Venkateswaran Subramanian

Endothelial Cell–Specific Deficiency of Ang II Type 1a Receptors Attenuates Ang II–Induced Ascending Aortic Aneurysms in LDL Receptor−/− Mice

Rationale: Human studies and mouse models have provided evidence for angiotensin II (Ang II)–based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that Ang II infusion induces aneurysmal pathology in the ascending aorta. Objective: The aim of this study was to define the role of angiotensin II type 1a (AT1a) receptors and their cellular location in Ang II–induced ascending aortic aneurysms (AAs). Methods and Results: Male LDL receptor−/− mice were fed a saturated fat–enriched diet for 1 week before osmotic mini-pump implantation and infused with either saline or Ang II (1000 ng/kg per minute) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT1a receptor deficiency ablated Ang II–induced ascending AAs (P<0.001). To determine the role of AT1a receptors on leukocytes, LDL receptor−/−×AT1a receptor+/+ or AT1a receptor−/− mice were irradiated and repopulated with bone marrow–derived cells isolated from either AT1a receptor+/+ or AT1a receptor−/− mice. Deficiency of AT1a receptors in bone marrow–derived cells had no effect on Ang II–induced ascending AAs. To determine the role of AT1a receptors on vascular wall cells, we developed AT1a receptor floxed mice with depletion on either smooth muscle or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT1a receptor deletion in smooth muscle cells had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology. Conclusions: Ang II infusion promotes aneurysms in the ascending aorta via stimulation of AT1a receptors that are expressed on endothelial cells.

β1 Integrins Modulate β-Adrenergic Receptor–Stimulated Cardiac Myocyte Apoptosis and Myocardial Remodeling

Sympathetic nerve activity increases in the heart during cardiac failure. Here, we hypothesized that &bgr;1 integrins play a protective role in chronic &bgr;-adrenergic receptor–stimulated cardiac myocyte apoptosis and heart failure. l-isoproterenol (iso; 400 &mgr;g/kg per hour) was infused in a group of wild-type (WT) and &bgr;1 integrin heterozygous knockout (hKO) mice. Left ventricular structural and functional remodeling was studied at 7 and 28 days of iso-infusion. Western blot analysis demonstrated reduced &bgr;1 integrin levels in the myocardium of hKO-sham. Iso-infusion increased heart weight:body weight ratios in both groups. However, the increase was significantly higher in WT-iso. M-mode echocardiography indicated increased left ventricular end-diastolic diameter, percentage of fractional shortening, and ejection fraction in the WT-iso group. The percentage of fractional shortening and ejection fraction were significantly lower in hKO-iso versus hKO-sham and WT-iso. Peak left ventricular developed pressure and left ventricular end-diastolic pressure measured using Langendorff–perfusion analyses were significantly higher in the WT-iso group (P<0.05 versus WT-sham and hKO-Iso). The number of TUNEL-positive myocytes was significantly higher in hKO-iso hearts 7 and 28 days after iso-infusion. The increase in myocyte cross-sectional area and fibrosis was higher in the WT-iso group. Matrix metalloproteinase-9 protein levels were significantly higher in WT-iso, whereas matrix metalloproteinase-2 levels were increased in hKO-iso hearts. Iso-infusion increased phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in both groups. The increase in c-Jun N-terminal kinase phosphorylation was significantly higher in hKO-iso (P<0.001 versus WT-iso). Thus, &bgr;1 integrins play a crucial role in &bgr;-adrenergic receptor–stimulated myocardial remodeling with effects on cardiac myocyte hypertrophy, apoptosis, and left ventricular function.

