This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ueba, H. Articles by Yaginuma, T. Search for Related Content PubMed PubMed Citation Articles by Ueba, H. Articles by Yaginuma, T.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1512-1516.)
© 1997 American Heart Association, Inc.

Articles

Shear Stress as an Inhibitor of Vascular Smooth Muscle Cell Proliferation

Role of Transforming Growth Factor-ß1 and Tissue-Type Plasminogen Activator

Hiroto Ueba; Masanobu Kawakami; ; Toshio Yaginuma

From the Division of Arteriosclerosis and Metabolism (M.K.), Department of Internal Medicine (H.U., T.Y.), Omiya Medical Center, Jichi Medical School, Japan.

Correspondence to M. Kawakami, Division of Arteriosclerosis and Metabolism, Department of Internal Medicine, Omiya Medical Center, Jichi Medical School, Amanuma-Cho 1-847, Omiya City 330, Japan.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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-ß1 (TGFß1), 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 TGFß1 and, interestingly, tissue-type plasminogen activator, which converts plasminogen to plasmin, an activator of TGFß1, increased twofold and fivefold, respectively. The levels of both latent and active forms of TGFß1 in conditioned media of VSMCs exposed to fluid flow increased significantly. An anti-TGFß1 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 TGFß1 in an autocrine manner. These data suggest that shear stress plays an important role as an inhibitor of atherogenesis in endothelium-desquamated lesions.

Key Words: atherosclerosis • shear stress • vascular smooth muscle cells • transforming growth factor-ß1 • tissue-type plasminogen activator

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
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 TGFß1, nitric oxide, and prostaglandins, were demonstrated to exert inhibitory effects on VSMC proliferation.⁶ Of these, TGFß1, 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} TGFß1 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 TGFß1 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 TGFß1 in an autocrine manner.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
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 8.7x10^-3 radian (0.5°) and 1.7x10^-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 () were computed using the following equations:

and

where r is the radius and the angular velocity of the cone, is the cone angle, 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 7.3x10^-4 Pa · s (0.73 cp) at 37°C. The average R and 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 1.7x10^-2 radian spinning at 25.1 radian/s (4 revolutions per second) and 8.7x10^-3 radian spinning at 31.4 radian/s (5 revolutions per second) were used to achieve average respective values of =1.4±0.11 Pa (14±1.1 dyne/cm²; average±SEM) with R=0.70±0.18 and =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).

View larger version (12K): [in this window] [in a new window]   Figure 1. Diagram of the cone-plate apparatus to expose VSMCs to shear stress on a collagen-coated 100-mm tissue-culture dish. The cone, with an angle of 8.7x10-3 radian or 1.7x10-2 radian, rotates in contact with the center of the tissue-culture dish. The ratio of the centrifugal to viscous forces (R) and shear stress () are computed as described in the "Methods" section.

Human VSMC Proliferation
VSMCs (5x10⁵) 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 TGFß1, 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 TGFß1
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 TGFß1 (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 TGFß1, 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 TGFß1. For the active TGFß1 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-TGFß1 Antibody
Experimental characterization of a rabbit polyclonal anti-human TGFß1 neutralizing antibody (TGFAb; R&D Systems Inc) demonstrated that TGFAb blocks TGFß1-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.

Statistical Analysis
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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Shear Stress on Human VSMC Proliferation
The number of VSMCs before loading of shear stress was 11.4±1.1x10⁵ cells per dish. Cell numbers increased to 19.2±1.4x10⁵ 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±0.9x10⁵ 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±1.4x10⁵ 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).

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of shear stress on human VSMC proliferation. VSMCs were exposed to shear stresses of 1.4 Pa and 2.8 Pa for 24 hours. Static controls were incubated under similar conditions without flow. Numbers of cells exposed to shear stresses were compared with that of static controls. Data shown are from six separate experiments. B indicates before shear stress exposure; C, static control; SS(1.4), shear stress of 1.4 Pa; and SS(2.8), shear stress of 2.8 Pa. *P<.05; #P<.01.

TGFß1 and TPA mRNA Levels
The static controls expressed TGFß1 and TPA mRNA constitutively. TGFß1 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).

