The Cardeza Center pursues a broad range of basic research that can be divided into three focus areas: basic research, clinical/translational and core laboratories. We also host a seminar series during the academic year.
Leonard Edelstein, PhD
Dr. Edelstein’s laboratory studies gene expression in megakaryocytes and platelets and their role in cardiovascular disease and thrombopoiesis. Current research includes:
Activated platelets release microparticles (MPs) which contain RNA and protein. These MPs have been shown to deliver miRNAs to endothelial cells in culture and alter the level of RNAs in these cells. We are studying this effect in a flow chamber in which the endothelial cells are cultured under shear stress and then analyzing the effect on genes regulated by inflammation or shear stress, two processes important to atherosclerotic plaque formation.
Genome wide association studies have identified ~50 loci in the human genome associated with heart attacks. However, neither the genetic variant responsible for the increased risk nor the cell type in which it functions has been identified. We are developing methods to identify which locus-linked genes are functional in platelets and the effect the common variants have on their expression and function.
Arterial hypertension is associated with thrombotic events due to platelet activation. The exact mechanism by which hypertensive platelets become more sensitive to agonist stimulation is unknown. We have collected platelet miRNA from healthy and pre-hypertensive subjects and found association between miRNA level and blood pressure. We will use this data to identify differentially expressed genes responsible for the increased function in platelets.
Leonard Edelstein, PhD
Research Assistant Professor
Xiango Kong, MD
Namrata Madan, PhD
Arie Horowitz, DSc
My laboratory conducts basic research in vascular biology. Our objective is to understand how blood vessels regulate the permeability of their walls. Specifically, we study how the junctions between adjacent endothelial cells (ECs) on the lumen of vessels are maintained, and how they respond to external stimuli, such as vascular endothelial growth factor, angiopoietin, or thrombin. We pursue these questions by probing intracellular signaling pathways and protein complexes that determine the behavior of the junctions. We use cell culture and genetically modified mouse models in combination with advanced optical imaging techniques.
In addition to membership in the Cardeza Center for Vascular Research, I am an adjunct faculty member in Cancer Biology, and a member of the Genetics, Genomics and Cancer Biology graduate program where I taught a course in Current Literature.
Figure 1: Scheme of Rab13-mediated trafficking of RhoA.
Regulation of cell junction dynamics by membrane traffic
We found that the GTPase Rab13, which recycles tight junction proteins, facilitates the translocation of RhoA and its guanine exchange factor PLEKHG5/Syx from cell junctions to the cell leading edge (Wu et al., 2011; Figure 1). This implicates Rab13 in cell migration, a previously unknown function of this protein. We are investigating the in vivo function of Rab13 using a new mouse model harboring an EC-specific deletion of Rab13. We previously found that global deletion of Rab13 is embryonic lethal. Our recent data indicates that deletion of Rab13 upon vascular endothelial cadherin (Cdh5) expression in the mouse embryo is not embryonic lethal. We have recently started phenotyping the rab13fl/fl Cdh5 mouse.
Figure 2: ECs grown on microcarrier beads and stained by a cytoplasmic (green) or nuclear (red) dyes.
High-throughput identification of genes involved in mediating the effects of junction disruptors and stabilizers on endothelial cell junctions.
We are leveraging CRISPR-dCas9 gRNA inhibitory and activating libraries in order to either silence or activate genes that code for proteins that are components of cell junction maintenance pathways. Our current focus is on thrombin, but we will pursue additional cell-junction modifying pathways, e.g. VEGF and angiopoietin. In order to generate sufficient coverage of the gRNA libraries, which code for all the annotated human genes, we miniaturized the permeability assay to 100-200 mm microcarrier beads. We use a fluorescently-conjugated probe that binds to the bead’s 1. surface which is exposed when the thrombin causes disassembly of the junctions between ECs (Figure 2). The resulting fluorescence signal facilitates sorting the beads to separate those where the EC junction response to thrombin is inhibited. In collaboration with Drs. Leonard Edelstein from the Cardeza Center and Eric Londin from the Computational Medicine Center of Thomas Jefferson University, we will identify genes that are required for mediating the effect of thrombin on cell junctions.
Figure 3: Micro-computed tomography images of the arterial coronary systems in mice of the indicated genotypes.
The role of membrane trafficking in EC junction homeostasis.
