Shear stress increases endothelial growth factor mRNA levels
platelet-derived
HSYUE-JEN HSIEH, NAN-QIAN LI, AND JOHN A. FRANGOS Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802
HSIEH, HSYUE-JEN, NAN-QIAN LI, AND JOHN A. FRANGOS. Shear stress increases endothelial platelet-derived growth factor mRNA levels. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H642H646, 1991.-We have investigated the effect of shear stress on platelet-derived growth factor (PDGF) A and B chain mRNA levels in cultured human umbilical vein endothelial cells (hUVEC). The levels of both PDGF A and B mRNA in hUVEC were increased by a physiological shear stress (16 dyn/ cm”), reaching a maximum -1.5-2 h after the onset of shear stress and returning almost to control values at 4 h. The peak levels showed a more than lo-fold enhancement for PDGF A mRNA and a 2- to 3-fold increase for PDGF B mRNA (P c 0.05). PDGF A mRNA also showed a shear-dependent increase from 0 to 6 dyn/cm” (P < 0.05) and then plateaued from 6 to 51 dyn/cm2. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm” then declined gradually to a minimum at 31 dyn/cm2 (P < 0.05) and increased again when shear stress rose to 51 dyn/cm2 (P < 0.05). PDGF, a potent smooth muscle cell mitogen and vasoconstrictor, released from the endothelium may regulate the blood flow in vivo. The shear stress-dependent elevation of PDGF A and B mRNA in endothelial cells may be involved in the adaptation of blood vessels to flow mediated by the endothelium.
flow effects; vascular
remodeling;
endothelial
cells
SEVERAL INVESTIGATIONS on atherosclerosis have proposed that wall shear stress in blood vessels may play an important role in the pathology of this disease (15, 29). Moreover, wall shear stress has been shown to be an important physiological factor in the adaptation of the vascular wall to blood flow and is maintained at an optimal level by an unknown mechanism (15, 29). The endothelium, which lines the inner surface of blood vessels and is in direct contact with blood flow, appears to be essential in this process (17). However the changes in blood vessel size can be achieved only by vascular smooth muscle cells (SMC). Therefore, one of the roles of the endothelium may be to senseblood flow and mediate any necessary response of the SMC (17). Platelet-derived growth factor (PDGF), a mitogen for SMC and a vasoconstrictor (3), is believed to be involved in this process. Endothelial cells secrete PDGF (24), which may bind to PDGF receptors on underlying SMC H642
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in vivo (24). PDGF genes are located on human chromosomes 7 and 22, encoding A chain and B chain polypeptides, respectively (4, 26), with amino acid sequence homology of -56% (4). Active PDGF is a dimer of two chains linked by disulfide bonds. Three possible forms of PDGF, i.e., A-A, A-B, and B-B dimers, have been identified (24), and four PDGF A transcripts (25) and one PDGF B transcript (8) were found in endothelial cells. In vitro studies indicate that exposure of endothelial cells to shear stress can induce prostacyclin production (11), increase the intracellular second messengersadenosine 3’,5’-cyclic monophosphate (22) and inositol trisphosphate (5, 6, 19, 20), decrease synthesis and release of fibronectin (l3), and increase tissue plasminogen activator (tPA) secretion (9). Furthermore, elevated levels of endothelin mRNA (28) and tPA mRNA (10) in endothelial cells subjected to shear stress have also been reported. These observations suggest that shear stress can cause a wide range of effects in endothelial cells. To demonstrate the possible role of PDGF in the hemodynamic response of vascular wall, we have examined the effect of flow on human umbilical vein endothelial cells (hUVEC) by measuring the levels of PDGF A and B mRNA. Our results show that flow induces a transient increase in the levels of both PDGF A and B mRNAs, with peaks occurring -1.5-2 h after the onset of shear stress. Moreover, PDGF A mRNA levels showed a shear-dependent increase from 0 to 6 dyn/cm’ and then plateaued from 6 to 51 dyn/cm’. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/ cm2 then declined gradually to a minimum at 31 dyn/ cm2 and increased again when shear stress rose to 51 dyn/cm2 MATERIALS
AND
METHODS
Cell culture and flow loop experiments. Primary cultures of hUVEC were harvested from human umbilical veins by collagenase treatment and were plated on glass slides (75 x 38 mm) as described elsewhere (11). The medium was changed 1 day before using the cultures in experiments. Cells were subjected to well-defined shear
0 1991 the American
Physiological
Society
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stress in a parallel plate flow chamber, where medium was circulated and driven by a constant-head flow loop (12). To eliminate possible effects due to the addition of fresh medium, each flow loop was primed with the same medium in which the cells were previously incubated in. RNA isolation and Northern blot analysis. Immediately after being subjected to shear stress, cells were lysed in a guanidinium solution. Total RNA was isolated from the cell lysate by centrifuging through a cesium chloride step gradient (2). After quantification by measuring absorbance at 260 nm, 3.5-7.5 pug of RNA samples (same amount per lane for each gel) were electrophoresed in 1.2% formaldehyde gels (2), transferred to nylon membranes (Nytran 0.45 pm, Schleicher & Schuell, Keene, NH) (2), and fixed by ultraviolet irradiation (user’s manual for Schleicher & Schuell Nytran membrane). The following cDNA fragments were labeled (Multiprime DNA labeling system, Amersham, Arlington Heights, IL) with deoxycytidine 5’-[a-32P]triphosphate (3,000 Ci/ mmol, Amersham) and used as probes: for PDGF A, 1.3 kb EcoR I fragment of PDGF-A-13.1 (received as a gift from C. Betsholtz, University Hospital, Uppsala, Sweden) (4); for PDGF B, 1.48 kb Pst I/BamH I fragment of pGGS2.7dBH (received as a gift from C. D. Rao, National Cancer Institute, Bethesda, MD) (21); for glyc-
A
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PDGF-A
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000511522534
m
OB 0
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FIG. 2. Normalized time course of PDGF A and B mRNA induction by shear stress. Intensity of each hybridization band on autoradiograms was determined by optical densitometry. Shear stress increased PDGF A mRNA >lO-fold (A) and PDGF B mRNA 2- to 3-fold (B) at 1.5-2 h (P < 0.05, by the Student’s t test). Each data point represents the mean of three experiments + SE. [n = 11 for stationary controls (0 h)].
