Transforming growth factor-β1 proliferated vascular smooth muscle cells from spontaneously hypertensive rats

AJH 1995; 8:160-166

Transforming Growth Factor- l Proliferated Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats Motohiko Sato, Yoshinobu Ohsaki, and Katsuyuki Tobise

To clarify whether the growth inhibitors, transforming growth factor-~l (TGF-~I), heparin, and interferon-~ (IFN-~/) contribute to the development of vascular hypertrophy in spontaneously hypertensive rats (SHR), the growth of vascular smooth muscle cells (VSMC) was evaluated both for cell numbers over a period of 4 days, and [3H]thymidine incorporation over 24 h. Heparin and IFN-~/ inhibited the proliferation of VSMC from SHR and Wistar-Kyoto (WKY) rats. TGF-~I enhanced SHR-VSMC proliferation by 16.6 - 8.9%; in contrast TGF-~I inhibited WKY-VSMC proliferation by 60.5 +-- 7.4%. There was no difference in affinity, number of binding sites, or subtype expression

of TGF-~ 1 receptor between SHR-VSMC and WKY-VSMC. This evidence suggests that the signal transduction system of TGF-~I either the receptor itself or downstream signaling molecules, may be altered in SHR-VSMC versus WKY-VSMC. This abnormal responsiveness to TGF-~I is involved in the proliferative characteristics of SHRVSMC. Therefore, TGF-~I could contribute to the development of hypertension or vascular hypertrophy in SHR. Am J Hypertens 1995;8:160-166

mooth muscle cells are an important component of vessel media that can cause vascular ,hypertrophy and atherosclerosis. 1"2 Growth and migration of smooth muscle cells are regulated by chemical mediators released from endothelial cells, lymphocytes, macrophages, and smooth muscle cells themselves. 1'2 The cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) have different growth characteristics from those from Wistar-Kyoto (WKY) rats. 3-5 One of these differences is hyper-response against growth stimulators such as serum, 3-5 platelet-derived growth factor, 3'5 epidermal growth factor (EGF), 3-5 or basic fibroblast growth factor, s Some kinds of medi-

ators occasionally cause growth inhibition on normal VSMC. VSMC grow normally w h e n the effect of these growth inhibitors is large enough to suppress the growth stimulatory action. Therefore, the abnormal response against growth inhibitors may cause vascular hypertrophy in SHR. However, the responsiveness to growth inhibitors has been reported in a few studies, 6~ despite the many reports about an enhanced responsiveness to growth stimulators in SHR-VSMC. Transforming growth factor-~ (TGF-~), 9"1° heparin, 6 and interferon ~ (IFN-~/)11 have been found to inhibit VSMC growth. SHR develops vascular hypertrophy before high blood pressure appears. 12'13 The responsiveness to these growth inhibitors may be genetically decreased in SHR-VSMC. To elucidate this, we studied the response to these growth inhibitors using cultured VSMC from SHR and WKY. Our results suggest that the decreased responsiveness of SHR-VSMC to growth inhibitors possibly contributes

S

Received May 23, 1994. Accepted September 27, 1994. From the First Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan. Address correspondence and reprint requests to Motohiko Sato, MD, First Department of Internal Medicine, Asahikawa Medical College, 4-5-3 Nishikagura, Asahikawa 078, Japan.

© 1995 by the American Journal of Hypertension, Ltd.

KEY WORDS: Transforming growth factor-~, interferon, heparin, growth inhibition, vascular smooth muscle, spontaneously hypertensive rat.

0895-7061/95/$9.50 0895-7061(94)00191-D

AJH-FEBRUARY 1995-VOL. 8, NO. 2

to the development of hypertension and vascular hypertrophy.

