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Angiotensin II induces proliferation of human cerebral artery smooth muscle cells through a basic fibroblast growth factor (bFGF) dependent mechanism
Neuroscience Letters 373 (2005) 38–41
Angiotensin II induces proliferation of human cerebral artery smooth muscle cells through a basic fibroblast growth factor (bFGF) dependent mechanism Zhongbiao Wanga,∗ , Pulipaka J. Raob , Samuel D. Shillcuttb , Walter H. Newmana,c a
b
Division of Basic Medical Science, Mercer University School of Medicine and Medical Center of Central Georgia, 1550 College Street, Macon, GA 31207, USA Department of Psychiatry and Behavioral Science, Mercer University School of Medicine and Medical Center of Central Georgia Macon, GA, USA c Department of Surgery, Mercer University School of Medicine and Medical Center of Central Georgia Macon, GA, USA Received 25 June 2004; received in revised form 24 September 2004; accepted 24 September 2004
Abstract Remodeling of cerebral arteries in hypertension produces thickened vessel walls associated with atherosclerotic plaque formation. In both thickening and plaque formation, proliferation of vascular smooth muscle cells is a hallmark. Genetically hypertensive rats treated with an angiotensin II (Ang II) AT1 receptor antagonist inhibited thickening of cerebral arteries suggesting a mitogenic action of Ang II on cerebral arterial VSMC (CVSMC). However, in studies using smooth muscle cells cultured from peripheral arteries, Ang II causes cell hypertrophy, but not proliferation. We determined the effect of Ang II on proliferation of cultured human CVSMC. CVSMC were cultured from the basilar artery obtained at autopsy. Ang II (10−7 M) stimulated proliferation determined by counting cells and mitochondrial activity assay. Synthesis and release of basic fibroblast growth factor (bFGF) was essential for Ang II-stimulated proliferation. These findings are consistent with the notion that Ang II stimulates CVSMC proliferation thereby contributing to vessel remodeling. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Angiotensin II; Cerebrovascular smooth muscle cells; Proliferation; Basic fibroblast growth factor
Chronic hypertension results in remodeled arteries with thickened walls and narrowed lumens and accelerated formation of atherosclerotic plaques in peripheral as well as cerebral arteries. The consequence of these changes in cerebral vessels is a three to four fold increase in the incidence of stroke. Angiotensin II (Ang II) contributes to hypertension through vasoconstriction and effects on vascular smooth muscle cell growth. Most studies in cells isolated from rat and human peripheral arteries (PVSMC), indicate that Ang II primarily stimulates protein synthesis resulting in cellular hypertrophy rather than increased cell proliferation [2,4,5]. Whether these results in peripheral cells apply to smooth muscle cells isolated from cerebral vessels (CVSMC) is unknown. Evidence suggests that Ang II plays a major role in hyper∗
Corresponding author. Tel.: +1 478 301 2095; fax: +1 478 301 2913. E-mail address: wang [email protected] (Z. Wang).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.09.068
tensive remodeling of cerebral vessels. For instance, studies with mice over expressing the renin–angiotensin system indicate that Ang II directly produces hypertrophy and remodeling of cerebral arterioles [1]. Additionally, chronic treatment with an Ang II subtype 1 (AT1) receptor blocker normalized cerebral autoregulation, reduced brain infarct size, and increased diameter and reduced wall thickness of cerebral vessels in spontaneously hypertensive rats [3,6,10]. One key feature associated with thickened walls of remodeled arteries is growth of vascular smooth muscle cells (VSMC). Therefore, we studied the effect of Ang II on CVSMC isolated from human basilar artery and found that Ang II stimulated proliferation by a mechanism involving basic fibroblast growth factor (bFGF). After obtaining consent for autopsy and use of tissue for research, the intracrainial basilar and coronary arteries from three donors were removed within 6 h of death. Primary cul-