Protein density in the cytoplasm next to EC junctions is one of the highest in the cell. These proteins, mostly organized in transient complexes, are constantly recycled in resting ECs, and translocate from and to the junctions when challenged by junction disruptors such as thrombin. Numerous junction transmembrane and cytoplasmic proteins are scaffolded by MPDZ, a large protein consisting of numerous protein-binding modules. We posit that MPDZ is the core of a large protein complex that changes its composition upon EC response to physiological cell disruptors. Our objective is to determine where this complex translocates to and how its composition changes during this process. We identified new binding partners of MPDZ that facilitate its membrane trafficking. In parallel, we are phenotyping a new Mpdz loss-of-function mouse model, to test the in vivo function of MPDZ. We found that the coronary (Fig. 3) and retina (Fig. 4) vessels are deformed in Mpdz-/- mice, and that EC junctions in the vessels of the retina are disorganized in comparison to wild type mice. Newborn Mpdz-/- mice on a C57BL/6J genetic background suffer from severe hydrocephalus and die prematurely. We are currently analyzing the mechanism responsible for this effect in collaboration with Dr. Richard Smeyne from Neuroscience. Brain tumors are often accompanied by intracranial edema, a condition that can be life threatening in itself. In collaboration with Dr. Craig Hooper from Cancer Biology, we are comparing the vasculature in brains and glioma tumors of wild type versus Mpdz-/-mice.
Erratum: Binding of internalized receptors to the PDZ domain of GIPC/synectin recruits myosin VI to endocytic vesicles (Proceedings of the National Academy of Sciences of the United States of America (August 22, 2006) 103, 34 (12735-12740) DOI: 10.1073/pnas.0605317103)
Arie Horowitz, DSc
Junning Yang, PhD
Claire Simonneau, PhD
Peisong Ma, PhD
Dr. Ma’s laboratory is involved with investigations in the areas of thrombosis and hemostasis, with a special emphasis on understanding GPCR (G-protein coupled receptor) and G-protein mediated platelet activation. Our current studies provide novel insights into the regulatory mechanisms that allow platelets to produce an optimal response to vascular injury. Using well-established vascular injury models, CRISRP-Cas9 genome-editing and biochemical approaches, we have provided strong evidence that defects in GPCR and G-protein signaling pathways translate into in vivo phenotypes. The following is a brief summary of major ongoing projects in the lab.
RGS-insensitive Gq (G188S) as probes of G protein Functions
We recently develop a mutant mouse line with a mutation (G188S) in Gq subunit that renders the G protein resistant to interaction with RGS (regulator of G protein signaling) proteins as a class. In contrast to enhanced Gi2 signaling in Gi2(G184S) mutant platelets, we have observed decreased platelet activation in Gq(G188S) mutant mice, suggesting that the negative feedback of Gq regulation is different from that of Gi2. An ongoing study is to fully characterize the effect of G188S mutation on platelet function and thrombus formation.
The signaling machinery that provides negative feedback regulation to G protein-dependent signaling during platelet activation
Using CRISPR-Cas9 genome editing, we have recently determined that multiple components of the platelet-signaling network are integrated to mediate GPCRs and G protein-dependent pathways. Ongoing studies are to characterize the mechanisms by which these molecules impact platelet functions, thrombus formation both in vitro and in vivo. To accomplish these goals, we make use of several recently generated mutant mouse lines, intravital microscopy approach and other biochemical techniques.
The regulatory networks that regulate platelet activation downstream of G protein signaling using Genome-wide screening
We established that αIIbβ3 activation as readout for genome-wide pooled CRISPR-Cas9 screen in primary megakaryocytes. We will identify novel positive regulators and negative regulators that control integrin activation in response to GPCR-coupled agonists.
Steven McKenzie, MD, PhD
Dr. McKenzie is a Hematology physician-scientist with clinical expertise in adult and pediatric non-malignant hematologic disorders and a scientific expertise in translational research. He works in the Cardeza Hemophilia and Thrombosis Center and also in the Hereditary Anemias Center. He is a member of the Sidney Kimmel Cancer Center (SKCC), in the Molecular Biology and Genetics Program. He is a member of two Graduate Programs:
Genetics, Genomics and Cancer Biology
Immunology and Microbial Pathogenesis
He is a physician mentor for Thomas Jefferson University MD/PhD program.
Dr. McKenzie directs two major laboratory research projects. The first project has a focus on immune-mediated thrombocytopenia and thrombosis syndromes (see McKenzie and Sachais, Current Opinion in Hematology, September 2014). This work led to the first and only mouse model of heparin-induced thrombocytopenia and thrombosis (HIT). The current work explores Novel Therapeutics in HIT, in an NIH-supported Program Project grant with Drs. Poncz and Rauova at Children’s Hospital of Philadelphia, Drs. Cines, Sachais and Cuker at University of Pennsylvania, and Dr. Arepally at Duke. In another subproject, Dr. McKenzie is co-funded on an NIH R01 with Dr. Bergmeier at UNC Chapel Hill and Dr. Woulfe at University of Delaware to explore platelet signaling mechanisms.
