Amaresh K. Ranjan

Multigenerational Undernutrition Increases Susceptibility to Obesity and Diabetes that Is Not Reversed after Dietary Recuperation

People in developing countries have faced multigenerational undernutrition and are currently undergoing major lifestyle changes, contributing to an epidemic of metabolic diseases, though the underlying mechanisms remain unclear. Using a Wistar rat model of undernutrition over 50 generations, we show that Undernourished rats exhibit low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age-/gender-matched control rats. Undernourished rats also have higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an 8-fold increased susceptibility to Streptozotocin-induced diabetes compared to control rats. Importantly, these metabolic abnormalities are not reversed after two generations of unrestricted access to commercial chow (nutrient recuperation). Altered epigenetic signatures in insulin-2 gene promoter region of Undernourished rats are not reversed by nutrient recuperation, and may contribute to the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations.

Cyclin A2 Induces Cardiac Regeneration After Myocardial Infarction Through Cytokinesis of Adult Cardiomyocytes

Cyclin A2 mediates cardiac regeneration of infarcted porcine hearts. A Change of Heart After Myocardial Infarction When blood flow is blocked off to the heart, the heart suffers permanent damage in part because cardiomyocytes are terminally differentiated and cannot proliferate. But what if these cells could be stimulated to divide? Some animals—like newts—have the ability to regenerate body parts when they are injured. Others—like zebrafish—can even regenerate heart tissue. Now, Shapiro et al. report that gene therapy can elicit a regenerative response in pig hearts. Cyclin A2 (Ccna2) has been shown to induce cardiac repair in small-animal models after myocardial infarction (MI). The authors have extended these studies by looking in the more translationally relevant pig model of MI. They found that Ccna2 delivered by an adenovirus improved heart function when compared with an adenoviral control. Cardiomyocytes in the pigs showed evidence of increased proliferation. If these data hold true in human studies, patients with MI can take heart. Cyclin A2 (Ccna2), normally silenced after birth in the mammalian heart, can induce cardiac repair in small-animal models of myocardial infarction. We report that delivery of the Ccna2 gene to infarcted porcine hearts invokes a regenerative response. We used a catheter-based approach to occlude the left anterior descending artery in swine, which resulted in substantial myocardial infarction. A week later, we performed left lateral thoracotomy and injected adenovirus carrying complementary DNA encoding CCNA2 or null adenovirus into peri-infarct myocardium. Six weeks after treatment, we assessed cardiac contractile function using multimodality imaging including magnetic resonance imaging, which demonstrated ~18% increase in ejection fraction of Ccna2-treated pigs and ~4% decrease in control pigs. Histologic studies demonstrate in vivo evidence of increased cardiomyocyte mitoses, increased cardiomyocyte number, and decreased fibrosis in the experimental pigs. Using time-lapse microscopic imaging of cultured adult porcine cardiomyocytes, we also show that Ccna2 elicits cytokinesis of adult porcine cardiomyocytes with preservation of sarcomeric structure. These data provide a compelling framework for the design and development of cardiac regenerative therapies based on cardiomyocyte cell cycle regulation.

Endothelial cells in pancreatic islet development and function

Endothelial cells represent one of the most abundant and widely found cell types in the mammalian embryo. These cells arise in close proximity with and often as an integral part of several organs such as the kidneys, lungs, liver and pancreas. In most of these organs, they play an instructive role to determine the fate of progenitor cells in the developing embryo. Studies carried out until now by Eckhard Lammert, Douglas Melton, Ken Zaret and colleagues have convincingly demonstrated the importance of endothelial cells in normal development of the pancreas. This article reviews the literature in development of endothelial and endodermal cells. Understanding these endothelium-derived signaling mechanisms that allow differentiation of endodermal cells to endocrine pancreatic lineage will help us develop strategies for making insulin-producing cells in vitro.

A Protocol for Measurement of Noncoding RNA in Human Serum

MicroRNAs (miRNAs) are small noncoding RNAs that act as regulators of gene expression by targeting mature messenger RNAs. Following the initial report of the presence of miRNAs in serum and plasma a number of studies have successfully demonstrated the use of these miRNAs as biomarkers of disease. Currently, there are many methods of isolating total RNA from liquid samples. Here, we describe a simple, cost effective method for extraction of RNA from human serum as well as subsequent real time PCR analysis of miRNA levels.

Simultaneous imaging of microRNA or mRNA territories with protein territory in mammalian cells at single cell resolution.

Cellular mechanisms that inhibit mRNA translation by regulatory molecules involving microRNAs (miRNAs), a class of noncoding RNAs (ncRNAs), are well recognized in recent days. However, methodologies that measure these changes in cell populations lack the capabilities to observe such effects at single cell resolution. This is mostly due to the low level of transcript abundance and the heterogeneity of cell populations, together with the inability to measure transcripts and proteins at the same time. Here, we combine an in situ TaqMan PCR method with immunostaining so as to amplify low abundance transcripts in cellular compartments and image these efficiently at single cell resolution. The method offers flexibility to end-users for further fine-tuning of this optimized protocol based on the number of PCR cycles for individual genes in any cell type. After immunostaining, confocal microscopy is performed to detect the fluorescence of TaqMan probes (representing amplified transcripts/miRNA) and fluorophores tagged to antibodies (representing proteins) simultaneously. The presented technique offers an important tool to understand functional genomics as well as molecular mechanism of transcriptional and translational regulation so as to map these at single cell resolution.

Cellular detection of multiple antigens at single cell resolution using antibodies generated from the same species

Biology at a cellular level comes with a great amount of heterogeneity. It is now evident that even clonally propagated cells in an in vitro population do not express the same set of cellular epitopes. The vascular endothelial as well as blood cells show a very high degree of heterogeneity in expression of specific proteins. Although several methods exist for identification of genome or transcriptome from a single cell, there is still limited advancement in detection of multiple cellular antigens in a single cell. This has been mainly due to the limited availability of different antibodies. Single-cell detection methods involving the use of multiple monoclonal antibodies generated in the same species would therefore provide with an important tool for cellular detection of antigens. Here, we describe a method to assess multiple proteins in a cell using different antibodies generated in the same species.

Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.