Z. Wang et al. / Neuroscience Letters 373 (2005) 38–41
tures of VSMC were isolated from explants of these arteries by a method described previously [12]. The cells in culture expressed smooth muscle-specific differentiation proteins, calponin, smooth muscle-myosin heavy chain, and smooth muscle ␣-actin, determined by Western blot analysis. Cells at passages 3 through 4 were used in the present work. To determine the effects of Ang II on proliferation, cells were added to 96-well plates in Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% calf serum (CS) for 24 h. Then they were growth arrested by incubation in DMEM containing 0.1% CS for an additional 2 days. Ang II (10−7 M) was then added and cell number was quantitated at 6, 12, 24, and 48 h. For comparison, an identical time course was determined using recombinant human bFGF (1 ng/ml). To evaluate the role of endogenous bFGF in Ang II-stimulated proliferation, cells were incubated as described above with Ang II or bFGF for 48 h in the presence or the absence of anti-bFGF antibodies (5 g/ml). Isotypematched nonimmune IgG was added to parallel cultures to assess the specificity of the anti-bFGF antibodies. Cell proliferation was assessed by the mitochondrial dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) to formazan as previously described [9]. Additionally, in identical experiments, cell proliferation was assayed by counting cells after staining with trypan blue. The effect of Ang II on expression of bFGF was determined in CVSMC by quantitation of mRNA in cell lysates with a Direct ProtectTM ribonuclease protection assay kit according to manufacturer’s instruction. Briefly, CVSMC were growth arrested as described above for 2 days, then stimulated with Ang II for 3 h and then lysed. Before assay, ␣-[32 P] UTP-labeled, anti-sense RNA probes were synthesized under the direction of T7 polymerase using the DNA templates containing human bFGF and ribosomal protein L32 cDNA, using in vitro transcription kit MAXIscriptTM . After purification, the labeled probes (5 × 104 CPM) were hybridized with the lysates of the cells overnight. Samples were co-assayed for L32 mRNA as an internal quantification standard. After digestion of the nonhybridizing species with a mixture of RNases A and T1, the protected cRNA fragments were separated by 5% polyacrylamide/8 M urea gel. The gels were then exposed to Kodak X-OMAT AR film with intensifying screens. The effect of Ang II on the release of bFGF into the culture medium by CVSMC was determined by slot-blotting. Briefly, the cells were growth arrested for 24 h as described and the medium was replaced with 0.1% CS-DMEM with or without Ang II (10−7 M). Levels of bFGF in the medium at 6, 12, and 24 h were determined with a monoclonal anti-bFGF antibody demonstrated by the manufacturer to be specific to bFGF and without cross reactivity to other cytokines including acid FGF. The blotted bFGF was visualized by ECL. Cell number was assessed with MTT assay as described above. The statistical evaluation of the data, expressed as mean ± S.D., was based on n = 9 (three donors, triplicate de-
39
Fig. 1. (A) Ang II and bFGF stimulated proliferation of cerebral vascular smooth muscle cells (CVSMC). CVSMC were seeded in 96-well plates (4000 per well) and cultured overnight to allow adherence. Cells were growth arrested and Ang II (10−7 M) or bFGF (1 ng/ml) was added. At 6, 12, 24, and 48 h cell proliferation was determined by MTT assay. Untreated cells served as control. The mean ± S.D. of optical densities for untreated cells was determined and defined as 100% ± S.D. Cell proliferation is expressed as percentage increase over untreated cells (Ctrl.). (B) bFGF but not Ang II stimulated cell proliferation in peripheral VSMC (PVSMC). Cells were growth arrested and Ang II (10−7 or 10−6 M) or bFGF (1 ng/ml) was added. Cell proliferation was determined at 48 h by MTT assay. P < 0.05 vs. control.
