Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling. Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling. Coronary collateralization or arteriogenesis is a vascular adaptive remodeling process that occurs in the context of arterial occlusion when preexisting collateral arterioles remodel to form larger diameter bypass arteries. The current arteriogenesis paradigm implicates local hemodynamics, recruitment/activation of monocytes, and structural remodeling of the vascular wall as key steps in collateralization (1Heil M. Schaper W. Coron. Artery Dis. 2004; 15: 373-378Crossref PubMed Scopus (34) Google Scholar). Two primary stages of collateralization have been described based predominantly on observations from femoral artery ligation models that induce collateral remodeling via an instantaneous increase in collateral flow. The initial stage of collateralization is characterized by a wave of monocyte-driven inflammation and rapid vasodilation, an immediate compensatory response to increased collateral flow. In the second phase of collateralization, the initial inflammatory response is resolved, and the newly formed conductance artery wall is stabilized (2Schaper W. Scholz D. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1143-1151Crossref PubMed Scopus (295) Google Scholar). To date, considerable attention has been dedicated to investigating the inflammatory regulation of this process (i.e. MCP-1 and GM-CSF), but despite evidence on the importance of the endothelium and endothelium-derived factors in flow-mediated arterial remodeling (3Langille B.L. O’Donnell F. Science. 1986; 231: 405-407Crossref PubMed Scopus (936) Google Scholar, 4Vita J.A. Holbrook M. Palmisano J. Shenouda S.M. Chung W.B. Hamburg N.M. Eskenazi B.R. Joseph L. Shapira O.M. Circulation. 2008; 117: 3126-3133Crossref PubMed Scopus (37) Google Scholar, 5Rudic R.D. Shesely E.G. Maeda N. Smithies O. Segal S.S. Sessa W.C. J. Clin. Invest. 1998; 101: 731-736Crossref PubMed Scopus (706) Google Scholar), less is known about how altered collateral vessel shear stress acting on the endothelium affects collateralization. Characterizing the specific role collateral flow plays in regulating functional endothelial cell phenotypes, and endothelium-dependent processes associated with adaptive remodeling should therefore provide a framework to better understand the establishment, maintenance, and remodeling of arteries. A number of signaling molecules have previously been characterized for their role in controlling smooth muscle cell phenotypes in both blood vessel maturation and vascular remodeling. For example, mice lacking platelet-derived growth factor B, S1P1 (sphingosine-1 phosphate-1), or KLF2 (Kruppel-like factor 2) are embryonic lethal due to defects in vascular wall formation and function (6Jain R.K. Nat. Med. 2003; 9: 685-693Crossref PubMed Scopus (2055) Google Scholar, 7Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (312) Google Scholar, 8Lee J.S. Yu Q. Shin J.T. Sebzda E. Bertozzi C. Chen M. Mericko P. Stadtfeld M. Zhou D. Cheng L. Graf T. MacRae C.A. Lepore J.J. Lo C.W. Kahn M.L. Dev. Cell. 2006; 11: 845-857Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 9Lindahl P. Hellstrom M. Kalen M. Betsholtz C. Curr. Opin. Lipidol. 1998; 9: 407-411Crossref PubMed Scopus (94) Google Scholar, 10Wu J. Bohanan C.S. Neumann J.C. Lingrel J.B. J. Biol. Chem. 2008; 283: 3942-3950Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Wani M.A. Means Jr., R.T. Lingrel J.B. Transgenic Res. 1998; 7: 229-238Crossref PubMed Scopus (117) Google Scholar). Models of arterial injury have also identified matrix-altering factors (namely transforming growth factor β (TGF-β) 4The abbreviations used are: TGF-β, transforming growth factor-β; ACC, adaptive remodeling coronary collateral (waveform); NCC, normal coronary collateral (waveform); μFD, microfluidic device; EC, endothelial cell; SMC, smooth muscle cell; EGM-2, endothelial growth medium-2; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione S-transferase. and bone morphogenic proteins) as critical in regulating smooth muscle cell proliferation and migration, and evidence exists indicating that the endothelium contributes to the in vivo production of these factors (12Majesky M.W. Lindner V. Twardzik D.R. Schwartz S.M. Reidy M.A. J. Clin. Invest. 1991; 88: 904-910Crossref PubMed Scopus (473) Google Scholar, 13Song R.H. Kocharyan H.K. Fortunato J.E. Glagov S. Bassiouny H.S. