Naveen K. Somanna

Interleukin-17A stimulates cardiac fibroblast proliferation and migration via negative regulation of the dual-specificity phosphatase MKP-1/DUSP-1.

The dual-specificity mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) inactivates MAP kinases by dephosphorylation. Here we show that the proinflammatory cytokine interleukin (IL)-17A induces adult mouse primary cardiac fibroblast (CF) proliferation and migration via IL-17 receptor A//IL-17 receptor C-dependent MKP-1 suppression, and activation of p38 MAPK and ERK1/2. IL-17A mediated p38 MAPK and ERK1/2 activation is inhibited by MKP-1 overexpression, but prolonged by MKP-1 knockdown. IL-17A induced miR-101 expression via PI3K/Akt, and miR-101 inhibitor reversed MKP-1 down regulation. Importantly, MKP-1 knockdown, pharmacological inhibition of p38 MAPK and ERK1/2, or overexpression of dominant negative MEK1, each markedly attenuated IL-17A-mediated CF proliferation and migration. Similarly, IL-17F and IL-17A/F heterodimer that also signal via IL-17RA/IL-17RC, stimulated CF proliferation and migration. These results indicate that IL-17A stimulates CF proliferation and migration via Akt/miR-101/MKP-1-dependent p38 MAPK and ERK1/2 activation. These studies support a potential role for IL-17 in cardiac fibrosis and adverse myocardial remodeling.

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.

Aldosterone-induced cardiomyocyte growth, and fibroblast migration and proliferation are mediated by TRAF3IP2

Sustained activation of the Renin-Angiotensin-Aldosterone System (RAAS) contributes to the pathogenesis of heart failure. Aldosterone (Aldo) is known to induce both myocardial hypertrophy and fibrosis through oxidative stress and proinflammatory pathways. Here we have investigated whether Aldo-mediated cardiomycocyte hypertrophy is dependent on TRAF3IP2, an upstream regulator of IKK and JNK. We also investigated whether the pro-mitogenic and pro-migratory effects of Aldo on cardiac fibroblasts are also mediated by TRAF3IP2. Aldo induced both superoxide and hydrogen peroxide in isolated adult mouse cardiomyocytes (CM), and upregulated TRAF3IP2 expression in part via the mineralocorticoid receptor and oxidative stress. Silencing TRAF3IP2 blunted Aldo-induced IKKβ, p65, JNK, and c-Jun activation, IL-18, IL-6 and CT-1 upregulation, and cardiomyocyte hypertrophy. In isolated adult mouse cardiac fibroblasts (CF), Aldo stimulated TRAF3IP2-dependent IL-18 and IL-6 production, CTGF, collagen I and III expression, MMP2 activation, and proliferation and migration. These in vitro results suggest that TRAF3IP2 may play a causal role in Aldo-induced adverse cardiac remodeling in vivo, and identify TRAF3IP2 as a potential therapeutic target in hypertensive heart disease.

The Nox1/4 Dual Inhibitor GKT137831 or Nox4 Knockdown Inhibits Angiotensin-II-Induced Adult Mouse Cardiac Fibroblast Proliferation and Migration. AT1 Physically Associates With Nox4