eraldehyde-3-phosphate dehydrogenase (GAPDH), 0.96 kb Hint II/Bgl I fragment of pHcGAP (deposited by R. Wu, received from American Tissue Cell Culture, Rockville, MD) (27). Membranes were hybridized with PDGF A probe first, after washes, exposed to Kodak XAR films at -70°C for autoradiography (2). Rehybridizations were done subsequently with PDGF B probe followed by GAPDH probe (user’s manual for Schleicher & Schuell Nytran membrane). Quantification of hybridization signals. The intensity of each hybridization band on XAR film was determined by optical densitometry (Quick Scan Jr. & Quick Quant III, Helena Laboratories, Beaumont, TX). Each GAPDH band served as the internal standard to normalize PDGF A and B signals, since GAPDH is constitutively expressed in hUVEC.
HOIXS
-
18S-
c
1
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j
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MRNA
ii E
HOUE
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FIG. 1. Time course of PDGF A and B mRNA induction by shear stress. hUVEC were subjected to steady shear stress of 16 dyn/cm* for 0.5, 1, 1.5, 2, 2.5, 3, and 4 h. Total RNA (3.6 fig/lane) was isolated from cells for Northern analysis of PDGF A (A), PDGF B (B), and GAPDH mRNA (C). Two major species (-2.5 and 3.0 kb) and two minor species (-1.7 and 3.8 kb) of PDGF A mRNA were induced. PDGF B mRNA (-3.7 kb) levels were also elevated. GAPDH mRNA (-1.5 kb) levels were relatively constant. Autoradiograms were exposed for 11 (A), 12 (B), and 4 h (C). See text for abbreviations.
RESULTS
hUVEC were exposed to steady shear stress (16 dyn/ cm”) for 0.5-4 h. The mRNA levels of PDGF A and B in sheared hUVEC showed a transient increase (Fig. 1, A and B). The increase in PDGF A mRNA by shear stress was more dramatic than PDGF B mRNA. In the stationary control hUVEC (0 h of duration of shear stress in Figs. 1A and 2A), PDGF A mRNA was rare. However, the onset of shear stress induced a dramatic increase of PDGF A mRNA level with a peak occurring around 1.5
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H644 A
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MHNA
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c
2as?Es---
icmJ
L
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GAPDH
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3. Effect of shear Stress on PDCF A and D mRNA levels. hlJVEC were exposed to steady shear stresses of 6, 16, 22,31, and 39 &n/cm’ for 2 h, Total RNA (3.5 pg/lane) was isolated for Northern anaIy&s of PDGF A (A,, PDCF B (8), and H mRNA (C). All five levels of shear stress increased PDGF H mRNA levels. Autoradiogrems were exposed for 11 (A), 12 (R), and 6 h (Cl. See text for abbreviations. FIG.
lowed by a gradual decline to near cant after 4 h. PDGF B mRNA appeared more abu PDGF A rnRNA in the stationary controls. Flow initiated a transient increase in PDGF RNA levels, with a peak between 1.6 and 2 h fool ed by a decrease. In contrast, GAPDH mRNA level ained relatively constant. of PDGF A and B mRNA were determetric scanning of each hybridization band. After normalization by the intensity of individual GAPDH mRNA bands, we found a more than 10-f&l transient increase of PDGF A mRNA levels by shear stress (Fig. 24) (P < 0.05). The PDGF B mRNA level was transiently enhanced two- to threefold by shear stress (Fig. 2B) (P < 0.05). Two major species ,5 and 3.0 kb) and two minor species (-1.7 and 3.8 of PDGF A mRNA, possibly were expressed (Fig. lA). For due to alternative spli PDGF B mRNA, only o;e band (-3.7 kb) was found (Fig. 123). GAPDH mRNA (-1.5 kb) (Fig. IC) was used as an internal standard. To elucidate if peak mRNA levels were also shearstress dependent, hUVE were subjected to steady shear stresses of 2, 6, 16, 22, 39, and 51 dyn/c The PDGF A mRNA level: in all shear-stres were more abundant than those in the stationary controls (Fig. 3A), PDGF A mRNA also showed a shear-depend-
20 straae
40
60
60
(dynwilom?
FIG. 4. Normalized effect of shear stress on PDGF A and B mRNA levels. Intensities of PDGF A and B mRNA signals were normalkd by GAPDH mRNA. PDGF A mRPdA levels were higher at 6 dyn/cm’ than those at 2 dyn/cm’ (1’ < 0.05), and mRNA levels at 2 dyn/rm’ were higher thnn those at 0 dyn/cm’ (P < 0.05). But no significant shear dependence of PDGF A mRNA levels was observed in range of 6-51 dyn/um2. In contrast, levels of PDGF R mRNA appeared to be varied within run* of O-51 dyn/cm*. In low shear regmn, there was an increase of PDGF R mRNA levels when shear stress was increased from 0 to 6 dyn/cm’. However, PDGF B mRNA levels at 6 dyn/cm’ were significantly higher than those at 16 and 22 dyn/cm’ (P < NKi). PDGF B mRNA levels at 16 and 22 dyn/cm’ were significantly hir;her tban those at 31 dyn/cml (P < MS). In addition, PDGF B mElNA levels at 51 dyn/cm’ were also kgnificantly higher than those at 31 dyn/cm” (P < 0.05). Data points represent mean of severai experiments f SE (IL = 16, 10, 5, 13, 8, 7, 5, and 7 for 0, 2, 6, 16, 22, 31, 39, and 51 dyn/cm2 shear stress points, respectively). All statistical analyses were perfbrmed with Student’s t test. See text for abbreviations.