The second major McKenzie lab project focuses on the Genomics and Molecular Genetics of inter-individual variation in human platelet activation via FcgammaRIIa. This molecule has dual functions, as a receptor for IgG immune complexes and as a transmembrane adapter in integrin “outside-in” signaling. In collaboration with Dr. Paul Bray, who led the PRAX1 study, our team has identified differentially expressed mRNAs and miRNAs as well as genomic variants that regulate platelet reactivity. Team members of our longstanding Platelet Interest Group are Drs. Bray, McKenzie, Holinstat, Edelstein and Naik at Thomas Jefferson University, Drs. Rigoutsos and Londin at Thomas Jefferson University Computational Medicine Center, Dr. Fortina of SKCC Genomics, Drs. Shaw and Simon at Baylor, and Dr. Kunapuli at Temple. Dr. McKenzie is also co-funded, with Dr. Holinstat as PI, on work that involves 12-LOX and diabetes vascular biology. Our foci in the McKenzie lab moving forward are novel molecular genetic pathways for determination of receptor levels, protein tyrosine phosphatase activity and oligo-ubiquitylation in platelet FcgammaRIIa functions.
Erratum: TULA-2 protein phosphatase suppresses activation of Syk through the GPVI platelet receptor for collagen by dephosphorylating Tyr(P)346, a regulatory site of Syk (The Journal of Biological Chemistry (2016) 291 (22427-22441) DOI: 10.1074/jbc.M116.743732)
Identification of a developmental gene expression signature, including hox genes, for the normal human colonic crypt stem cell niche: Overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis
Shaji Abraham, PhD
Ulhas P. Naik, PhD
The Naik laboratory is focused on developing therapeutic strategies to interrupt the progress of cardiovascular diseases and cancer, which are the leading causes of death in the western world. In this regard, the team has identified several novel gene products that play key regulatory roles in the progression of these diseases, with an emphasis on how these genes affect platelet functions (since platelet activity often potentiates these diseases). Using cell and molecular biological approaches, the team has characterized the potential role of calcium- and integrin-binding (CIB) protein family and junctional adhesion molecule (JAM) family members in physiological and pathological settings. Cutting edge technologies, such as the yeast two-hybrid system, siRNA, transgenic mouse models, CRISPR/Cas9, and in vivo disease models are routinely employed in the laboratory. Dr. Naik is also the Director of the Cardeza Center for Vascular Biology, Director of Integrative Physiology Graduate Program, and a member of the following Graduate Programs:
Genetics, Genomics and Cancer Biology
Cell Biology and Regenerative Medicine
Biochemistry and Molecular Pharmacology
Our three major ongoing projects are:
Positive and negative regulatory mechanisms of platelet activation during thrombosis.
Our team has identified numerous novel regulators of platelet activity genetic ablation of which has shown protection from thrombosis (e.g. Cib1 [Naik MU, et al., 2009] and Ask1 [Naik MU, et al., 2017]) or amplification of platelet function (e.g. JAM-A [Naik MU, et al., 2012]). For example, the adjacent image from Naik MU, et al., 2017 shows how in a model of pulmonary thromboembolism, whereby clots are induced to form in the tail vein and subsequently lodge in the lungs leading to breathing cessation, Ask1 knockout mice have a much higher survival rate than their wildtype counterparts; hence deletion of Ask1 protects from thrombosis. Currently, several other genes products are being investigated through exciting in vitro, ex vivo, and in vivo methods, including carotid artery injury, cremaster injury, stroke, and Deep Vein Thrombosis models.
Regulation of new blood vessel formation (angiogenesis) by Junctional Adhesion Molecule A (JAM-A).
We have cloned and characterized a novel junctional adhesion molecule, JAM-A (Naik, U.P., et al., Biochem. J. 1995; Naik, U.P., et al., J. Cell Sci, 2001; Naik, U.P. and Eckfeld, K. J. Biol. Regul. Homeost. Agents, 2003). JAM-A is expressed in endothelial and epithelial cells and resides at the tight junctions. We were the first to demonstrate that JAM-A regulates bFGF-induced angiogenesis through its interaction with integrin alphavbeta3 (Naik, M., et. al., Blood, 2003; Naik, M. and Naik, U.P., J. Cell Sci. 2006). Using siRNA and Jam-A knockout mouse, we have shown that JAM-A is essential for bFGF-induced angiogenesis (Naik, M., et al., Arterioscler Thromb. Vasc. Biol, 2003,; Cooke, et al., Arterioscler Thromb. Vasc. Biol, 2006). We extended this work further to demonstrate that JAM-A suppresses VEGF/VEGFR2 expression on endothelial cells, thus regulating vascular permeability and angiogenesis.
Mechanism of breast and prostate cancer cell metastasis.
Recently, we have found that JAM-A expression is inversely related to the metastatic ability of breast cancer cells (Naik, M., et al., Cancer Res. 2008). Overexpression of JAM-A in highly metastatic cells reduced their invasiveness; conversely, the knock-down of JAM-A in low metastatic cells increased their invasiveness. Studies are now ongoing to elucidate the molecular mechanism of this regulation. Furthermore, in collaboration with Justin Lathia of Cleveland Clinic, it has been shown that JAM-A regulates cancer stem cell function (Lathia, et al., Cell Rep, 2014).
Fraunhofer-UD Research Grant
Kalyan Golla, PhD
Meghna U. Naik, MSc
Pravin Patel, BS