Mechanical Strain Induces Specific Changes in the Synthesis and Organization of Proteoglycans by Vascular Smooth Muscle Cells

In the mechanically active environment of the artery, cells sense mechanical stimuli and regulate extracellular matrix structure. In this study, we explored the changes in synthesis of proteoglycans by vascular smooth muscle cells in response to precisely controlled mechanical strains. Strain increased mRNA for versican (3.2-fold), biglycan (2.0-fold), and perlecan (2.0-fold), whereas decorin mRNA levels decreased to a third of control levels. Strain also increased versican, biglycan, and perlecan core proteins, with a concomitant decrease in decorin core protein. Deformation did not alter the hydrodynamic size of proteoglycans as evidenced by molecular sieve chromatography but increased sulfate incorporation in both chondroitin/dermatan sulfate proteoglycans and heparan sulfate proteoglycans (p < 0.05 for both). Using DNA microarrays, we also identified the gene for the hyaluronan-linking protein TSG6 as mechanically induced in smooth muscle cells. Northern analysis confirmed a 4.0-fold increase in steady state mRNA for TSG6 following deformation. Size exclusion chromatography under associative conditions showed that versican-hyaluronan aggregation was enhanced following deformation. These data demonstrate that mechanical deformation increases specific vascular smooth muscle cell proteoglycan synthesis and aggregation, indicating a highly coordinated extracellular matrix response to biomechanical stimulation. In the mechanically active environment of the artery, cells sense mechanical stimuli and regulate extracellular matrix structure. In this study, we explored the changes in synthesis of proteoglycans by vascular smooth muscle cells in response to precisely controlled mechanical strains. Strain increased mRNA for versican (3.2-fold), biglycan (2.0-fold), and perlecan (2.0-fold), whereas decorin mRNA levels decreased to a third of control levels. Strain also increased versican, biglycan, and perlecan core proteins, with a concomitant decrease in decorin core protein. Deformation did not alter the hydrodynamic size of proteoglycans as evidenced by molecular sieve chromatography but increased sulfate incorporation in both chondroitin/dermatan sulfate proteoglycans and heparan sulfate proteoglycans (p < 0.05 for both). Using DNA microarrays, we also identified the gene for the hyaluronan-linking protein TSG6 as mechanically induced in smooth muscle cells. Northern analysis confirmed a 4.0-fold increase in steady state mRNA for TSG6 following deformation. Size exclusion chromatography under associative conditions showed that versican-hyaluronan aggregation was enhanced following deformation. These data demonstrate that mechanical deformation increases specific vascular smooth muscle cell proteoglycan synthesis and aggregation, indicating a highly coordinated extracellular matrix response to biomechanical stimulation. The vascular smooth muscle cell plays a prominent role in development and maintenance of arterial structure. Vascular smooth muscle cells are the primary source of arterial extracellular matrix (ECM), 1The abbreviations used are: ECMextracellular matrixASMCarterial smooth muscle cellsPAGEpolyacrylamide gel electrophoresisLDLlow density lipoprotein including collagens, elastic fibers, and several proteoglycans (1Wight T.N. Fuster V. Ross R. Topol E.J. Atherosclerosis and Coronary Artery Disease. Lippincott-Raven Publishers, Philadelphia, PA1996: 421-440Google Scholar). Proteoglycans serve several functions in the artery wall, including regulation of cell adhesion, migration, and proliferation (2Wight T.N. Kinsella M.G. Qwarnstrom E.E. Curr. Opin. Cell Biol. 1992; 4: 793-801Crossref PubMed Scopus (344) Google Scholar, 3Wight T.N. Arteriosclerosis. 1989; 9: 1-20Crossref PubMed Google Scholar, 4Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Abstract Full Text PDF PubMed Scopus (1223) Google Scholar). The major proteoglycan in the arterial ECM synthesized by arterial smooth muscle cells (ASMC) is the large chondroitin sulfate proteoglycan versican, also known as PG-M (5Yao L.Y. Moody C. Schonherr E. Wight T.N. Sandell L.J. Matrix Biol. 1994; 14: 213-225Crossref PubMed Scopus (106) Google Scholar, 6Lemire J.M. Braun K.R. Maurel P. Kaplan E.D. Schwartz S.M. Wight T.