termination of one culture of each donor). Analysis was performed by ANOVA with P values < 0.05 considered to be significant. As seen in Fig. 1A, Ang II (10−7 M) stimulated proliferation of CVSMC. Forty-eight hours after the addition of Ang II cell number had increased 56% over control cells by MTT measurement. In cell counting experiments, a similar increase in cell number (48%) was determined at 48 h after Ang II (10−7 M). In comparison, bFGF (1 ng/ml) increased cell number by over 100% at 48 h. In contrast, Ang II at 10−7 M and at a concentration 10-fold higher (10−6 M) than in CVSMC had no detectable effect on proliferation of PVSMC at 48 h (Fig. 1B). These cells isolated from a peripheral artery, however, were capable of proliferating as seen by the 140% increase in cell number following the addition of bFGF. To determine a potential role of endogenous bFGF in Ang II-induced CVSMC proliferation, we first examined Ang II
40
Z. Wang et al. / Neuroscience Letters 373 (2005) 38–41
Fig. 2. Ang II stimulated transcription of bFGF gene and release of bFGF from CVSMC. CSMC were growth-arrested as described in Fig. 1 and then stimulated with Ang II (10−7 M). (A) 3 h after the stimulation, cells were lysed. bFGF and L32 mRNAs were detected using ribonuclease protection assay. (B) 6, 12, and 24 h after stimulation with Ang II, cell culture medium was collected, and analyzed for bFGF protein by immuno-slot-blot assay. Medium from untreated cells was used as controls. Shown is one representative of results from three experiments.
stimulated bFGF gene transcription. As shown in Fig. 2A, mRNA for bFGF was just detectable in control cells. At 3 h post stimulation with Ang II there was a marked increase in transcription of the growth factor gene. Next, release of bFGF into the culture medium of CVSMC was determined. Media was collected at different time intervals after stimulation of Ang II. Media from untreated cells served as controls. bFGF released from untreated cells was detectable at 24 but not at 6 and 12 h (Fig. 2B). Stimulation with Ang II (10−7 M) increased release of bFGF compared to the corresponding control at 12 and 24 h. To evaluate possible roles of AT1 receptor signaling and bFGF released by the cells in Ang II-stimulated proliferation of CVSMC, a selective AT1 receptor antagonist (ZD 7155, Tocris Cookson Inc., Ballwin, MO) and a neutralizing antibody was, respectively, added to the culture medium prior to stimulation with Ang II. As seen in Fig. 3, addition of ZD 7155 (10 nM) or bFGF neutralizing antibody (5 g/ml) completely blocked Ang IIinduced cell proliferation measured by MTT. Cell counting experiments showed a similar effect of neutralization of bFGF. The antibody also blocked cell proliferation induced by the addition of exogenous bFGF (1 ng/ml). Isotypematched nonimmune IgG was added to parallel cultures at same concentration had no effect on the proliferation (data not shown). These results indicate that Ang II stimulates proliferation of CVSMC but not VSMC isolated from a peripheral vessel, coronary artery. Further, Ang II stimulated proliferation in CVSMC by inducing gene transcription, synthesis, and release of bFGF. bFGF released into the culture medium acted in an autocrine manner to stimulate proliferation. The proliferative signal of Ang II in the CSMC was possibly transduced by AT1 receptor. Our results in the coronary artery cells agree with a previous report using human coronary cells where Ang II did not induce cell proliferation but induced
hypertrophy by increasing protein synthesis [5]. Similarly in smooth muscle cells derived for human saphenous vein, Ang II failed to induce proliferation [8]. In most reports using cells isolated from peripheral vessels of the rat, cell proliferation in response to Ang II was not detected [2,4] although there is one report to the contrary known to us [13]. These authors found that proliferation stimulated by Ang II correlated with increased expression of the growth factors TGF1 and PDGF A-chain. However, addition of antibodies to these two factors failed to inhibit Ang II-stimulated prolifer-
Fig. 3. A neutralizing antibody to bFGF inhibited Ang II-stimulated CVSMC proliferation. CVSMC were incubated with a monoclonal neutralizing antibody to bFGF (5 g/ml) for 30 min before stimulation with Ang II (10−7 M) or bFGF (1 ng/ml). Forty-eight hours later, cell proliferation was determined by MTT assay. The statistical evaluation was performed as described in Fig. 1. P < 0.05 vs. control.
