Shear Stress as an Inhibitor of Vascular Smooth Muscle Cell Proliferation
Role of Transforming Growth Factor-β1 and Tissue-Type Plasminogen Activator
Abstract We examined whether shear stress can inhibit vascular smooth muscle cell (VSMC) proliferation in vitro directly. Human VSMCs were exposed to fluid flow for 24 hours using a cone-plate apparatus, and their proliferation was inhibited significantly by shear stresses of 1.4 and 2.8 Pa (14 and 28 dyne/cm2), according to the magnitude. Next, we investigated whether transforming growth factor-β1 (TGFβ1), which is known to be an important cytokine that suppresses VSMC proliferation, is the predominant mediator of shear-induced inhibition of VSMC growth. After exposure of VSMCs to shear stress (2.8 Pa) for 24 hours, gene expression of TGFβ1 and, interestingly, tissue-type plasminogen activator, which converts plasminogen to plasmin, an activator of TGFβ1, increased twofold and fivefold, respectively. The levels of both latent and active forms of TGFβ1 in conditioned media of VSMCs exposed to fluid flow increased significantly. An anti-TGFβ1 antibody reversed shear-induced inhibition of VSMC growth significantly. We concluded that shear stress inhibited VSMC proliferation in vitro and this inhibition was mediated predominantly by TGFβ1 in an autocrine manner. These data suggest that shear stress plays an important role as an inhibitor of atherogenesis in endothelium-desquamated lesions.
- shear stress
- vascular smooth muscle cells
- transforming growth factor-β1
- tissue-type plasminogen activator
- Received August 26, 1996.
- Accepted October 24, 1996.
Shear stress, the frictional force acting in the direction of blood flow on the inner surface of blood vessels, has been demonstrated to play a pivotal role in determining the focal localization of atherosclerotic lesions.1 In endothelium-preserved lesions, shear stress has been shown to exert an inhibitory effect on intimal thickening of the carotid artery2 and suppress VSMC proliferation via endothelial cell–derived inhibitory factors in the vascular grafts.3 However, in endothelium-desquamated lesions, such as the balloon injury site after PTCA, little is known about the direct effect of shear stress on VSMCs.
After PTCA, the rate of restenosis due to VSMC proliferation was reported to be significantly lower in widely dilated lesions with intimal dissection than insufficiently dilated lesions,4 even though intense mechanical injury with dissection could activate the VSMC proliferative response further.5 This observation suggests that increased laminar shear stress induced by eliminating poststenotic lesions with low shear stress or vortical flow6 can inhibit VSMC proliferation directly, despite the lack of endothelium.
Several factors, including TGFβ1, nitric oxide, and prostaglandins, were demonstrated to exert inhibitory effects on VSMC proliferation.6 Of these, TGFβ1, a 25-kD homodimeric protein produced by various cells, including VSMCs,7 8 is notable, as it has been reported to cause cell-cycle arrest at the late G1 phase, similar to the manner in which cell growth is inhibited by shear stress.9 10 TGFβ1 is known to be secreted in a latent, biologically inactive form, which is activated by plasmin in VSMCs.11 12 Shear stress has been documented to induce TPA gene expression in endothelial cells.13 Taking these findings together, it is conceivable that shear stress induces TGFβ1 and TPA gene expression in VSMCs and inhibits their proliferation directly.
In this study, we examined the effect of shear stress on human VSMC proliferation in the absence of endothelial cells in vitro and tested our hypothesis that shear stress inhibits VSMC proliferation and that this inhibition is mediated by TGFβ1 in an autocrine manner.