ent increase from 0 to 6 dyn/cm” (P < 0.05) and then plateaued from 6 to 51 dyn/cm” (Fig. 4A ). In contrast, the levels of PDGF B mRNA appeared to be varied from 0 to 51 dyn/cm” (Fig. 3B), PDGF 3 mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm’ then declined gradually to a minimum at 31 dyn/cm’ (P < 0.05) and increased again when shear stress rose to 51 dyn/cm’ (P < 0.05) (Fig. 4R). DISCUSSION
B mRNA expression can be induced in endothelial cells by thrombin, transforming growth factor-p (TGF-P), and ph bol myristate acetate (PMA) (251, whereas basic fibr last growth factor (bFGF) decreases the PDCF I3 mRNA levels in hUVEC (16). Our results indicate that levels of PDGF A and B mRNA in hUVEC are enhanced by shear stress, whereas GAPDH
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mRNA levels remained relatively constant (Figs. 1 and 3). Thus it appears that PDGF expression can be regulated by physical factors as well as humoral factors. Collins and co-workers (8) reported that the mRNA levels of PDGF A and B in cultured hUVEC were nearly the same; however, our results showed that PDGF B mRNA was significantly more abundant than PDGF A mRNA (Fig. 1). The difference may be attributed to the fact that Collins et al. (8) used passaged hUVEC, whereas we used primary cultures. Collins et al. (8) also reported that only PDGF A-A dimers were released from hUVEC. However, Bowen-Pope et al. (7) observed that -15% of PDGF secreted by hUVEC was in the form of B-B dimers, whereas the rest was in the form of A-A and/or A-B dimers (7). The inconsistency between the PDGF gene transcription and protein secretion observations may be due to either cell association of PDGF B translational products (i.e., B-B, A-B dimers) (8), rapid degradation (8), or storage of an inactive precursor (18), resulting in reduced secretion of B-B and A-B dimers from hUVEC. Previous work has shown that shear stress stimulates phosphoinositide turnover in endothelial cells, producing the second messengers inositol trisphosphate and diacylglycerol (5, 6, 19, 20). It has been hypothesized that this signal transduction pathway, by activating protein kinase C, may regulate many of the observed effects of shear stress on endothelial cells (6). The release of PDGF from endothelial cells has been reported to be greatly stimulated by thrombin (14), endotoxin (I), and PMA (25), the first two of which lead to inositol phospholipid turnover. PMA, like diacylglycerol, is an activator of protein kinase C. Therefore, shear-stress induction of PDGF expression appears consistent with a signal transduction mechanism involving protein kinase C activation. In addition, the message levels for both PDGF A and B peaked at the same time (-1.5-2 h), suggesting a similar induction mechanism for expression. Kamiya and Togawa (15) and Zarins et al. (29) have suggested that the blood vessel wall adapts to changes in blood flow by autoregulating the wall shear stress. They point to evidence that the average wall shear stress in arterial vessels, independent of blood flow rate, size, location, and even species, is between 15 and 20 dyn/ cm2. These calculations are based on average flow rates and vessel diameters and assume Poiseuille flow. Our data indicate that the expression levels of PDGF B are at their lowest in the range of 15-31 dyn/cm2 (in cells subjected to shear). Since PDGF is both a mitogen for SMC and fibroblasts (24) and a potent vasoconstrictor (3), it may be involved in the autoregulation of wall shear stress. This hypothesis is consistent with in vivo observations. Normal blood vessels respond to shear stress and adapt their blood vessel diameter at two levels: acutely through vasoactive mechanisms (flow-dependent dilation), and chronically by adjusting vascular caliber (15, 29). If the decrease in PDGF B expression levels with increasing shear translates into decreased PDGF protein secretion, a vasodilatory (decreased vasoconstrictory) signal will result. The role of PDGF in flow-dependent vasomotor control would be in conjunction with other
GROWTH
FACTOR
H645
MRNA
known mediators of flow-dependent dilation, such as endothelial-derived relaxing factor and prostacyclin. Chronically, altered PDGF secretion may regulate the chronic changes in vascular caliber in the process of autoregulating wall shear stress. The role of flow-dependent PDGF secretion is not so clear in this case. A reduction in vascular wall shear stress leads to a reduction in vessel diameter, which is not due to growth of vessel wall tissue but rather to a change in vessel size (17). In contrast, shear stress of 51 dyn/cm2, which is higher than the average arterial shear stress (15-31 dyn/ cm2), also induced increased expression of PDGF B (Fig. 4B). This may partly explain how increased blood flow leads to increased media cross-sectional area (29). It is likely, however, that flow-induced vascular remodeling involves several other interacting factors. The increased expression of PDGF B with decreasing shear may also be involved in atherogenic mechanisms. Intimal thickening or proliferation has been found in certain regions of low shear stress in blood vessels (29), which may coincide with areas of vascular lesions. On the other hand, the elevated PDGF B mRNA levels induced by high shear stresses (39-51 dyn/cm2) (Fig. 4B) seem consistent with the “response-to-injury hypothesis of atherogenesis” proposed by Ross (23). High shear stress may be seen as an injury to the endothelium, which may stimulate endothelial cells to secrete growth factors including PDGF, possibly involved in the atherogenesis. Great care, however, must be taken in extrapolating in vitro data to in vivo pathology. In summary, our results indicate that both PDGF A and B mRNA levels are elevated transiently by shear stress, whereas the increase of PDGF A mRNA levels is more dramatic than PDGF B mRNA. Moreover, PDGF A mRNA levels showed a shear-dependent increase from 0 to 6 dyn/cm’ and then plateaued from 6 to 51 dyn/cm2. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm2 then declined gradually to a minimum at 31 dyn/cm2 and increased again when shear stress rose to 51 dyn/cm2. The flow-dependent elevation of PDGF A and B mRNA in endothelial cells may be involved in the flow-mediated adaptation of blood vessels. We thank C. Betsholtz for providing the PDGF A chain cDNA plasmid and C. D. Rao for providing the PDGF B chain cDNA plasmid. This investigation was supported by National Heart, Lung, and Blood Institute Grant HL-40696. J. A. Frangos is a recipient of the National Science Foundation Presidential Young Investigator Award. Address for reprint requests: J. A. Frangos, Dept. of Chemical Engineering, 149 Fenske Laboratory, The Pennsylvania State University, University Park, PA 16802. Received
18 July
1990; accepted
in final
form
9 November
1990.