MATERIALS AND METHODS Materials Cell culture materials and media were obtained from GIBCO (Bethesda, MD). Fetal bovine serum was purchased from Cell Culture Laboratories (Cleveland, OH). Heparin and TGF-~I (human, recombinant) were from WAKO Corp. (Osaka, Japan). Recombinant mouse interferon-~/was obtained from Genozyme (Cambridge, MA). Phenylmethylsulfonyl fluoride, leupeptin, and pepstatin were obtained from Sigma Chemical Corp. (St. Louis, MO). [3H]thymidine (925 GBq/mmol) and 125I-TGF-~1 (human, recombinant) were purchased from Amersham Japan (Tokyo, Japan). Animals Male s p o n t a n e o u s l y hypertensive rats (SHRs/NCrj) and their normotensive counterparts Wistar-Kyoto (WKY/NCrj) rats were obtained from Charles River Japan (Shizuoka, Japan). We used male SHR and WKY at 8 weeks of age. After body weight measurements, the animals' systolic blood pressures were measured in an unanesthetized condition by the indirect tail-cuff method. The average weight of SHRs (mean -- SD) was less than that of WKYs (221 + 16 g v 237 + 15 g; n = 18, P > .05). Systolic blood pressure in SHR was significantly higher than WKY (153 -+ 16 m m H g v 1 0 7 _ + 9 m m H g ; n = 18, P ~ . 0 1 ) .

TGF-[~1 RESPONSIVENESS IN SHR-VSMC

161

absence of growth inhibitor. The culture medium of each plate was replaced every day to maintain the activity of growth inhibitors. The doubling time was calculated from cell numbers on days 0 and 4.

DNA Synthesis Measurement DNA synthesis was measured by the incorporation of [3H]thymidine into TCA-perceptible material. Briefly, growth-arrested VSMC at approximate density 1.5 x 104 cells/cm 2 were exposed to 5% FBS-DMEM or 5% FBS-DMEM containing growth inhibitors as well as 0.5 ~Ci/mL [3H]thymidine. VSMC were incubated for another 24 h. At the end of the labeling period, the cells were washed twice with a 4°C PBS. After washing, 10% TCA was added to each well. Residual TCA-precipitated fractions were extracted using 0.2% N NaOH, and the level of radioactivity was measured using the liquid scintillation counter (LS1801, Beckman, Fullerton, CA).

Receptor-Binding Assay The TGF-~I receptorbinding assay was p e r f o r m e d as previously described. ~5 The cells from SHR or WKY were seeded in 24-well culture plates. When subconfluency (approximately 1 x 10S/cm 2) was obtained, the cells were washed using a binding buffer (DMEM, 1% BSA, 20 mmol/L HEPES, pH 7.4), and incubated in 1.0 mUwell of the same buffer for 2 h at 37°C to dissociate bound endogenous TGF-~I. After washing, binding buffer, containing various concentrations of the 125I-TGF-~l (30 Vascular Smooth Muscle Cell Culture We used the to 500 pmol/L), was added in the presence or absence of modified explant method described by Kawabe et a114 100-fold excess unlabeled TGF-~I. The cells were incuin which VSMC were established from thoracic aortas bated at 4°C, and rocked gently for 3 h. At the end of of SHR and WKY. Briefly, the thoracic aorta was di- incubation, the medium was removed by aspiration, gested with collagenase to remove the endothelium cells were washed four times using the binding buffer, and the adventitia. The medial tissue was minced and free ligand concentration was determined by meainto small pieces, explants, that were then placed on surement of radioactivity in both removed and washed a culture dish and incubated. Culture cells were media. The cells were solubilized using Triton solution VSMC, because they had typical hill and valley ap- (1% Triton X-100, 10% glycerol, 20 mmolFL HEPES, pH pearances as well as containing ot-actin fibers; this 7.4). Aliquots were then counted using a gamma was confirmed by immunohistochemical staining. counter (model 1282, LKB-Wallac Turku, Finland). BeCells from the fourth to the eighth passage were used fore the solubilization step, cell numbers were deterin the present experiment. mined. Binding data w e r e a n a l y z e d u s i n g the Growth Arrest of VSMC Cultured VSMCs in the "LIGAND" program by Munson and Rodbard. 16 normal growth medium were placed in 0.2% FBSAffinity Cross Linking Assay In the cross linking DMEM for 48 h. Flow microfluorimetry confirmed assays, subconfluent cells in 6-well plates (approxithat the cells were arrested in G o (G1), because >95% mately 1 x 105/cm 2) were w a s h e d using binding of VSMC had G O (G1) DNA content. buffer, then incubated in 2.0 mL of the same buffer Doubling Time Measurement Cell growth was ar- for 2 h at 37°C. After washing with binding buffer, rested at an approximate density of 0.5 x 104 cells/ cells were incubated with 0.8 mL of binding buffer cm 2. Cells were then incubated under standard con- containing 125 pmol/L 125I-TGF-~l in the presence or ditions for 4 days in 24 multiwell plates containing 5% absence of unlabeled TGF-~I (5 nmol/L) at 4°C for 3 h. FBS-DMEM with or without growth inhibitors. After After the medium was removed, cells were washed disaggregation using trypsin, the cells were counted four times with Hank's-buffer saline without BSA. in triplicate every second day using a hemocytome- Cells were incubated at 4°C for 15 rain with Hank's ter. Growth inhibition was evaluated by a relative buffer containing disuccinimidyl suberate (DSS) unvalue that was compared to that of the controls in the der constant rotation. The reaction was stopped by

