Phani K. Patibandla

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

Fibrinogen Induces Alterations of Endothelial Cell Tight Junction Proteins

We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin‐associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell‐substrate impedance system, 8‐well chambered, or in 12‐well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen‐activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti‐ICAM‐1 antibody or BQ788 (endothelin type B receptor blocker), endothelin‐1, endothelin‐1 with BQ788, or medium alone for 24 h. Fg induced a dose‐dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT‐PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden‐1 (ZO‐1), and zona occluden‐2 (ZO‐2) in ECs. Fg‐induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti‐ICAM‐1 antibody. While BQ788 inhibited endothelin‐1‐induced decrease in TEER, it did not affect Fg‐induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO‐1, and ZO‐2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM‐1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. J. Cell. Physiol. 221: 195–203, 2009.

Fibrinogen-induced endothelin-1 production from endothelial cells

We previously demonstrated that fibrinogen (Fg) binding to the vascular endothelial intercellular adhesion molecule-1 (ICAM-1) leads to microvascular constriction in vivo and in vitro. Although a role of endothelin-1 (ET-1) in this Fg-induced vasoconstriction was suggested, the mechanism of action was not clear. In the current study, we tested the hypothesis that Fg-induced vasoconstriction results from ET-1 production by vascular endothelial cells (EC) and is mediated by activation of extracellular signal-regulated kinase -1/2 (ERK-1/2). Confluent, rat heart microvascular endothelial cells (RHMECs) were treated with one of the following: Fg (2 or 4 mg/ml), Fg (4 mg/ml) with ERK-1/2 kinase inhibitors (PD-98059 or U-0126), Fg (4 mg/ml) with an antibody against ICAM-1, or medium alone for 45 min. The amount of ET-1 formed and the concentration of released von Willebrand factor (vWF) in the cell culture medium were measured by ELISAs. Fg-induced exocytosis of Weibel-Palade bodies (WPBs) was assessed by immunocytochemistry. Phosphorylation of ERK-1/2 was detected by Western blot analysis. Fg caused a dose-dependent increase in ET-1 formation and release of vWF from the RHMECs. This Fg-induced increase in ET-1 production was inhibited by specific ERK-1/2 kinase inhibitors and by anti-ICAM-1 antibody. Immunocytochemical staining showed that an increase in Fg concentration enhanced exocytosis of WPBs in ECs. A specific endothelin type B receptor blocker, BQ-788, attenuated the enhanced phosphorylation of ERK-1/2 in ECs caused by increased Fg content in the culture medium. The presence of an endothelin converting enzyme inhibitor, SM-19712, slightly decreased Fg-induced phosphorylation of ERK-1/2, but inhibited production of Fg-induced ET-1 production. These results suggest that Fg-induced vasoconstriction may be mediated, in part, by activation of ERK-1/2 signaling and increased production of ET-1 that further increases EC ERK-1/2 signaling. Thus, an increased content of Fg may enhance vasoconstriction through increased production of ET-1.

Enhancing complement control on endothelial barrier reduces renal post-ischemia dysfunction.

BACKGROUND Excessive complement activation is an integral part of ischemia and reperfusion (IR) injury (IRI) of organs. In kidney transplantation, the pathologic consequence of IRI and complement activation can lead to delayed graft function, which in turn is associated with acute rejection. Previous strategies to reduce complement-induced IRI required systemic administration of agents, which can lead to increased susceptibility to infections/immune diseases. The objective of this study was to determine whether an increase in complement control defenses of rat kidney endothelium reduces IRI. We hypothesized that increased complement control on the endothelial barrier reduces IR-mediated complement activation and reduces kidney dysfunction. MATERIALS AND METHODS Fischer 344 rats underwent left kidney ischemia for 45 min and treatment with a novel fusogenic lipid vesicle (FLVs) delivery system to decorate endothelial cells with vaccinia virus complement control protein (VCP), followed by reperfusion for 24 h. Assessment included renal function by serum creatinine and urea, myeloperoxidase assay for neutrophil infiltration, histopathology, and quantification of C3 production in kidneys. RESULTS Animals in which the kidney endothelium was bolstered by FLVs+VCP treatment had better renal function with a significant reduction in serum creatinine compared with vehicle controls (P < 0.05). Also, C3 production was significantly reduced (P < 0.05) in treated animals compared with vehicle controls. CONCLUSION Increasing complement control at the endothelial barrier with FLVs+VCP modulates complement activation/production during the first 24 h, reducing renal dysfunction following IRI.

Evaluation of the effect of diminished pulsatility as seen in continuous flow ventricular assist devices on arterial endothelial cell phenotype and function.

Evaluation of the effect of diminished pulsatility as seen in continuous flow ventricular assist devices on arterial endothelial cell phenotype and function Phani K. Patibandla, PhD, Namakkal S. Rajasekaran, PhD, Sandeep B. Shelar, PhD, Guruprasad A. Giridharan, PhD, Silvio H. Litovsky, MD, and Palaniappan Sethu, PhD From the Division of Cardiovascular Disease, Department of Medicine and Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama; Department of Bioengineering, Speed School of Engineering, University of Louisville, Louisville, Kentucky; Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama; Division of Cardiovascular Medicine, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah; and the Department of Biomedical Engineering, School of Engineering, University of Alabama at Birmingham, Birmingham, Alabama

Transcriptional Profile of Immediate Response to Ionizing Radiation Exposure

Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose–course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBIs Gene Expression Omnibus (GEO), accession GSE64375.

Elucidation of dose-dependent transcriptional events immediately following ionizing radiation exposure

Long duration space missions expose astronauts to ionizing radiation events associated with highly energetic and charged heavy particles. Such exposure can result in chromosomal aberrations increasing the likelihood of the development of cancer. Early detection and mitigation of these events is critical in providing positive outcomes. In order to aid in the development of portable devices used to measure radiation exposure, we constructed a genome-wide screen to detect transcriptional changes in peripheral blood lymphocytes shortly after (approximately 1 hour) radiation exposure at low (0.3 Gy), medium (1.5 Gy) and high (3.0 Gy) doses compared to control (0.0 Gy) using Affymetrix® Human Gene 1.0 ST v1 microarrays. Our results indicate a number of sensitive and specific transcriptional profiles induced by radiation exposure that can potentially be implemented as biomarkers for radiation exposure as well as dose effect. For overall immediate radiation exposure, KDELC1, MRPS30, RARS, and HEXIM1 were determined to be effective biomarkers while PRDM9, CHST4, and SLC26A10 were determined to be biomarkers specific to 0.3 Gy exposure; RPH, CCDC96, WDYHV1, and IFNA16 were identified for 1.5 Gy exposure; and CWC15, CHCHD7, and DNAAF2 were determined to be sensitive and specific to 3.0 Gy exposure. The resulting raw and analyzed data are publicly available through NCBIs Gene Expression Ominibus via accession GSE64375.

A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (?45?min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8?min following which the mixture is introduced into an inertial washing module to obtain ?analysis-ready? samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ?holding time? to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the conventional procedure (45?min) and our microfluidic approach (12?min).