Z. Wang et al. / Neuroscience Letters 373 (2005) 38–41
ation [13]. The addition of a general growth factor inhibitor, suramin, blocked proliferation suggesting that the Ang II effect was growth factor dependent. It also has been reported that stimulation with Ang II induces expression of bFGF in PVSMC [7]. In this regard, Parenti et al. [11] reported that Ang II (10−7 M) alone stimulated expression of bFGF mRNA, slightly increased cellular bFGF but with no cellular proliferation in rat aortic SMC. However, Ang II combined with norepinephrine synergistically induced bFGF expression and cellular proliferation which was inhibited by antibodies to bFGF [11]. Here we found that in CVSMC Ang II-induced proliferation was entirely dependent on induced expression of bFGF. The mechanism(s) by which CVSMC and PVSMC respond differently to Ang II is not defined in the present work. Because CSMC and PVSMC were from same donors, this difference in response seems related to different phenotypes of VSMC. Expression of membrane receptors, such as Ang II receptors, may vary from phenotype to phenotype. Ang II-induced changes in VSMC function are controlled by complex signals transduced through two receptor subtypes, AT1 and AT2, in which AT1 transduces mitogenic signals while AT2 is anti-mitogenic and pro-apoptotic. It is possible that CVSMC and PVSMC express different levels of these receptors or that the interplay between signals initiated by the two receptors is different between the phenotypes. Although we do not define the exact mechanism, the new findings in this present study are consistent with a role for Ang II in the remodeling of cerebral arteries caused by hypertension where it contributes to arterial wall thickening by inducing an increase in smooth muscle cell number.
Acknowledgments This work was supported in part by a grant from the MedCen Foundation and the Clinical Research Center of the Medical Center of Central Georgia. We thank Dr. Jerome P. Tift, Department of Pathology, Mercer University School of Medicine, for providing the vessel samples.
41
References [1] G.L. Baumbach, C.D. Sigmund, F.M. Faraci, Cerebral arteriolar structure in mice overexpressing human renin and angiotensinogen, Hypertension 41 (2003) 50–55. [2] B.C. Berk, V. Vekshtein, H.M. Gordon, T. Tsuda, Angiotensin IIstimulated protein synthesis in cultured vascular smooth muscle cells, Hypertension 13 (1989) 305–314. [3] J.M. Chillon, G.L. Baumbach, Effects of an angiotensin-converting enzyme inhibitor and a -blocker on cerebral arteriolar dilatation in hypertensive rats, Hypertension 37 (2001) 1388–1393. [4] A.A. Geisterfer, M.J. Peach, G.K. Owens, Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells, Circ. Res. 62 (1988) 749–756. [5] S. Hafizi, A.H. Chester, S.P. Allen, K. Morgan, M.H. Yacoub, Growth response of human coronary smooth muscle to angiotensin II and influence of angiotensin AT1 receptor blockade, Coron. Artery Dis. 9 (1998) 167–175. [6] T. Ito, H. Yamakawa, C. Bregonzio, J.A. Terron, A. Falcon-Neri, J.M. Saavedra, Protection against ischemia and improvement of cerebral blood flow in genetically hypertensive rats by chronic pretreatment with an angiotensin AT1 antagonist, Stroke 33 (2002) 2297– 2303. [7] H. Itoh, M. Mukoyama, R.E. Pratt, G.H. Gibbons, V.J. Dzau, Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II, J. Clin. Invest. 91 (1993) 2268–2274. [8] S. Mii, J.A. Ware, S.A. Mallette, K.C. Kent, Effect of angiotensin II on human vascular smooth muscle cell growth, J. Surg. Res. 57 (1994) 174–178. [9] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [10] Y. Nishimura, T. Ito, J.M. Saavedra, Angiotensin II AT1 blockade normalizes cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats, Stroke 31 (2000) 2478–2486. [11] A. Parenti, L. Brogelli, S. Donnini, M. Ziche, F. Ledda, ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) 99–107. [12] Z. Wang, P.J. Rao, S.D. Shillcutt, W.H. Newman, Phenotypic diversity of smooth muscle cells isolated from human intracranial basilar artery, Neurosci. Lett. 351 (2003) 1–4. [13] H. Weber, D.S. Taylor, C.J. Molloy, Angiotensin II induces delayed mitogenesis and cellular proliferation in rat aortic smooth muscle cells. Correlation with the expression of specific endogenous growth factors and reversal by suramin, J. Clin. Invest. 93 (1994) 788–798.