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1630-1639Crossref PubMed Scopus (76) Google Scholar, 7Gutierrez P. O'Brien K.D. Ferguson M. Nikkari S.T. Alpers C.E. Wight T.N. Cardiovasc. Pathol. 1997; 6: 271-278Crossref PubMed Scopus (55) Google Scholar, 8Schonherr E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar, 9Wight T.N. Lara S. Riessen R. Le Baron R. Isner J. Am. J. Pathol. 1997; 151: 963-973PubMed Google Scholar). Versican is a member of a family of proteoglycans including brevican, neurocan, and aggrecan that can bind hyaluronan and form large aggregates (10Margolis R.K. Margolis R.U. Methods Enzymol. 1994; 245: 105-126Crossref PubMed Scopus (90) Google Scholar, 11Zimmermann D.R. Iozzo R.V. Proteoglycans, Structure, Biology and Molecular Interactions. Marcel Dekker, NY2000: 327-342Google Scholar). These large aggregates contribute to tissue mechanical properties, providing a hydrated sponge-like matrix that resists or cushions against deformation (12Grodzinsky A.J. Crit. Rev. Biomed. Eng. 1983; 9: 133-199PubMed Google Scholar). Arteries also contain smaller proteoglycans that contain dermatan sulfate glycosaminoglycans such as decorin and biglycan, which interact with other ECM proteins and with macromolecules that enter the vascular wall such as low density lipoproteins (13Chait A. Wight T.N. Curr. Opin. Lipid. 2000; 11: 457-463Crossref PubMed Scopus (70) Google Scholar). In addition, blood vessels contain perlecan, which is a heparan sulfate proteoglycan associated with basal lamina surrounding ASMC (2Wight T.N. Kinsella M.G. Qwarnstrom E.E. Curr. Opin. Cell Biol. 1992; 4: 793-801Crossref PubMed Scopus (344) Google Scholar). extracellular matrix arterial smooth muscle cells polyacrylamide gel electrophoresis low density lipoprotein Vascular smooth muscle cells are under dynamic mechanical stresses from arterial pressure, and their responses to mechanical stimuli have therefore been of long-standing interest. In the past decade, improvements in bioengineering have provided much more precise and uniform methods of cell deformation (14Brown T.D. J. Biomech. 2000; 33: 3-14Crossref PubMed Scopus (472) Google Scholar). Using DNA microarrays and a device that provides a precise and uniform biaxial strain profile, we have shown that small mechanical deformations, well below the amplitudes that cause cell injury, induce highly specific molecular events in ASMC (15Feng Y. Yang J.H. Huang H. Kennedy S.P. Turi T.G. Thompson J.F. Libby P. Lee R.T. Circ. Res. 1999; 85: 1118-1123Crossref PubMed Scopus (128) Google Scholar). These events include induction of several genes that may affect arterial extracellular matrix, including tenascin-C and plaminogen activator inhibitor-1. In addition, small deformations specifically suppress matrix metalloproteinase-1, an enzyme that can initiate degradation of fibrillar collagen (16Yang J.H. Briggs W.H. Libby P. Lee R.T. J. Biol. Chem. 1998; 273: 6550-6555Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These studies indicate that deformation regulates smooth muscle cell ECM metabolism and suggest that ASMCs may modify their biomechanical environment in a manner that limits potential biomechanical injury. Because proteoglycans are ECM molecules that can play a prominent role in tissue mechanics, this study was designed to determine whether mechanical strain induced specific changes in the synthesis of proteoglycans and altered their ability to interact with other ECM molecules. ASMC were prepared from explants from excess aortic tissue from the donor at the time of organ harvest for orthotopic cardiac transplantation at Brigham and Women's Hospital. ASMC were maintained in Dulbecco's modified essential medium, 10% fetal calf serum and 1% penicillin/streptomycin sulfate (16Yang J.H. Briggs W.H. Libby P. Lee R.T. J. Biol. Chem. 1998; 273: 6550-6555Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) at 37 °C, 5% CO2 up to passage 6–7 for experiments. The Brigham and Women's Hospital Committee for Human Research approved the protocol. Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured, an approach that provides a nearly homogeneous biaxial strain profile. Each culture dish consists of a plastic cylinder and a circular silicone elastometric membrane, which is the culture surface. The membrane undergoes cyclic tensile deformation as the platen assembly moves sinusoidally. We have previously measured membrane strains with a high-resolution video device (17Cheng G.C. Briggs W.H. Gerson D.S. Libby P. Grodzinsky A.J. Gray M.L. Lee R.T. Circ. Res. 1997; 80: 28-36Crossref PubMed Scopus (121) Google Scholar); for this study, all experiments were performed with 4% cyclic strain, a magnitude of strain that does not lead to cell injury but reproducibly induces a restricted set of genes (15Feng Y. Yang J.H. Huang H. Kennedy S.P. Turi T.G. Thompson J.F. Libby P. Lee R.T. Circ. Res. 1999; 85: 1118-1123Crossref PubMed Scopus (128) Google Scholar). Culture membranes were precoated with 2 μg/ml serum fibronectin in 13 ml of Hank's solution for 24 h at 4 °C and then washed twice with 10 ml of phosphate-buffered saline. ASMC were plated on the coated membrane dish at a density of 6 × 105 cells/dish in 13 ml of Dulbecco's modified essential medium containing 10% fetal bovine serum and incubated for 24 h. Before mechanical strain was applied, 10 ml of fresh medium was exchanged. To eliminate the variable of time-dependent changes because of cell age or effects of adhesion to fibronectin or the membrane in each experiment, all cells were cultured on the membrane for an identical time period, and cells and media from all samples were harvested at the same time. For example, in a time course experiment with strain, the time point represents the time prior to harvest that strain was initiated, such that the strain sample and control sample were harvested at the same time. To assess proteoglycan synthesis, cells were labeled with 100 μCi per ml Na235SO4 for various times. The medium was combined with a 0.1 volume of 10× protease inhibitors dissolved in 8 m urea buffer (8 m urea, 2 mm EDTA, 0.25 m NaCl, 50 mmTris-HCl, and 2% Triton X-100 detergent, pH 7.4, Ref. 8Schonherr E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar). The cell layer was washed with phosphate-buffered saline and scraped into 8m urea buffer with 1× protease inhibitors (5 mm benzamidine, 10 mm 6-aminohexanoic acid, and 1 mm phenylmethylsulfonyl fluoride, Ref. 18Kinsella M.G. Wight T.N. J. Biol. Chem. 1988; 263: 19222-19231Abstract Full Text PDF PubMed Google Scholar). Total 35Ssulfate incorporation into proteoglycans was determined by CPC precipitation (19Wasteson A. Uthne K. Westermark B. Biochem. J. 1973; 136: 1069-1074Crossref PubMed Scopus (156) Google Scholar). Medium and cell layer extracts were purified and concentrated by ion exchange chromatography on DEAE-Sephacel in 8 m urea buffer and eluted with 8m urea buffer containing 3 m NaCl (20Olin K.L. Potter-Perigo S. Barrett P.H. Wight T.N. Chait A. J. Biol. Chem. 1999; 274: 34629-34636Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Aliquots of DEAE-purified material containing 30,000 dpm 35S were precipitated in 80% ethanol and 1.3% potassium acetate. The resulting pellet was then applied directly to SDS-PAGE or digested by incubation prior to chromatography with 2.3 units per ml chondroitin ABC lyase (Sigma) in Tris-buffered solution (45 mm Tris, 0.09 mg/ml bovine serum albumin, 2.7 mm sodium acetate, pH 8.0, Ref.21Saito H. Yamagata T. Suzuki S. J. Biol. Chem. 1968; 243: 1536-1542Abstract Full Text PDF PubMed Google Scholar), or in heparitinase I and II (Sigma) in Tris-buffered solution (45 mm Tris, 0.09 mg/ml bovine serum albumin, 10 mmcalcium acetate, pH 7, Ref. 22Chang M.Y. Olin K.L. Tsoi C. Wight T.N. Chait A. J. Biol. Chem. 1998; 273: 15985-15992Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). To determine the size classes of 35Ssulfate-labeled proteoglycans synthesized and secreted by the cells, medium and cell layer extracts, purified and concentrated over DEAE-Sephacel, were applied to 8 mm × 113 cm-Sepharose CL2B molecular sieve column in 4 m guanidine buffer (4 m guanidine, 10 mm EDTA, 0.5% Triton X-100 detergent, 50 mm sodium acetate, pH 7.4) and collected in 0.5-ml fractions (23Hascall V.C. Calabro A. Midura R.J. Yanagishita M. Methods Enzymol. 