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 923-930Crossref PubMed Scopus (37) Google Scholar, 14Corriere M.A. Rogers C.M. Eliason J.L. Faulk J. Kume T. Hogan B.L. Guzman R.J. J. Surg. Res. 2008; 145: 142-149Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Furthermore, constitutively active angiopoietin 1 signaling through its endothelial receptor Tie2 has been shown to lead to vascular wall hyperplasia (15Sullivan C.C. Du L. Chu D. Cho A.J. Kido M. Wolf P.L. Jamieson S.W. Thistlethwaite P.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12331-12336Crossref PubMed Scopus (126) Google Scholar). 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To the of how collateral flow acting on the endothelium affects adaptive remodeling we previously of the shear stress waveforms in coronary and used these waveforms to the effect of collateral flow on EC gene expression and the subsequent flow-mediated endothelial paracrine effects on SMC molecular using a three-dimensional microfluidic that vascular cell we that collateral flow-mediated EC genes significantly influence SMC migration, a critical process the stages of collateralization C. S. D. Scholz D. A. Schaper W. Schaper J. J. 1998; Full Text PDF PubMed Scopus Google Scholar). of lumped parameter hemodynamic model was to simulate shear stress waveforms acting the coronary collateral and remodeling coronary collateral vessels A number of were for the based on is known the of the coronary collateral a was in the coronary collateral vessels and to the of occlusion in the vessel vessel in A and collateral has been in of with coronary J. Circulation. PubMed Scopus Google Scholar). both the collateral and (i.e. number of collateral vessels a have been to in and of J. Circulation. PubMed Scopus Google Scholar), we a collateral diameter of is with human with flow a coronary artery was based on the of a critical and is to B.R. Rogers L. T. Circulation. PubMed Scopus Google Scholar). from previously was used to for the T. R.D. J. PubMed Scopus Google Scholar). These results were then used to the instantaneous vessel for both the and and subsequently the wall shear stress acting the collateral vessel by the the vessel and is the vessel for of the human EC were and to shear stress as previously described S. S. B.R. R.D. Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar). and growth factor medium was used for the shear experiments with and with a of stress to EC using the and shear stress waveforms was using a flow S. S. B.R. R.D. Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar). For the of KLF2 EC were a cell of and the were with a or a control a of of for the connective tissue growth factor EC were a of and The cells were with of a by of the to by of M.A. J. 2004; PubMed Scopus Google and for using the EC were with the KLF2 or control as described an the was and of vascular SMC were and in growth medium to the For experiments in conditioned medium flow-mediated endothelium-derived factors was to SMC, SMC were on number to to conditioned medium and in medium growth medium with the and the of and shear stress waveforms on EC were in the flow 1 to to the downstream SMC via control experiments were with SMC and in the but in the of and of for EC transcriptional profiling were to the for the using distinct genes and as previously described Jr., M.A. J. Clin. Invest. 2006; PubMed Scopus Google Scholar). The were for and using regulation of and flow-mediated genes was then in for using experiments and gene regulation for J. S. Jr., M.A. 2004; PubMed Scopus Google Scholar). regulated genes were then by function using the on the and with the on the The were as a to the number and from EC and SMC used for was using and using the to the was by and was by an with was using a The was in of and to a microfluidic was of using with as described by of 1997; 7: Scopus Google Scholar). The were and then for in and with number 1 to form microfluidic The were for to and then a was the and and the were with and for the was the three-dimensional and for in to form a three-dimensional for cell the was then the microfluidic and and for to the for cell The was then by cell medium and to for cell and EC were via of the three-dimensional a of in endothelial growth and for to SMC in the microfluidic was for co-culture medium with and endothelial cell growth in to the three-dimensional to with the co-culture SMC were subsequently with the fluorescent to the and in the the endothelial a of The co-culture was then and for and of SMC For connective tissue growth factor migration the were and the was as described SMC were on the as in the co-culture medium described SMC three-dimensional for both co-culture and migration experiments was by cell with of the cells were in for with for and in of cell and SMC were then for the three-dimensional using an and a the of of the SMC be from the EC, with the of SMC the three-dimensional The of cell number three-dimensional and number of cells of co-culture was using this the a assay was used to the and were from of the that the J.