Quantitative Proteomic Analysis of Sokotrasterol Sulfate-stimulated Primary Human Endothelial Cells

The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting. The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting. Angiogenesis, the formation of new blood vessels from existing vessels, is a complex process requiring modulation of multiple endothelial cell functions (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). Endothelial cells line the interior of blood vessels and are responsible for initiating the growth of new vessels (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). The formation of capillary sprouts from the existing microvasculature occurs secondary to an inciting stimulus resulting in increased vascular permeability, accumulation of extravascular fibrin, and local proteolytic degradation of the basement membrane (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). The endothelial cells overlying the disrupted region become activated, change shape, and extend elongated processes into the surrounding tissue (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). Directed migration toward the angiogenic stimulus results in the formation of a column of endothelial cells (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). Just proximal to the migrating tip of the column is a region of proliferating cells that cause the sprout to increase in length (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). Proximal to the proliferative zone, the endothelial cells undergo a second shape change, adhere tightly to each other, and begin to form a lumen resulting in a capillary plexus (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). Fusion of individual sprouts at their tips closes the loop and circulates blood into the vascularized area (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). Throughout this process the expression of various proteins is tightly regulated (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 2Karsan A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). The development of new blood vessels is required in various pathological and physiological processes including embryogenesis, organogenesis, ovarian follicle development, and tumor growth and metastasis (1Carmeliet P. Angiogenesis in health and disease..Nat. Med. 2003; 9: 653-660Google Scholar, 3Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease..Nat. Med. 1995; 1: 27-31Google Scholar). Ischemic coronary artery disease is a major cause of morbidity and the leading cause of mortality in the industrialized world (4Helisch A. Ware J.A. Therapeutic angiogenesis for ischemic heart disease..Adv. Exp. Med. Biol. 2000; 476: 327-350Google Scholar). The major reason for cardiovascular disease is the occlusion of blood vessels due to atherosclerosis (4Helisch A. Ware J.A. Therapeutic angiogenesis for ischemic heart disease..Adv. Exp. Med. Biol. 2000; 476: 327-350Google Scholar). Neovascularization, or new blood vessel growth, is also required to bypass the occlusion of arteries caused by atherosclerosis (4Helisch A. Ware J.A. Therapeutic angiogenesis for ischemic heart disease..Adv. Exp. Med. Biol. 2000; 476: 327-350Google Scholar). The effects of neovascularization in ischemic disease may be both beneficial and detrimental to the patient (5Dor Y. Keshet E. Ischemia-driven angiogenesis..Trends Cardiovasc. Med. 1997; 7: 289-294Google Scholar, 6Ware J.A. Simons M. Angiogenesis in ischemic heart disease..Nat. Med. 1997; 3: 158-164Google Scholar). Lack of adequate neovascularization when arteries narrow can result in clinical symptoms of ischemia, and strategies to promote angiogenesis may improve symptoms and tissue function (7Kutryk M.J. Stewart D.J. Angiogenesis of the heart..Microsc. Res. Tech. 2003; 60: 138-158Google Scholar). In contrast, neovascularization of an atherosclerotic plaque can result in growth of the plaque and further luminal stenosis, or plaque hemorrhage and rupture (8Celletti F.L. Waugh J.M. Amabile P.G. Brendolan A. Hilfiker P.R. Dake M.D. Vascular endothelial growth factor enhances atherosclerotic plaque progression..Nat. Med. 2001; 7: 425-429Google Scholar, 9Simons M. Ware J.A. Therapeutic angiogenesis in cardiovascular disease..Nat. Rev. Drug Discov. 2003; 2: 863-871Google Scholar). While the beneficial effects of using angiogenic drugs to promote neovascularization in ischemic disease remains controversial, several clinical trials are under way to evaluate the benefits (9Simons M. Ware J.A. Therapeutic angiogenesis in cardiovascular disease..Nat. Rev. Drug Discov. 2003; 2: 863-871Google Scholar). In an attempt to identify small molecules with the ability to stimulate blood vessel growth for potential use in therapeutic angiogenesis, we recently screened a library of crude marine extracts and identified a sulfated steroid called sokotrasterol sulfate (10Makarieva T.N. Shubina L.K. Kalinovsky A.I. Stonik V.A. Elyakov G.B. Steroids in Porifera. II. Steroid derivatives from two sponges of the family Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern of side chain alkylation..Steroids. 1983; 42: 267-281Google Scholar). 1A. Karsan, I. Pollet, T. P. Conrads, R. Andersen, and T. Veenstra, manuscript in preparation. 