Clinical and Experimental Pharmacology and Physiology (1992) 19,396-399
SHORT COMMUNICATION
DIFFERENTIAL REGULATION BY TRANSFORMING GROWTH FACTOR-PI OF PLATELET-DERIVED GROWTH FACTOR-STIMULATED PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS FROM SHR AND WKY RATS John Saltis, Alex Agrotis and Alex Bobik Baker Medical Research Institute and Alfred-Baker Medical Unit, A l f e d Hospital, Prahran, Victoria, Australia (Received 13 December 1991)
SUMMARY 1. This study has examined and compared the time-course of action of transforming growth factor-pl (TGF-PI) on platelet-derived growth factor-BB-stimulated proliferation of vascular smooth muscle cells isolated from normotensive Wistar-Kyoto (WKY)rats and spontaneously hypertensive rats (SHR). 2. Transforming growth factor-& inhibited vascular smooth muscle cell proliferation stimulated by platelet-derived growth factor-BB in WKY rats by approximately 60-75%. 3. In contrast, transforming growth factor-pi potentiated an 8-35% growth factor action on cell proliferation in the SHR. 4. Defects in transforming growth factor-PI action may be part of the molecular mechanism responsible for the development of vascular hypertrophy in the SHR. Key words: spontaneously hypertensive rat, transforming growth factor-81, vascular smooth muscle.
INTRODUCTION Vascular hypertrophy is known to be a major contribulor to the elevated blood pressure in the spontaneously hypertensive rat (SHR Korner et al. 1989). In this strain vascular hypertrophy has been generally attributed to genetic defects in the smooth muscle (Owens 1989). To date, however, the precise nature of these defects has not been characterized. A
number of studies in cell culture have shown that vascular smooth muscle cells (VSMC) from SHR proliferate more rapidly than VSMC from normotensive Wistar-Kyoto (WKY) rats (Hadrava et al. 1989; Scott-Burden et d. 1989; Saltis et a/. 1991). Since vascular growth in vivo would be expected to be a balance between positive and negative regulators of
Correspondence: Dr John Saltis, Baker Medical Research Institute, Alfred Hospital, Commercial Road, Prahran, Vic. 3 181, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
Transforming growth factor-PI
proliferation, it is also possible that defects exist in mechanisms that attenuate vascular smooth muscle growth in the SHR. During the past few years, TGF-PI has been shown to be an important regulator of vascular smooth muscle growth. TGF-PI is secreted by both VSMC and endothelial cells and thus has the potential to exert its effects on VSMC in both an autocrine and paracrine fashion (Antonelli-Orlidge et al. 1989). Importantly, TGF-PI can exert multiple effects on VSMC, including the induction of cellular hypertrophy and polyploidy (Owens et al. 1988), as well as stimulating or inhibiting cell proliferation (Majack 1987; Battegay et al. 1990). These effects are the consequence of TGF-PI binding to specific receptors on the surface of vascular smooth muscle cells (Massague 1990). In a recent study it was demonstrated that serumstimulated proliferation of VSMC from SHR is less susceptible to the growth inhibitory effect of TGF-pl when compared with VSMC from WKY rats (Saltis & Bobik 1992). Since the growth factors that interacted with TGF-PI in this study were poorly characterized, the present study examined whether a similar effect of TGF-Pl was observed when VSMC from SHR were stimulated with plateletderived growth factor-BB (PDGF-BB), a growth factor known to be secreted by cells within the vascular wall (Ross et al. 1986). Hence the aim of this study was to examine and compare the actions of transforming growth factor-pl on PDGF-BB-stimulated proliferation of VSMC from SHR and WKY rats.
METHODS Isolation of aortic smooth muscle cells Primary cultures of vascular smooth muscle cells were prepared by enzyme digestion of aortic media from 12-14 week old SHR and WKY rats as described by Saltis el al. (1991). Briefly, thoracic aortae were dissected under sterile conditions and placed in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal calf serum (FCS) and 60 pg/mL penicillin G. The vessels were cleared of fat and connective tissue, cut longitudinally and then incubated at 37" C for 30 min with 3 mg/ mL collagenase type I (Sigma, St. Louis, MO, USA) in DMEM to remove the endothelium. The aortae were transferred to fresh 10% FCS/ DMEM and the media plus intima peeled from the adventitia using watchmaker forceps. The muscle layer was then cut into 1-2 mm strips and
397
incubated at 37°C for approximately 3 h in the presence of collagenase and elastase (1.O mg/ aorta) in DMEM. Isolated vascular smooth muscle cells were grown in 10%FCS/DMEM and passaged every 5-7 days.