162

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AJH-FEBRUARY 1995-VOL. 8, NO. 2

3

- - o - wKY

y

-----0-- SHR

0

2

4

days

FIGURE 1.

Proliferation of smooth muscle cells in 5% FBSD M E M . Values are expressed as mean +- SD (n = 18). **P < .01 as compared with W K Y by unpaired t test.

the addition of d e t a c h m e n t buffer (10 mmol/L Tris, p H 7.4, 1 mmol/L EDTA, 10% glycerol, 0.3 mmol/L p h e n y l m e t h y l s u l f o n y l fluoride, 1 p~g/mL pepstatin, 1 ~,g/mL leupeptin), cells were h a r v e s t e d by scraping. After centrifugation at 2000 g for 15 min at 4°C, the cells were r e s u s p e n d e d in the presence of solubilization buffer (125 mmol/L NaC1, 10 mmol/L Tris, p H 7.4, 1 mmol/L EDTA, 1% Triton X-100, 0.3 mmol/L p h e n y l m e t h y l s u l f o n y l fluoride, 1 p,g/mL pepstatin, 1 ~g/mL leupeptin). The d e t e r g e n t - s o l u b l e fractions were electrophoresed on 8% SDS-polyacrylamide gel in the presence of dithiothreitol. The gels were dried and exposed using Kodak X-ray film at -80°C. Values are expressed as m e a n + SD. The level of significance of differences b e t w e e n the m e a n s was evaluated by u n p a i r e d t test, and onew a y or t w o - w a y analysis of variance (ANOVA) with multiple comparisons. Statistical A n a l y s i s

RESULTS Characteristics of Vascular S m o o t h M u s c l e Proliferation We c o m p a r e d the proliferation of VSMC from

SHR with that from WKY over 4 days in 5% FBSDMEM. Quiescent VSMC started proliferation after r e p l a c e m e n t of the m e d i u m of serum-free DMEM with 5% FBS-DMEM. On the second day, the n u m b e r

of SHR-VSMC was larger than that of WKY-VSMC, and the difference b e t w e e n WKY and SHR cell n u m bers was significant on the fourth day. Doubling time of SHR-VSMC was 38.4 -+ 6.7 h, and that of WKYVSMC was 41.4 -+ 2.5 h (mean + SD, n = 18, P < .05). SHR-VSMC had an accelerated cell proliferation c o m p a r e d to that of WKY (Figure 1). We also e x a m i n e d the possibility that autocrine proliferation of VSMC influences the results of our e x p e r i m e n t s . S u b c o n f l u e n t SHR-VSMC or WKYVSMC was incubated in DMEM for 48 h, and the m e d i u m was collected as a conditioned m e d i u m . We cultured SHR-VSMC and WKY-VSMC u n d e r 5% FBS in the presence or absence of the c o n d i t i o n e d medium; the conditioned m e d i u m did not s h o w a n y effect on the proliferation of either VSMC (Table 1). Effects of Growth Inhibitors on Cell Proliferation

