Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are highly contractile and mechanosensitive to aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo respectively results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics. Synopsis: In this study, we show the role of lipogenic transcription factors sterol regulatory element binding proteins (SREBPs) in the regulation of intraocular pressure (IOP). ( Synopsis Figure - Created using Biorender.com ) SREBPs are involved in the sensing of changes in mechanical stress on the trabecular meshwork (TM). SREBPs aid in transducing the mechanical signals to induce actin polymerization and filopodia/lamellipodia formation.SREBPs inactivation lowered genes and enzymes involved in lipogenesis and modified lipid levels in TM.SREBPs activity is a critical regulator of ECM engagement to the matrix sites.Inactivation of SCAP-SREBP pathway lowered IOP via actin relaxation and decreasing ECM production and deposition in TM outflow pathway signifying a novel relationship between SREBP activation status and achieving IOP homeostasis.

Galectin-3 S-glutathionylation regulates its effect on adipocyte insulin signaling.

Protein-S-glutathionylation promotes redox signaling in physiological and oxidative distress conditions. Galectin-3 (Gal-3) promotes insulin resistance by down-regulating adipocyte insulin signaling, however, its S-glutathionylation and significance is not known. In this context, we report reversible S-glutathionylation of Gal-3. Site-directed mutagenesis established Gal-3 Cys187 as the putative S-glutathionylation site. Glutathionylated Gal-3 prevents Gal-3(WT)-Insulin Receptor interaction and facilitates insulin-induced murine adipocyte p-IRS1(tyr895) and p-AKT(ser473) signaling and glucose uptake in a Gal-3 Cys187 glutathionylation dependent manner in murine adipocytes, as assessed by Western blotting and 2-NBDG uptake assay respectively. Pre-glutathionylated Gal-3 at Cys187 resisted irreversible oxidation by H2O2. M2 macrophages showed enhanced Gal-3 S-glutathionylation when compared to M1 phenotype. Serum and stromal vascular fraction (SVF) isolated from control mice showed increased Gal-3 S-glutathionylation as compared to db/db mice. A significant increase in Gal-3 S-glutathionylation was observed in metformin-treated db/db mice when compared to db/db mice alone. Similar to murine, enhanced Gal-3 S-glutathionylation is observed in primary human monocyte derived M2 macrophages when compared to the M1 macrophage phenotype and Gal-3 regulates primary human adipocyte insulin signaling in a glutathionylation dependent manner. Collectively, we identified Gal-3 S-glutathionylation as a protective phenomenon, which relieves its inhibitory effect on adipocyte insulin signaling.

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.

Lin28B Regulates Angiotensin II-Mediated Let-7c/miR-99a MicroRNA Formation Consequently Affecting Macrophage Polarization and Allergic Inflammation.

Angiotensin-II (Ang-II) receptor plays a role in allergic airway inflammation; however, the underlying mechanism and role of macrophages need better understanding. In the present study, angiotensin-II infusion (1 µg/kg/min) in ovalbumin-induced airway inflammation mice model significantly decreased immune cell infiltration, goblet cell hyperplasia, and eosinophil numbers in lungs. Ang-II infusion increased M1 and decreased M2 macrophage population in bronchoalveolar lavage fluid and respective macrophage markers in lung macrophages. Similarly, in vitro Ang-II treatment in murine bone marrow-derived macrophages (BMDMs) induced M1 and reduced M2 macrophage phenotype with enhanced bactericidal activity. Mechanistically, Ang-II inhibits Let-7c and miR-99a expression in BMDMs and in vivo as well. Lentiviral overexpression of Let-7c and miR-99a miRNAs in BMDMs abrogated Ang-II-induced M1 phenotype activation and promoted M2 phenotype, which is governed by targeting TNFα by miR-99a. In lung macrophages, ovalbumin-induced TNFα inhibition was rescued after Ang-II treatment. In BMDMs, knockdown of TNFα abrogated Ang-II-induced M2 to M1 macrophage phenotype switch and associated bactericidal activity. Ang-II affects mature miRNA formation by enhancing Lin28B levels in macrophages in vivo and in vitro. Furthermore, Lin28B knockdown prevented Ang-II-mediated inhibition of mature Let-7c/miR-99a miRNA formation, M2 to M1 macrophage phenotype switch, and increased bactericidal activity. Therefore, present study suggests a role of Lin28B in Ang-II-induced Let-7c/miR-99a miRNA formation that consequently affects TNFα production, M1 phenotype activation, and allergic airway inflammation. Graphical Abstract Ovalbumin inhibits LIN28B expression thereby fails to inhibit premature to mature Let-7c/miR-99a miRNA formation. Mature miR-99a miRNA that inhibits TNFα consequently promotes M2 polarization and allergic airway inflammation. While Ang-II induces Lin28B, which inhibits Let-7c/miR-99a miRNA processing and mature miRNA formation, this results in increased TNFα levels that lead to M1 polarization and allergic airway inflammation inhibition.

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.