Troy Stevens, Ph.D.
||Colorado State University
||University of Colorado
The Stevens lab studies the endothelium, with a particular interest in endothelial cell heterogeneity. Considerable work in the past 10 years has demonstrated that pulmonary artery, capillary and vein endothelial cells are highly specialized in order to perform the physiological functions that are relevant to their vascular location. The mechanisms responsible for establishing such heterogeneity are still poorly understood, and are the focus of our ongoing work.
Endothelium forms a semi-permeable barrier that separates blood from underlying tissue. Strength of this barrier, and the nature of cell-cell adhesion, changes dramatically from the pulmonary artery to the capillaries, where capillary endothelial cells form an especially tight barrier. During the course of inflammation, endothelial cells transiently decrease cell-cell adhesion and form intercellular gaps. Calcium entry through ion channels on the plasma membrane is an important signal that triggers cytoskeletal reorganization and promotes gap formation.
Our group has worked to identify calcium channels that provide the calcium source responsible for inter-endothelial cell gap formation. We have found that proteins belonging to the canonical transient receptor potential family of proteins (TRPC) contribute subunits to this channel. More specifically, TRPC1, TRPC3 and TRPC4 appear to interact to form the channel pore. TRPC4, in particular, is linked directly to the cytoskeleton. TRPC4 binds to protein 4.1, which tethers the channel to the spectrin membrane skeleton and the cortical actin rim. Ongoing studies suggest that this linkage establishes a privileged niche, where calcium entering through the channel serves as a catalyst to reorganize peripheral actin and enable intercellular gap formation. It will be important to continue resolving the molecular anatomy of this channel, to better understand how it is activated, and how calcium permeation through the channel impacts endothelial shape during the cell's response to inflammation.
While calcium entry through a TRPC channel disrupts the endothelial cell barrier, the second messenger adenosine 3'5'-cyclic monophosphate (cAMP) strengthens the barrier. cAMP activates two downstream signals, including protein kinase A and exchange protein activated by cAMP (EPAC), which collectively strengthen the cortical actin rim and support junctional complexes. Hormones such as epinephrine increase endothelial cAMP, and therefore have anti-inflammatory actions.
In contrast to the anti-inflammatory actions of epinephrine, pathogenic bacteria, such as Pseudomonas aeruginosa, have evolved mechanisms to utilize adenylyl cyclase toxins to generate cAMP and disrupt the endothelial cell barrier. P. aeruginosa utilizes a type III secretion system to inject exotoxin Y (exoY) into host cells, including the endothelium. Once in the host cell, exoY binds a mammalian cofactor and becomes an active enzyme, generating cAMP. Unlike the cAMP that is synthesized by the host cell, which is primarily produced at the plasma membrane, exoY generates cAMP in the cytosol. This cytosolic cAMP does not strengthen the cortical actin rim, but rather, disassembles microtubules leading to intercellular gap formation. Hence, studies on the function of exoY have revealed previously unappreciated mechanisms of cAMP signal transduction, cytoskeletal regulation of endothelial cell barrier, and mechanisms of bacterial pathogenesis. It will be important to continue these studies, to better understand how exoY becomes localized to target microtubule structures, and how the toxin serves to impact bacterial dissemination and pathogenicity.
- Wu S, Moore TM, Brough G, Li M, and Stevens T. Cyclic nucleotide gated channels mediate membrane depolarization following activation of store operated Ca2+ entry in endothelial cells. J. Biol. Chem., 275: 18887-18896, 2000.
- Stevens T, Garcia JGN, Shasby DM, Bhattacharya J, and Malik AB. Mechanisms regulating endothelial cell barrier function. Am. J. Physiol., 279: L419-L422, 2000.
- Moore T, Norwood N, Creighton J, Babal P, Brough G, Shasby D, and Stevens T. Receptor-dependent activation of store-operated Ca2+ entry increases endothelial cell permeability. Am. J. Physiol., 279: L691-L699, 2000.
- Norwood N, Moore TM, Dean D, Bhattacharjee R, Creighton J, Babal P, and Stevens T. Store operated Ca2+ and endothelial cell permeability. Am. J. Physiol., 279: L815-L824, 2000.
- Stevens T. Pulmonary vasoconstriction induced by Gq agonists. Is there a role for store operated calcium entry? Am. J. Physiol., 280: L866-L869, 2001.
- Brough G, Wu S, Moore TM, Li M, Dean N, and Stevens T. Contribution of endogenously expressed Trp-1 to a Ca2+ selective store operated Ca2+ entry pathway. FASEB J., 15: 1727-1738, 2001.