1994; 230: 390-417Crossref PubMed Scopus (91) Google Scholar). Total RNA was isolated using guanidine isothiocyanate solubilization followed by phenol extraction at pH 4 and subsequent precipitation in isopropyl alcohol (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65696) Google Scholar). Purified samples were fractionated on 1.0% formaldehyde-agarose gels, alkali denatured in 50 mm NaOH, 10 mm NaCl, and transferred to nylon blotting membranes (Zeta Probe; Bio-Rad Laboratories, Richmond, CA). Blots were hybridized with cDNA probes to the following matrix molecules: human versican, clone 7 (25Zimmerman D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (535) Google Scholar); human biglycan (26Fisher L.W. Heegaard A.M. Vetter U. Vogel W. Just W. Termine J.D. Young M.F. J. Biol. Chem. 1991; 266: 14371-14377Abstract Full Text PDF PubMed Google Scholar), clone p16; human collagen type I, clone HF677 (27Chu M.L. Myers J.C. Bernard M.P. Ding J.F. Ramirez F. Nucleic Acids Res. 1982; 10: 5925-5934Crossref PubMed Scopus (410) Google Scholar); human perlecan, clone HS-1 (28Cohen I.R. Grassel S. Murdoch A.D. Iozzo R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar), and bovine decorin Termine J.D. Young Biochem. J. 1987; PubMed Scopus Google Scholar). These were from Research of of Philadelphia, and of For of mRNA levels were using and to the of RNA as by Cell were harvested as for proteoglycan analysis into 8 m urea buffer with protease concentrated over DEAE-Sephacel, and precipitated with were then digested with chondroitin ABC lyase or heparitinase I and applied to SDS-PAGE and transferred to membranes and using a Cell for of biglycan and Versican and were transferred using a Bio-Rad Cell. The transferred proteins were then with a of primary and enhanced with specific for biglycan and decorin P. L.W. Young M.F. Termine J.D. J. PubMed Scopus Google Scholar) were a from Research of to versican J. Full Text PDF PubMed Scopus Google Scholar) and perlecan Ref. C. Yang W. A.D. J. Biochem. PubMed Scopus Google Scholar) were The of at and of to TSG6 were the of of W.H. J.D. J. 1994; 33: PubMed Scopus Google Scholar). Medium containing 35Ssulfate-labeled proteoglycans from cells to 4% strain for or was into an associative buffer mm sodium acetate, detergent, pH by passage a × size exclusion column of The was to eliminate into and applied to a × 50 molecular sieve column in the same associative to of the was digested with 2 units (Sigma) at 37 °C for 24 h. associative proteoglycans containing aggregates in the of of the for by the of the proteoglycans to a T.N. V.C. J. Cell Biol. 1983; PubMed Scopus (90) Google Scholar, J.H. V.C. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Human ASMC were to 4% strain for or h prior to harvest of all samples at the same with media changes in the Northern analysis a time-dependent increase in mRNA levels for versican, biglycan, perlecan, and type I collagen The increase in versican mRNA at h. of versican and biglycan mRNA were at h whereas of perlecan were at 24 indicating that the synthesis of proteoglycans may by In decorin mRNA by h the of strain and low the In strain we changes in gene the h of To the of strain on proteoglycan synthesis, ASMC were labeled with 35Ssulfate for the h of or h of 4% increase in incorporation of 35Ssulfate into as determined by CPC at h < The increase in proteoglycans was in both the cell layer and the The and heparan sulfate of proteoglycans were determined by chondroitin ABC lyase of the cell and medium followed by CPC precipitation 2 The increase in chondroitin/dermatan sulfate synthesis was in the medium at h < whereas that of the heparan sulfate proteoglycans in the medium was at h < with the increase in perlecan mRNA levels In the cell heparan sulfate incorporation a at h < Because the heparan sulfate of the medium was twice that of the cell the of cell and medium heparan sulfate at h. To determine whether strain altered the or size of the classes of proteoglycan synthesized by proteoglycans collected h strain were isolated by ion exchange and to molecular sieve chromatography on major eluted at and in the medium and and in the cell layer The major of the large is versican and the smaller is a of biglycan and decorin