Edema; Immune System; Permeability; NF-kappa B; Sepsis; Tight Junctions; Adherens Junctions; Microvessels
Public Health Interests
Cardiovascular Disease; Sepsis
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.
Specialized Terms: Sepsis; Vascular Hyperpermeability (leak); Tumor Necrosis Factor (TNF); Interleukin-1 (IL-1); Claudin-5; Microvascular Endothelial Cells; NF-κB signaling
Extensive Research Description
Vascular hyper-permeability (or “leak”) is a life threatening consequence of systemic inflammatory disease in bacterial sepsis, in viral hemorrhagic disease or in pathogenic-free immune responses such as SIRS. A better understanding of endothelial cell inflammation is needed for developing effective therapies that may prevent the loss of selective permeability, an important feature of the healthy microvascular endothelium present in bodily organs. We are currently using system based approaches like RNA-Seq and siRNA library screening to identify new molecular targets and to define gene expression patterns by which the inflammatory cytokine tumor necrosis factor (TNF) destabilizes blood vessel barriers. This work employs our in vitro model of tight junction-dependent endothelial barrier function, which models how capillary leak leads to organ failure in inflammatory disease.
In a pilot project award to Martin S. Kluger funded by the Yale Center for Molecular Discovery we have performed a phenotypic screen in cultured microvascular endothelial cells of the entire human kinome to identify kinases regulating TNF leak. We are now characterizing these results, which may form the basis of novel therapeutic targets. We anticipate this approach, performed in collaboration with the inventors of ECIS at Applied BioPhysics, Inc. (Troy, NY) will be applicable to ECIS-based high throughput functional screening of diverse adherent cell types using different inflammatory mediators, e.g., other cytokines, PAMPs, DAMPs or toxins, or by replacing siRNA with CRISPR (by methods reported in our publication Abrahimi, et al., Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9. 2015. Circulation Research. 117: 121-128).
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 have focused on how inflammatory cytokines such as TNF, IL-1b and LPS act on tight junction (TJ) proteins because of their essential role in the permeability barriers of capillaries, which form the largest anatomical interface separating blood from vascularized organs and are the microvascular segment most responsible for preventing harmful hyperpermeability responses. In this work we have characterized signaling pathways affecting the organization of TJs in human microvascular endothelial cells, with an emphasis on the role of claudin-5, and have studied how TJ proteins interact with the actin cytoskeleton.
- 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.
- 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.
- Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells. *D’Alessio, A., * Kluger, M.S (*co-First Author), Li, J.H., Al-Lamki, R., Bradley, J.R., and Pober. J.S. J Biol Chem. 2010 Jul 30;285(31):23868-79. doi: 10.1074/jbc.M110.122853. Epub 2010 May 28.
- 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: 18292573
- 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 transendothelial migration and arterial recruitment of T cells. 2006. Circulation 114, 1293-1300 PMID: 16940188.
- 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. Transplantation 82, S9-14 PMID: 16829798.
- 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