- Wu S, Sangerman J, Li M, Brough G, Goodman S, and Stevens T. Essential control of an endothelial cell ISOC by the spectrin membrane skeleton. J. Cell Biol., 154: 1225-1233, 2001.
- Stevens T, Rosenberg B, Aird W, Quertermous T, Garcia JGN, Hebbel R, Tuder R, Garfinkel S. NHLBI workshop report: Endothelial cell phenotypes in heart, lung and blood diseases. Am. J. Physiol., 281: C1422-C1433, 2001.
- Parker J, Stevens T, Randall J, Sybert C, Yoshikawa S, and Penton A. Hydraulic conductance of segmental endothelial phenotypes in pulmonary circulation. Proc. 7th World Cong. Microcirc., pp. 505-511, 2002.
- Cioffi D, Moore TM, Schaack J, Creighton J, Cooper DMF and Stevens T. Dominant regulation of inter-endothelial cell gap formation by the calcium-inhibited type 6 adenylyl cyclase. J. Cell Biol., 157: 1267-1278, 2002.
- Stevens T. Bronchial endothelial cell phenotypes and the form:function relationship. Am. J. Physiol. 283: L518-L519, 2002.
- Wang Q, Pfeiffer GR, Stevens T, and Doerschuk CM. Pulmonary microvascular and arterial endothelial cells differ in their responses to ICAM-1 ligation. Am. Rev. Resp. Crit. Care Med., 166: 872-877, 2002.
- Norwood N and Stevens T. Molecular motors in control of store operated Ca2+ entry. Cell Biochem. Biophys., 37: 53-70, 2002.
- Creighton J, Masada N, Cooper DMF, and Stevens T. Coordinate regulation of membrane cAMP by calcium inhibited adenylyl cyclase (type 6) and phosphodiesterase (type 4) activities. Am. J. Physiol., 284: L100-L107, 2003.
- Cioffi DL, Wu S, and Stevens T. On the endothelial cell ISOC. Cell Calcium. 33: 323-336, 2003.
- Wu S, Haynes J, Taylor J, Brough GH, Li M, and Stevens T. Cav3.1 (α1G) T-type Ca2+ channels mediate vaso-occlusion of sickled erythrocytes in lung microcirculation. Circ. Res., 93: 346-353, 2003.
- Chatterjee S, Al-Mehdi A, Levitan I, Stevens T, and Fisher AB. Shear stress increases expression of a KATP channel in rat pulmonary microvascular endothelial cells. Am. J. Physiol., 285: C959-967, 2003.
- King J, Hamil T, Creighton J, Wu S, Bhat P, McDonald F, and Stevens T. Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes. Microvasc. Res., 67: 139-151, 2004.
- Li J, Zheng M, Tang ZL, Stevens T, Pitt B, and Li S. CpG DNA-mediated immune response in pulmonary endothelial cells. Am. J. Physiol., 286: L808-L816, 2004.
- Gebb SA and Stevens T. On lung endothelial cell heterogeneity. Microvasc. Res., 68: 1-12, 2004.
- King JA, Ofori-Acquah SF, Stevens T, Al-Mehdi A., Fodstad O. and Jiang WG. Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator. Breast Canc. Res., 6: 478-487, 2004.
- Ihida-Stansbury K, Gebb SA, McKean DM, Martin JF, Stevens T, Kaplan-Alburquerque N, and Jones PL. The paired-related homeobox gene transcription factor Prx1 is required for lung vascularization and normal alveolar development. Circ. Res., 94: 1507-1514, 2004.
- Newman JH, Fanburg BL, Archer SL, Badesch DB, Barst RJ, Garcia JGN, Kao PN, Knowles JA, Loyd JE, McGoon MD, Morse JH, Nichols WC, Rabinovitch M, Rodman DM, Stevens T, Tuder RM, Voelkel NF, and Gail DB. Pulmonary arterial hypertension: future directions. Report of a National Heart, Lung and Blood Institute/Office of Rare Diseases Workshop. Circulation, 109: 2947-2952, 2004.
- Sayner SL, Frank DW, King J, Chen H, VandeWaa J, and Stevens T. Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by P. aeruginosa ExoY. Circ. Res., 95: 196-203, 2004.
- Metze B, Ofori-Acquah S, Stevens T, and Balczon R. Stat3 activity is required for centrosome duplication in Chinese hamster ovary cells. J. Biol. Chem., 279:41801-41806, 2004.
- Sayner S and Stevens T. Adenylyl cyclase and cAMP regulation of the endothelial barrier. In: Perspectives on Lung Endothelial Barrier Function. Editor: C. E. Patterson. Elsevier Science, ISBN: 0-444-51834-7, pg. 139-164, 2005.