Human VSMCs were explanted from the media of umbilical artery as described previously.14 Primary cells were grown in MCDB131 (Chlorella Co) supplemented with 20% FCS and 40 μg/mL gentamicin at 37°C, under air containing 5% CO2 in a humidified incubator. Cells were subcultured in the same medium containing 10% FCS, and cells from passages 5 to 10 were used for the experiments. VSMCs were identified by their “hill-and-valley” pattern in confluent culture and immunofluorescence staining with HHF35, a muscle-actin–specific monoclonal antibody.15
Shear Stress Apparatus
The cone-plate viscometer described by Sdougos et al16 was modified to expose VSMCs to shear stress in the humidified incubator under the conditions described above. Briefly, two interchangeable cones with cone angles of 8.7×10−3 radian (0.5°) and 1.7×10−2 radian (1°) were made of acetal homopolymer. The acrylic plate was adjusted to accommodate a col- lagen-coated 100-mm polystyrene tissue-culture dish (Iwaki Glass Co) on which VSMCs were grown. Fig 1⇓ shows a diagram of the apparatus. The ratio of the centrifugal to viscous forces (R) and shear stress (τ) were computed using the following equations: and where r is the radius and ω the angular velocity of the cone, α is the cone angle, ν the kinematic viscosity, and μ the static viscosity of the medium.16 The kinematic viscosity was measured using a Cannon-Fenske–type viscometer (Kusano Scientific Instrument Mfg Co Ltd). The static viscosity obtained by multiplying the kinematic viscosity and the density of the medium was 7.3×10−4 Pa · s (0.73 cp) at 37°C. The average R and τ were obtained by integrating their respective values over the area of the tissue-culture dish and dividing these values by the total surface area of the dish. Cones of 1.7×10−2 radian spinning at 25.1 radian/s (4 revolutions per second) and 8.7×10−3 radian spinning at 31.4 radian/s (5 revolutions per second) were used to achieve average respective values of τ=1.4±0.11 Pa (14±1.1 dyne/cm2; average±SEM) with R=0.70±0.18 and τ=2.8±0.059 Pa (28±0.59 dyne/cm2) with R=0.22±0.056. Dye-flow studies showed steady laminar flow in the shear stress range used in these experiments. Measurement of the lactic dehydrogenase levels in the culture media of cells exposed to shear stress demonstrated no significant increase compared with control cell levels, indicating that shear stress induced no cell injury (data not shown).
Human VSMC Proliferation
VSMCs (5×105) were seeded onto collagen-coated 100-mm polystyrene tissue-culture dishes, allowed to reach confluence for 48 hours, and then exposed to shear stresses (1.4 and 2.8 Pa) for 24 hours in medium supplemented with 10% FCS. Under these conditions, the cells were confirmed to be in the logarithmic proliferating phase. Cells were removed by trypsinization, the total cell count was obtained, and the trypan blue exclusion test was performed using a hemocytometer. Static control cells were incubated under similar conditions without being subjected to shear stress and were harvested 48 and 72 hours after seeding. Cell detachment due to increased shear stress was examined in two ways: microscopic examination and counting of cells in the culture medium.
RNA Isolation and Northern Blot Analysis
After exposure to shear stress (2.8 Pa) in serum-free medium for 2, 4, 8, and 24 hours, the total cellular RNA was isolated by the modified acid guanidinium-phenol-chloroform method17 using RNAzol B (Biotecx Laboratories Inc), according to the manufacturer’s instructions. Aliquots (20 μg) of the RNA were electrophoresed in 1.2% agarose/2.2 mol/L formaldehyde gels, transferred onto nylon membranes by capillary elution, and immobilized by baking. Prehybridization and hybridization were performed by using standard procedures. 32P-labeled 40-mer oligonucleotide probes (Oncogene Science) specific for human TGFβ1, TPA, and GAPDH were used for hybridization. After washing, autoradiography and densitometric analysis were performed with a bioimaging analyzer (Fuji Photo Film Co). GAPDH was employed as the internal standard, because the concentration of its mRNA is not affected by shear stress.18
ELISA for TGFβ1
Confluent cells in serum-free medium were exposed to shear stress (2.8 Pa) for 24 hours, then clarified conditioned medium was obtained by centrifugation at 2250g for 20 minutes and stored at −20°C. Assays were performed using an ELISA kit for TGFβ1 (Morinaga Seikagaku Lab Co), according to the manufacturer’s instructions. The detection range of this assay is 156 to 5000 pg/mL. To quantify the total TGFβ1, the latent form was converted to the active form by acidification with 1N HCl for 1 hour at 4°C and then neutralized with 1N NaOH, because the ELISA kit used can detect only the active form of TGFβ1. For the active TGFβ1 assay, samples were concentrated 23-fold to 31-fold in Centriprep-10 and Microcon-10 units (Amicon Inc) without acidification. Conditioned media from static control cultures were assayed in the same manner.
Effect of Anti-TGFβ1 Antibody
Experimental characterization of a rabbit polyclonal anti-human TGFβ1 neutralizing antibody (TGFAb; R&D Systems Inc) demonstrated that TGFAb blocks TGFβ1-induced in- hibition of VSMC proliferation specifically.8 19 TGFAb (100 μg/mL) was added to the medium and the cells were exposed to shear stress (2.8 Pa) for 24 hours, and then they and the static controls were harvested and the cells counted as described above. Cells in medium without antibody or with rabbit IgG (100 μg/mL; Sigma Chemical Co) were subjected to shear stress and treated identically.
The data are expressed as mean±SEM; group means were compared using paired Student’s t test, and differences at P<.05 were considered statistically significant.
