Siva S.V.P. Sakamuri

Angiotensin II enhances AT1-Nox1 binding and stimulates arterial smooth muscle cell migration and proliferation through AT1, Nox1, and interleukin-18

The redox-sensitive transcription factors NF-κB and activator protein-1 (AP-1) are critical mediators of ANG II signaling. The promitogenic and promigratory factor interleukin (IL)-18 is an NF-κB- and AP-1-responsive gene. Therefore, we investigated whether ANG II-mediated smooth muscle cell (SMC) migration and proliferation involve IL-18. ANG II induced rat carotid artery SMC migration and proliferation and IL-18 and metalloproteinase (MMP)-9 expression via ANG II type 1 (AT(1)) receptor. ANG II-induced superoxide generation, NF-κB and AP-1 activation, and IL-18 and MMP-9 induction were all markedly attenuated by losartan, diphenyleneiodonium chloride (DPI), and Nox1 knockdown. Similar to ANG II, addition of IL-18 also induced superoxide generation, activated NF-κB and AP-1, and stimulated SMC migration and proliferation, in part via Nox1, and both ANG II and IL-18 induced NOX1 transcription in an AP-1-dependent manner. AT(1) physically associates with Nox1 in SMC, and ANG II enhanced this binding. Interestingly, exogenous IL-18 neither induced AT(1) binding to Nox1 nor enhanced the ANG II-induced increase in AT(1)/Nox1 binding. Importantly, IL-18 knockdown, or pretreatment with IL-18 neutralizing antibodies, or IL-18 binding protein, all attenuated the migratory and mitogenic effects of ANG II. Continuous infusion of ANG II for 7 days induced carotid artery hyperplasia in rats via AT(1) and was associated with increased AT(1)/Nox1 binding (despite lower AT(1) levels); increased DPI-inhibitable superoxide production; increased phospho-IKKβ, JNK, p65, and c-Jun; and induction of IL-18 and MMP-9 in endothelium-denuded carotid arteries. These results indicate that IL-18 amplifies the ANG II-induced, redox-dependent inflammatory cascades by activating similar promitogenic and promigratory signal transduction pathways. The ANG II/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis.

Angiotensin II stimulates cardiac fibroblast migration via the differential regulation of matrixins and RECK

Sustained induction and activation of matrixins (matrix metalloproteinases or MMPs), and the destruction and deposition of extracellular matrix (ECM), are the hallmarks of cardiac fibrosis. The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a unique membrane-anchored endogenous MMP regulator. We hypothesized that elevated angiotensin II (Ang II), which is associated with fibrosis in the heart, differentially regulates MMPs and RECK both in vivo and in vitro. Continuous infusion of Ang II into male C57Bl/6 mice for 2weeks resulted in cardiac fibrosis, with increased expressions of MMPs 2, 7, 9 and 14, and of collagens Ia1 and IIIa1. The expression of RECK, however, was markedly suppressed. These effects were inhibited by co-treatment with the Ang II type 1 receptor (AT1) antagonist losartan. In vitro, Ang II suppressed RECK expression in adult mouse cardiac fibroblasts (CF) via AT1/Nox4-dependent ERK/Sp1 activation, but induced MMPs 2, 14 and 9 via NF-κB, AP-1 and/or Sp1 activation. Further, while forced expression of RECK inhibits, its knockdown potentiates Ang II-induced CF migration. Notably, RECK overexpression reduced Ang II-induced MMPs 2, 9 and 14 activation, but enhanced collagens Ia1 and IIIa1 expression and soluble collagen release. These results demonstrate for the first time that Ang II suppresses RECK, but induces MMPs both in vivo and in vitro, and RECK overexpression blunts Ang II-induced MMP activation and CF migration in vitro. Strategies that upregulate RECK expression in vivo have the potential to attenuate sustained MMP expression, and blunt fibrosis and adverse remodeling in hypertensive heart diseases.