View larger version (44K): [in this window] [in a new window]   Figure 3. Northern blot analysis and mRNA levels of TGFß1 (A and B) and TPA (C and D). Total RNA (20 µg) from VSMCs exposed to shear stress (2.8 Pa) for the indicated time and static controls were hybridized with an oligonucleotide probe of TGFß1 or TPA and the membrane was washed and rehybridized with GAPDH probe. TGFß1 and TPA mRNA levels were normalized by GAPDH mRNA level. The ratios of the normalized mRNA levels of cells exposed to shear stress to that of static controls are also shown. Figures are representative of four (A) and three (C) separate experiments and data on bar graphs are from three (B) and two (D) separate experiments. C indicates static control and SS, shear stress.

TGFß1 Protein Levels
The amount of total TGFß1 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 TGFß1 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 TGFß1 in medium supplemented with 10% FCS were in the range of 1.1 to 1.7 ng/mL (n=6).

View larger version (13K): [in this window] [in a new window]   Figure 4. Total (A) and active (B) TGFß1 concentration quantified by ELISA. VSMCs were exposed to shear stress (2.8 Pa) for 24 hours in serum-free medium. To quantify the total TGFß1, conditioned medium was centrifuged and acidified to convert latent TGFß1 to the active form. For the active TGFß1 assay, conditioned medium was centrifuged and concentrated without acidification. Assay was performed using an ELISA kit for TGFß1. Data shown are from five (A) and three (B) separate experiments. C indicates static control and SS, shear stress. *P<.05.

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).

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of TGFAb on shear-induced inhibition of VSMC growth. VSMCs were exposed to shear stress (2.8 Pa) for 24 hours in medium containing 10% FCS without antibody and with control IgG or TGFAb. Static controls were incubated under similar conditions without flow. The number of cells in medium with TGFAb was compared with that of cells in medium without antibody and with control IgG. Data shown are from four separate experiments. C indicates static control; SS, shear stress without antibody; SS+IgG, shear stress with control IgG (100 µg/mL); and SS+TGFAb, shear stress with anti-TGFß1 antibody (100 µg/mL). *P<.01 against both SS and SS+IgG.

Both TGFAb and control IgG exerted no significant effect on cell growth in static conditions (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 TGFß1, 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 TGFß1 production and activation and plays an important role in the inhibition of VSMC growth.

As a possible mechanism by which the active form of TGFß1 was increased, the induction of TPA was demonstrated at the level of mRNA, in addition to the increase of total TGFß1. The latent form of TGFß1 exists in circulation. In our experimental conditions, a small but substantial amount of TGFß1 also exists in the culture medium containing serum. Accordingly, the relative contribution of TGFß1 in serum to the increase of the active form of TGFß1 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 TGFß1, 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 TGFß1 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 TGFß1 = transforming growth factor-ß1 TPA = tissue-type plasminogen activator VSMC = vascular smooth muscle cell

Received August 26, 1996; accepted October 24, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Davies PF, Robotewskyj A, Griem ML, Dull RO, Polacek DC. Hemodynamic forces and vascular cell communication in arteries. Arch Pathol Lab Med.. 1992;116:1301-1306.[Medline] [Order article via Infotrieve]

2. 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.[Abstract/Free Full Text]

3. 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.[Abstract/Free Full Text]

4. 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.[Abstract/Free Full Text]

5. 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.

6. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med.. 1994;330:1431-1438.[Free Full Text]

7. Goustin AS, Leof EB, Shipley G, Moses HL. Growth factors and cancer. Cancer Res.. 1986;46:1015-1029.[Abstract/Free Full Text]

8. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy versus hyperplasia: autocrine transforming growth factor-ß1 expression determines growth response to angiotensin II. J Clin Invest.. 1992;90:456-461.

9. Sterpetti AV, Cucina A, Santoro L, Cardillo B, Cavallaro A. Modulation of arterial smooth muscle cell growth by haemodynamic forces. Eur J Vasc Surg.. 1992;6:16-20.[Medline] [Order article via Infotrieve]

10. Reddy KB, Howe PH. Transforming growth factor-ß1–mediated inhibition of smooth muscle cell proliferation is associated with a late G1 cell cycle arrest. J Cell Physiol.. 1993;156:48-55.[Medline] [Order article via Infotrieve]

11. Flaumenhaft R, Abe M, Sato Y, Miyazono K, Harpel J, Heldin C-H, Rifkin DB. Role of the latent TGF-ß binding protein in the activation of latent TGF-ß by co-cultures of endothelial and smooth muscle cells. J Cell Biol.. 1993;120:995-1002.[Abstract/Free Full Text]