1A. Karsan, I. Pollet, T. P. Conrads, R. Andersen, and T. Veenstra, manuscript in preparation. Sokotrasterol sulfate stimulation causes sprouting of endothelial cells in vitro in a dose-dependent manner and induces the development of new blood vessels in an in vivo chick chorioallantoic membrane model of angiogenesis. To ensure the safe use of angiogenic therapies, however, a thorough understanding of the mechanisms of neovascularization is important. To understand how sokotrasterol sulfate affects the endothelial cell and potentially understand its mechanism of angiogenic action, differential protein analysis using cleavable isotope-coded affinity tag (cICAT) 2The abbreviations used are: cICAT, cleavable isotope-coded affinity tag; MS, mass spectrometry; HUVEC, human umbilical vein endothelial cell; SCXLC, strong cation exchange liquid chromatography; μRP, microcapillary reversed-phase; ESI, electrospray ionization; PBS, phosphate-buffered saline; LC, liquid chromatography; MS/MS, tandem mass spectrometry; ALCAM, activated leucocyte cell adhesion molecule. 2The abbreviations used are: cICAT, cleavable isotope-coded affinity tag; MS, mass spectrometry; HUVEC, human umbilical vein endothelial cell; SCXLC, strong cation exchange liquid chromatography; μRP, microcapillary reversed-phase; ESI, electrospray ionization; PBS, phosphate-buffered saline; LC, liquid chromatography; MS/MS, tandem mass spectrometry; ALCAM, activated leucocyte cell adhesion molecule. reagents combined with mass spectrometry (11Cao Y. O’Reilly M.S. Marshall B. Flynn E. Ji R.W. Folkman J. Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases..J. Clin. Investig. 1998; 101: 1055-1063Google Scholar) was used to quantitate protein expression differences in primary human endothelial cells stimulated with sokotrasterol sulfate. In addition, a statistical analysis was performed to determine what magnitude of change in relative abundance between proteins extracted from the treated and control human umbilical vein endothelial cells (HUVECs) was significant. Previous studies using ICAT have arbitrarily selected a specific cut-off as a measure of significance; however, the results presented in this study show that the level of significance is likely to be different for every data set analyzed in this manner. In particular, membrane proteins such as Tie-1 and the αv integrin that are known to be required in the developing vasculature were shown to be up-regulated at the borderline level, bolstering the requirement for choosing optimal cut-off levels for up- and down-regulated proteins. Several other proteins, such as Dysferlin and E2F4, demonstrated borderline but significant up-regulation by sokotrasterol sulfate implicating these proteins in the process of angiogenesis induced by sokotrasterol sulfate. Ammonium bicarbonate (NH4HCO3), ammonium formate (NH4HCO2), guanidine hydrochloride, dibasic sodium phosphate (Na2HPO4), monobasic sodium phosphate (NaH2PO4), sodium chloride (NaCl), ammonium hydroxide (NH4OH), Tris, sodium fluoride (NaF), sodium orthovanadate (Na3VO4), Triton X-100, and phenylmethylsulfonyl fluoride were purchased from Sigma. Trifluoroacetic acid and formic acid were obtained from Fluka (Milwaukee, WI). High performance liquid chromatography grade acetonitrile (CH3CN) and methanol (CH3OH) were obtained from EM Science (Darmstadt, Germany). UltraLink™ immobilized monomeric avidin, Tris(2-carboxyethyl)phosphine hydrochloride, ImmunoPure d-biotin, and bicinchonic acid (BCA) protein assay reagent kit were purchased from Pierce. Water was purified by a Barnstead Nanopure system (Dubuque, IA). Cleavable ICAT reagents were purchased from Applied Biosystems, Inc. (Foster City, CA). Primary were and as A. E. Harlan J.M. of a human human in endothelial Scholar). tissue of were treated for with sokotrasterol sulfate or were with and the with Triton X-100, and phenylmethylsulfonyl and from each was into a and of protein extracts were with the or the of the reagent using a method from that by the T. of the isotope-coded affinity tag to Res. 3: Scholar). of each cell protein was in of guanidine in was by of Tris(2-carboxyethyl)phosphine by in a for The were to or in of and at 37 for The two were into using a column and with at 37 using an to protein of The was by the in a for and phenylmethylsulfonyl fluoride to a of A immobilized monomeric column was in a and with sodium The column was with in PBS, the was from the of the column to the and the column was with PBS, The were for to the and for at the column with each of PBS, PBS, and the were using formic acid and to The was from the by treatment with the reagents by the for at 37 and to The were in of formic and a strong cation exchange liquid chromatography column The was used to the from the column at a of for by a increase to in a increase to in a increase to in and at for A was and was were every for was and in of acid to microcapillary were line with an mass to the extracted from the control and sokotrasterol To the were to a tip were using a The were a to an capillary system was used to A formic acid in and formic acid in of the were at a of using a of for and for The