Acetylsalicylic acid inhibits IL-18-induced cardiac fibroblast migration through the induction of RECK

The pathogenesis of cardiac fibrosis and adverse remodeling is thought to involve the ROS‐dependent induction of inflammatory cytokines and matrix metalloproteinases (MMPs), and the activation and migration of cardiac fibroblasts (CF). Here we investigated the role of RECK (reversion‐inducing‐cysteine‐rich protein with Kazal motifs), a unique membrane‐anchored MMP regulator, on IL‐18‐induced CF migration, and the effect of acetylsalicylic acid (ASA) on this response. In a Matrigel invasion assay, IL‐18‐induced migration of primary mouse CF was dependent on both IKK/NF‐κB‐ and JNK/AP‐1‐mediated MMP9 induction and Sp1‐mediated RECK suppression, mechanisms that required Nox4‐dependent H2O2 generation. Notably, forced expression of RECK attenuated IL‐18‐induced MMP9 activation and CF migration. Further, therapeutic concentrations of ASA inhibited IL‐18‐induced H2O2 generation, MMP9 activation, RECK suppression, and CF migration. The salicylic acid moiety of ASA similarly attenuated IL‐18‐induced CF migration. Thus, ASA may exert potential beneficial effect in cardiac fibrosis through multiple protective mechanisms. J. Cell. Physiol. 229: 845–855, 2014.

Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration

The omega-3 polyunsaturated fatty acids (ω-3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω-3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.

Cardiac-Restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis and Dysfunction

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPβ and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPβ, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.

TRAF3IP2 mediates atherosclerotic plaque development and vulnerability in ApoE(-/-) mice.

BACKGROUND AND AIMS Atherosclerosis is a major cause of heart attack and stroke. Inflammation plays a critical role in the development of atherosclerosis. Since the cytoplasmic adaptor molecule TRAF3IP2 (TRAF3-Interacting Protein 2) plays a causal role in various autoimmune and inflammatory diseases, we hypothesized that TRAF3IP2 mediates atherosclerotic plaque development. METHODS TRAF3IP2/ApoE double knockout (DKO) mice were generated by crossing TRAF3IP2(-/-) and ApoE(-/-) mice. ApoE(-/-) mice served as controls. Both DKO and control mice were fed a high-fat diet for 12 weeks. Plasma lipids were measured by ELISA, atherosclerosis by en face analysis of aorta and plaque cross-section measurements at the aortic valve region, plaque necrotic core area, collagen and smooth muscle cell (SMC) content by histomorphometry, and aortic gene expression by RT-qPCR. RESULTS The plasma lipoprotein profile was not altered by TRAF3IP2 gene deletion in ApoE(-/-) mice. While total aortic plaque area was decreased in DKO female, but not male mice, the plaque necrotic area was significantly decreased in DKO mice of both genders. Plaque collagen and SMC contents were increased significantly in both female and male DKO mice compared to respective controls. Aortic expression of proinflammatory cytokine (Tumor necrosis factor α, TNFα), chemokine (Chemokine (C-X-C motif) Ligand 1, CXCL1) and adhesion molecule (Vascular cell adhesion molecule 1, VCAM1; and Intercellular adhesion molecule 1, ICAM1) gene expression were decreased in both male and female DKO mice. In addition, the male DKO mice expressed markedly reduced levels of extracellular matrix (ECM)-related genes, including TIMP1 (Tissue inhibitor of metalloproteinase 1), RECK (Reversion-Inducing-Cysteine-Rich Protein with Kazal Motifs) and ADAM17 (A Disintegrin And Metalloproteinase 17). CONCLUSIONS TRAF3IP2 plays a causal role in atherosclerotic plaque development and vulnerability, possibly by inducing the expression of multiple proinflammatory mediators. TRAF3IP2 could be a potential therapeutic target in atherosclerotic vascular diseases.

Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.

A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice

Cerebral blood flow (CBF) is uniquely regulated by the anatomical design of the cerebral vasculature as well as through neurovascular coupling. The process of directing the CBF to meet the energy demands of neuronal activity is referred to as neurovascular coupling. Microvasculature in the brain constitutes the critical component of the neurovascular coupling. Mitochondria provide the majority of ATP to meet the high-energy demand of the brain. Impairment of mitochondrial function plays a central role in several age-related diseases such as hypertension, ischemic brain injury, Alzheimer’s disease, and Parkinson disease. Interestingly, microvessels and small arteries of the brain have been the focus of the studies implicating the vascular mechanisms in several age-related neurological diseases. However, the role of microvascular mitochondrial dysfunction in age-related diseases remains unexplored. To date, high-throughput assay for measuring mitochondrial respiration in microvessels is lacking. The current study presents a novel method to measure mitochondrial respiratory parameters in freshly isolated microvessels from mouse brain ex vivo using Seahorse XFe24 Analyzer. We validated the method by demonstrating impairments of mitochondrial respiration in cerebral microvessels isolated from old mice compared to the young mice. Thus, application of mitochondrial respiration studies in microvessels will help identify novel vascular mechanisms underlying a variety of age-related neurological diseases.