Angiotensin II induces proliferation of human cerebral artery smooth muscle cells through a basic fibroblast growth factor (bFGF) dependent mechanism Zhongbiao Wanga,∗ , Pulipaka J. Raob , Samuel D. Shillcuttb , Walter H. Newmana,c a
b
Division of Basic Medical Science, Mercer University School of Medicine and Medical Center of Central Georgia, 1550 College Street, Macon, GA 31207, USA Department of Psychiatry and Behavioral Science, Mercer University School of Medicine and Medical Center of Central Georgia Macon, GA, USA c Department of Surgery, Mercer University School of Medicine and Medical Center of Central Georgia Macon, GA, USA Received 25 June 2004; received in revised form 24 September 2004; accepted 24 September 2004
Abstract Remodeling of cerebral arteries in hypertension produces thickened vessel walls associated with atherosclerotic plaque formation. In both thickening and plaque formation, proliferation of vascular smooth muscle cells is a hallmark. Genetically hypertensive rats treated with an angiotensin II (Ang II) AT1 receptor antagonist inhibited thickening of cerebral arteries suggesting a mitogenic action of Ang II on cerebral arterial VSMC (CVSMC). However, in studies using smooth muscle cells cultured from peripheral arteries, Ang II causes cell hypertrophy, but not proliferation. We determined the effect of Ang II on proliferation of cultured human CVSMC. CVSMC were cultured from the basilar artery obtained at autopsy. Ang II (10−7 M) stimulated proliferation determined by counting cells and mitochondrial activity assay. Synthesis and release of basic fibroblast growth factor (bFGF) was essential for Ang II-stimulated proliferation. These findings are consistent with the notion that Ang II stimulates CVSMC proliferation thereby contributing to vessel remodeling. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Angiotensin II; Cerebrovascular smooth muscle cells; Proliferation; Basic fibroblast growth factor
Chronic hypertension results in remodeled arteries with thickened walls and narrowed lumens and accelerated formation of atherosclerotic plaques in peripheral as well as cerebral arteries. The consequence of these changes in cerebral vessels is a three to four fold increase in the incidence of stroke. Angiotensin II (Ang II) contributes to hypertension through vasoconstriction and effects on vascular smooth muscle cell growth. Most studies in cells isolated from rat and human peripheral arteries (PVSMC), indicate that Ang II primarily stimulates protein synthesis resulting in cellular hypertrophy rather than increased cell proliferation [2,4,5]. Whether these results in peripheral cells apply to smooth muscle cells isolated from cerebral vessels (CVSMC) is unknown. Evidence suggests that Ang II plays a major role in hyper∗
Corresponding author. Tel.: +1 478 301 2095; fax: +1 478 301 2913. E-mail address: wang [email protected] (Z. Wang).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.09.068
tensive remodeling of cerebral vessels. For instance, studies with mice over expressing the renin–angiotensin system indicate that Ang II directly produces hypertrophy and remodeling of cerebral arterioles [1]. Additionally, chronic treatment with an Ang II subtype 1 (AT1) receptor blocker normalized cerebral autoregulation, reduced brain infarct size, and increased diameter and reduced wall thickness of cerebral vessels in spontaneously hypertensive rats [3,6,10]. One key feature associated with thickened walls of remodeled arteries is growth of vascular smooth muscle cells (VSMC). Therefore, we studied the effect of Ang II on CVSMC isolated from human basilar artery and found that Ang II stimulated proliferation by a mechanism involving basic fibroblast growth factor (bFGF). After obtaining consent for autopsy and use of tissue for research, the intracrainial basilar and coronary arteries from three donors were removed within 6 h of death. Primary cul-