Cell proliferation Vascular smooth muscle cells (1-2 X 104) were plated onto 24-well tissue culture dishes in 1.0 mL of 10% FCS/DMEM. The following day, the medium was replaced with 1.0 mL of DMEM alone and cells were incubated for a further 24 h. Transforming growth factor-pl (British Bio-Technology Ltd, Oxford, UK) and platelet-derived growth factor-BB (Amersham, Melbourne, Australia) were then added in 0.5 mL of serum-free medium containing transferrin and insulin (4% Monomed A; Commonwealth Serum Laboratories, Melbourne, Australia) in DMEM. This medium containing the growth factors was changed every 2 days. Cell numbers were determined at the indicated times using a Coulter Counter. Vascular smooth muscle cells passaged once were used in this study. Growth rates were calculated using the formula: (PZ--PI)/PI (t2-t
1)
where PI and PZare the cell numbers at respective times tl and tz. Statistical significance was tested with an unpaired t-test analysis and differences were accepted as significant at the P
SHORT COMMUNICATION
DIFFERENTIAL REGULATION BY TRANSFORMING GROWTH FACTOR-PI OF PLATELET-DERIVED GROWTH FACTOR-STIMULATED PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS FROM SHR AND WKY RATS John Saltis, Alex Agrotis and Alex Bobik Baker Medical Research Institute and Alfred-Baker Medical Unit, A l f e d Hospital, Prahran, Victoria, Australia (Received 13 December 1991)
SUMMARY 1. This study has examined and compared the time-course of action of transforming growth factor-pl (TGF-PI) on platelet-derived growth factor-BB-stimulated proliferation of vascular smooth muscle cells isolated from normotensive Wistar-Kyoto (WKY)rats and spontaneously hypertensive rats (SHR). 2. Transforming growth factor-& inhibited vascular smooth muscle cell proliferation stimulated by platelet-derived growth factor-BB in WKY rats by approximately 60-75%. 3. In contrast, transforming growth factor-pi potentiated an 8-35% growth factor action on cell proliferation in the SHR. 4. Defects in transforming growth factor-PI action may be part of the molecular mechanism responsible for the development of vascular hypertrophy in the SHR. Key words: spontaneously hypertensive rat, transforming growth factor-81, vascular smooth muscle.
INTRODUCTION Vascular hypertrophy is known to be a major contribulor to the elevated blood pressure in the spontaneously hypertensive rat (SHR Korner et al. 1989). In this strain vascular hypertrophy has been generally attributed to genetic defects in the smooth muscle (Owens 1989). To date, however, the precise nature of these defects has not been characterized. A
number of studies in cell culture have shown that vascular smooth muscle cells (VSMC) from SHR proliferate more rapidly than VSMC from normotensive Wistar-Kyoto (WKY) rats (Hadrava et al. 1989; Scott-Burden et d. 1989; Saltis et a/. 1991). Since vascular growth in vivo would be expected to be a balance between positive and negative regulators of
Correspondence: Dr John Saltis, Baker Medical Research Institute, Alfred Hospital, Commercial Road, Prahran, Vic. 3 181, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
Transforming growth factor-PI
proliferation, it is also possible that defects exist in mechanisms that attenuate vascular smooth muscle growth in the SHR. During the past few years, TGF-PI has been shown to be an important regulator of vascular smooth muscle growth. TGF-PI is secreted by both VSMC and endothelial cells and thus has the potential to exert its effects on VSMC in both an autocrine and paracrine fashion (Antonelli-Orlidge et al. 1989). Importantly, TGF-PI can exert multiple effects on VSMC, including the induction of cellular hypertrophy and polyploidy (Owens et al. 1988), as well as stimulating or inhibiting cell proliferation (Majack 1987; Battegay et al. 1990). These effects are the consequence of TGF-PI binding to specific receptors on the surface of vascular smooth muscle cells (Massague 1990). In a recent study it was demonstrated that serumstimulated proliferation of VSMC from SHR is less susceptible to the growth inhibitory effect of TGF-pl when compared with VSMC from WKY rats (Saltis & Bobik 1992). Since the growth factors that interacted with TGF-PI in this study were poorly characterized, the present study examined whether a similar effect of TGF-Pl was observed when VSMC from SHR were stimulated with plateletderived growth factor-BB (PDGF-BB), a growth factor known to be secreted by cells within the vascular wall (Ross et al. 1986). Hence the aim of this study was to examine and compare the actions of transforming growth factor-pl on PDGF-BB-stimulated proliferation of VSMC from SHR and WKY rats.
METHODS Isolation of aortic smooth muscle cells Primary cultures of vascular smooth muscle cells were prepared by enzyme digestion of aortic media from 12-14 week old SHR and WKY rats as described by Saltis el al. (1991). Briefly, thoracic aortae were dissected under sterile conditions and placed in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal calf serum (FCS) and 60 pg/mL penicillin G. The vessels were cleared of fat and connective tissue, cut longitudinally and then incubated at 37" C for 30 min with 3 mg/ mL collagenase type I (Sigma, St. Louis, MO, USA) in DMEM to remove the endothelium. The aortae were transferred to fresh 10% FCS/ DMEM and the media plus intima peeled from the adventitia using watchmaker forceps. The muscle layer was then cut into 1-2 mm strips and
397
incubated at 37°C for approximately 3 h in the presence of collagenase and elastase (1.O mg/ aorta) in DMEM. Isolated vascular smooth muscle cells were grown in 10%FCS/DMEM and passaged every 5-7 days.
Cell proliferation Vascular smooth muscle cells (1-2 X 104) were plated onto 24-well tissue culture dishes in 1.0 mL of 10% FCS/DMEM. The following day, the medium was replaced with 1.0 mL of DMEM alone and cells were incubated for a further 24 h. Transforming growth factor-pl (British Bio-Technology Ltd, Oxford, UK) and platelet-derived growth factor-BB (Amersham, Melbourne, Australia) were then added in 0.5 mL of serum-free medium containing transferrin and insulin (4% Monomed A; Commonwealth Serum Laboratories, Melbourne, Australia) in DMEM. This medium containing the growth factors was changed every 2 days. Cell numbers were determined at the indicated times using a Coulter Counter. Vascular smooth muscle cells passaged once were used in this study. Growth rates were calculated using the formula: (PZ--PI)/PI (t2-t
1)
where PI and PZare the cell numbers at respective times tl and tz. Statistical significance was tested with an unpaired t-test analysis and differences were accepted as significant at the P