mass was in a in the molecular in the were and selected for using a of The for the molecular was using two for the mass of and the for the was using In this analysis the were the was for each of the and with a of for and for the in between was The and for the capillary of the were set at and The data the mass were using the data from the The data in the analysis was using from the and for were set by mass of the for the and for the in a were set as for for and for and as for the of within the The identified were using the relative abundances in this data of the area of their extracted steroid have shown to have or but the mechanism by these function remains to be J.M. and a Biol. 2001; Scholar). is that functions by the and of other angiogenic to promote neovascularization J. levels of vascular endothelial growth factor in vivo in murine J. 2003; Scholar, steroid of vascular endothelial growth factor in the human for angiogenesis the and in the of Clin. Scholar). The of a novel function for the sulfated steroid sokotrasterol sulfate to attempt to understand its mechanism of is the of sokotrasterol sulfate the molecular of cell the sprouting of new vessels is initiated at the level of the endothelial primary human endothelial cells were treated with sokotrasterol sulfate or to quantitate differential protein expression between these two protein extracts from treated with as a control or with sokotrasterol sulfate for were and with the of the reagents and of the reagent a and a by a region in the in of in the the protein extracts were combined and with and the were using immobilized The were into by that were analyzed by to measurements is the ability to identify of proteins. is that or is of the complex of that results from a proteolytic of a two or the of the and results in of identified from complex analysis of the by protein 2001; Scholar). is a of that a mass can mass measurements several for a of these to be selected for the of to increase the of that may be in is the of the from a of the and proteins identified in each analysis of the is between the of identified and the of each of the In to proteins, from treated with or sokotrasterol sulfate were quantitated in this study the analysis in identified of these may identify protein due to proteins we have a of the and proteins identified to for these effects and that are to identify a protein and identify protein protein due to in protein in the data with increased expression levels in response to sokotrasterol increase in protein of identified is shown in protein protein protein family protein complex factor protein protein A phosphate protein protein region protein factor complex protein protein protein protein cDNA of protein protein protein protein protein protein protein protein protein protein αv chain chain protein protein complex chain protein protein protein protein and protein to protein factor complex protein factor factor Tie-1 of identified is shown in in a new The ability to identify a protein the to the identified a proteins in the data The of in is with strong every of the the and of the proteolytic the and of the mass the used to the the of the and the and of the data is that data from the be to to determine the of the A analysis of the was by the data from the of the an data for This analysis in the of from the data as with from the human data a of of for the data set that the of the and used in this The relative abundances of the were the area of their extracted from the molecular of the of each can be for what in relative abundance as by statistically significant in abundance the of multiple These such as the to the of was the relative of of each of the from the the and of the and the of molecular each to determine the and the abundance were statistically significant within this data The of the of identified within abundance was the of the ICAT be a as is the at a is as the of the total of and is The is as a and can be obtained from the data of the and the of the The abundance an increase or in abundance be as statistically significant is as or the these we that the a abundance a statistically significant increase in abundance be to be and the a abundance a statistically significant in abundance be to be for these specific these 93 proteins were significantly and 37 proteins were significantly in to of treatment with sokotrasterol sulfate and with expression levels in response to sokotrasterol increase in protein of identified is shown in protein acid factor protein protein A protein factor complex protein protein to cDNA protein protein protein protein of protein complex protein factor protein of identified is shown in in a new To this two proteins were Dysferlin and the factor E2F4, that at the of what be significant a increase and the tandem mass of the and ICAT of the of the of Dysferlin molecular the tandem mass of the of the of E2F4, the of the molecular of is presented in by that both Dysferlin and are up-regulated by sokotrasterol sulfate at and and to a protein that change in demonstrated a of the of and at as with the by the extracted from the expression of proteins as by were treated with or sokotrasterol sulfate for and as cell were and