- Thorin E, Stevens T, and Patterson CE. Heterogeneity of Lung Endothelial Cells. In: Perspectives on Lung Endothelial Barrier Function. Editor: C. E. Patterson. Elsevier Science, ISBN: 0-444-51834-7, pg. 277-310, 2005.
- Wu S, Cioffi E, Alvarez D, Sayner SL, Chen H, Cioffi DL, King J, Creighton JR, Townsley M, Goodman SR, and Stevens T. Essential role of a Ca2+ selective store operated current (ISOC) in endothelial cell permeability: determinants of the vascular leak site. Circ. Res., 96(8):856-863, 2005.
- Stevens T. Calcium inhibited adenylyl cyclase (AC6) controls endothelial cell barrier function. In: Proinflammatory Signaling Mechanisms in the Pulmonary Circulation. Editor: J. Bhattacharya. Humana Press, pg. 203-215, 2005.
- Wu S, Cioffi D, and Stevens T. Receptor-operated Ca2+ channels. In: Ion Channels in the Pulmonary Vasculature. Editor: J. X. Yuan. Taylor and Francis, Boca Raton, Florida pg. 99-124, 2005.
- Cioffi D, Wu S, and Stevens T. Transient receptor potential cation channels and store-operated Ca2+ channels. In: Ion Channels in the Pulmonary Vasculature. Editor: J. X. Yuan. Taylor and Francis, Boca Raton, Florida, pg. 125-146, 2005.
- Al-Mehdi A, Schaphorst KL and Stevens T. Lung endothelial heterogeneity. In: Encyclopedia of the Microvasculature. Editor: J.G.N. Garcia. Elsevier Science, pg. 465-470, 2005.
- Cioffi DL, Wu S, Alexeyev M, Goodman SR, Zhu MX, and Stevens T. Activation of the Endothelial Store-Operated ISOC Ca2+ Channel Requires Interaction of Protein 4.1 With TRPC4. Circ. Res., 97:1164-1172, 2005.
- Stevens T. Molecular and cellular determinants of lung endothelial cell heterogeneity. Chest, 128: 558S - 564S, 2005.
- Stevens T, Kasper M, Cool C, and Voelkel NF. Pulmonary circulation and pulmonary hypertension. In: Endothelial Cells in Health and Disease. Editor: W. C. Aird. Informa Healthcare, pages 417-438, 2005.
- Zhu B, Strada S, Stevens T. Cyclic GMP-specific phosphodiesterase 5 regulates cell growth and apoptosis in pulmonary endothelial cells. Am. J. Physiol., 289:L196-206, 2005.
- Matthay MA, Christman JW, Pitt BR, Schwiebert LM, Stevens T, and Ware LB. Perspectives, translational research, and letters to the editor. Am. J. Physiol., 290:L621, 2006. [Editorial]
- He F, Li J, Mu Y, Kuruba R, Ma Z, Wilson A, Alber D, Jian Y, Stevens T, Watkins S, Pitt B, Xie W, and Li S. Downregulation of endothelin-1 farnesoid X receptor in vascular endothelial cells. Circ. Res., 98:192-199, 2006.
- Sayner S, Alexeyev M, Dessauer CW, and Stevens T. Soluble adenylyl cyclase reveals the significance of cAMP compartmentation on pulmonary microvascular endothelial cell barrier. Circ. Res., 98:675-681, 2006.
- Masedunskas A, King JA, Tan F, Cochran R, Stevens T, Sviridov D and Ofori-Acquah SF. Activated leukocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration. FEBS Lett., 580:2637-45, 2006.
- Sayner S and Stevens T. Soluble adenylate cyclase reveals the significance of compartmentalized cAMP on endothelial cell barrier function. Biochemical Society Transactions, 34:492-494, 2006.
- Parker J, Stevens T, Randall J, Weber D, and King J. Hydraulic conductance of pulmonary microvascular and macrovascular endothelial cell monolayers. Am. J. Physiol., 291:L30-7, 2006.
- Cioffi D and Stevens T. Regulation of endothelial cell barrier function by store operated calcium entry. Microcirculation, 13:709-723, 2006.
- Zhang L, Croix C, Cao R, Wasserloos K, Watkins SC, Stevens T, Li S, Tyurin V, Kagan VE and Pitt BR. Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells. Exp. Biol. Med. 231:1507-15, 2006.
