Macrophage p47phox regulates pressure overload-induced left ventricular remodeling by modulating IL-4/STAT6/PPARγ signaling.

NADPH oxidase (Nox) mediates ROS production and contributes to cardiac remodeling. However, macrophage p47phox, a Nox subunit regulating cardiac remodeling, is unclear. We aimed to investigate the role of macrophage p47phox in hypertensive cardiac remodeling. Pressure-overload induced by Angiotensin II (AngII) for two weeks in young adult male p47phox deficient (KO) mice showed aggravated cardiac dysfunction and hypertrophy as indicated from echocardiographic and histological studies in comparison with wild-type littermates (WT). Additionally, LV of AngII-infused KO mice showed augmented interstitial fibrosis, collagen deposition and, myofibroblasts compared to AngII-infused WT mice. Moreover, these changes in AngII-infused KO mice correlated well with the gene analysis of hypertrophic and fibrotic markers. Similar results were also found in the transverse aortic constriction model. Further, AngII-infused KO mice showed elevated circulating immunokines and increased LV leukocytes infiltration and CD206+ macrophages compared to AngII-infused WT mice. Likewise, LV of AngII-infused KO mice showed upregulated mRNA expression of anti-inflammatory/pro-fibrotic M2 macrophage markers (Ym1, Arg-1) compared to AngII-infused WT mice. AngII and IL-4 treated bone marrow-derived macrophages (BMDMs) from KO mice showed upregulated M2 macrophage markers and STAT6 phosphorylation (Y641) compared to AngII and IL-4 treated WT BMDMs. These alterations were at least partly mediated by macrophage as bone marrow transplantation from KO mice into WT mice aggravated cardiac remodeling. Mechanistically, AngII-infused KO mice showed hyperactivated IL-4/STAT6/PPARγ signaling and downregulated SOCS3 expression compared to AngII-infused WT mice. Our studies show that macrophage p47phox limits anti-inflammatory signaling and extracellular matrix remodeling in response to pressure-overload.

MicroRNA-99a mimics inhibit M1 macrophage phenotype and adipose tissue inflammation by targeting TNFα.

In human adipose tissue and obesity, miR-99a expression is negatively correlated with inflammation. Therefore, the present study investigated the role of miR-99a in macrophage phenotype activation and adipose tissue inflammation. M2 BMDMs showed a significant increase in miR-99a expression when compared to the M0 and M1 phenotypes. Phenotype-switching experiments established an association between upregulated miR-99a expression and the M2 phenotype. Overexpression of miR-99a prevented M1 phenotype activation and attenuated bactericidal activity. Likewise, knockdown of miR-99a abolished M2 phenotype activation. By means of in silico target prediction tools and a luciferase reporter assay, TNFα was identified as a direct target of miR-99a. Knockdown of TNFα recapitulated the effect of miR-99a overexpression in M1 BMDMs. In a db/db mice model, miR-99a expression was reduced in eWAT and F4/80+ ATMs. Systemic overexpression of miR-99a in db/db mice attenuated adipocyte hypertrophy with increased CD301 and reduced CD86 immunostaining. Flow cytometry analysis also showed an increased M2 and a reduced M1 macrophage population. Mimics of miR-99a also improved the diabetic dyslipidemia and insulin signaling in eWAT and liver, with an attenuated expression of gluconeogenesis and cholesterol metabolism genes in the liver. Furthermore, adoptive transfer of miR-99a-overexpressing macrophages in the db/db mice recapitulated in vivo miR-99a mimic effects with increased M2 and reduced M1 macrophage populations and improved systemic glucose, insulin sensitivity, and insulin signaling in the eWAT and liver. The present study demonstrates that miR-99a mimics can regulate macrophage M1 phenotype activation by targeting TNFα. miR-99a therapeutics in diabetic mice reduces the adipose tissue inflammation and improves insulin sensitivity.

Cilostazol ameliorates heart failure with preserved ejection fraction and diastolic dysfunction in obese and non-obese hypertensive mice.