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), with of the a and of the in The were as to to to to and to The and were to To the of were with of human KLF2 to glutathione with in the of 1 for the were by and to a was with the was by of for experiments with and were by for of less were for the is described of Coronary lumped parameter model was to the shear stress waveforms on coronary collateral endothelium A and The of the model was to simulate collateral vessel shear stress waveforms in the normal coronary collateral when is and in the adaptive remodeling coronary collateral to a reduction of the coronary previously W. Neumann F. W. J. Full Text PDF PubMed Scopus Google or (i.e. vascular and to vessel of the coronary circulation and the T. R.D. J. PubMed Scopus Google and to the and models distinct collateral vessel waveforms and These previously collateral waveforms that provide an of the shear stress by both a normal collateral vessel and a collateral vessel adaptive remodeling. coronary collateral waveforms were applied to cultured human EC for and differences in the resulting EC molecular were by genome-wide transcriptional In genes were to have as by J. S. Jr., M.A. 2004; PubMed Scopus Google the regulation of endothelial genes that have previously been shown to influence both and vascular in the and the collateral flow-mediated endothelial gene expression the regulation of and transcription factor flow up-regulated expression of genes and endothelial and for arterial and and of and transcription factors and flow endothelial genes with growth factor and inflammation morphogenic and morphogenic transcription and expression of collateral flow-mediated endothelial genes identified by transcriptional profiling and associated with adaptive genes are previously transcriptional targets of KLF2 protein; factor transcription of 1 growth factor growth tissue growth genes are previously transcriptional targets of KLF2 Jr., M.A. J. Clin. Invest. 2006; PubMed Scopus Google in a The collateral flow-mediated transcriptional profiling with previously endothelial from also that a of these collateral flow-mediated both and are transcriptional targets of the endothelial transcription factor KLF2 in Jr., M.A. J. Clin. Invest. 2006; PubMed Scopus Google Scholar). transcription factors were also to be differentially regulated by the collateral waveforms and of the up-regulated by the displayed as evidence as KLF2 for controlling the formation and of the vessel wall C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (312) Google Scholar, 10Wu J. Bohanan C.S. Neumann J.C. Lingrel J.B. J. Biol. Chem. 2008; 283: 3942-3950Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). KLF2 from the as a collateral transcription factor that as a of endothelium-dependent events critical for adaptive arterial remodeling. The of KLF2 by the was by with several genes that have previously been shown to be on KLF2 including the of and and the down-regulation of the vascular genes and Jr., M.A. J. Clin. Invest. 2006; PubMed Scopus Google Scholar). Collectively, these transcriptional expression demonstrate that collateral flow a distinct endothelial molecular associated with arterial vessels and provide a of collateral flow-mediated endothelial genes that a role in adaptive remodeling. on the to the collateral flow waveforms acting on EC have paracrine to differentially smooth muscle gene we a that the of human smooth muscle cells (SMC) flow-mediated EC conditioned medium The SMC was the of the collateral flow experiments and EC and SMC, effects by interactions. this system, of SMC to flow-mediated EC conditioned medium for in increased SMC expression of a transcriptional for the expression of SMC B.R. 2004; PubMed Scopus Google Scholar, R.D. J.M. Arterioscler. Thromb. Vasc. 