TGF-61 inhibited the serum-stimulated proliferation of WKY-VSMC by 60.5 -+ 7.4% at the m a x i m u m . In contrast, it stimulated the proliferation of SHR-VSMC by 16.6 + 8.9% (Figure 2A). H e p a r i n inhibited the g r o w t h of WKY-VSMC after the second day (Figure 3A), and it also inhibited the SHR-VSMC proliferation in a d o s e - d e p e n d e n t fashion. H o w e v e r , the inhibition of SHR-VSMC by heparin was significantly smaller than that of WKY-VSMC on the second day in the concentration of 50 ~g/mL or 100 ~g/mL (SHRVSMC v WKY-VSMC: 93.2 + 10.6% a n d 63.0 + 15.2% in 50 ~,g/mL, 85.9 -+ 8.4% and 59.2 + 11.5% in 100 ~g/mL, respectively; P < .01). There was no statistical difference in the inhibitory ratio at the h i g h e r concentration of 200 p,g/mL. IFN-~ inhibited the proliferation of both VSMCs in a d o s e - d e p e n d e n t fashion. The inhibitory ratio was similar in SHR-VSMC and WKY-VSMC at 46.1 -+ 7.5% and 47.7 -+ 12.3%, respectively, at the m a x i m u m (Figure 4A). The biological activity of IFN-y differs a m o n g species. In this study, we used m o u s e IFN-y that h a d an activity equivalent to rat IFN-y. 17 Alt h o u g h TGF-[31 s u p p r e s s e d D N A synthesis on WKYVSMC in a d o s e - d e p e n d e n t fashion, it e n h a n c e d Effects of Growth Inhibitors on D N A S y n t h e s i s

TABLE 1. EFFECT OF CONDITIONED MEDIUM ON VSMC GROWTH

Cell

WKY

SHR

Conditioned Medium From

-WKY SHR -WKY SHR

Cell Number (/well x 10 -3) Day 2

Day 0

36.6 39.0 35.0 33.1 37.5 34.2

+ 3.0 + 4.8 + 3.4 + 4.3 + 4.4 -+ 4.4

NS NS NS NS

99.0 103.5 101.7 122.7 124.7 123.2

NS, not significant compared with normal growth medium group by unpaired t test,n = 6.

+ 4.9 + 6.1 NS + 13.0 NS + 13.1 + 14.7 NS -+ 12.8 NS

Day 4

178.6 189.3 176.5 239.0 227.0 241.7

-+ 14.0 -+ 12.2 + 16.5 + 14.8 + 14.2 + 24.2

NS NS NS NS

TGF-f~1 RESPONSIVENESSIN SHR-VSMC 163

AJH-FEBRUARY 1995-VOL. 8, NO. 2

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FIGURE 3. (A) Effect of heparin on proliferation of smooth

muscle cells stimulated by 5% FBS. Values are expressed as mean + SD (n -= 6) of representative experiments (out of four experiments that gave similar results). *P < .05, **P < .01 as compared with WKY by unpaired t test. (B) Effect of TGF-~I on [3H]thymidine incorporation into DNA of smooth muscle cells stimulated by 5% FBS. Values are expressed as mean +- SD (n = 6 in duplicate) of representative experiments (ou~ of three experiments that gave similar results). **P < .01 as compared with WKY by unpaired t test.

muscle cells stimulated by 5% FBS. Values are expressed as mean +- SD (n = 6) of representative experiments (out of four experiments that gave similar results). **P < .01 as compared with WKY by unpaired t test. (B) Effect of heparin on [3H]thymidine incorporation into DNA of smooth muscle cells stimulated by 5% FBS. Values are expressed as mean +- SD (n = 6 in duplicate) of representative experiments (out of three experiments that gave similar results). *P < .05, **P < .01 as compared with WKY by unpaired t test.

DNA synthesis in SHR-VSMC at concentrations of 1, 5, and 10 ng/mL (Figure 2B). Heparin suppressed DNA synthesis in a dose-dependent fashion in SHRVSMC and WKY-VSMC. Although the inhibitory effects in SHR-VSMC were smaller at the concentration of 50 p,g/mL or 100 t~g/mL than those in WKY-VSMC (P < .05 in 50 t~g/mL, and P < .01 in 100 p~g/mL), they were equivalent at 200 t~g/mL in both VSMCs (Figure 3B). IFN-~ inhibited DNA synthesis in VSMC from both strains in a dose-dependent fashion. The inhibition of IFN-~/was not significant in SHR-VSMC and WKY-VSMC (Figure 4B).

of high-affinity binding sites as WKY-VSMC (ie, 3500 sites/cell with an apparent K d of 17 pmol/L in SHRVSMC and 2400 sites/cell with an apparent K d of 14 pmol/L in WKY-VSMC). There seemed to be no difference in the number of high-affinity receptors between SHR-VSMC and WKY-VSMC.