Z. Wang et al. / Neuroscience Letters 373 (2005) 38–41
tures of VSMC were isolated from explants of these arteries by a method described previously [12]. The cells in culture expressed smooth muscle-specific differentiation proteins, calponin, smooth muscle-myosin heavy chain, and smooth muscle ␣-actin, determined by Western blot analysis. Cells at passages 3 through 4 were used in the present work. To determine the effects of Ang II on proliferation, cells were added to 96-well plates in Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% calf serum (CS) for 24 h. Then they were growth arrested by incubation in DMEM containing 0.1% CS for an additional 2 days. Ang II (10−7 M) was then added and cell number was quantitated at 6, 12, 24, and 48 h. For comparison, an identical time course was determined using recombinant human bFGF (1 ng/ml). To evaluate the role of endogenous bFGF in Ang II-stimulated proliferation, cells were incubated as described above with Ang II or bFGF for 48 h in the presence or the absence of anti-bFGF antibodies (5 g/ml). Isotypematched nonimmune IgG was added to parallel cultures to assess the specificity of the anti-bFGF antibodies. Cell proliferation was assessed by the mitochondrial dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) to formazan as previously described [9]. Additionally, in identical experiments, cell proliferation was assayed by counting cells after staining with trypan blue. The effect of Ang II on expression of bFGF was determined in CVSMC by quantitation of mRNA in cell lysates with a Direct ProtectTM ribonuclease protection assay kit according to manufacturer’s instruction. Briefly, CVSMC were growth arrested as described above for 2 days, then stimulated with Ang II for 3 h and then lysed. Before assay, ␣-[32 P] UTP-labeled, anti-sense RNA probes were synthesized under the direction of T7 polymerase using the DNA templates containing human bFGF and ribosomal protein L32 cDNA, using in vitro transcription kit MAXIscriptTM . After purification, the labeled probes (5 × 104 CPM) were hybridized with the lysates of the cells overnight. Samples were co-assayed for L32 mRNA as an internal quantification standard. After digestion of the nonhybridizing species with a mixture of RNases A and T1, the protected cRNA fragments were separated by 5% polyacrylamide/8 M urea gel. The gels were then exposed to Kodak X-OMAT AR film with intensifying screens. The effect of Ang II on the release of bFGF into the culture medium by CVSMC was determined by slot-blotting. Briefly, the cells were growth arrested for 24 h as described and the medium was replaced with 0.1% CS-DMEM with or without Ang II (10−7 M). Levels of bFGF in the medium at 6, 12, and 24 h were determined with a monoclonal anti-bFGF antibody demonstrated by the manufacturer to be specific to bFGF and without cross reactivity to other cytokines including acid FGF. The blotted bFGF was visualized by ECL. Cell number was assessed with MTT assay as described above. The statistical evaluation of the data, expressed as mean ± S.D., was based on n = 9 (three donors, triplicate de-
39
Fig. 1. (A) Ang II and bFGF stimulated proliferation of cerebral vascular smooth muscle cells (CVSMC). CVSMC were seeded in 96-well plates (4000 per well) and cultured overnight to allow adherence. Cells were growth arrested and Ang II (10−7 M) or bFGF (1 ng/ml) was added. At 6, 12, 24, and 48 h cell proliferation was determined by MTT assay. Untreated cells served as control. The mean ± S.D. of optical densities for untreated cells was determined and defined as 100% ± S.D. Cell proliferation is expressed as percentage increase over untreated cells (Ctrl.). (B) bFGF but not Ang II stimulated cell proliferation in peripheral VSMC (PVSMC). Cells were growth arrested and Ang II (10−7 or 10−6 M) or bFGF (1 ng/ml) was added. Cell proliferation was determined at 48 h by MTT assay. P < 0.05 vs. control.