REFERENCES S. M., J. A. ELIAS, E. M. LEVINE, AND J. A. KERN. Endotoxin stimulates platelet-derived growth factor production from cultured human pulmonary endothelial cells. Am. J. Physiol. 257 (Lung CeZl. Mol. Physiol 1): L65-L70, 1989. AUSUBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN, J. A. SMITH, AND K. STRUHL. Current Protocols in Molecular Biology. New York: Wiley, 1987, p. 4.2.1-4.2.5, p. 4.9.14.9. BERK, B. C., R. W. ALEXANCER, T. A. BROCK, M. A. GIMBRONE, ALBELDA,
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H646
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JR., AND R. C. WEBB. Vasoconstriction:
4.
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a new activity for plateletderived growth factor. Science Wash. DC 232: 87-90, 1986. BETSHOLTZ, C., A. JOHNSSON, C.-H. HELDIN, B. WESTERMARK, P. LIND, M. S. URDEA, R. EDDY, T. B. SHOWS, K. PHILPOTT, A. L. MELLOR, T. J. KNOTT, AND J. SCOTT. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumor cell lines. Nature Land. 320: 695-699,1986. BHAGYALAKSHMI, A., AND J. A. FRANGOS. Mechanism of shearinduced prostacyclin production in endothelial cells. Biochem. Biophys. Res. Commun. 158: 33-37, 1989. BHAGYALAKSHMI, A., AND J. A. FRANGOS. Membrane phospholipid metabolism in sheared endothelial cells. Proceedings 2nd International Symposium on Biofluid Mechanics and Biorheology, Munich, FRG, June 1989, 1989, p. 249. BOWEN-POPE, D. F., C. E. HART, AND R. A. SEIFERT. Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which bind to different classes of PDGF receptor. J. Biol. Chem. 264: 2502-2508, 1989. COLLINS, T., J. S. POBER, M. A. GIMBRONE, JR., A. HAMMACHER, C. BETSHOLTZ, B. WESTERMARK, AND C.-H. HELDIN. Cultured human endothelial cells express platelet-derived growth factor A chain. Am. J. Pathol. 127: 7-12, 1987. DIAMOND, S. L., S. G. ESKIN, AND L. V. MCINTIRE. Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science Wash. DC 243: 1483-1485, 1989. DIAMOND, S. L., J. B. SHAREFKIN, C. DIFFENBACH, K. F-SCOTT, L. V. MCINTIRE, AND S. G. ESKIN. Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. J. Cell. Physiol. 143: 364-371, 1990. FRANGOS, J. A., S. G. ESKIN, L. V. MCINTIRE, AND C. L. IVES. Flow effectson prostacyclin production by cultured human endothelial cells. Science Wash. DC 227: 1477-1479, 1985. FRANGOS, J. A., L. V. MCINTIRE, AND S. G. ESKIN. Shear stress induced stimulation of mammalian cell metabolism. Biotechnol. Bioeng. 32: 1053-1060,1988. GUPTE, A., AND J. A. FRANGOS. Effects of flow on the synthesis and releaseof fibronectin by endothelial cells. In Vitro Cell. Dev. Biol. 26: 57-60, 1990. HARLAN, J. M., P. J. THOMPSON, R. R. Ross, AND D. F. BOWENPOPE. cu-Trombin induces release of platelet-derived growth factorlike molecule(s) by cultured human endothelial cells. J. Cell Biol. 103: 1129-1133,1986. KAMIYA, A., AND T. TOGAWA. Adaptive regulation of wall shear stress to flow change in the canine carotid artery. Am. J. Physiol. 239 (IIeart Circ. Phvsiol. 8): Hl4-H21. 1980.
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16. KOUREMBANAS, S., AND D. V. FALLER. Platelet-derived growth factor production by human umbilical vein endothelial cells is regulated by basic fibroblast growth factor. J. Biol. Chem. 264: 4456-4459,1989. 17. LANGILLE, B. L., AND F. O’DONNELL. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science Wash. DC 231: 405-407, 1986. timulus-secretion coupling 18. NEWBY, A. C., AND A. H. HENDERSON. in vascular endothelial cells. Annu. Rev. Physiol. 52: 661-674,199O. 19. NOLLERT, M. U., S. G. ESKIN, AND L. V. MCINTIRE. Shear stress increases inositol trisphosphate levels in human endothelial cells. Biochem. Biophys. Res. Commun. 170: 281-287, 1990. 20. PRASAD, A. R. S., R. M. NEREM, C. J. SCHWARTZ, AND E. A. SPRAGUE. Stimulation of phosphoinositide hydrolysis in bovine aortic endothelial cells exposed to elevated shear stress (Abstract). J. Cell Biol. 109: 313A, 1989. 21. RAO, C. D., H. IGARASHI, I. M. CHIU, K. C. ROBBINS, AND S. A. AARONSON. Structure and sequence of the human c-sislplateletderived growth factor 2 (SISIPDFG2) transcriptional unit. Proc. Natl. Acad. Sci. USA 83: 2392-2396, 1986. 22. REICH, K. M., C. V. GAY, AND J. A. FRANGOS. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J. Cell. Physiol. 143: 100-104, 1990. 23. ROSS, R. The pathogenesis of atherosclerosis: an update. New Engl. J. Med. 314: 488-500, 1986. 24. Ross, R., E. W. RAINES, AND D. F. BOWEN-POPE. The biology of platelet-derived growth factor. Cell 46: 155-169, 1986. 25. STARKSEN, N. F., G. R. HARSH, V. C. GIBBS, AND L. T. WILLIAMS. Regulated expression of the platelet-derived growth factor A chain gene in microvascular endothelial cells. J. Biol. Chem. 262: 1438114384, 1987. 26. SWAN, D. C., 0. W. MCBRIDE, K. C. ROBBINS, D. A. KEITHLEY, E. P. REDDY, AND S. A. AARONSON. Chromosomal mapping of the simian sarcoma virus one gene analogue in human cells. Proc. Natl. Acad. Sci. USA 79: 4691-4695, 1982. 27. Tso, J. Y., X. H. SUN, T. H. KAO, K. S. REECE, AND R. Wu. Isolation and characterization of rat and human glyceraldehyde-3phosphate dehydrogenase cDNAs. Nucleic Acids Res. 13: 24852502,1985. 28. YOSHIZUMI, M., H. KURIHARA, T. SUGIYAMA, F. TAKAKU, M. YANAGISAWA, T. MASAKI, AND Y. YAZAKI. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem. Biophys. Res. Commun. 161: 859-864, 1989. 29. ZARINS, C. K., M. A. ZATINA, D. P. GIDDENS, D. N. Ku, AND S. GLAGOV. Shear stress regulation of artery lumen diameter in exnerimental atherogenesis. J. Vast. Sure. 5: 413-420. 1987.