TGF-~ Receptor-Binding Assay To d e t e r m i n e whether the proliferative effect of TGF-~I on SHRVSMC is due to receptor properties, we examined the binding characteristics of 125 I-TGF-~I using subconfluent SHR-VSMC and WKY-VSMC. Scatchard plot analysis showed a linear plot in both VSMCs (Figure 5). SHR-VSMC had approximately the same number

Affinity Cross-Linking Assay of TGF-f~ Receptor To determine the characteristics of TGF-~ receptors in SHR-VSMC further, we examined the subtype expression of TGF-~ receptors using affinity cross-linking assay. We confirmed TGF-~I binding as bands with calculated molecular mass of 65,000, 85,000, and 280,000 daltons, corresponding to the types I, II, and III TGF-~ receptors, respectively (Figure 6). Both SHR-VSMC and WKY-VSMC expressed all three types of receptors. The binding was specific, because these bands were absent in the presence of 5 nmol/L unlabeled TGF-~I. Therefore, the subtype expression of TGF-~ receptors in SHR-VSMC was identical with WKY-VSMC.

164

SATO ET AL

AJH-FEBRUARY 1995-VOL. 8, NO. 2

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FIGURE 4. (A) Effect of lFN-~/ on proliferation of smooth muscle cells stimulated by 5% FBS. Values are expressed as mean +SD (n = 6) of representative experiments (out of four experiments that gave similar results). (B) Effect of IFN-'y on [3H]thymidine incorporation into DNA of smooth muscle cells stimulated by 5% FBS. Values are expressed as mean + SD (n = 6 in duplicate) of representative experiments (out of three experiments that gave similar results).

DISCUSSION

The response to growth inhibitors, such as heparin and TGF-~I, using SHR-VSMC has been reported in only a few articles. 6~ There has been no report describing SHR-VSMC growth response to IFN-~/. In contrast, the growth response of cultured VSMC from SHR to various growth stimulators has been described in many reports. 3-5 These chemical mediators exist in an atherosclerotic plaque, and probably regulate the growth of VSMC. Therefore, the responses to these growth inhibitors are very important in studying the mechanisms of vascular hypertrophy. TGF-[33 stimulated SHR-VSMC growth at low cell density (1.5 x 104 cell/cm2), whereas TGF-[31 suppressed WKY-VSMC growth at the same cell density. TGF-~ is released by endothelial cells, is smooth muscle cells, 19 and macrophages. 2° TGF-~ induces the hypertrophy and polyploidy of VSMC, 9 and stimulates extracellular matrix synthesis in fibroblasts, m This evidence suggests that TGF-6~ probably contributes to the development of hypertension and vascular hy-

~ lO ~m° o

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0

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6

FIGURE 5. Scatchard analysis of 125I-TGF-~l binding to vascular smooth muscle cells from SHR (A) and WKY (B) using the "'LIGAND'" program developed by Munson and Rodbard. 16 Specifically bound and free ligands were measured as described in Materials and Methods.

pertrophy. In fact, hypertension and aging cause the increased expression of TGF-~ 3 mRNA in the aorta in vivo. 22 In VSMC, TGF-[3 usually acts as an inhibitor of proliferation under serum stimulation in vitro. 9'1° In the SHR-VSMC system, Hamet et al 7 reported that TGF-[31 significantly increased D N A synthesis in SHR-VSMC at a high cell density (approximately 1.2 x 105 cell/cm2); in contrast, there is no effect on proliferation in WKY-VSMC. Saltis et al 8 observed that the combination of TGF-~I and EGF stimulated DNA synthesis in SHR-VSMC. In their report, the synergic interaction between TGF-~I and EGF was independent of initial cell density at 1.0 x 104, 1.0 x 10s, and 2.5 x 10s cell/cm 2. This evidence shows that the proliferative effect of TGF-~I in SHR is independent of cell density. There seemed to be no difference in the TGF-~ receptor number and affinity between SHR-VSMC and WKY-VSMC (Figure 5). The different response in SHR could be explained by the alteration of TGF-[3 receptor subtypes. Boyd and Massagu623 reported that the MvlLu epithelial mutants, which have selec-