termination of one culture of each donor). Analysis was performed by ANOVA with P values < 0.05 considered to be significant. As seen in Fig. 1A, Ang II (10−7 M) stimulated proliferation of CVSMC. Forty-eight hours after the addition of Ang II cell number had increased 56% over control cells by MTT measurement. In cell counting experiments, a similar increase in cell number (48%) was determined at 48 h after Ang II (10−7 M). In comparison, bFGF (1 ng/ml) increased cell number by over 100% at 48 h. In contrast, Ang II at 10−7 M and at a concentration 10-fold higher (10−6 M) than in CVSMC had no detectable effect on proliferation of PVSMC at 48 h (Fig. 1B). These cells isolated from a peripheral artery, however, were capable of proliferating as seen by the 140% increase in cell number following the addition of bFGF. To determine a potential role of endogenous bFGF in Ang II-induced CVSMC proliferation, we first examined Ang II
40
Z. Wang et al. / Neuroscience Letters 373 (2005) 38–41
Fig. 2. Ang II stimulated transcription of bFGF gene and release of bFGF from CVSMC. CSMC were growth-arrested as described in Fig. 1 and then stimulated with Ang II (10−7 M). (A) 3 h after the stimulation, cells were lysed. bFGF and L32 mRNAs were detected using ribonuclease protection assay. (B) 6, 12, and 24 h after stimulation with Ang II, cell culture medium was collected, and analyzed for bFGF protein by immuno-slot-blot assay. Medium from untreated cells was used as controls. Shown is one representative of results from three experiments.
stimulated bFGF gene transcription. As shown in Fig. 2A, mRNA for bFGF was just detectable in control cells. At 3 h post stimulation with Ang II there was a marked increase in transcription of the growth factor gene. Next, release of bFGF into the culture medium of CVSMC was determined. Media was collected at different time intervals after stimulation of Ang II. Media from untreated cells served as controls. bFGF released from untreated cells was detectable at 24 but not at 6 and 12 h (Fig. 2B). Stimulation with Ang II (10−7 M) increased release of bFGF compared to the corresponding control at 12 and 24 h. To evaluate possible roles of AT1 receptor signaling and bFGF released by the cells in Ang II-stimulated proliferation of CVSMC, a selective AT1 receptor antagonist (ZD 7155, Tocris Cookson Inc., Ballwin, MO) and a neutralizing antibody was, respectively, added to the culture medium prior to stimulation with Ang II. As seen in Fig. 3, addition of ZD 7155 (10 nM) or bFGF neutralizing antibody (5 g/ml) completely blocked Ang IIinduced cell proliferation measured by MTT. Cell counting experiments showed a similar effect of neutralization of bFGF. The antibody also blocked cell proliferation induced by the addition of exogenous bFGF (1 ng/ml). Isotypematched nonimmune IgG was added to parallel cultures at same concentration had no effect on the proliferation (data not shown). These results indicate that Ang II stimulates proliferation of CVSMC but not VSMC isolated from a peripheral vessel, coronary artery. Further, Ang II stimulated proliferation in CVSMC by inducing gene transcription, synthesis, and release of bFGF. bFGF released into the culture medium acted in an autocrine manner to stimulate proliferation. The proliferative signal of Ang II in the CSMC was possibly transduced by AT1 receptor. Our results in the coronary artery cells agree with a previous report using human coronary cells where Ang II did not induce cell proliferation but induced
hypertrophy by increasing protein synthesis [5]. Similarly in smooth muscle cells derived for human saphenous vein, Ang II failed to induce proliferation [8]. In most reports using cells isolated from peripheral vessels of the rat, cell proliferation in response to Ang II was not detected [2,4] although there is one report to the contrary known to us [13]. These authors found that proliferation stimulated by Ang II correlated with increased expression of the growth factors TGF1 and PDGF A-chain. However, addition of antibodies to these two factors failed to inhibit Ang II-stimulated prolifer-
Fig. 3. A neutralizing antibody to bFGF inhibited Ang II-stimulated CVSMC proliferation. CVSMC were incubated with a monoclonal neutralizing antibody to bFGF (5 g/ml) for 30 min before stimulation with Ang II (10−7 M) or bFGF (1 ng/ml). Forty-eight hours later, cell proliferation was determined by MTT assay. The statistical evaluation was performed as described in Fig. 1. P < 0.05 vs. control.
