Sheeja Rajasingh

MicroRNA: A new therapeutic strategy for cardiovascular diseases.

Myocardial infarction, atherosclerosis, and hypertension are the most common heart-related diseases that affect both the heart and the blood vessels. Multiple independent risk factors have been shown to be responsible for cardiovascular diseases. The combination of a healthy diet, exercise, and smoking cessation keeps these risk factors in check and helps maintain homeostasis. The dynamic monolayer endothelial cell integrity and cell-cell communication are the fundamental mechanisms in maintaining homeostasis. Recently, it has been revealed that small noncoding RNAs (ncRNAs) play a critical role in regulation of genes involved in either posttranscriptional or pretranslational modifications. They also control diverse biological functions like development, differentiation, growth, and metabolism. Among ncRNAs, the short interfering RNAs (siRNAs), and microRNAs (miRNAs) have been extensively studied, but their specific functions remain largely unknown. In recent years, miRNAs are efficiently studied as one of the important candidates for involvement in most biological processes and have been implicated in many human diseases. Thus, the identification and the respective targets of miRNAs may provide novel molecular insight and new therapeutic strategies to treat diseases. This review summarizes the recent developments and insight on the role of miRNAs in cardiovascular disease prognosis, diagnostic and clinical applications.

Epigenetic modifiers reduce inflammation and modulate macrophage phenotype during endotoxemia-induced acute lung injury

ABSTRACT Acute lung injury (ALI) during sepsis is characterized by bilateral alveolar infiltrates, lung edema and respiratory failure. Here, we examined the efficacy the DNA methyl transferase (DNMT) inhibitor 5-Aza 2-deoxycytidine (Aza), the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), as well as the combination therapy of Aza and TSA (Aza+TSA) provides in the protection of ALI. In LPS-induced mouse ALI, post-treatment with a single dose of Aza+TSA showed substantial attenuation of adverse lung histopathological changes and inflammation. Importantly, these protective effects were due to substantial macrophage phenotypic changes observed in LPS-stimulated macrophages treated with Aza+TSA as compared with untreated LPS-induced macrophages or LPS-stimulated macrophages treated with either drug alone. Further, we observed significantly lower levels of pro-inflammatory molecules and higher levels of anti-inflammatory molecules in LPS-induced macrophages treated with Aza+TSA than in LPS-induced macrophages treated with either drug alone. The protection was ascribed to dual effects by an inhibition of MAPK–HuR–TNF and activation of STAT3–Bcl2 pathways. Combinatorial treatment with Aza+TSA reduces inflammation and promotes an anti-inflammatory M2 macrophage phenotype in ALI, and has a therapeutic potential for patients with sepsis. Summary: Combinatorial treatment with Aza+TSA reduces inflammation and promotes an anti-inflammatory M2 macrophage phenotype in ALI, and has a therapeutic potential for patients with sepsis and ALI.

Macrophage polarization in response to epigenetic modifiers during infection and inflammation.

Macrophages are a heterogeneous population of phagocytic cells present in all tissues. Recently, several drugs that target the epigenetic machinery have emerged as attractive molecules for treating infection and inflammation by modulating macrophages. Treatment of lipopolysaccharide (LPS)-challenged macrophages with epigenetic modifiers leads to phenotype switching. This could provide stimulatory/destructive (M1) or suppressive/protective (M2) therapeutic strategies, which are crucial in the cytokine milieu in which the macrophages reside. In this review, we provide an overview of macrophage functional diversity during various diseases, including infection, as well as the current status in the development and clinical utility of epigenetic modifiers.

Combinatorial Therapy with Acetylation and Methylation Modifiers Attenuates Lung Vascular Hyperpermeability in Endotoxemia-Induced Mouse Inflammatory Lung Injury

Impairment of tissue fluid homeostasis and migration of inflammatory cells across the vascular endothelial barrier are crucial factors in the pathogenesis of acute lung injury (ALI). The goal for treatment of ALI is to target pathways that lead to profound dysregulation of the lung endothelial barrier. Although studies have shown that chemical epigenetic modifiers can limit lung inflammation in experimental ALI models, studies to date have not examined efficacy of a combination of DNA methyl transferase inhibitor 5-Aza 2-deoxycytidine and histone deacetylase inhibitor trichostatin A (herein referred to as Aza+TSA) after endotoxemia-induced mouse lung injury. We tested the hypothesis that treatment with Aza+TSA after lipopolysaccharide induction of ALI through epigenetic modification of lung endothelial cells prevents inflammatory lung injury. Combinatorial treatment with Aza+TSA mitigated the increased endothelial permeability response after lipopolysaccharide challenge. In addition, we observed reduced lung inflammation and lung injury. Aza+TSA also significantly reduced mortality in the ALI model. The protection was ascribed to inhibition of the eNOS-Cav1-MLC2 signaling pathway and enhanced acetylation of histone markers on the vascular endothelial-cadherin promoter. In summary, these data show for the first time the efficacy of combinatorial Aza+TSA therapy in preventing ALI in lipopolysaccharide-induced endotoxemia and raise the possibility of an essential role of DNA methyl transferase and histone deacetylase in the mechanism of ALI.