Both oxidative stress and inflammation contribute to chronic hypertension‐induced myocardial fibrosis and adverse cardiac remodeling. Here we investigated whether angiotensin (Ang)‐II‐induced fibroblast proliferation and migration are NADPH oxidase (Nox) 4/ROS and IL‐18 dependent. Our results show that the potent induction of mouse cardiac fibroblast (CF) proliferation and migration by Ang‐II is markedly attenuated by Nox4 knockdown and the Nox inhibitor DPI. Further, Nox4 knockdown and DPI pre‐treatment attenuated Ang‐II‐induced IL‐18, IL‐18Rα and collagen expression, and MMP9 and LOX activation. While neutralization of IL‐18 blunted Ang‐II‐induced CF proliferation and migration, knockdown of MMP9 attenuated CF migration. The antioxidant NAC and the cell‐permeable SOD mimetics Tempol, MnTBAP, and MnTMPyP attenuated oxidative stress and inhibited CF proliferation and migration. The Nox1/Nox4 dual inhibitor GKT137831 also blunted Ang‐II‐induced H2O2 production and CF proliferation and migration. Further, AT1 bound Nox4, and Ang‐II enhanced their physical association. Notably, GKT137831 attnuated the AT1/Nox4 interaction. These results indicate that Ang‐II induces CF proliferation and migration in part via Nox4/ROS‐dependent IL‐18 induction and MMP9 activation, and may involve AT1/Nox4 physical association. Thus, either (i) neutralizing IL‐18, (ii) blocking AT1/Nox4 interaction or (iii) use of the Nox1/Nox4 inhibitor GKT137831 may have therapeutic potential in chronic hypertension‐induced adverse cardiac remodeling. J. Cell. Physiol. 231: 1130–1141, 2016.

Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

Histone acetylation/deacetylation plays a key role in the epigenetic regulation of multiple pro-fibrotic genes. Here we investigated the effects of histone deacetyltransferase (HDAC) inhibition on angiotensin (Ang)-II-induced pro-fibrotic changes in adult mouse cardiac fibroblasts (CF). CF express class I HDACs 1 and 2, and Ang-II induces their activation. Notably, silencing HDAC1 or HDAC2 attenuated Ang-II induced CF proliferation and migration. Under basal conditions, HDAC1 dimerizes with HDAC2 in CF and Ang-II reversed this interaction. Treatment with Trichostatin A (TSA), a broad-spectrum HDAC inhibitor, restored their physical association, and attenuated Ang-II-induced MMP9 expression, IL-18 induction, and extracellular matrix (collagen I, collagen III and fibronectin) production. Further, TSA inhibited Ang-II-induced MMP9 and Il18 transcription by blocking NF-κB and AP-1 binding to their respective promoter regions. By inhibiting Sp1 binding to RECK promoter, TSA reversed Ang-II-induced RECK suppression, collagen and fibronectin expression, and CF migration and proliferation. The class I-specific HDAC inhibitor Mocetinostat (MGCD) recapitulated TSA effects on Ang-II-treated CF. Together, these results demonstrate that targeting HDACs attenuates the pro-inflammatory and pro-fibrotic effects of Ang-II on CF.

Intratracheal administration of cyclooxygenase-1-transduced adipose tissue-derived stem cells ameliorates monocrotaline-induced pulmonary hypertension in rats.

The effect of intratracheal administration of cyclooxygenase-1 (COX-1)-modified adipose stem cells (ASCs) on monocrotaline-induced pulmonary hypertension (MCT-PH) was investigated in the rat. The COX-1 gene was cloned from rat intestinal cells, fused with a hemagglutanin (HA) tag, and cloned into a lentiviral vector. The COX-1 lentiviral vector was shown to enhance COX-1 protein expression and inhibit proliferation of vascular smooth muscle cells without increasing apoptosis. Human ASCs transfected with the COX-1 lentiviral vector (ASCCOX-1) display enhanced COX-1 activity while exhibiting similar differentiation potential compared with untransduced (native) ASCs. PH was induced in rats with MCT, and the rats were subsequently treated with intratracheal injection of ASCCOX-1 or untransduced ASCs. The intratracheal administration of ASCCOX-1 3 × 10(6) cells on day 14 after MCT treatment significantly attenuated MCT-induced PH when hemodynamic values were measured on day 35 after MCT treatment whereas administration of untransduced ASCs had no significant effect. These results indicate that intratracheally administered ASCCOX-1 persisted for at least 21 days in the lung and attenuate MCT-induced PH and right ventricular hypertrophy. In addition, vasodilator responses to the nitric oxide donor sodium nitroprusside were not altered by the presence of ASCCOX-1 in the lung. These data emphasize the effectiveness of ASCCOX-1 in the treatment of experimentally induced PH.

