Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

[Figure: see text].

Inhibition of RUNX1 blocks the differentiation of lung fibroblasts to myofibroblasts.

Pathological fibrosis contributes to progression of various diseases, for which the therapeutic options are limited. Idiopathic pulmonary fibrosis (IPF) is one such progressive and fatal interstitial fibrotic disease that is often characterized by excessive accumulation of extracellular matrix (ECM) proteins leading to stiff lung tissue and impaired gas exchange. However, the molecular mechanisms underlying IPF progression remain largely unknown. In this study, we determined the role of Runt-related transcription factor 1 (RUNX1), an evolutionarily conserved transcription factor, in the differentiation of human lung fibroblasts (HLFs) in vitro and in an animal model of bleomycin (BLM)-induced lung fibrosis. We observed that the expression of RUNX1 was significantly increased in the lungs of BLM-injected mice as compared to saline-treated mice. Furthermore, HLFs stimulated with transforming growth factor ß (TGF-ß) showed significantly higher RUNX1 expression at both mRNA and protein levels, and compartmentalization in the nucleus. Inhibition of RUNX1 in HLFs (using siRNA) showed a significant reduction in the differentiation of fibroblasts into myofibroblasts as evidenced by reduced expression of alpha-smooth muscle actin (α-SMA), TGF-ß and ECM proteins such as fibronectin 1 (FN1), and collagen 1A1 (COL1A1). Mechanistic studies revealed that the increased expression of RUNX1 in TGF-ß-stimulated lung fibroblasts is due to enhanced mRNA stability of RUNX1 through selective interaction with the RNA-binding profibrotic protein, human antigen R (HuR). Collectively, our data demonstrate that increased expression of RUNX1 augments processes involved in lung fibrosis including the differentiation of fibroblasts into collagen-synthesizing myofibroblasts. Our study suggests that targeting RUNX1 could limit the progression of organ fibrosis in diseases characterized by abnormal collagen deposition.

Increased m6A-RNA methylation and FTO suppression is associated with myocardial inflammation and dysfunction during endotoxemia in mice.

Endotoxemia triggers life-threatening immune and cardiovascular response that leads to tissue damage, multi-organ failure, and death. The understanding of underlying molecular mechanisms is still evolving. N6-methyladenosine (m6A)-RNA modification plays key regulatory role in numerous biological processes. However, it remains unclear whether endotoxemia alters RNA methylation in the myocardium. In the current study, we investigated the effect of lipopolysaccharide (LPS)-induced endotoxemia on m6A-RNA methylation and its implications on myocardial inflammation and left ventricular (LV) function. Following LPS administration, mice showed increases in m6A-RNA methylation in the myocardium with a corresponding decrease in the expression of fat mass and obesity-associated protein (FTO, an m6A eraser/demethylase). The changes were associated with a significant increase in expression of myocardial inflammatory cytokine genes, such as IL-6, TNF-α, IL-1ß, and reduced LV function. Moreover, rat cardiomyoblasts (H9c2) exposed to LPS showed similar changes (with increase in m6A-RNA methylation and inflammatory cytokine genes, whereas downregulation of FTO). Furthermore, methylated RNA immunoprecipitation assay showed hypermethylation and increase in the expression of IL-6 and TNF-α genes in LPS-treated H9c2 cells as compared to untreated cells. Interestingly, FTO knockdown in cardiomyocytes mimicked the above effects. Taken together, these data suggest that endotoxemia-induced m6A methylation might play a critical role in expression of cardiac proinflammatory cytokines, and modulation of m6A methylation might limit myocardial inflammation and dysfunction during endotoxemia.

MicroRNA-181c-5p modulates phagocytosis efficiency in bone marrow-derived macrophages.

OBJECTIVE AND DESIGN: Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. MATERIALS AND METHODS: miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively. RESULTS: miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. CONCLUSION: Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.

Evaluation of therapeutic potential of recombinant buffalo lactoferrin N-lobe expressed in E. coli.

Lactoferrin (Lf) is a multifunctional bi-lobate iron-binding glycoprotein belonging to transferrin family with a mass of approximately 80 kD. Being ubiquitously present in almost all biological secretions, it performs important biological functions. One of the earliest and very well-documented functions of Lf is the antibacterial effect against broad spectrum Gram-negative and Gram-positive bacteria. In this study, buffalo Lf N-lobe cDNA was amplified, cloned and expressed as a fusion protein in Escherichia coli cells using pQE30 expression vector. After post-induction confirmation of expressed protein by SDS-PAGE, purification of recombinant protein using Ni-NTA was attempted and the yield of recombinant buffalo N-lobe Lf was estimated to be 1 mg/ml. Antibacterial activity of recombinant buffalo Lf N-lobe was assessed on pathogenic E. coli and Staphylococcus aureus strains. Peptic digest of recombinant N-lobe buffalo Lf showed antibacterial activity comparable to commercially available bovine Lf. The successful expression and characterization of functional recombinant N-lobe of buffalo Lf expressed in E. coli opens new vistas for developing alternate therapeutics, particularly against the diseases caused by Gram-negative microbes such as septicemia and diarrhea in newborn calves and mastitis in dairy animals.

