Metformin alleviates lung-endothelial hyperpermeability by regulating cofilin-1/PP2AC pathway.

Background: Microvascular endothelial hyperpermeability is an earliest pathological hallmark in Acute Lung Injury (ALI), which progressively leads to Acute Respiratory Distress Syndrome (ARDS). Recently, vascular protective and anti-inflammatory effect of metformin, irrespective of glycemic control, has garnered significant interest. However, the underlying molecular mechanism(s) of metformin's barrier protective benefits in lung-endothelial cells (ECs) has not been clearly elucidated. Many vascular permeability-increasing agents weakened adherens junctions (AJ) integrity by inducing the reorganization of the actin cytoskeleton and stress fibers formation. Here, we hypothesized that metformin abrogated endothelial hyperpermeability and strengthen AJ integrity via inhibiting stress fibers formation through cofilin-1-PP2AC pathway. Methods: We pretreated human lung microvascular ECs (human-lung-ECs) with metformin and then challenged with thrombin. To investigate the vascular protective effects of metformin, we studied changes in ECs barrier function using electric cell-substrate impedance sensing, levels of actin stress fibers formation and inflammatory cytokines IL-1ß and IL-6 expression. To explore the downstream mechanism, we studied the Ser3-phosphorylation-cofilin-1 levels in scramble and PP2AC-siRNA depleted ECs in response to thrombin with and without metformin pretreatment. Results: In-vitro analyses showed that metformin pretreatment attenuated thrombin-induced hyperpermeability, stress fibers formation, and the levels of inflammatory cytokines IL-6 and IL-ß in human-lung-ECs. We found that metformin mitigated Ser3-phosphorylation mediated inhibition of cofilin-1 in response to thrombin. Furthermore, genetic deletion of PP2AC subunit significantly inhibited metformin efficacy to mitigate thrombin-induced Ser3-phosphorylation cofilin-1, AJ disruption and stress fibers formation. We further demonstrated that metformin increases PP2AC activity by upregulating PP2AC-Leu309 methylation in human-lung-ECs. We also found that the ectopic expression of PP2AC dampened thrombin-induced Ser3-phosphorylation-mediated inhibition of cofilin-1, stress fibers formation and endothelial hyperpermeability. Conclusion: Together, these data reveal the unprecedented endothelial cofilin-1/PP2AC signaling axis downstream of metformin in protecting against lung vascular endothelial injury and inflammation. Therefore, pharmacologically enhancing endothelial PP2AC activity may lead to the development of novel therapeutic approaches for prevention of deleterious effects of ALI on vascular ECs.

Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a-/-) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146-/- podocytes from these animals also showed a basally upregulated TGFß-Smad3 signaling in vitro. Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFß, and linked downstream signaling.

CD11b agonists offer a novel approach for treating lupus nephritis.

Lupus nephritis (LN) develops in more than a third of all systemic lupus erythematosus (SLE) patients and is the strongest predictor of morbidity and mortality. Increased circulating levels of type I interferon (IFN I) and anti-double stranded DNA (anti-dsDNA) and anti-RNA binding protein (anti-RNP) antibodies lead to increased glomerular injury via leukocyte activation and glomerular infiltration. Uncontrolled Toll-like receptor (TLR) signaling in leukocytes results in increased production of IFN I and anti-dsDNA antibodies. ITGAM gene codes for integrin CD11b, the α-chain of integrin heterodimer CD11b/CD18, that is highly expressed in leukocytes and modulates TLR-dependent pro-inflammatory signaling. Three nonsynonymous SNPs in the ITGAM gene strongly correlate with increased risk for SLE and LN and with IFN I levels. Here we review the literature on the role of CD11b on leukocytes in LN. We also incorporate conclusions from several recent studies that show that these ITGAM SNPs result in a CD11b protein that is less able to suppress TLR-dependent pro-inflammatory pathways in leukocytes, that activation of CD11b via novel small molecule agonists suppresses TLR-dependent pathways, including reductions in circulating levels of IFN I and anti-dsDNA antibodies, and that CD11b activation reduces LN in model systems. Recent data strongly suggest that integrin CD11b is an exciting new therapeutic target in SLE and LN and that allosteric activation of CD11b is a novel therapeutic paradigm for effectively treating such autoimmune diseases.

Upregulated Angiogenesis Is Incompetent to Rescue Dilated Cardiomyopathy Phenotype in Mice.

Dilated cardiomyopathy (DCM) is characterized by pathologic cardiac remodeling resulting in chambers enlargement and impaired heart contractility. Previous reports and our in-silico analysis support the association of DCM phenotype and impaired tissue angiogenesis. Here, we explored whether the modulation in cardiac angiogenesis partly intervenes or rescues the DCM phenotype in mice. Here, a DCM mouse model [α-tropomyosin 54 (α-TM54) mutant] was crossbred with microRNA-210 transgenic mice (210-TG) to develop microRNA-210 (miR-210) overexpressing α-TM54 mutant mice (TMx210). Contrary to wild-type (WT) and 210-TG mice, a significant increase in heart weight to body weight ratio in aged mixed-gender TMx210 and DCM mice was recorded. Histopathological analysis revealed signs of pathological cardiac remodeling such as myocardial disarray, myofibrillar loss, and interstitial fibrosis in DCM and TMx210 mice. Contrary to WT and DCM, a significant increase in angiogenic potential was observed in TMx210 and 210-TG mice hearts which is reflected by higher blood vessel density and upregulated proangiogenic vascular endothelial growth factor-A. The echocardiographic assessment showed comparable cardiac dysfunction in DCM and TMx210 mice as compared to WT and 210-TG. Overall, the present study concludes that miR-210 mediated upregulated angiogenesis is not sufficient to rescue the DCM phenotype in mice.