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.
- Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9. Abrahimi, P., Chang, WG., Kluger, MS., Qyang, Y., Tellides, G., Saltzman, WM., and Pober, J.S. Circulation Research. 2015. 117: 121-128
- Tumor Necrosis Factor Disrupts Claudin-5 Endothelial Tight Junction Barriers in Two Distinct NF-kappaB-Dependent Phases. Clark P.R., Kim R.K., Pober J.S., and Kluger M.S. 2015. PLOS ONE, PLoS ONE 10(3): e0120075. doi:10.1371/journal.pone.0120075
- Pericytes modulate endothelial sprouting. Chang, W.G., Andrejecsk, J.W., Kluger, M. S., Saltzman, W.M., and Pober, J.S. 2013. Cardiovasc Res 100(3):492-500
- In vitro assembly of human pericyte-supported endothelial microvessels in three-dimensional co-culture: A simple model for interrogating endothelial-pericyte interactions. Waters, J.P., Kluger, M. S., Graham, M. Chang, W.G., Bradley J.R. and Pober, J.S. 2013. J Vasc Res 50(4):324-331
- Claudin-5 controls intercellular barriers of human dermal microvascular but not human umbilical vein endothelial cells. Kluger, M. S., Clark, P. R., Gerke, V. and Pober, J.S. 2013. Arterioscler Thromb Vasc Biol. 33:489-500
- MEK5 is activated by shear stress, activates ERK5 and induces KLF4 to modulate TNF responses in human dermal microvascular endothelial cells. Clark, P. R., Jensen, T., Kluger, M. S., Morelock, M., Hanidu, A., Qi, Z., Tatake, R. J., and Pober, J. S. 2011. Microcirculation, 18(2), 102–117
- Functional analyses of the bone marrow kinase in the X chromosome in vascular endothelial growth factor-induced lymphangiogenesis. Jones, D., Xu, Z., Zhang, H., He, Y., Kluger, M. S., Chen, H., and Min, W. 2010. Arterioscler Thromb Vasc Biol 30, 2553-2561
- Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells. D'Alessio, A., Kluger, M. S., Li, J. H., Al-Lamki, R., Bradley, J. R., and Pober, J. S. 2010. J Biol Chem 285, 23868-23879
- Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells. Madge, L. A., Kluger, M. S., Orange, J. S., and May, M. J. 2008. J Immunol 180, 3467-3477
- Regulation of arterial-venous differences in tumor necrosis factor responsiveness of endothelial cells by anatomic context. Liu, M., Kluger, M. S., D'Alessio, A., Garcia-Cardena, G., and Pober, J. S. 2008. Am J Pathol 172, 1088-1099
- Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect. Clark, P. R., Pober, J. S., and Kluger, M. S. 2008. Nucleic Acids Res 36, 1081-1097
- IL-10 inhibits endothelium-dependent T cell costimulation by up-regulation of ILT3/4 in human vascular endothelial cells. Gleissner, C. A., Zastrow, A., Klingenberg, R., Kluger, M. S., Konstandin, M., Celik, S., Haemmerling, S., Shankar, V., Giese, T., Katus, H. A., and Dengler, T. J. 2007. Eur J Immunol 37, 177-192
- Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations. Clark, P. R., Manes, T. D., Pober, J. S., and Kluger, M. S. 2007. J Invest Dermatol 127, 762-774
- 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. Ranjbaran, H., Wang, Y., Manes, T. D., Yakimov, A. O., Akhtar, S., Kluger, M. S., Pober, J. S., and Tellides, G. 2006. Circulation 114, 1293-1300
- 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. Manes, T. D., Pober, J. S., and Kluger, M. S. 2006. Transplantation 82, S9-14
- Interferon-gamma Augments CD95(APO-1/Fas) and Pro-Caspase-8 Expression and Sensitizes Human Vascular Endothelial Cells to CD95-Mediated Apoptosis. Li, J. H., Kluger, M. S., Madge, L. A., Zheng, L., Bothwell, A. L., and Pober, J. S. 2002. Am J Pathol 161, 1485-1495
- Internalization of transduced E-Selectin by cultured human endothelial cells: Comparison of dermal microvascular and umbilical vein cells and identification of a phosphoserine-type di-leucine motif. Kluger, M. S., Shiao, S. L., Bothwell, A. L. M., and Pober, J. S. 2002. Cutting Edge: J Immunol 168, 2091-2095
- The death domain of tumor necrosis factor receptor 1 is necessary but not sufficient for Golgi retention of the receptor and mediates receptor desensitization. Gaeta, M. L., Johnson, D. R., Kluger, M. S., and Pober, J. S. 2000. Lab Invest 80, 1185-1194
- In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. Schechner, J. S., Nath, A. K., Zheng, L., Kluger, M. S., Hughes, C. C., Sierra-Honigmann, M. R., Lorber, M. I., Tellides, G., Kashgarian, M., Bothwell, A. L., and Pober, J. S. 2000. Proc Natl Acad Sci U S A 97, 9191-9196
- Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant bcl-2 without alterations in growth or activation responses. Zheng, L., Dengler, T. J., Kluger, M. S., Madge, L. A., Schechner, J. S., Maher, S. E., Pober, J. S., and Bothwell, A. L. 2000. J Immunol 164, 4665-4671
- Mechanism of sustained E-selectin expression in cultured human dermal microvascular endothelial cells. Kluger, M. S., Johnson, D. R., and Pober, J. S. 1997. J Immunol 158, 887-896
- Human endothelial cell presentation of antigen and the homing of memory/effector T cells to skin. Pober, J. S., Kluger, M. S., and Schechner, J. S. 2001. Ann N Y Acad Sci 941, 12-25
- Vascular endothelial cell adhesion and signaling during leukocyte recruitment. Kluger, M. S. 2004. Adv Dermatol 20, 163-201
- Lymphangiogenesis linked to VEGF-C from tumor-associated macrophages: accomplices to metastasis by cutaneous squamous cell carcinoma? Kluger, M. S., and Colegio, O. R. 2011. J Invest Dermatol 131, 17-19