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility.

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.

Exosomes: new molecular targets of diseases

Extracellular vesicles (EVs) comprise apoptotic bodies, microvesicles and exosomes, and they perform as key regulators in cell-to-cell communication in normal as well as diseased states. EVs contain natural cargo molecules, such as miRNA, mRNA and proteins, and transfer these functional cargos to neighboring cells or more distant cells through circulation. These functionally active molecules then affect distinct signaling cascades. The message conveyed to the recipient cells is dependent upon the composition of the EV, which is determined by the parent cell and the EV biogenesis. Because of their properties such as increased stability in circulation, biocompatibility, low immunogenicity and toxicity, EVs have drawn attention as attractive delivery systems for therapeutics. This review focuses on the functional use of exosomes in therapy and the potential advantages and challenges in using exosomes for therapeutic purposes.

Optical-flow based non-invasive analysis of cardiomyocyte contractility

Characterization of cardiomyocyte beat patterns is needed for quality control of cells intended for surgical injection as well as to establish phenotypes in disease modeling or toxicity studies. Optical-flow based analysis of videomicroscopic recordings offer a manipulation-free and efficient characterization of contractile cycles, an important characteristics of cardiomyocyte phenotype. We demonstrate that by appropriate computational analysis of optical flow data one can identify distinct contractile centers and distinguish active cell contractility from passive elastic tissue deformations. Our proposed convergence measure correlates with myosin IIa immuno-localization and is capable to resolve contractile waves and their synchronization within maturing, unlabeled induced pluripotent stem cell-derived cardiomyocyte cultures.

Exosomes: Outlook for Future Cell-Free Cardiovascular Disease Therapy

Cardiovascular diseases are the number one cause of death globally with an estimated 7.4 million people dying from coronary heart disease. Studies have been conducted to identify the therapeutic utility of exosomes in many diseases, including cardiovascular diseases. It has been demonstrated that exosomes are immune modulators, can be used to treat cardiac ischemic injury, pulmonary hypertension and many other diseases, including cancers. Exosomes can be used as a biomarker for disease and cell-free drug delivery system for targeting the cells. Many studies suggest that exosomes can be used as a cell-free vaccine for many diseases. In this chapter, we explore the possibility of future therapeutic potential of exosomes in various cardiovascular diseases.

Biology and clinical relevance of noncoding sno/scaRNAs

Small nucleolar RNAs (snoRNAs) are a group of noncoding RNAs that perform various biological functions, including biochemical modifications of other RNAs, precursors of miRNA, splicing, and telomerase activity. The small Cajal body-associated RNAs (scaRNAs) are a subset of the snoRNA family and collect in the Cajal body where they perform their canonical function to biochemically modify spliceosomal RNAs prior to maturation. Failure of sno/scaRNAs have been implicated in pathology such as congenital heart anomalies, neuromuscular disorders, and various malignancies. Thus, understanding of sno/scaRNAs demonstrates the clinical value.

DNMT and HDAC inhibitors together abrogate endotoxemia mediated macrophage death by STAT3-JMJD3 signaling

Acute lung injury (ALI) is a common complication of sepsis that often leads to fatal lung disease without effective therapies. It is known that bone marrow derived macrophages are important in resolving the inflammation and maintaining tissue homeostasis. Here, we hypothesize that treatment in combination of DNA methyl transferase inhibitor (DNMTi) 5-Aza 2-deoxycytidine (Aza) and histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) mitigates the inflammation induced pyroptosis and apoptosis during endotoxemia induced ALI. To test this hypothesis, the mice challenged with a sublethal dose of LPS followed by one-hour post-treatment with a single dose of Aza and TSA intraperitoneally showed a substantial attenuation of apoptosis and inflammation. Importantly, we observed significant changes in the mitochondrial membrane structure, and lower levels of DNA fragmentation, reduced expression of apoptotic and pyroptotic genes both transcriptionally and translationally in LPS induced BMDMs treated by a combination of Aza and TSA than in LPS-induced BMDMs treated with either drug alone. The protection was mediated by an inhibition of JNK-ERK and STAT3-JMJD3 activated pathways. Thus, targeting these important signaling pathways with the combination of Aza and TSA would be a good treatment modality for ALI.