to with E2F4, or were to and the increase in protein expression of Dysferlin and in response to treatment with sokotrasterol sulfate or was by in cells the of To further proteins regulated by sokotrasterol sulfate, proteins were to the of molecular and Sokotrasterol proteins in are in and down-regulated proteins are in in this proteins involved in to and in cell including cell adhesion were likely to be in response to sokotrasterol sulfate. at protein with cell tumor factor was down-regulated by sokotrasterol sulfate. The of proteins within was and between proteins that were and that were that as membrane proteins were to be up-regulated, consistent with the to The up-regulated membrane proteins molecules such as to have with angiogenesis, as as other proteins, such as the Tie-1 and the αv integrin are both known to be required for vascular development but have shown to be up-regulated by steroid A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google of proteins down-regulated in endothelial cells by sokotrasterol sulfate. is the of proteins to the of molecular function and process Ischemic coronary artery disease is a major cause of morbidity and the leading cause of mortality in the world (4Helisch A. Ware J.A. Therapeutic angiogenesis for ischemic heart disease..Adv. Exp. Med. Biol. 2000; 476: 327-350Google Scholar). therapeutic for with ischemic heart disease or coronary by coronary or bypass (4Helisch A. Ware J.A. 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Scholar). proteins are in both vascular and development of vascular cell by Cardiovasc. Med. 2003; Scholar). In the shown to be for in a of is in a of and expression and Scholar). to this study of in or involved in endothelial sprouting or angiogenesis. data sokotrasterol expression of new to be further in angiogenic findings have in a the endothelial cells were in a tissue the process of vascular sprouting is a of the molecules by differential protein analysis a for in in vivo using in vitro data also that of the findings may be to angiogenesis secondary to other inciting other proteins may be specific to sokotrasterol angiogenesis. The of what magnitude of change a significant increase or in protein expression as using ICAT reagents from a to differential increase or have selected in studies to a statistically significant change in protein abundance M.D. of cells using isotope-coded affinity and mass Scholar, P.R. I. J. R. analysis of the cell response to Biol. Scholar). such an may proteins that increased in may significant the other under a differential expression may be to proteins that a significant change in In this study a statistical method was used to determine the cut-off levels for proteins in response to sokotrasterol sulfate, and we have that cut-off level of increase in expression level is by the up-regulation of is consistent with a of sokotrasterol sulfate. of the family are known to be for the the cell also shown to be for growth and to be required for of cell growth in response to growth factor the and of growth cell Biol. Scholar). This potential between and vascular The second is the membrane of cells and is for the of the This protein is in and shown to be in endothelial cells Dysferlin and the membrane in Biol. Scholar). While the of this membrane protein remains to be is to that Dysferlin may be required to the of the membrane the of angiogenesis the cell is membrane protein expression and at two other proteins that are up-regulated by sokotrasterol sulfate, Tie-1 and αv are for angiogenesis A. Harlan J.M. Hematology: Basic Principles and Practice. Churchill Livingstone, New York1999: 1770-1782Google Scholar). integrin are clinical trials as in however, Tie-1 shown to be up-regulated by steroid J. of J. Med. 1995; Scholar). These findings have a cut-off of increase This of significantly or other proteins at this borderline level that may be potentially important in vascular studies have to analysis of endothelial cells B. B. J. M. Y. I. and protein expression of human endothelial cells activated with tumor Res. Res. 2003; Scholar, of human endothelial cell and using and mass Scholar, A. A. J. M. B. study of human umbilical vein endothelial cells in 2003; 3: Scholar). In to differential analysis that we this is the analysis of primary endothelial cells in a of primary using proteins were identified A. A. J. M. B. study of human umbilical vein endothelial cells in 2003; 3: Scholar). In a to identify of endothelial and proteins were identified of human endothelial cell and using and mass Scholar). In this we identified of these proteins of human endothelial cell and using and mass Scholar). also identified multiple other membrane proteins known to be in endothelial such as Dysferlin and ALCAM, further for the as in this these findings be important to in angiogenesis but also to involved in angiogenesis. to angiogenesis to tumor growth and metastasis have recently in and and have for a of this area of M. for 2003; Scholar, P. D.J. A of an endothelial growth factor for J. Med. 2003; Scholar). The of molecules that are up-regulated angiogenesis potential toward therapeutic can be

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