Pioglitazone-Induced Reductions in Atherosclerosis Occur via Smooth Muscle Cell–Specific Interaction With PPARγ

Rationale: Peroxisome proliferator-activated receptor (PPAR)&ggr; agonists attenuate atherosclerosis and abdominal aortic aneurysms (AAAs). PPAR&ggr;, a nuclear receptor, is expressed on many cell types including smooth muscle cells (SMCs). Objective: To determine whether a PPAR&ggr; agonist reduces angiotensin II (Ang II)–induced atherosclerosis and AAAs via interaction with SMC-specific PPAR&ggr;. Methods and Results: Low-density lipoprotein receptor (LDLR)−/− mice with SMC-specific PPAR&ggr; deficiency were developed using PPAR&ggr; floxed (PPAR&ggr;f/f) and SM22 Cre+ mice. PPAR&ggr;f/f littermates were generated that did not express Cre (Cre0/0) or were hemizygous for Cre (Cre+/0). To assess the contribution of SMC-specific PPAR&ggr; in ligand-mediated attenuation of Ang II–induced atherosclerosis and AAAs, both male and female Cre0/0 and Cre+/0 mice were fed a fat-enriched diet with or without the PPAR&ggr; agonist pioglitazone (Pio) (20 mg/kg per day) for 5 weeks. After 1 week of feeding modified diets, mice were infused with Ang II (1000 ng/kg per minute) for 4 weeks. SMC-specific PPAR&ggr; deficiency or Pio administration had no effect on plasma cholesterol concentrations. Pio administration attenuated Ang II–increased systolic blood pressure equivalently in both Cre0/0 and Cre+/0 groups. SMC-specific PPAR&ggr; deficiency increased atherosclerosis in male mice. Pio administration reduced atherosclerosis in only the Cre0/0 mice, but not in mice with SMC-specific PPAR&ggr; deficiency. SMC-specific PPAR&ggr; deficiency or Pio administration had no effect on Ang II–induced AAA development. Pio also did not attenuate Ang II–induced monocyte chemoattractant protein-1 production in PPAR&ggr;-deficient SMCs. Conclusions: Pio attenuates Ang II–induced atherosclerosis via the interaction with SMC-specific PPAR&ggr;, but has no effect on the development of AAAs.

Angiotensin II Induces a Region-Specific Hyperplasia of the Ascending Aorta Through Regulation of Inhibitor of Differentiation 3

Rationale: Angiotensin II (Ang II) has diverse effects on smooth muscle cells (SMCs). The diversity of effects may relate to the regional location of this cell type. Objective: The aim of this study was to define whether Ang II exerted divergent effects on smooth muscle cells in the aorta and determine the role of blood pressure and specific oxidant mechanisms. Methods and Results: Ang II (1000 ng/kg per minute) infusion for 28 days into mice increased systolic blood pressure and promoted medial expansion of equivalent magnitude throughout the entire aorta. Both effects were ablated by angiotensin II type 1a (AT1a) receptor deficiency. Similar increases in systolic blood pressure by administration of norepinephrine promoted no changes in aortic medial thickness. Increased medial thickness was attributable to SMC expansion owing to hypertrophy in most aortic regions, with the exception of hyperplasia of the ascending aorta. Deficiency of the p47phox component of NADPH oxidase ablated Ang II–induced medial expansion in all aortic regions. Analysis of mRNA and protein throughout the aorta revealed a much higher abundance of the inhibitor of differentiation 3 (Id3) in the ascending aorta compared to all other regions. A functional role was demonstrated by Id3 deficiency inhibiting Ang II–induced SMC hyperplasia of the ascending aorta. Conclusions: In conclusion, Ang II promotes both aortic medial hypertrophy and hyperplasia in a region-specific manner via an oxidant mechanism. The ascending aortic hyperplasia is dependent on Id3.

Deficiency of β1 integrins results in increased myocardial dysfunction after myocardial infarction