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In to the effect of endothelial KLF2 expression on SMC migration, we a three-dimensional assay to SMC migration in response to factors in co-culture or in response to exogenous factors in The three-dimensional used was a flow by three-dimensional that were with a cells the flow can the three-dimensional and via paracrine the V. J. Chung S. on a 2008; PubMed Scopus Google Scholar). The effect of EC KLF2 expression on SMC migration was characterized by SMC with KLF2 EC in of the three-dimensional SMC migration and the three-dimensional was compared co-culture endothelium control and of three-dimensional SMC migration for the co-culture that a significantly number of SMC the three-dimensional co-culture with compared with both the endothelium and co-culture as a of cell number migration as as the number of SMC the three-dimensional was when SMC migration in co-culture with EC to control EC These three-dimensional SMC migration results demonstrate that the effect of KLF2 expression in EC to reduce SMC migration and provide the molecular used by endothelial cells to control SMC sought to a endothelium-derived factor that for the in three-dimensional SMC migration and on is an EC gene that was to be by the has been shown by to migration in cell A. M. D. D.R. M. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar, F. D. D. M. C. F. 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Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). In this we with molecular and functional vascular cell and were to that collateral flow endothelial gene expression to adaptive remodeling and that the resulting endothelial have the to both smooth muscle gene expression and of the genes characterized by genome-wide transcriptional profiling to be differentially regulated by the coronary collateral waveforms have previously been to critical processes with adaptive remodeling. the increased the expression of genes important for vessel and vascular and and of the arterial and of collateral vessels has been to be on endothelial the initial stage of collateral S. E. S. S. S. M. Schaper W. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar), in vivo evidence a role for the and in both arterial and adaptive coronary remodeling Lu M.M. J.A. Proc. Natl. Acad. Sci. U. S. A. 2008; PubMed Scopus Google Scholar, N. 2000; PubMed Scopus Google Scholar, A. M. D. T. A. 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Furthermore, when we flow-mediated EC conditioned medium to cultured SMC, the SMC displayed gene expression with the that collateral flow vascular wall In particular, we expression of and increased expression of SMC as as the transcriptional targets and The that collateral flow acting on the endothelium results in paracrine of the SMC an important associated with adaptive remodeling. The transcription factor KLF2 from the collateral flow-mediated EC transcriptional as a key in regulating an adaptive remodeling endothelial response based on evidence of its flow and to the function of the vessel A of the of transcription has been shown to be regulated by flow Jr., M.A. J. Clin. Invest. 2006; PubMed Scopus Google Scholar, R.J. S. R.D. S. E. A.J. 100: PubMed Scopus Google Scholar, N. S. Lingrel J.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. N. Lingrel J.B. Biol. Chem. 2004; PubMed Scopus Google and critical for controlling endothelial phenotypes Jr., M.A. J. 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Cell. 2006; 11: 845-857Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), a by J. Bohanan C.S. Neumann J.C. Lingrel J.B. J. Biol. Chem. 2008; 283: 3942-3950Abstract Full Text Full Text PDF PubMed Scopus (74) Google that the of vessels of mice is and that SMC of these mice a with when compared with of these however, displayed that the vascular associated with KLF2 via the of smooth muscle associated with vessel wall and evidence to the functional effect of EC KLF2 expression on with SMC important for adaptive remodeling. Using three-dimensional SMC migration as a functional SMC results that EC KLF2 expression SMC migration and that this effect can be rescued by the exogenous of an factor expression is by KLF2 the transcriptional Furthermore, using a we that KLF2 can the to of the was of that in the a these the that KLF2 has a role in adaptive remodeling by controlling SMC function and that regulation of SMC migration to the vascular wall defects in KLF2 Furthermore, the three-dimensional migration assay as used in these experiments for cell EC and SMC, indicating that the effect of EC KLF2 expression on SMC migration is due to the regulation and of and to be identified EC the results demonstrate that collateral flow events critical for adaptive including endothelial gene expression and endothelium-derived paracrine that smooth muscle gene expression and functional In particular, endothelial genes regulated by collateral flow were to control smooth muscle migration, a that has important for how influence endothelial with smooth muscle cells. these that an endothelial response to flow adaptive remodeling critical events to the blood vessel wall and and for in for the and of the endothelial cells used in these of for on the flow their stages of and and Chen of for the