Cilostazol (Ciloz) a potent Type III phosphodiesterase inhibitor is effective against inflammation, insulin resistance and cardiomyopathy. However, the effect of Ciloz on obesity-associated left ventricular diastolic dysfunction has not been explored yet. Hence, we examined the effect of Ciloz on cardiac remodelling and dysfunction in non-obese and obese-insulin resistant mice infused with AngiotensinII (AngII). Male C57BL/6 J mice were initially subjected to 19 weeks of chow or high fat diet (HFD) regimen and thereafter animals were randomised for AngII (1500 ng/kg/min, s.c) infusion or saline and Ciloz (50 mg/kg, p.o) for another 1 week. Obese and non-obese mice infused with AngII exhibited significant diastolic dysfunction and features of heart failure with preserved ejection fraction (HFpEF) since a decrease in fractional shortening and no change in ejection fraction were observed when compared with respective controls. Administration of AngII and Ciloz in HFD fed mice significantly improved the left ventricular function compared with AngII infused HFD mice as evinced from the echocardiographic data. Further, Ciloz treatment significantly reduced cardiomyocyte area, interstitial and perivascular fibrosis; and collagen deposition. Moreover, Ciloz reduced the inflammatory milieu in the heart as evinced by decreased F4/80+ and CD68+ cells; IL-1ß and IL-6 gene transcripts. Quantitative assessment of the expression levels revealed substantial upregulation of MMP-9 (pro- and mature-forms) and α-SMA in the left ventricle of AngII infused HFD-fed mice, which was considerably suppressed by Ciloz regimen. The beneficial effect of Ciloz was associated with the normalization in gene expression of hypertrophic and fibrotic markers. Likewise, Ciloz administration markedly reduced the AngII and HFD induced TGF-ß1/SMAD3 and Akt/mTOR signalling. Additionally, AngII administered and HFD-fed mice showed increased glycolytic flux, which was considerably diminished by Ciloz treatment as indicated from suppressed PKM2, HK-2, PDK-2, HIF-1α mRNA and GLUT-1 protein expression. Taken together, Ciloz might be therapeutically exploited against AngII and obesity-associated diastolic dysfunction thereby preventing overt heart failure.

Coagulin-L ameliorates TLR4 induced oxidative damage and immune response by regulating mitochondria and NOX-derived ROS.

Withanolides possess diverse biological and pharmacological activity but their immunomodulatory function is less realized. Hence, coagulin-L, a withanolide isolated from Withania coagulans Dunal has been studied for such an effect in human and murine cells, and mice model. Coagulin-L (1, 3, 10µM) exhibited immunomodulatory effect by suppressing TLR4 induced immune mediators such as cytokines (GMCSF, IFNα, IFNγ, IL-1α, IL-1Rα, IL-1ß, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17), chemokines (IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10, KC, MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, RANTES/CCL5, eotaxin/CCL11), growth factors (FGF-basic, VEGF), nitric oxide and intracellular superoxide. Mechanistically, coagulin-L abrogated LPS induced total and mitochondrial ROS generation, NOX2, NOX4 mRNA expression, IRAK and MAPK (p38, JNK, ERK) activation. Coagulin-L also attenuated IκBα degradation, which prevented NFκB downstream iNOS expression and pro-inflammatory cytokine release. Furthermore, coagulin-L (10, 25, 50mg/kg, p.o.), undermined the LPS (10mg/kg, i.p.) induced endotoxemia response in mice as evinced from diminished cytokine release, nitric oxide, aortic p38 MAPK activation and endothelial tissue impairment besides suppressing NOX2 and NOX4 expression in liver and aorta. Moreover, coagulin-L also alleviated the ROS mediated oxidative damage which was assessed through protein carbonyl, lipid hydroperoxide, 8-isoprostane and 8-hydroxy-2-deoxyguanosine quantification. To extend, coagulin-L also suppressed carrageenan-induced paw edema and thioglycollate-induced peritonitis in mice. Therefore, coagulin-L can be of therapeutic importance in pathological conditions induced by oxidative damage.