12. Antonelli-Orlidge A, Saunders KB, Smith SR, D'Amore PA. An activated form of transforming growth factor-ß is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci U S A.. 1989;86:4544-4548.[Abstract/Free Full Text]

13. Resnick N, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J.. 1995;9:874-882.[Abstract]

14. 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.[Abstract]

15. Tsukada T, Tippens D, Gordon D, Ross R, Gown AM. HHF35, a muscle-actin–specific monoclonal antibody. Am J Pathol.. 1987;126:51-60.[Abstract]

16. Sdougos HP, Bussolari SR, Dewey CF. Secondary flow and turbulence in a cone-and-plate device. J Fluid Mech.. 1984;138:379-404.

17. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem.. 1987;162:156-159.[Medline] [Order article via Infotrieve]

18. 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.[Abstract/Free Full Text]

19. McCaffrey TA, Falcone DJ, Brayton CF, Agarwal LA, Welt FGP, Weksler BB. Transforming growth factor-ß activity is potentiated by heparin via dissociation of the transforming growth factor-ß/₂ macroglobulin inactive complex. J Cell Biol.. 1989;109:441-448.[Abstract/Free Full Text]

20. Reinhart WH. Shear-dependence of endothelial functions. Experientia.. 1994;50:87-93.[Medline] [Order article via Infotrieve]

21. 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.

22. 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.[Abstract/Free Full Text]

23. 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.

24. Nollert MU, Panaro NJ, Mcintire LV. Regulation of genetic expression in shear stress–stimulated endothelial cells. Ann N Y Acad Sci.. 1992;665:94-104.[Medline] [Order article via Infotrieve]

25. Hsieh HJ, Li NQ, Frangos JA. Pulsatile and steady flow induces c-fos expression in human endothelial cells. J Cell Physiol.. 1993;154:143-151.[Medline] [Order article via Infotrieve]

26. 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.[Abstract/Free Full Text]

27. 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.[Abstract/Free Full Text]

28. 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.

29. Loesberg C, Van Wijk R, Zandbergen J, Van Aken WG, Van Mourik JA, De Groot PHG. Cell cycle–dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1. Exp Cell Res.. 1985;160:117-125.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				V. Rizzo Enhanced interstitial flow as a contributing factor in neointima formation: (shear) stressing vascular wall cell types other than the endothelium Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1196 - H1197. [Full Text] [PDF]

				C. M Kielty, S. Stephan, M. J Sherratt, M. Williamson, and C. A. Shuttleworth Applying elastic fibre biology in vascular tissue engineering Phil Trans R Soc B, August 29, 2007; 362(1484): 1293 - 1312. [Abstract] [Full Text] [PDF]

				C. P. Ng, B. Hinz, and M. A. Swartz Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro J. Cell Sci., October 15, 2005; 118(20): 4731 - 4739. [Abstract] [Full Text] [PDF]

				J. S. Garanich, M. Pahakis, and J. M. Tarbell Shear stress inhibits smooth muscle cell migration via nitric oxide-mediated downregulation of matrix metalloproteinase-2 activity Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2244 - H2252. [Abstract] [Full Text] [PDF]

				T. M. Razzaq, R. Bass, D. J. Vines, F. Werner, S. A. Whawell, and V. Ellis Functional Regulation of Tissue Plasminogen Activator on the Surface of Vascular Smooth Muscle Cells by the Type-II Transmembrane Protein p63 (CKAP4) J. Biol. Chem., October 24, 2003; 278(43): 42679 - 42685. [Abstract] [Full Text] [PDF]

				L. Wang, M. Andersson, L. Karlsson, M.-A. Watson, D. J. Cousens, S. Jern, and D. Erlinge Increased Mitogenic and Decreased Contractile P2 Receptors in Smooth Muscle Cells by Shear Stress in Human Vessels With Intact Endothelium Arterioscler Thromb Vasc Biol, August 1, 2003; 23(8): 1370 - 1376. [Abstract] [Full Text] [PDF]

				A. Pasipoularides, M. Shu, A. Shah, M. S. Womack, and D. D. Glower Diastolic right ventricular filling vortex in normal and volume overload states Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1064 - H1072. [Abstract] [Full Text] [PDF]