Objective: To study the role of β1 integrins in left ventricular (LV) remodelling after myocardial infarction (MI). Methods and results: LV structural and functional alterations were determined in wild-type (WT) and β1 integrin heterozygous knockout (hKO) mice one month after MI. MI increased β1 integrin expression in both groups; however, the increase was lower in hKO. Infarct size was similar in WT and hKO mice, whereas lung wet weight to dry weight ratio was increased in the hKO-MI mice (5.17 (SE 0.13) v 4.60 (0.15) in WT-MI, p < 0.01). LV end systolic and end diastolic diameters were significantly higher and percentage fractional shortening was significantly lower in hKO-MI. The ratio of peak velocity of early LV filling (E wave) to that of the late LV filling (A wave) and the isovolumic relaxation time (IVRT) were increased in both MI groups but the increase in IVRT was significantly higher in hKO-MI group than in WT-MI mice. Langendorff perfusion analysis indicated reduced peak LV developed pressure and increased LV end diastolic pressure in both MI groups. The reduction in peak LV developed pressure (36.7 (2.2) v 53.4 (1.9) mm Hg, p < 0.05) and increase in LV end diastolic pressure was higher in hKO-MI than in WT-MI. Increase in fibrosis was not different between the two MI groups. The increase in myocyte circumference was higher in the hKO-MI group (p < 0.001 v WT-MI). The number of apoptotic myocytes was significantly higher in hKO-MI than in WT-MI mice (p < 0.005) three days after MI. The number of necrotic myocytes was not different between the two MI groups. Conclusion: β1 integrins are crucial in post-MI remodelling with effects on LV function, hypertrophy and apoptosis.

Calpain inhibition attenuates angiotensin II-induced abdominal aortic aneurysms and atherosclerosis in low-density lipoprotein receptor-deficient mice.

Abstract Chronic infusion of angiotensin II (AngII) augments atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. AngII-induced AAAs are associated with medial macrophage accumulation and matrix metalloproteinase (MMP) activation. Inhibition of calpain, a calcium-activated neutral cysteine protease, by overexpression of its endogenous inhibitor, calpastatin, attenuates AngII-induced leukocyte infiltration, perivascular inflammation, and MMP activation in mice. The purpose of this study was to define whether pharmacological inhibition of calpain influences AngII-induced AAAs in hypercholesterolemic mice. Male low-density lipoprotein receptor−/− mice were fed a fat-enriched diet and administered with either vehicle or a calpain-specific inhibitor, BDA-410 (30 mg/kg per day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1000 ng/kg per minute) for 4 weeks. AngII-infusion profoundly increased aortic calpain protein and activity. BDA-410 administration had no effect on plasma cholesterol concentrations or AngII-increased systolic blood pressure. Calpain inhibition significantly attenuated AngII-induced AAA formation and atherosclerosis development. BDA-410 administration attenuated activation of MMP12, proinflammatory cytokines (IL-6, monocyte chemoattractant protein-1), and macrophage infiltration into the aorta. BDA-410 administration significantly attenuated thioglycolate-elicited macrophage accumulation in the peritoneal cavity. We conclude that calpain inhibition using BDA-410 attenuated AngII-induced AAA formation and atherosclerosis development in low-density lipoprotein receptor−/− mice.

Urokinase-Type Plasminogen Activator Deficiency in Bone Marrow–Derived Cells Augments Rupture of Angiotensin II–Induced Abdominal Aortic Aneurysms

Objective—Abdominal aortic aneurysms (AAAs) are associated with fragmentation of extracellular matrix during development of aortic dilation and rupture. Therefore, it is important to identify specific protease systems involved in extracellular matrix degradation during AAA formation. The present study determined the contribution of the urokinase system to AAA formation and rupture. Methods and Results—Angiotensin II (Ang II)–induced AAAs were associated with increased aortic abundance of both urokinase-type plasminogen activator receptor (uPAR) and urokinase-type plasminogen activator (uPA) proteins. However, this increased presence was unrelated to AAA formation because deficiencies of either uPAR or uPA had no effect on either the incidence or size of Ang II–induced AAAs in both normolipidemic mice and low-density lipoprotein receptor−/− mice fed a saturated fat–enriched diet. Although uPA deficiency did not affect development of AAAs, there was an effect of increasing mortality rate from AAA rupture in hypercholesterolemic mice. Bone marrow transplantation demonstrated that enhanced aneurysmal rupture was attributable to deficiency of uPA in leukocytes. uPA deficiency led to an increased propensity for impaired resolution of the thrombotic material within the aneurysmal tissue. Neither uPAR nor uPA deficiency had any effect on Ang II–induced atherosclerosis in low-density lipoprotein receptor−/− mice. Conclusion—The uPA-uPAR axis has no effect on the formation of Ang II–induced AAAs, but uPA deficiency promotes aneurysmal rupture.