Z. Wang et al. / Neuroscience Letters 373 (2005) 38–41
ation [13]. The addition of a general growth factor inhibitor, suramin, blocked proliferation suggesting that the Ang II effect was growth factor dependent. It also has been reported that stimulation with Ang II induces expression of bFGF in PVSMC [7]. In this regard, Parenti et al. [11] reported that Ang II (10−7 M) alone stimulated expression of bFGF mRNA, slightly increased cellular bFGF but with no cellular proliferation in rat aortic SMC. However, Ang II combined with norepinephrine synergistically induced bFGF expression and cellular proliferation which was inhibited by antibodies to bFGF [11]. Here we found that in CVSMC Ang II-induced proliferation was entirely dependent on induced expression of bFGF. The mechanism(s) by which CVSMC and PVSMC respond differently to Ang II is not defined in the present work. Because CSMC and PVSMC were from same donors, this difference in response seems related to different phenotypes of VSMC. Expression of membrane receptors, such as Ang II receptors, may vary from phenotype to phenotype. Ang II-induced changes in VSMC function are controlled by complex signals transduced through two receptor subtypes, AT1 and AT2, in which AT1 transduces mitogenic signals while AT2 is anti-mitogenic and pro-apoptotic. It is possible that CVSMC and PVSMC express different levels of these receptors or that the interplay between signals initiated by the two receptors is different between the phenotypes. Although we do not define the exact mechanism, the new findings in this present study are consistent with a role for Ang II in the remodeling of cerebral arteries caused by hypertension where it contributes to arterial wall thickening by inducing an increase in smooth muscle cell number.
Acknowledgments This work was supported in part by a grant from the MedCen Foundation and the Clinical Research Center of the Medical Center of Central Georgia. We thank Dr. Jerome P. Tift, Department of Pathology, Mercer University School of Medicine, for providing the vessel samples.
41
References [1] G.L. Baumbach, C.D. Sigmund, F.M. Faraci, Cerebral arteriolar structure in mice overexpressing human renin and angiotensinogen, Hypertension 41 (2003) 50–55. [2] B.C. Berk, V. Vekshtein, H.M. Gordon, T. Tsuda, Angiotensin IIstimulated protein synthesis in cultured vascular smooth muscle cells, Hypertension 13 (1989) 305–314. [3] J.M. Chillon, G.L. Baumbach, Effects of an angiotensin-converting enzyme inhibitor and a -blocker on cerebral arteriolar dilatation in hypertensive rats, Hypertension 37 (2001) 1388–1393. [4] A.A. Geisterfer, M.J. Peach, G.K. Owens, Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells, Circ. Res. 62 (1988) 749–756. [5] S. Hafizi, A.H. Chester, S.P. Allen, K. Morgan, M.H. Yacoub, Growth response of human coronary smooth muscle to angiotensin II and influence of angiotensin AT1 receptor blockade, Coron. Artery Dis. 9 (1998) 167–175. [6] T. Ito, H. Yamakawa, C. Bregonzio, J.A. Terron, A. Falcon-Neri, J.M. Saavedra, Protection against ischemia and improvement of cerebral blood flow in genetically hypertensive rats by chronic pretreatment with an angiotensin AT1 antagonist, Stroke 33 (2002) 2297– 2303. [7] H. Itoh, M. Mukoyama, R.E. Pratt, G.H. Gibbons, V.J. Dzau, Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II, J. Clin. Invest. 91 (1993) 2268–2274. [8] S. Mii, J.A. Ware, S.A. Mallette, K.C. Kent, Effect of angiotensin II on human vascular smooth muscle cell growth, J. Surg. Res. 57 (1994) 174–178. [9] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [10] Y. Nishimura, T. Ito, J.M. Saavedra, Angiotensin II AT1 blockade normalizes cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats, Stroke 31 (2000) 2478–2486. [11] A. Parenti, L. Brogelli, S. Donnini, M. Ziche, F. Ledda, ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) 99–107. [12] Z. Wang, P.J. Rao, S.D. Shillcutt, W.H. Newman, Phenotypic diversity of smooth muscle cells isolated from human intracranial basilar artery, Neurosci. Lett. 351 (2003) 1–4. [13] H. Weber, D.S. Taylor, C.J. Molloy, Angiotensin II induces delayed mitogenesis and cellular proliferation in rat aortic smooth muscle cells. Correlation with the expression of specific endogenous growth factors and reversal by suramin, J. Clin. Invest. 93 (1994) 788–798.