				D. J. Tschumperlin, J. D. Shively, T. Kikuchi, and J. M. Drazen Mechanical Stress Triggers Selective Release of Fibrotic Mediators from Bronchial Epithelium Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 142 - 149. [Abstract] [Full Text] [PDF]

				S. Tateshima, Y. Murayama, J. P. Villablanca, T. Morino, K. Nomura, K. Tanishita, and F. Vinuela In Vitro Measurement of Fluid-Induced Wall Shear Stress in Unruptured Cerebral Aneurysms Harboring Blebs Stroke, January 1, 2003; 34(1): 187 - 192. [Abstract] [Full Text] [PDF]

				S. Kinlay, J. Grewal, D. Manuelin, J. C. Fang, A. P. Selwyn, J. A. Bittl, and P. Ganz Coronary Flow Velocity and Disturbed Flow Predict Adverse Clinical Outcome After Coronary Angioplasty Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1334 - 1340. [Abstract] [Full Text] [PDF]

				B. C. Berk Vascular Smooth Muscle Growth: Autocrine Growth Mechanisms Physiol Rev, July 1, 2001; 81(3): 999 - 1030. [Abstract] [Full Text] [PDF]

				M. Negishi, D. Lu, Y.-Q. Zhang, Y. Sawada, T. Sasaki, T. Kayo, J. Ando, T. Izumi, M. Kurabayashi, I. Kojima, et al. Upregulatory Expression of Furin and Transforming Growth Factor-{beta} by Fluid Shear Stress in Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 785 - 790. [Abstract] [Full Text] [PDF]

				M. R. Ward, P. S. Tsao, A. Agrotis, R. J. Dilley, G. L. Jennings, and A. Bobik Low Blood Flow After Angioplasty Augments Mechanisms of Restenosis : Inward Vessel Remodeling, Cell Migration, and Activity of Genes Regulating Migration Arterioscler Thromb Vasc Biol, February 1, 2001; 21(2): 208 - 213. [Abstract] [Full Text] [PDF]

				S. Wang and J. M. Tarbell Effect of Fluid Flow on Smooth Muscle Cells in a 3-Dimensional Collagen Gel Model Arterioscler Thromb Vasc Biol, October 1, 2000; 20(10): 2220 - 2225. [Abstract] [Full Text] [PDF]

				R. Palumbo, C. Gaetano, G. Melillo, E. Toschi, A. Remuzzi, and M. C. Capogrossi Shear Stress Downregulation of Platelet-Derived Growth Factor Receptor-{beta} and Matrix Metalloprotease-2 Is Associated With Inhibition of Smooth Muscle Cell Invasion and Migration Circulation, July 11, 2000; 102(2): 225 - 230. [Abstract] [Full Text] [PDF]

				S. Tada and J. M. Tarbell Interstitial flow through the internal elastic lamina affects shear stress on arterial smooth muscle cells Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1589 - H1597. [Abstract] [Full Text] [PDF]

				R. H. Song, H. K. Kocharyan, J. E. Fortunato, S. Glagov, and H. S. Bassiouny Increased Flow and Shear Stress Enhance In Vivo Transforming Growth Factor-{beta}1 After Experimental Arterial Injury Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 923 - 930. [Abstract] [Full Text] [PDF]

				F. Werner, T. M. Razzaq, and V. Ellis Tissue Plasminogen Activator Binds to Human Vascular Smooth Muscle Cells by a Novel Mechanism. EVIDENCE FOR A RECIPROCAL LINKAGE BETWEEN INHIBITION OF CATALYTIC ACTIVITY AND CELLULAR BINDING J. Biol. Chem., July 30, 1999; 274(31): 21555 - 21561. [Abstract] [Full Text] [PDF]

				M. Papadaki, J. Ruef, K. T. Nguyen, F. Li, C. Patterson, S. G. Eskin, L. V. McIntire, and M. S. Runge Differential Regulation of Protease Activated Receptor-1 and Tissue Plasminogen Activator Expression by Shear Stress in Vascular Smooth Muscle Cells Circ. Res., November 16, 1998; 83(10): 1027 - 1034. [Abstract] [Full Text] [PDF]

				S. Tada and J. M. Tarbell Flow through internal elastic lamina affects shear stress on smooth muscle cells (3D simulations) Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H576 - H584. [Abstract] [Full Text] [PDF]

This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ueba, H. Articles by Yaginuma, T. Search for Related Content PubMed PubMed Citation Articles by Ueba, H. Articles by Yaginuma, T.