro-collagen type Ia1, pro-collagen type IIIa1, TGFb1 and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was normalized by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1–7 levels. rhACE2 treatment reversed Ang II-mediated activation of protein kinase C (PKC)α and PKCβ1 protein levels and phosphorylation of the ERK1/2, JAK2 and STAT3 signaling pathways in WT mice. A subpressor dose of Ang II (0.15 mg.kg−1.d−1) resulted in a milder phenotype which was also completed suppressed by rhACE2 (2 mg.kg−1.d−1; i.p). In cultured adult murine ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production and ERK1/2 signaling were suppressed by rhACE2 in an Ang 1–7 dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wildtype mice. Conclusions: Elevated Ang II induces hypertension, myocardial hypertrophy, fibrosis and diastolic dysfunction which were exacerbated by ACE2 deficiency while rhACE2 prevents this Ang II-induced and pressure-overload induced myocardial adverse remodeling. Hence, ACE2 as an important negative regulator of Ang II-induced heart disease can have therapeutic potentials for patients with heart disease.