E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar). major were in the of control or strain The of material secreted into the medium was in the indicating increased synthesis in response to is with increased levels of versican mRNA The of a in hydrodynamic size in the versican that the increased of 35Ssulfate-labeled material is a of increased synthesis of the proteoglycan core proteins and their of as been for the synthesis of proteoglycans by ASMC by E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar). small in the of the in the medium to indicating a hydrodynamic was because of an increase in the of biglycan to decorin in that because biglycan is a E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar). is by the increase in mRNA and protein of biglycan by strain 1 and and the decrease in mRNA and protein for decorin 1 and Proteoglycans from cells to strain for h were applied to and biglycan as in the gel whereas versican in the gel E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar). in the of biglycan by this the other the of the decorin in the medium was to of the control is with of decorin mRNA by cells to strain analysis was performed on medium and cell layer extracts from ASMC to strain for h 4 were in the levels of versican, biglycan, and Versican core protein was increased to of control levels in both medium and cell was increased to and of control levels in the medium and cell The increase in biglycan levels not determined by because levels were in medium of In the of decorin core protein was to 13 and of control levels in medium and cell In DNA experiments with the that were at the same time as we that the gene for the protein TSG6 was a small set of genes increased by 4% strain in Northern analysis confirmed that mRNA for TSG6 was reproducibly increased by strain and strain to increases in a by in a analysis under conditions not to a size that is the molecular of TSG6 in the cell or a at are with with the identical used in this that secreted TSG6 can form with other molecules W.H. 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The of material at the volume of a molecular sieve column under associative conditions in the control medium proteoglycans that volume in medium from cells 6 the of a of hyaluronan and of the samples with prior to chromatography all of the from cells to the volume of the whereas on the column with control These demonstrate that mechanical strain induces proteoglycan aggregation with In this study, we that highly controlled biomechanical deformation induces the synthesis of specific vascular proteoglycans including versican, biglycan, and perlecan, whereas decorin was We also increased of the protein mechanical strain increased proteoglycan aggregation, that the response to deformation is highly Because DNA experiments demonstrate that genes are altered by mechanical strain in cells (15Feng Y. Yang J.H. Huang H. Kennedy S.P. Turi T.G. Thompson J.F. Libby P. Lee R.T. Circ. Res. 1999; 85: 1118-1123Crossref PubMed Scopus (128) Google Scholar), experiments the restricted and specific molecular regulation of ASMC by biomechanical Vascular smooth muscle cells a role in arterial conditions such as the vascular smooth muscle cell sense the changes and These responses include changes in the extracellular matrix, and prior studies have that mechanical strain increases collagen and proteoglycan synthesis by cells S. PubMed Scopus Google Scholar, A.J. 1988; PubMed Scopus Google Scholar, W. J. 2000; 14: PubMed Scopus Google Scholar). the molecular of the proteoglycan increase not been Because proteoglycans can have we that not all vascular proteoglycans These experiments demonstrate that decorin is specifically decreased by Because decorin to collagen D.R. J.D. R. Iozzo R.V. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and regulates collagen H. H. Iozzo R.V. J. 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Pathol. 1994; Google Scholar). whereas of proteoglycans may contribute to mechanical of the artery, may also a for In this study, we demonstrate that biomechanical strain of biglycan and versican, vascular proteoglycans that can serve as enhanced proteoglycan synthesis may as an against mechanical that can as the