96 ANALYSIS OF NATRIURETIC PEPTIDE RECEPTOR A INTERNALIZATION BY RIBONUCLEIC ACID INTERFERENCE.

Guanylyl cyclase/natriuretic peptide receptor A (GC-A/NPRA) is considered the principal receptor for both atrial and brain natriuretic peptides (ANP and BNP). Binding of these hormones to NPRA leads to the generation of the intracellular second messenger cGMP, which, in turn, maintains blood pressure and cardiovascular homeostasis. The objective of the present study was to determine whether ANP-NPRA complexes are internalized to release the bound ANP from the receptor in the intracellular compartments. In these studies, we used human embronic kidney-293 (HEK-293) cells stably transfected with murine NPRA cDNA. Cells were cultured in Dulbecco9s Modified Eagle9s Medium (DMEM) supplemented with 10% bovine fetal serum and maintained at 37°C under an atmosphere of 5% CO 2 /95% O 2 . Receptor internalization studies were performed using 125 I-ANP in intact HEK-293 cells. All hormonal treatments and binding assays were performed in DMEM serum-free medium containing 0.1% bovine serum albumin. We used vector-based artificial microRNA for expression of short-hairpin RNA (shRNA) to more potently silence the target gene expression of NPRA. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses were applied to confirm the specificity and the efficiency of the constructs. The results showed that the expression of shRNA accounted for more than 80% down-regulation of NPRA. Furthermore, the results revealed that the internalized 125 I-ANP radioactivities were drastically reduced by almost 80% in cells depleted with NPRA compared with wild-type control cells. The results demonstrate that NPRA undergoes internalization as part of its normal trafficking and metabolism. The findings of this study are important toward the understanding of the signaling mechanisms and the functional role of NPRA to maintain the cardiovascular homeostasis.

DISRUPTION OF NATRIURETIC PEPTIDE RECEPTOR A GENE INCREASES ADRENAL ANGIOTENSIN II AND ALDOSTERONE LEVELS.: 264

The disruption of guanylyl cyclase/natriuretic peptide receptor A (GC-A/NPRA) gene (Npr1) leads to elevated arterial blood pressure, cardiac hypertrophy, congestive heart failure, and sudden death in mice lacking NPRA. ANP-NPRA signaling is known to counteract the renin-angiotensin-aldosterone system (RAAS). We studied whether Npr1 gene copy number affects adrenal angiotensin II (Ang II) and aldosterone (ALDO) levels in a gene-dose dependent manner in Npr1 gene-targeted mice. Adrenal Ang II and ALDO levels increased in one-copy (gene-disrupted heterozygous allele, 15%, p

INHIBITION OF NUCLEAR FACTOR κB INDUCES REGRESSION OF CARDIAC HYPERTROPHY IN MICE LACKING NATRIURETIC PEPTIDE RECEPTOR A.: 270

Guanylyl cyclase A/natriuretic peptide receptor A (GC-A/NPRA) is the principal receptor for the cardiac hormones atrial and brain natriuretic peptides (ANP and BNP). Mice carrying targeted disruption of Npr1 gene (encoding for GC-A/NPRA) exhibit hypertension, marked cardiac hypertrophy, and congestive heart failure with sudden death. The objective of the present study was to determine whether disruption of NPRA activates the nuclear factor κB (NF-κB) and further to evaluate whether the inhibition of NF-κB signaling can attenuate cardiac hypertrophy in mice lacking NPRA. We analyzed hypertrophy marker genes expression, NF-κB nuclear translocation, IKK-kinase activity, and IκB-α phosphorylation status in the ventricular tissues of 16-week-old male Npr1 gene-disrupted (Npr1−/−) and wild-type (Npr1+/+) mice. Electrophoretic mobility shift assay (EMSA) was performed with NF-[203}B-specific oligonucleotides in the nuclear extracts isolated from the ventricular tissues of Npr1−/−and Npr1+/+mice. NF-κB subunits (p65 and p50) protein levels and IκB-α phosphorylation status were analyzed using specific antibodies. Heart weight/body weight ratio and hypertrophy marker genes c-fos (p