- Stevens T. Microheterogeneity of lung endothelium. In: Endothelial Biomedicine. Editor: William C. Aird, Cambridge University Press, pages 1161-1170, 2007.
- Lowe K, Alvarez D, King J, and Stevens T. Phenotypic heterogeneity in lung capillary and extra - alveolar endothelial cells. Increased extra-alveolar endothelial permeability is sufficient to decrease compliance. J. Surg. Res. 143:170-177, 2007.
- Creighton J, Zhu B, Alexeyev M, and Stevens T. Spectrin-anchored phosphodiesterase 4D4 restricts cAMP from disrupting microtubules and inducing endothelial cell gap formation. J. Cell Sci. 121:110-119, 2007.
- Stevens T, Gillespie MN. The hyperproliferative endothelial cell phenotype in idiopathic pulmonary arterial hypertension. Am. J. Physiol., 293:L546-547, 2007.
- Wu S, Chen H, Alexeyev MF, King JAC, Moore TM, Stevens T, and Balczon RD. Microtubule motors regulate ISOC activation necessary to increase endothelial cell permeability. J. Biol. Chem., 282:34801-34808, 2007.
- Clark J, Alvarez D, Alexeyev M, King J, Huang L, Yoder M, and Stevens T. Regulatory role for nucleosome assembly protein-1 in the proliferative and vasculogenic phenotype of pulmonary endothelium. Am. J. Physiol., 294:L431-L439, 2008.
- Alvarez D, Huang L, King J, Yoder M, and Stevens T. Lung microvascular endothelium is enriched with progenitor cells that exhibit rapid vasculogenic capacity. Am. J. Physiol., 294:L419-L430, 2008.
- Stevens T. Epithelium: sticking it out, together. Am. J. Physiol., 294:L440-L441, 2008.
- Haynes Jr J, Obiako B, Hester RB, Baliga BS, Stevens T. Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium. Am J. Physiol., 294:H379-H385, 2008.
- Ofori-Acquah SF, King J, Voelkel N, Schaphorst KL, and Stevens T. Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions. Microvas. Res., 75:391-402, 2008.
- King J, Agarwal S, Syklawer E, Prasain N, Chen H, Resmondo J, McDonald F, Bauer N, Alvarez D, Wu S, Stevens T, Shevde L, Moore T, and Townsley M. Quantum Dots - Utilization in TEM. Microsc. Microanal., 14(Suppl2): 702-703, 2008.
- Franks TJ, Colby TV, Travis WD, Tuder RM, Reynolds HY, Brody AR, Cardoso WV, Crystal RG, Drake CJ, Englehardt J, Frid M, Herzog E, Mason R, Phan SH, Randell SH, Rose MC, Stevens T, et al. NHLBI Workshop Summaries on Resident Cellular Components of the Human Lung. Proc. Am. Thorac. Soc., 5:763-766, 2008.
- Stevens T, Phan S, Frid MG, Alvarez D, Herzog E, and Stenmark KR. NIH workshop on Lung Vascular Cell Heterogeneity: endothelium, smooth muscle and fibroblasts. Proc. Am. Thorac. Soc., 5:783-791, 2008.
- Rai PR, Cool CD, King JAC, Stevens T. Burns N, Winn RA, Kasper M, and Voelkel NF. The cancer paradigm of severe angioproliferative pulmonary hypertension (SAPPH). AM. J. Resp. Crit. Care Med., 178:558-564, 2008.
- Zhu B, Zhang L, Alexeyev M, Alvarez DF, Strada SJ, and Stevens T. Type 5 phosphodiesterase expression is a critical determinant of the endothelial cell angiogenic phenotype. Am. J. Physiol., 296: L220-228, 2009.
- Prasain N and Stevens T. The actin cytoskeleton in endothelial phenotypes. Microvasc. Res., 77: 53-63, 2009.
- Prasain N, Alexeyev M, and Stevens T. Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction. Am. J. Physiol., 297: L73-L83, 2009.
- Tuder RM, Abman SH, Braun T, Capron F, Stevens T, Thistlethwaite PA, and Haworth SG. Development and pathology of pulmonary hypertension. J. Am. Coll. Cardiol., 54: S3-S9, 2009.
- Cioffi D, Lowe K, Alvarez DF, Barry C, and Stevens T. TRPing on the lung endothelium. Antiox. Redox. Signal., In Press.
- Cioffi D, Barry C, and Stevens T. Studies on the structure and function of the calcium selective store operated calcium entry current. In: Membrane Receptors, Channels and Transporters in the Pulmonary Circulation. Editors: Jeremy P.T. Ward and Jason X.-J. Yuan, Humana Press, In Press.