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platelet-derived
HSYUE-JEN HSIEH, NAN-QIAN LI, AND JOHN A. FRANGOS Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802
HSIEH, HSYUE-JEN, NAN-QIAN LI, AND JOHN A. FRANGOS. Shear stress increases endothelial platelet-derived growth factor mRNA levels. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H642H646, 1991.-We have investigated the effect of shear stress on platelet-derived growth factor (PDGF) A and B chain mRNA levels in cultured human umbilical vein endothelial cells (hUVEC). The levels of both PDGF A and B mRNA in hUVEC were increased by a physiological shear stress (16 dyn/ cm”), reaching a maximum -1.5-2 h after the onset of shear stress and returning almost to control values at 4 h. The peak levels showed a more than lo-fold enhancement for PDGF A mRNA and a 2- to 3-fold increase for PDGF B mRNA (P c 0.05). PDGF A mRNA also showed a shear-dependent increase from 0 to 6 dyn/cm” (P < 0.05) and then plateaued from 6 to 51 dyn/cm2. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm” then declined gradually to a minimum at 31 dyn/cm2 (P < 0.05) and increased again when shear stress rose to 51 dyn/cm2 (P < 0.05). PDGF, a potent smooth muscle cell mitogen and vasoconstrictor, released from the endothelium may regulate the blood flow in vivo. The shear stress-dependent elevation of PDGF A and B mRNA in endothelial cells may be involved in the adaptation of blood vessels to flow mediated by the endothelium.
flow effects; vascular
remodeling;
endothelial
cells
SEVERAL INVESTIGATIONS on atherosclerosis have proposed that wall shear stress in blood vessels may play an important role in the pathology of this disease (15, 29). Moreover, wall shear stress has been shown to be an important physiological factor in the adaptation of the vascular wall to blood flow and is maintained at an optimal level by an unknown mechanism (15, 29). The endothelium, which lines the inner surface of blood vessels and is in direct contact with blood flow, appears to be essential in this process (17). However the changes in blood vessel size can be achieved only by vascular smooth muscle cells (SMC). Therefore, one of the roles of the endothelium may be to senseblood flow and mediate any necessary response of the SMC (17). Platelet-derived growth factor (PDGF), a mitogen for SMC and a vasoconstrictor (3), is believed to be involved in this process. Endothelial cells secrete PDGF (24), which may bind to PDGF receptors on underlying SMC H642
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in vivo (24). PDGF genes are located on human chromosomes 7 and 22, encoding A chain and B chain polypeptides, respectively (4, 26), with amino acid sequence homology of -56% (4). Active PDGF is a dimer of two chains linked by disulfide bonds. Three possible forms of PDGF, i.e., A-A, A-B, and B-B dimers, have been identified (24), and four PDGF A transcripts (25) and one PDGF B transcript (8) were found in endothelial cells. In vitro studies indicate that exposure of endothelial cells to shear stress can induce prostacyclin production (11), increase the intracellular second messengersadenosine 3’,5’-cyclic monophosphate (22) and inositol trisphosphate (5, 6, 19, 20), decrease synthesis and release of fibronectin (l3), and increase tissue plasminogen activator (tPA) secretion (9). Furthermore, elevated levels of endothelin mRNA (28) and tPA mRNA (10) in endothelial cells subjected to shear stress have also been reported. These observations suggest that shear stress can cause a wide range of effects in endothelial cells. To demonstrate the possible role of PDGF in the hemodynamic response of vascular wall, we have examined the effect of flow on human umbilical vein endothelial cells (hUVEC) by measuring the levels of PDGF A and B mRNA. Our results show that flow induces a transient increase in the levels of both PDGF A and B mRNAs, with peaks occurring -1.5-2 h after the onset of shear stress. Moreover, PDGF A mRNA levels showed a shear-dependent increase from 0 to 6 dyn/cm’ and then plateaued from 6 to 51 dyn/cm’. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/ cm2 then declined gradually to a minimum at 31 dyn/ cm2 and increased again when shear stress rose to 51 dyn/cm2 MATERIALS
AND
METHODS
Cell culture and flow loop experiments. Primary cultures of hUVEC were harvested from human umbilical veins by collagenase treatment and were plated on glass slides (75 x 38 mm) as described elsewhere (11). The medium was changed 1 day before using the cultures in experiments. Cells were subjected to well-defined shear
0 1991 the American
Physiological
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stress in a parallel plate flow chamber, where medium was circulated and driven by a constant-head flow loop (12). To eliminate possible effects due to the addition of fresh medium, each flow loop was primed with the same medium in which the cells were previously incubated in. RNA isolation and Northern blot analysis. Immediately after being subjected to shear stress, cells were lysed in a guanidinium solution. Total RNA was isolated from the cell lysate by centrifuging through a cesium chloride step gradient (2). After quantification by measuring absorbance at 260 nm, 3.5-7.5 pug of RNA samples (same amount per lane for each gel) were electrophoresed in 1.2% formaldehyde gels (2), transferred to nylon membranes (Nytran 0.45 pm, Schleicher & Schuell, Keene, NH) (2), and fixed by ultraviolet irradiation (user’s manual for Schleicher & Schuell Nytran membrane). The following cDNA fragments were labeled (Multiprime DNA labeling system, Amersham, Arlington Heights, IL) with deoxycytidine 5’-[a-32P]triphosphate (3,000 Ci/ mmol, Amersham) and used as probes: for PDGF A, 1.3 kb EcoR I fragment of PDGF-A-13.1 (received as a gift from C. Betsholtz, University Hospital, Uppsala, Sweden) (4); for PDGF B, 1.48 kb Pst I/BamH I fragment of pGGS2.7dBH (received as a gift from C. D. Rao, National Cancer Institute, Bethesda, MD) (21); for glyc-
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FIG. 2. Normalized time course of PDGF A and B mRNA induction by shear stress. Intensity of each hybridization band on autoradiograms was determined by optical densitometry. Shear stress increased PDGF A mRNA >lO-fold (A) and PDGF B mRNA 2- to 3-fold (B) at 1.5-2 h (P < 0.05, by the Student’s t test). Each data point represents the mean of three experiments + SE. [n = 11 for stationary controls (0 h)].
