Progesterone Regulates Proliferation of Endothelial Cells
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.2185
ISSN1083-351X
AutoresFrancisca Vázquez, Juan Carlos Rodrı́guez-Manzaneque, John P. Lydon, Dean P. Edwards, Bert W. O’Malley, M. Luisa Iruela‐Arispe,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoThe use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. Although the contribution of estradiol to endothelial cell function has been addressed, little information is available on the effect of progestins on this cell type. Here, we provide direct evidence for the presence of functional nuclear progesterone receptor in endothelial cells and demonstrate that physiological levels of progesterone inhibit proliferation through a nuclear receptor-mediated mechanism. The effects of progesterone were blocked by pretreatment with a progesterone receptor antagonist, and progesterone receptor-deficient endothelial cells failed to respond to the hormone. We evaluated the effect of progesterone by analysis of aorta re-endothelialization experiments in wild-type and progesterone receptor knockout mice. The rate of re-endothelialization was significantly decreased in wild-type mice when in the presence of progesterone, whereas there was no difference between control and progesterone-treated progesterone receptor knockout mice. FACS analysis showed that progestins arrest endothelial cell cycle in G1. The lag in cell cycle progression involved reduction in cyclin-dependent kinase activity, as shown by down-regulation in retinoblastoma protein phosphorylation. In addition, treatment of endothelial cells with progestins altered the expression of cyclin E and A in accordance with G1 arrest. These results have important implications to our current knowledge of the effect of steroids on endothelial cell function and to the overall contribution of progesterone to vascular repair. The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. Although the contribution of estradiol to endothelial cell function has been addressed, little information is available on the effect of progestins on this cell type. Here, we provide direct evidence for the presence of functional nuclear progesterone receptor in endothelial cells and demonstrate that physiological levels of progesterone inhibit proliferation through a nuclear receptor-mediated mechanism. The effects of progesterone were blocked by pretreatment with a progesterone receptor antagonist, and progesterone receptor-deficient endothelial cells failed to respond to the hormone. We evaluated the effect of progesterone by analysis of aorta re-endothelialization experiments in wild-type and progesterone receptor knockout mice. The rate of re-endothelialization was significantly decreased in wild-type mice when in the presence of progesterone, whereas there was no difference between control and progesterone-treated progesterone receptor knockout mice. FACS analysis showed that progestins arrest endothelial cell cycle in G1. The lag in cell cycle progression involved reduction in cyclin-dependent kinase activity, as shown by down-regulation in retinoblastoma protein phosphorylation. In addition, treatment of endothelial cells with progestins altered the expression of cyclin E and A in accordance with G1 arrest. These results have important implications to our current knowledge of the effect of steroids on endothelial cell function and to the overall contribution of progesterone to vascular repair. progesterone receptor PR knockout fetal calf serum basic fibroblast growth factor human dermal endothelial cell hormone replacement therapy vascular endothelial growth factor retinoblastoma protein cyclin-dependent kinase fluorescence-activated cell sorter progesterone-responsive element. Progesterone receptor (PR)1 is a member of a family of nuclear receptors capable of regulating gene expression upon binding to the appropriate hormones (1Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 2Edwards D.P. Altmann M. DeMarzo A. Zhang Y. Weigel N.L. Beck C.A. J. Steroid Biochem. Mol. Biol. 1995; 53: 449-458Crossref PubMed Scopus (69) Google Scholar, 3Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar, 4Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar). As such, PR is considered a transcription factor with known ability to influence development and morphogenesis (5Lydon J.P. DeMayo F.J. Cindee R.F. Mani S.K. Hughes A.R. Montgomery Jr C.A. Shyamala G. Conneely O.M. O'Malley B.W. Genes Dev. 1995; 9: 2266-2278Crossref PubMed Scopus (1495) Google Scholar). Aside from its expression in several cell types of the mammary gland, uterus, and ovary, PR has also been identified in brain and vascular tissue (6Apostolakis E.M. Garai J. Clark J.H. O'Malley B.W. Mol. Endocrinol. 1996; 10: 1595-1604Crossref PubMed Scopus (35) Google Scholar, 7Perrot-Applanat M. Groyer-Picard M.T. Garcia E. Lorenzo F. Milgrom E. Endocrinology. 