Effect of Shear Stress on Human VSMC Proliferation
The number of VSMCs before loading of shear stress was 11.4±1.1×105 cells per dish. Cell numbers increased to 19.2±1.4×105 cells per dish during the next 24 hours (n=6; Fig 2⇓) in the culture medium without shear stress (static control), while the number of cells exposed to a shear stress of 1.4 Pa for 24 hours was 14.0±0.9×105 cells per dish. The decrease in cell proliferation by shear stress was significant compared with the static controls (n=6, P<.05; Fig 2⇓). A higher shear stress (2.8 Pa) further suppressed cell proliferation (12.3±1.4×105 cells per dish, n=6, P<.01; Fig 2⇓). The shear stresses we used were within the range of those encountered in human arteries. The absence of cell detachment was confirmed microscopically in each experiment. Cell numbers in the culture medium after shear stress exposure were also confirmed to be similar to that of static controls (<1% of total cells). The trypan blue exclusion test showed that the viabilities of cells exposed to fluid flow and static control cells did not differ significantly (data not shown).
TGFβ1 and TPA mRNA Levels
The static controls expressed TGFβ1 and TPA mRNA constitutively. TGFβ1 mRNA expression by cells exposed to shear stress (2.8 Pa) for 2 hours increased approximately twofold compared with the static controls, and this shear-induced increase was maintained for almost 24 hours (Fig 3A⇓ and 3B⇓). The TPA mRNA level increased slowly as a result of the exposure to shear stress and after 24 hours reached five times the static control level (Fig 3C⇓ and 3D⇓).
TGFβ1 Protein Levels
The amount of total TGFβ1 in the serum-free conditioned medium after exposure to shear stress (2.8 Pa) for 24 hours increased significantly compared with the static control level (2.0±0.1 versus 1.4±0.2 ng/mL, n=5, P<.05; Fig 4A⇓). The active TGFβ1 level in the serum-free culture medium of the cells subjected to shear stress was also elevated significantly (19.4±1.4 versus 7.8±1.8 pg/mL, n=3, P<.05; Fig 4B⇓). The levels of latent TGFβ1 in medium supplemented with 10% FCS were in the range of 1.1 to 1.7 ng/mL (n=6).
Effect of TGFAb on Shear-Induced Inhibition of VSMC Growth
TGFAb reversed the shear-induced inhibition of VSMC growth significantly. As mentioned above, the number of cells exposed to shear stress (2.8 Pa) for 24 hours was 62.0±3.7% of that of controls. Suppressed cell proliferation due to the shear stress was partially reversed by addition of TGFAb (cell number=87.0±2.3% of control, n=4, P<.01; Fig 5⇓). Control IgG did not show this reversal of effect (cell number=59.5±1.6% of control, n=4; Fig 5⇓).
Both TGFAb and control IgG exerted no significant effect on cell growth in static conditions (data not shown).
In the present study we demonstrated that VSMCs of human arterial origin responded to shear stress with slowed proliferation. This effect was mediated predominantly by TGFβ1, which was produced by VSMCs in response to shear stress and acted in an autocrine fashion. Furthermore, we showed that shear stress markedly increased TPA gene expression of VSMCs. These findings are interesting, as they suggest that shear stress modulates TGFβ1 production and activation and plays an important role in the inhibition of VSMC growth.
As a possible mechanism by which the active form of TGFβ1 was increased, the induction of TPA was demonstrated at the level of mRNA, in addition to the increase of total TGFβ1. The latent form of TGFβ1 exists in circulation. In our experimental conditions, a small but substantial amount of TGFβ1 also exists in the culture medium containing serum. Accordingly, the relative contribution of TGFβ1 in serum to the increase of the active form of TGFβ1 compared with that produced by VSMCs remains to be elucidated. However, it is still very clear that shear stress induces the production and activation of TGFβ1, which plays the central role in the inhibition of VSMC proliferation.
Shear stress has been observed to exert inhibitory effects on atherogenesis by suppressing VSMC proliferation at the site of lesions.6 20 However, most of the relevant studies focused predominantly on the functions of endothelial cells showing a variety of biological responses to shear stress, which could modulate VSMC proliferation.21 22 23 24 25 Nevertheless, the clinical consequences of atherogenesis, particularly that of restenosis after PTCA, cannot be explained only by these endothelium-modulated responses. It has been documented that after balloon catheterization, injured intima can remain uncovered by regenerating endothelium for weeks or months.26 Accordingly, PTCA sites can be in an endothelium-desquamated condition for weeks or even longer. Even under these conditions during which VSMCs are exposed directly to blood flow, laminar shear stress appears to inhibit VSMC proliferation, as widely dilated PTCA sites, in which laminar shear stresses are increased by eliminating residual stenoses that cause low shear stress and vortical flow in their distal sites, showed significantly lower rates of restenosis development due to VSMC proliferation.4 A direct inhibitory effect of shear stress on VSMC proliferation was also suggested by the result of experiments on the rat endothelium-desquamated common carotid artery.27 In their study on bovine cells, Sterpetti et al9 observed inhibition of VSMC proliferation by shear stress in proportion to its magnitude with a range lower than that encountered in arteries,9 but they did not establish the mechanism responsible.