eraldehyde-3-phosphate dehydrogenase (GAPDH), 0.96 kb Hint II/Bgl I fragment of pHcGAP (deposited by R. Wu, received from American Tissue Cell Culture, Rockville, MD) (27). Membranes were hybridized with PDGF A probe first, after washes, exposed to Kodak XAR films at -70°C for autoradiography (2). Rehybridizations were done subsequently with PDGF B probe followed by GAPDH probe (user’s manual for Schleicher & Schuell Nytran membrane). Quantification of hybridization signals. The intensity of each hybridization band on XAR film was determined by optical densitometry (Quick Scan Jr. & Quick Quant III, Helena Laboratories, Beaumont, TX). Each GAPDH band served as the internal standard to normalize PDGF A and B signals, since GAPDH is constitutively expressed in hUVEC.
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FIG. 1. Time course of PDGF A and B mRNA induction by shear stress. hUVEC were subjected to steady shear stress of 16 dyn/cm* for 0.5, 1, 1.5, 2, 2.5, 3, and 4 h. Total RNA (3.6 fig/lane) was isolated from cells for Northern analysis of PDGF A (A), PDGF B (B), and GAPDH mRNA (C). Two major species (-2.5 and 3.0 kb) and two minor species (-1.7 and 3.8 kb) of PDGF A mRNA were induced. PDGF B mRNA (-3.7 kb) levels were also elevated. GAPDH mRNA (-1.5 kb) levels were relatively constant. Autoradiograms were exposed for 11 (A), 12 (B), and 4 h (C). See text for abbreviations.
RESULTS
hUVEC were exposed to steady shear stress (16 dyn/ cm”) for 0.5-4 h. The mRNA levels of PDGF A and B in sheared hUVEC showed a transient increase (Fig. 1, A and B). The increase in PDGF A mRNA by shear stress was more dramatic than PDGF B mRNA. In the stationary control hUVEC (0 h of duration of shear stress in Figs. 1A and 2A), PDGF A mRNA was rare. However, the onset of shear stress induced a dramatic increase of PDGF A mRNA level with a peak occurring around 1.5
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3. Effect of shear Stress on PDCF A and D mRNA levels. hlJVEC were exposed to steady shear stresses of 6, 16, 22,31, and 39 &n/cm’ for 2 h, Total RNA (3.5 pg/lane) was isolated for Northern anaIy&s of PDGF A (A,, PDCF B (8), and H mRNA (C). All five levels of shear stress increased PDGF H mRNA levels. Autoradiogrems were exposed for 11 (A), 12 (R), and 6 h (Cl. See text for abbreviations. FIG.
lowed by a gradual decline to near cant after 4 h. PDGF B mRNA appeared more abu PDGF A rnRNA in the stationary controls. Flow initiated a transient increase in PDGF RNA levels, with a peak between 1.6 and 2 h fool ed by a decrease. In contrast, GAPDH mRNA level ained relatively constant. of PDGF A and B mRNA were determetric scanning of each hybridization band. After normalization by the intensity of individual GAPDH mRNA bands, we found a more than 10-f&l transient increase of PDGF A mRNA levels by shear stress (Fig. 24) (P < 0.05). The PDGF B mRNA level was transiently enhanced two- to threefold by shear stress (Fig. 2B) (P < 0.05). Two major species ,5 and 3.0 kb) and two minor species (-1.7 and 3.8 of PDGF A mRNA, possibly were expressed (Fig. lA). For due to alternative spli PDGF B mRNA, only o;e band (-3.7 kb) was found (Fig. 123). GAPDH mRNA (-1.5 kb) (Fig. IC) was used as an internal standard. To elucidate if peak mRNA levels were also shearstress dependent, hUVE were subjected to steady shear stresses of 2, 6, 16, 22, 39, and 51 dyn/c The PDGF A mRNA level: in all shear-stres were more abundant than those in the stationary controls (Fig. 3A), PDGF A mRNA also showed a shear-depend-
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FIG. 4. Normalized effect of shear stress on PDGF A and B mRNA levels. Intensities of PDGF A and B mRNA signals were normalkd by GAPDH mRNA. PDGF A mRPdA levels were higher at 6 dyn/cm’ than those at 2 dyn/cm’ (1’ < 0.05), and mRNA levels at 2 dyn/rm’ were higher thnn those at 0 dyn/cm’ (P < 0.05). But no significant shear dependence of PDGF A mRNA levels was observed in range of 6-51 dyn/um2. In contrast, levels of PDGF R mRNA appeared to be varied within run* of O-51 dyn/cm*. In low shear regmn, there was an increase of PDGF R mRNA levels when shear stress was increased from 0 to 6 dyn/cm’. However, PDGF B mRNA levels at 6 dyn/cm’ were significantly higher than those at 16 and 22 dyn/cm’ (P < NKi). PDGF B mRNA levels at 16 and 22 dyn/cm’ were significantly hir;her tban those at 31 dyn/cml (P < MS). In addition, PDGF B mElNA levels at 51 dyn/cm’ were also kgnificantly higher than those at 31 dyn/cm” (P < 0.05). Data points represent mean of severai experiments f SE (IL = 16, 10, 5, 13, 8, 7, 5, and 7 for 0, 2, 6, 16, 22, 31, 39, and 51 dyn/cm2 shear stress points, respectively). All statistical analyses were perfbrmed with Student’s t test. See text for abbreviations.