Deficiency of receptor-associated protein attenuates angiotensin II-induced atherosclerosis in hypercholesterolemic mice without influencing abdominal aortic aneurysms

OBJECTIVE Receptor-associated protein (RAP) was initially described as a regulator of low density lipoprotein receptor-related protein 1 (LRP1), but is now known to regulate many proteins. Since the direct effects of RAP on vascular pathologies have not been studied, this study determined whether RAP deficiency influenced angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. METHODS AND RESULTS Male LDL receptor -/- mice that were either RAP +/+ or -/- were infused with AngII (500 ng/kg/min) for 4 weeks while consuming a saturated fat-enriched diet. RAP deficiency had no effects on body weight or AngII-induced increases of systolic blood pressure. Despite increased plasma cholesterol concentrations, RAP deficiency reduced atherosclerotic lesion size in aortic arches, while having no effect on AngII-induced AAAs. RAP deficiency profoundly reduced LRP1 protein abundance in macrophages, but did not change its abundance in aortic smooth muscle cells. Also, RAP deficiency had no effects on mRNA abundance of LRP1 or lipoprotein lipase in macrophages. To determine whether RAP deficiency in leukocytes influenced AngII-induced atherosclerosis, irradiated male LDL receptor -/- mice were repopulated with bone marrow-derived cells from either RAP +/+ or -/- male mice. The chimeric mice were infused with AngII (500 ng/kg/min) for 4 weeks while fed the saturated fat-enriched diet. RAP deficiency in bone marrow-derived cells did not influence either plasma cholesterol concentrations or atherosclerotic lesion size. CONCLUSIONS Whole body RAP deficiency attenuated atherosclerosis without influencing AAAs in hypercholesterolemic mice infused with AngII. The anti-atherogenic effect was not attributable to RAP deficiency in bone marrow-derived cells.

Regulation of Peroxisome Proliferator–Activated Receptor-γ by Angiotensin II Via Transforming Growth Factor-β1–Activated p38 Mitogen-Activated Protein Kinase in Aortic Smooth Muscle Cells

Objective—Peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) ligands attenuate angiotensin II (Ang II)–induced atherosclerosis through interactions with vascular smooth muscle cell (VSMC)–specific PPAR&ggr; in hypercholesterolemic mice. Therefore, the purpose of this study was to determine the mechanism of Ang II–mediated intracellular regulation of PPAR&ggr; in VSMCs. Methods and Results—Incubation of cultured mouse aortic VSMCs with Ang II for 24 hours reduced abundance of PPAR&ggr; protein, mRNA, and transcriptional activity (P<0.001). This effect was attenuated by an angiotensin type 1 receptor antagonist, losartan. Ang II–induced PPAR&ggr; reduction was dependent on stimulation of transforming growth factor (TGF)-&bgr;1 as demonstrated using either a neutralizing antibody or small interfering RNA (siRNA). Ang II–induced TGF-&bgr;1 secretion was dependent on epidermal growth factor receptor kinase activation through reactive oxygen species production. Inhibition of p38 mitogen-activated protein kinase by SB203580 or siRNA inhibited both Ang II– and TGF-&bgr;1–induced PPAR&ggr; reduction. Blockade of TGF-&bgr;1 decreased p38 phosphorylation induced by Ang II. siRNA–mediated inhibition of histone deacetylase 3 attenuated p38-mediated reductions in PPAR&ggr; abundance. Conclusion—These findings suggest that Ang II decreases PPAR&ggr; abundance in cultured VSMCs via an angiotensin type 1 receptor–dependent secretion of TGF-&bgr;1 via phosphorylation of p38 mitogen-activated protein kinase and histone deacetylase 3.

Calpain-2 compensation promotes angiotensin II-induced ascending and abdominal aortic aneurysms in calpain-1 deficient mice.

Background and Objective Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methodology/Results To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr −/− mice that were either calpain-1 +/+ or −/− were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and −/− mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta. Conclusion Calpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.