TLR4-induced NF-κB and MAPK signaling regulate the IL-6 mRNA stabilizing protein Arid5a.

The AT-rich interactive domain-containing protein 5a (Arid5a) plays a critical role in autoimmunity by regulating the half-life of Interleukin-6 (IL-6) mRNA. However, the signaling pathways underlying Arid5a-mediated regulation of IL-6 mRNA stability are largely uncharacterized. Here, we found that during the early phase of lipopolysaccharide (LPS) stimulation, NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA. Subsequently, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) promotes translocation of AU-rich element RNA-binding protein 1 (AUF-1) from the nucleus to the cytoplasm, where it destabilizes Arid5a mRNA by binding to AU-rich elements in the 3΄ UTR. This results in downregulation of IL-6 mRNA expression. During the late phase of LPS stimulation, p38 MAPK phosphorylates Arid5a and recruits the WW domain containing E3 ubiquitin protein ligase 1 (WWP1) to its complex, which in turn ubiquitinates Arid5a in a K48-linked manner, leading to its degradation. Inhibition of Arid5a phosphorylation and degradation increases production of IL-6 mRNA. Thus, our data demonstrate that LPS-induced NF-κB and MAPK signaling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. This study therefore substantially increases our understanding of the mechanisms by which IL-6 is regulated.

The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis.

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps to chromosome 2 (locus 2q31.1), near Titin (TTN), which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in failing human heart tissue (HF) compared to non-failing hearts (NF). Using the Sanger sequencing method, we characterized the human OLA1 gene and screened for mutations in the OLA1 gene in patients with failing and non-failing hearts. Among failing and non-failing heart patients, we found 15 different mutations in the OLA1 gene, including two transversions, one substitution, one deletion, and eleven transitions. All mutations were intronic except for a non-synonymous 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results demonstrate that this PCR test can effectively screen for OLA1 mutation-associated cardiomyopathy in human patients using easily accessible cells or tissues, such as blood cells. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

Obg-like ATPase 1 Genetic Deletion Leads to Dilated Cardiomyopathy in Mice and Structural Changes in Drosophila Heart.

Cardiomyopathy, disease of the heart muscle, is a significant contributor to heart failure. The pathogenesis of cardiomyopathy is multifactorial and involves genetic, environmental, and lifestyle factors. Identifying and characterizing novel genes that contribute to cardiac pathophysiology are crucial for understanding cardiomyopathy and effective therapies. In this study, we investigated the role of a novel gene, Obg-like ATPase 1 ( Ola1 ), in cardiac pathophysiology using a cardiac-specific knockout mouse model as well as a Drosophila model. Our previous work demonstrated that OLA1 modulates the hypertrophic response of cardiomyocytes through the GSK-beta/beta-catenin signaling pathway. Furthermore, recent studies have suggested that OLA1 plays a critical role in organismal growth and development. For example, Ola1 null mice exhibit increased heart size and growth retardation. It is not known, however, if loss of function for Ola1 leads to dilated cardiomyopathy. We generated cardiac-specific Ola1 knockout mice (OLA1-cKO) to evaluate the role of OLA1 in cardiac pathophysiology. We found that Ola1 -cKO in mice leads to dilated cardiomyopathy (DCM) and left ventricular (LV) dysfunction. These mice developed severe LV dilatation, thinning of the LV wall, reduced LV function, and, in some cases, ventricular wall rupture and death. In Drosophila, RNAi-mediated knock-down specifically in developing heart cells led to the change in the structure of pericardial cells from round to elongated, and abnormal heart function. This also caused significant growth reduction and pupal lethality. Thus, our findings suggest that OLA1 is critical for cardiac homeostasis and that its deficiency leads to dilated cardiomyopathy and dysfunction. Furthermore, our study highlights the potential of the Ola1 gene as a therapeutic target for dilated cardiomyopathy and heart failure.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps on chromosome 2, at the locus 1q31, close to the Titin (TTN) gene, which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in human failing heart tissue (HF) as compared to in non-failing heart tissues (NF). Moreover, using the Sanger sequencing method, we characterized the human OLA1 gene and screened genetic mutations in patients with heart-failing and non-failing. Among failing and non-failing heart patients, we found a total of 15 mutations, including two transversions, one substitution, one indel, and eleven transition mutations in the OLA1 gene. All the mutations were intronic except for a non-synonymous mutation, 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen for the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results show that this test can be used as a genetic screening tool for human cardiomyopathy. These findings have important implications for the diagnosis and treatment of cardiomyopathy.