ent increase from 0 to 6 dyn/cm” (P < 0.05) and then plateaued from 6 to 51 dyn/cm” (Fig. 4A ). In contrast, the levels of PDGF B mRNA appeared to be varied from 0 to 51 dyn/cm” (Fig. 3B), PDGF 3 mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm’ then declined gradually to a minimum at 31 dyn/cm’ (P < 0.05) and increased again when shear stress rose to 51 dyn/cm’ (P < 0.05) (Fig. 4R). DISCUSSION
B mRNA expression can be induced in endothelial cells by thrombin, transforming growth factor-p (TGF-P), and ph bol myristate acetate (PMA) (251, whereas basic fibr last growth factor (bFGF) decreases the PDCF I3 mRNA levels in hUVEC (16). Our results indicate that levels of PDGF A and B mRNA in hUVEC are enhanced by shear stress, whereas GAPDH
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mRNA levels remained relatively constant (Figs. 1 and 3). Thus it appears that PDGF expression can be regulated by physical factors as well as humoral factors. Collins and co-workers (8) reported that the mRNA levels of PDGF A and B in cultured hUVEC were nearly the same; however, our results showed that PDGF B mRNA was significantly more abundant than PDGF A mRNA (Fig. 1). The difference may be attributed to the fact that Collins et al. (8) used passaged hUVEC, whereas we used primary cultures. Collins et al. (8) also reported that only PDGF A-A dimers were released from hUVEC. However, Bowen-Pope et al. (7) observed that -15% of PDGF secreted by hUVEC was in the form of B-B dimers, whereas the rest was in the form of A-A and/or A-B dimers (7). The inconsistency between the PDGF gene transcription and protein secretion observations may be due to either cell association of PDGF B translational products (i.e., B-B, A-B dimers) (8), rapid degradation (8), or storage of an inactive precursor (18), resulting in reduced secretion of B-B and A-B dimers from hUVEC. Previous work has shown that shear stress stimulates phosphoinositide turnover in endothelial cells, producing the second messengers inositol trisphosphate and diacylglycerol (5, 6, 19, 20). It has been hypothesized that this signal transduction pathway, by activating protein kinase C, may regulate many of the observed effects of shear stress on endothelial cells (6). The release of PDGF from endothelial cells has been reported to be greatly stimulated by thrombin (14), endotoxin (I), and PMA (25), the first two of which lead to inositol phospholipid turnover. PMA, like diacylglycerol, is an activator of protein kinase C. Therefore, shear-stress induction of PDGF expression appears consistent with a signal transduction mechanism involving protein kinase C activation. In addition, the message levels for both PDGF A and B peaked at the same time (-1.5-2 h), suggesting a similar induction mechanism for expression. Kamiya and Togawa (15) and Zarins et al. (29) have suggested that the blood vessel wall adapts to changes in blood flow by autoregulating the wall shear stress. They point to evidence that the average wall shear stress in arterial vessels, independent of blood flow rate, size, location, and even species, is between 15 and 20 dyn/ cm2. These calculations are based on average flow rates and vessel diameters and assume Poiseuille flow. Our data indicate that the expression levels of PDGF B are at their lowest in the range of 15-31 dyn/cm2 (in cells subjected to shear). Since PDGF is both a mitogen for SMC and fibroblasts (24) and a potent vasoconstrictor (3), it may be involved in the autoregulation of wall shear stress. This hypothesis is consistent with in vivo observations. Normal blood vessels respond to shear stress and adapt their blood vessel diameter at two levels: acutely through vasoactive mechanisms (flow-dependent dilation), and chronically by adjusting vascular caliber (15, 29). If the decrease in PDGF B expression levels with increasing shear translates into decreased PDGF protein secretion, a vasodilatory (decreased vasoconstrictory) signal will result. The role of PDGF in flow-dependent vasomotor control would be in conjunction with other
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known mediators of flow-dependent dilation, such as endothelial-derived relaxing factor and prostacyclin. Chronically, altered PDGF secretion may regulate the chronic changes in vascular caliber in the process of autoregulating wall shear stress. The role of flow-dependent PDGF secretion is not so clear in this case. A reduction in vascular wall shear stress leads to a reduction in vessel diameter, which is not due to growth of vessel wall tissue but rather to a change in vessel size (17). In contrast, shear stress of 51 dyn/cm2, which is higher than the average arterial shear stress (15-31 dyn/ cm2), also induced increased expression of PDGF B (Fig. 4B). This may partly explain how increased blood flow leads to increased media cross-sectional area (29). It is likely, however, that flow-induced vascular remodeling involves several other interacting factors. The increased expression of PDGF B with decreasing shear may also be involved in atherogenic mechanisms. Intimal thickening or proliferation has been found in certain regions of low shear stress in blood vessels (29), which may coincide with areas of vascular lesions. On the other hand, the elevated PDGF B mRNA levels induced by high shear stresses (39-51 dyn/cm2) (Fig. 4B) seem consistent with the “response-to-injury hypothesis of atherogenesis” proposed by Ross (23). High shear stress may be seen as an injury to the endothelium, which may stimulate endothelial cells to secrete growth factors including PDGF, possibly involved in the atherogenesis. Great care, however, must be taken in extrapolating in vitro data to in vivo pathology. In summary, our results indicate that both PDGF A and B mRNA levels are elevated transiently by shear stress, whereas the increase of PDGF A mRNA levels is more dramatic than PDGF B mRNA. Moreover, PDGF A mRNA levels showed a shear-dependent increase from 0 to 6 dyn/cm’ and then plateaued from 6 to 51 dyn/cm2. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm2 then declined gradually to a minimum at 31 dyn/cm2 and increased again when shear stress rose to 51 dyn/cm2. The flow-dependent elevation of PDGF A and B mRNA in endothelial cells may be involved in the flow-mediated adaptation of blood vessels. We thank C. Betsholtz for providing the PDGF A chain cDNA plasmid and C. D. Rao for providing the PDGF B chain cDNA plasmid. This investigation was supported by National Heart, Lung, and Blood Institute Grant HL-40696. J. A. Frangos is a recipient of the National Science Foundation Presidential Young Investigator Award. Address for reprint requests: J. A. Frangos, Dept. of Chemical Engineering, 149 Fenske Laboratory, The Pennsylvania State University, University Park, PA 16802. Received
18 July
1990; accepted
in final
form
9 November
1990.