In the present study, we used cells of human arterial origin and shear stresses within the range encountered in human arteries. Therefore, even though this was an in vitro experiment, the results could be relevant to clinical conditions. The data obtained are consistent with and supplement those reported previously,4 9 27 suggesting that shear stress plays an important role as an inhibitor of VSMC proliferation in endothelium-desquamated lesions.
The effect of TGFAb provided evidence that TGFβ1 is a predominant mediator of shear-induced inhibition of human VSMC proliferation. However, the participation of other factors cannot be excluded, as the reversal of shear-induced suppression of VSMC proliferation by TGFAb was not complete. Synthesis of nitric oxide and prostaglandins by VSMCs has been documented to be induced,28 29 although whether this occurs in response to shear stress, as it does in endothelial cells,1 13 remains to be determined.
Selected Abbreviations and Acronyms
|FCS||=||fetal calf serum|
|PTCA||=||percutaneous transluminal coronary angioplasty|
|TGFβ1||=||transforming growth factor-β1|
|TPA||=||tissue-type plasminogen activator|
|VSMC||=||vascular smooth muscle cell|
Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low and oscillatory shear stress. Arteriosclerosis.. 1985;5:293-302.
Kraiss LW, Kirkman TR, Kohler TR, Zierler B, Clowes AW. Shear stress regulates smooth muscle proliferation and neointimal thickening in porous polytetrafluoroethylene grafts. Arterioscler Thromb.. 1991;11:1844-1852.
Leimgruber PP, Roubin GS, Hallman J, Cotsonis GA, Meier B, Douglas JS, King SB III, Gruentzig AR. Restenosis after successful coronary angioplasty in patients with single-vessel disease. Circulation.. 1986;73:710-717.
Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III-47-III-52.
Goustin AS, Leof EB, Shipley G, Moses HL. Growth factors and cancer. Cancer Res.. 1986;46:1015-1029.
Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy versus hyperplasia: autocrine transforming growth factor-β1 expression determines growth response to angiotensin II. J Clin Invest.. 1992;90:456-461.
Flaumenhaft R, Abe M, Sato Y, Miyazono K, Harpel J, Heldin C-H, Rifkin DB. Role of the latent TGF-β binding protein in the activation of latent TGF-β by co-cultures of endothelial and smooth muscle cells. J Cell Biol.. 1993;120:995-1002.
Antonelli-Orlidge A, Saunders KB, Smith SR, D’Amore PA. An activated form of transforming growth factor-β is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci U S A.. 1989;86:4544-4548.
Resnick N, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J.. 1995;9:874-882.
Macleod DC, Strauss BH, de Jong M, Escaned J, Umans VA, van Suylen RA, Verkerk A, de Feyter P-J, Serruys PW. Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions. J Am Coll Cardiol.. 1994;23:59-65.
Malek AM, Greene AL, Izumo S. Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP. Proc Natl Acad Sci U S A.. 1993;90:5999-6003.
McCaffrey TA, Falcone DJ, Brayton CF, Agarwal LA, Welt FGP, Weksler BB. Transforming growth factor-β activity is potentiated by heparin via dissociation of the transforming growth factor-β/α2 macroglobulin inactive complex. J Cell Biol.. 1989;109:441-448.
Ohno M, Cooke JP, Dzau VJ, Gibbons GH. Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production. J Clin Invest.. 1995;95:1363-1369.
Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF Jr, Gimbrone MA Jr. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress–responsive element. Proc Natl Acad Sci U S A.. 1993;90:4591-4595.
Malek AM, Gibbons GH, Dzau VJ, Izumo S. Fluid shear stress differentially modulates expression of genes encoding basic fibroblast growth factor and platelet-derived growth factor B chain in vascular endothelium. J Clin Invest.. 1993;92:2013-2021.
Lindoner V, Giachelli CM, Schwartz SM, Reidy MA. A subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-B chain mRNA. Circ Res.. 1995;76:951-957.
Tohda K, Masuda H, Kawamura K, Shozawa T. Difference in dilatation between endothelium-preserved and -desquamated segments in the flow-loaded rat common carotid artery. Arterioscler Thromb.. 1992;12:519-528.
Fukuo K, Inoue T, Morimoto S, Nakahashi T, Yasuda O, Kitano S, Sasada R, Ogihara T. Nitric oxide mediates cytotoxicity and basic fibroblast growth factor release in cultured vascular smooth muscle cells: a possible mechanism of neovascularization in atherosclerotic plaques. J Clin Invest.. 1995;95:669-676.