Edema; Permeability; NF-kappa B; Sepsis; Tight Junctions; Adherens Junctions; Tumor Necrosis Factor Decoy Receptors; Microvessels
Endothelial cells form an inner lining to human microvessels that serve as a systemic organ for regulating the access of fluid, macromolecules and cells from circulating blood into all vascularized tissues. We focus on how to maintain stable endothelial cell-cell junctions during activation by inflammatory mediators (cytokines). This is important to systemic sepsis, atherosclerosis and ischemia reperfusion injury. We are also interested in how endothelial cells and pericytes form new microvessels (angiogenesis). This process is relevant to wound healing, to tumor (lymph)angiogenesis and to the engineering of vascularized synthetic tissues.
Extensive Research DescriptionWe are defining how the inflammatory cytokine tumor necrosis factor (TNF) destabilizes blood vessel barrier integrity by increasing their paracellular permeability to fluid and (macro)molecules in circulating blood. We study how transmembrane adhesion molecules found at cell-cell junctions (such as claudin-5, JAM-A, occludin and VE-cadherin) regulate paracellular permeability by knocking down and overexpressing these genes in cultured primary human endothelial cells (EC). A second research goal with applications to biomedical engineering of replacement organs is in defining how nascent microvessels become stabilized when EC interact with accessory cells (pericytes) during angiogenesis. In this work, we explore the molecular mechanisms of microvascular tube formation by EC and pericytes in 3-D collagen/fibronectin matrices.
1. In RO1 grant-supported research funded by he NIH-NHLBI (Proteins of the Endothelial Cell Surface 2R01HL036003, Kluger, MS and Pober, J.S co-Principal Investigators) we focus on the role of tight junction (TJ) proteins, especially claudin 5, in human capillary ECs, and how TJs (and claudin 5) are affected by the pro-inflammatory cytokine, tumor necrosis factor (TNF). Our specific aims are first, to characterize the organization of TJs, with emphasis on the role of claudin 5, in human continuous capillary ECs and determine how TJ proteins interact with each other and with the actin cytoskeleton. Second, to determine how TNF affects the organization of human capillary TJ proteins and identify those proteins whose expression levels are changed by TNF, resulting in TJ disruption and capillary leak. Third, to determine how KLF4 regulates TNF-induced changes in capillary ECs and if this can be exploited to control TNF-induced pathological changes.
2. In other NIH-NHLBI-funded research (Optimizing Revascularization by Endothelial Cell Transplantation RO1-HL085416, Saltzman, W.M. and Pober, J.S. Co-PIs; Kluger, M.S., Co-Investigator) we introduce into SCID-beige immunodeficient mice human EC of wild type and mutant forms of Bcl-2, TNF-R2 and Etk for revascularization within scaffolds; we optimize the conditions for transplantation of transduced EC by incorporation of wild type or modified human pericytes (or vascular smooth muscle cells), by modifications of the scaffold composition; we also introduce controlled release of agents into the scaffold design such as a inhibitory microRNA aimed at modulating cord formation by EC and pericytes/SMCs.
- Abrahimi, P., Chang, WG., Kluger, MS., Qyang, Y., Tellides, G., Saltzman, WM., and Pober, J.S. Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9. 2015. Circulation Research (Accepted for Publication)
- Clark P.R., Kim R.K., Pober J.S., and Kluger M.S. Tumor Necrosis Factor Disrupts Claudin-5 Endothelial Tight Junction Barriers in Two Distinct NF-kappaB-Dependent Phases. 2015. PLOS ONE, PLoS ONE 10(3): e0120075. doi:10.1371/journal.pone.0120075
- Kluger MS, Clark PR, Tellides G, Gerke V, Pober JS. Claudin-5 Controls Intercellular Barriers of Human Dermal Microvascular but not Human Umbilical Vein Endothelial Cells. 2013. Arteriosclerosis, Thrombosis, and Vascular Biology 33:489-500. Click to view:
- Kluger, M. S., and Colegio, O. R. Lymphangiogenesis linked to VEGF-C from tumor-associated macrophages: accomplices to metastasis by cutaneous squamous cell carcinoma? 2011. J Invest Dermatol 131, 17-19 PMID: 21157425.
- *D’Alessio, A., * Kluger, M.S (*co-First Author), Li, J.H., Al-Lamki, R., Bradley, J.R., and Pober. J.S. Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells. 2010. Journal of Biological Chemistr
- Madge, L. A., Kluger, M. S., Orange, J. S., and May, M. J. Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells. 2008. J Immunol 180, 3467-3477 MID: 18292
- Liu, M., Kluger, M. S., D'Alessio, A., Garcia-Cardena, G., and Pober, J. S. Regulation of arterial-venous differences in tumor necrosis factor responsiveness of endothelial cells by anatomic context. 2008. Am J Pathol 172, 1088-1099 PMID: 18292233.
- Clark, P.R., Manes, T.D., Pober, J.S. and Kluger, M.S. Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations. 2007. Journal of Investigative Dermatology. 127: 762-774.
- Ranjbaran, H., Wang, Y., Manes, T. D., Yakimov, A. O., Akhtar, S., Kluger, M. S., Pober, J. S., and Tellides, G. Heparin displaces interferon-gamma-inducible chemokines (IP-10, I-TAC, and Mig) sequestered in the vasculature and inhibits the transendotheli
- Manes, T. D., Pober, J. S., and Kluger, M. S. Endothelial cell-T lymphocyte interactions: IP-10 stimulates rapid transendothelial migration of human effector but not central memory CD4+ T cells. Requirements for shear stress and adhesion molecules. 2006.
- Kluger, M. S. Vascular endothelial cell adhesion and signaling during leukocyte recruitment. 2004. Adv Dermatol 20, 163-201 PMID: 15544200
- Pober, J. S., Kluger, M. S., and Schechner, J. S. Human endothelial cell presentation of antigen and the homing of memory/effector T cells to skin. 2001. Ann N Y Acad Sci 941, 12-25 PMID: 11594565