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JR., AND R. C. WEBB. Vasoconstriction:
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a new activity for plateletderived growth factor. Science Wash. DC 232: 87-90, 1986. BETSHOLTZ, C., A. JOHNSSON, C.-H. HELDIN, B. WESTERMARK, P. LIND, M. S. URDEA, R. EDDY, T. B. SHOWS, K. PHILPOTT, A. L. MELLOR, T. J. KNOTT, AND J. SCOTT. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumor cell lines. Nature Land. 320: 695-699,1986. BHAGYALAKSHMI, A., AND J. A. FRANGOS. Mechanism of shearinduced prostacyclin production in endothelial cells. Biochem. Biophys. Res. Commun. 158: 33-37, 1989. BHAGYALAKSHMI, A., AND J. A. FRANGOS. Membrane phospholipid metabolism in sheared endothelial cells. Proceedings 2nd International Symposium on Biofluid Mechanics and Biorheology, Munich, FRG, June 1989, 1989, p. 249. BOWEN-POPE, D. F., C. E. HART, AND R. A. SEIFERT. Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which bind to different classes of PDGF receptor. J. Biol. Chem. 264: 2502-2508, 1989. COLLINS, T., J. S. POBER, M. A. GIMBRONE, JR., A. HAMMACHER, C. BETSHOLTZ, B. WESTERMARK, AND C.-H. HELDIN. Cultured human endothelial cells express platelet-derived growth factor A chain. Am. J. Pathol. 127: 7-12, 1987. DIAMOND, S. L., S. G. ESKIN, AND L. V. MCINTIRE. Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science Wash. DC 243: 1483-1485, 1989. DIAMOND, S. L., J. B. SHAREFKIN, C. DIFFENBACH, K. F-SCOTT, L. V. MCINTIRE, AND S. G. ESKIN. Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. J. Cell. Physiol. 143: 364-371, 1990. FRANGOS, J. A., S. G. ESKIN, L. V. MCINTIRE, AND C. L. IVES. Flow effectson prostacyclin production by cultured human endothelial cells. Science Wash. DC 227: 1477-1479, 1985. FRANGOS, J. A., L. V. MCINTIRE, AND S. G. ESKIN. Shear stress induced stimulation of mammalian cell metabolism. Biotechnol. Bioeng. 32: 1053-1060,1988. GUPTE, A., AND J. A. FRANGOS. Effects of flow on the synthesis and releaseof fibronectin by endothelial cells. In Vitro Cell. Dev. Biol. 26: 57-60, 1990. HARLAN, J. M., P. J. THOMPSON, R. R. Ross, AND D. F. BOWENPOPE. cu-Trombin induces release of platelet-derived growth factorlike molecule(s) by cultured human endothelial cells. J. Cell Biol. 103: 1129-1133,1986. KAMIYA, A., AND T. TOGAWA. Adaptive regulation of wall shear stress to flow change in the canine carotid artery. Am. J. Physiol. 239 (IIeart Circ. Phvsiol. 8): Hl4-H21. 1980.
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16. KOUREMBANAS, S., AND D. V. FALLER. Platelet-derived growth factor production by human umbilical vein endothelial cells is regulated by basic fibroblast growth factor. J. Biol. Chem. 264: 4456-4459,1989. 17. LANGILLE, B. L., AND F. O’DONNELL. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science Wash. DC 231: 405-407, 1986. timulus-secretion coupling 18. NEWBY, A. C., AND A. H. HENDERSON. in vascular endothelial cells. Annu. Rev. Physiol. 52: 661-674,199O. 19. NOLLERT, M. U., S. G. ESKIN, AND L. V. MCINTIRE. Shear stress increases inositol trisphosphate levels in human endothelial cells. Biochem. Biophys. Res. Commun. 170: 281-287, 1990. 20. PRASAD, A. R. S., R. M. NEREM, C. J. SCHWARTZ, AND E. A. SPRAGUE. Stimulation of phosphoinositide hydrolysis in bovine aortic endothelial cells exposed to elevated shear stress (Abstract). J. Cell Biol. 109: 313A, 1989. 21. RAO, C. D., H. IGARASHI, I. M. CHIU, K. C. ROBBINS, AND S. A. AARONSON. Structure and sequence of the human c-sislplateletderived growth factor 2 (SISIPDFG2) transcriptional unit. Proc. Natl. Acad. Sci. USA 83: 2392-2396, 1986. 22. REICH, K. M., C. V. GAY, AND J. A. FRANGOS. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J. Cell. Physiol. 143: 100-104, 1990. 23. ROSS, R. The pathogenesis of atherosclerosis: an update. New Engl. J. Med. 314: 488-500, 1986. 24. Ross, R., E. W. RAINES, AND D. F. BOWEN-POPE. The biology of platelet-derived growth factor. Cell 46: 155-169, 1986. 25. STARKSEN, N. F., G. R. HARSH, V. C. GIBBS, AND L. T. WILLIAMS. Regulated expression of the platelet-derived growth factor A chain gene in microvascular endothelial cells. J. Biol. Chem. 262: 1438114384, 1987. 26. SWAN, D. C., 0. W. MCBRIDE, K. C. ROBBINS, D. A. KEITHLEY, E. P. REDDY, AND S. A. AARONSON. Chromosomal mapping of the simian sarcoma virus one gene analogue in human cells. Proc. Natl. Acad. Sci. USA 79: 4691-4695, 1982. 27. Tso, J. Y., X. H. SUN, T. H. KAO, K. S. REECE, AND R. Wu. Isolation and characterization of rat and human glyceraldehyde-3phosphate dehydrogenase cDNAs. Nucleic Acids Res. 13: 24852502,1985. 28. YOSHIZUMI, M., H. KURIHARA, T. SUGIYAMA, F. TAKAKU, M. YANAGISAWA, T. MASAKI, AND Y. YAZAKI. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem. Biophys. Res. Commun. 161: 859-864, 1989. 29. ZARINS, C. K., M. A. ZATINA, D. P. GIDDENS, D. N. Ku, AND S. GLAGOV. Shear stress regulation of artery lumen diameter in exnerimental atherogenesis. J. Vast. Sure. 5: 413-420. 1987.
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