TGF-13] RESPONSIVENESS IN SHR-VSMC 165

AJH-FEBRUARY 1995-VOL. 8, NO. 2

TGF, B I ~ -

~ III

FIGURE 6. Affinity labeling of the TGF-~I receptor of SHR-

explains that there is no difference in the heparin binding at this high concentration. Although SHRVSMC kept quiescent in the concentrations above 200 ~g/mL, it started to proliferate easily at concentrations below 200 ~g/mL. This concentration may be derived from the destruction of vessel walls at the beginning of vascular hypertrophy. IFN-% which exists in the edge of atherosclerotic plaque, B°is a cytokine released from activated T cells. IFN-~/ suppressed the vascular hypertrophy inhibiting VSMC proliferation. 11 In the present study, the growth inhibition of IFN-~/ did not differ between SHR-VSMC and WKY-VSMC (Figure 4). Although details of IFN-~/signal transduction pathway remain unclear, the response to IFN-~/ hardly explains vascular hypertrophy in SHR. ACKNOWLEDGMENT

VSMC and WKY-VSMC. Cells were incubated with ~zSI-TGF-f31 alone ( - ) or in the presence of 5 nmol/L unlabeled TGF-~I (+), then treated with DSS as described in Materials and Methods. The positions of the three labeled TGF-f31 receptor types are indicated at the right of the figure. Molecular mass standards are indicated in thousands on the left.

We thank Prof. Kenjiro Kikuchi for discussion and critical review, Dr. Sokichi Onodera for advice on the manuscript, Dr. Jun-ich Kawabe for helpful collaboration, and Simon Bayley for revision of the English.

tively lost the expression of type I receptor, were resistant to the growth inhibitory action of TGFqB. We examined the subtype expression of TGF-~ receptors to explain the different response between SHR and WKY to TGF-~I. Type I receptor is a major component of TGFq3 actions. 23'24 Both type I and II receptors are necessary to respond to TGF-~I biologically.2~ Type III, the most abundant cell surface receptor, modified the binding properties in type I and II receptors. 2B'24 In the present study, type I, If, and III subtype receptors were equally expressed in both VSMCs (Figure 6). That is a novel observation. TGF[31 suppressed the expression of c-myc mRNA keratinocyte26 and HL-60 cells27 when TGFq31 was added to the culture media and inhibited the cell proliferation. However, TGFq31 enhanced the c-myc mRNA expression in SHR-VSMC. s This suggests that TGF-~I enhanced the proliferation in SHR-VSMC through alteration of the signal transduction system, either at the level of the receptor itself (receptor turnover or p r i m a r y structure) or d o w n s t r e a m signaling molecules. Heparin is an element of extracellular matrix, which is released by endothelial cells as and V S M C . 29 Heparin keeps normal VSMC quiescent in vivo. SHRVSMC had a decreased responsiveness to heparin, which may be attributable to a low binding capacity of [3H]heparin to the extracellular surface. 6 Our results at concentrations of 50 ~g/mL and 100 ~g/mL supported the report of Resink et al. 6 However, no statistical difference was observed at the concentration of 200 ~g/mL (Figure 3). This evidence possibly

1. Ross R: The pathogenesis of atherosclerosis--an update. N Engl J Med 1986;314:488-500. 2. Bondjers G, Glukhova M, Hansson GK, et ah Hypertension and atherosclerosis; cause and effect, or two effects with one unknown cause? Circulation 1991; 84(suppl VI):VI-2-VI-16. 3. Hadrava V, Tremblay J, Hamet P: Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension 1989; 13:589-597. 4. Scott-Burden T, Resink TJ, Baur U, et al: Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension 1989;13:295-304. 5. SaltisJ, Agrotis A, Bobik A: Difference in growth characteristics of vascular smooth muscle from spontaneously hypertensive and Wistar-Kyoto rats are growth factor dependent. J Hypertens 1993;11:629-637. 6. Resink TJ, Scott-Burden T, Baur U, et al: Decreased susceptibility of cultured smooth muscle cells from SHR rats to growth inhibition by heparin. J Cell Physiol 1989;138:137-144. 7. Hamet P, Hadrava V, Kruppa U, Tremblay J: Transforming growth factor ~1 expression and effect in aortic smooth muscle cells from spontaneously hypertensive rats. Hypertension 1991;17:896-901. 8. Saltis J, Agrotis A, Bobik A: TGF-f31 potentiates growth factor-stimulated proliferation of vascular smooth muscle cells in genetic hypertension. Am J Physiol 1992;263 (Cell Physiol 32):C420-C428. 9. Owens GK, Geisterfer AAT, Yang YW-H, Komoriya A: Transforming growth factor-p-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. J Cell Biol 1988;107:771-780. 10. Majack RA: Beta-type transforming growth factor

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