1988; 123: 1511-1519Crossref PubMed Scopus (185) Google Scholar, 8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar). The presence of PR in blood vessels has relevance because of the increase use of progestins in hormone replacement therapy (HRT).HRT with estrogen and progestins has become a well accepted treatment regimen for postmenopausal women, particularly those with an absence of familial breast cancer history. A large body of literature has demonstrated that the benefits of HRT extend beyond the amelioration of symptoms associated with menopause. HRT also aids in the prevention of osteoporosis and in the reduction of cardiovascular disease (9Lobo R.A. Obstet. Gynecol. 1990; 75: 18S-25SCrossref PubMed Scopus (129) Google Scholar, 10Sullivan J.M. Circulation. 1996; 94: 2699-2702Crossref PubMed Scopus (21) Google Scholar, 11Rossouw J.E. Circulation. 1996; 94: 2982-2985Crossref PubMed Scopus (51) Google Scholar). Most of these effects have been attributed to estradiol, as demonstrated by several experimental and epidemiological studies. In fact, estradiol has been shown to diminish the incidence of cardiovascular disease up to 45% (12Stampfer M.J. Colditz G.A. Prev. Med. 1991; 20: 47-63Crossref PubMed Scopus (1496) Google Scholar, 13Sullivan J.M. Prog. Cardiovasc. Dis. 1995; 38: 211-222Crossref PubMed Scopus (17) Google Scholar, 14Sullivan J.M. Fowlkes L.P. Cardiol. Clin. 1996; 14: 105-116Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The contribution of progesterone to the overall effect of HRT is less clear. Historically, progestins were included to counteract endometrial dysplasia caused by estradiol (11Rossouw J.E. Circulation. 1996; 94: 2982-2985Crossref PubMed Scopus (51) Google Scholar, 15Whitehead M. Lobo R.A. Lancet. 1988; 2: 1243-1244Abstract PubMed Google Scholar). Although the inclusion of progestins to HRT has recognized value, dosage levels and frequency have been the object of much controversy, mostly due to collateral effects (16Fraser I.S. Diczfalusy E. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 384-413Google Scholar, 17Sivin I. Stud. Fam. Plan. 1988; 19: 81-94Crossref PubMed Scopus (224) Google Scholar, 18Shoupe D. Mishell D.R.J. Bopp B.L. Fielding M. Obstet. Gynecol. 1991; 77: 256-260Crossref PubMed Scopus (71) Google Scholar).In blood vessels, PR has been localized in smooth muscle cells of the tunica media (7Perrot-Applanat M. Groyer-Picard M.T. Garcia E. Lorenzo F. Milgrom E. Endocrinology. 1988; 123: 1511-1519Crossref PubMed Scopus (185) Google Scholar, 19Colburn P. Buonassisi V. Science. 1978; 201: 817-819Crossref PubMed Scopus (219) Google Scholar, 20Ingegno M.D. Money S.M. Thelmo W. Greene G.L. Davidian M. Jaffe B.M. Pertschuk L.P. Lab. Invest. 1988; 59: 353-356PubMed Google Scholar) and has been shown to suppress smooth muscle cell proliferation in vitro (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar). Nevertheless, neither the presence of functional PR nor the effect of progesterone have been addressed on endothelial cells. Because constant levels of exogenous progestins can be associated with the breakdown of vessels and irregular endometrial bleedings (21Macqueo M. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 138-152Google Scholar), we hypothesized that this hormone might play a direct role in endothelial function. Because progesterone mediates signals through its receptor (2Edwards D.P. Altmann M. DeMarzo A. Zhang Y. Weigel N.L. Beck C.A. J. Steroid Biochem. Mol. Biol. 1995; 53: 449-458Crossref PubMed Scopus (69) Google Scholar, 3Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar, 4Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar), we investigated the presence of PR in endothelial cells from several organs and addressed the effect of the ligand on several aspects of endothelial function.DISCUSSIONIn this study, we have provided functional support to the presence of progesterone receptor on vascular endothelium. Although expression of progesterone receptor has been previously reported in the intima of blood vessels using biochemical and immunocytochemical approaches (19Colburn P. Buonassisi V. Science. 1978; 201: 817-819Crossref PubMed Scopus (219) Google Scholar,20Ingegno M.D. Money S.M. Thelmo W. Greene G.L. Davidian M. Jaffe B.M. Pertschuk L.P. Lab. Invest. 1988; 59: 353-356PubMed Google Scholar), to our knowledge, this is the first study that performs a broad-based analysis on several endothelial types and that provides a functional relevance to this receptor on endothelial biology.The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. However, the contribution of progesterone to the replacement regime has been controversial. Although experimental studies evaluating intimal thickness (37Hanke H. Hanke S. Bruck B. Brehme U. Gugel N. Finking G. Muck A.O. Schmahl F.W. Hombach V. Haasis R. Atherosclerosis. 1996; 121: 129-138Abstract Full Text PDF PubMed Scopus (86) Google Scholar) and bromodeoxyuridine incorporation (38Hanke H. Hanke S. Finking G. Muhic-Lohrer A. Muck A.O. Schmahl F.W. Haasis R. Hombach V. Circulation. 1996; 94: 175-181Crossref PubMed Scopus (78) Google Scholar) conclude that progesterone directly inhibits the atheroprotective effect of estrogen, several epidemiological and in vivo studies demonstrate otherwise (39Grodstein F. Stampfer M.J. Manson J.E. Colditz G.A. Willett W.C. Rosner B. Speizer F.E. Hennekens C.H. N. Engl. J. Med. 1996; 335: 453-461Crossref PubMed Scopus (1285) Google Scholar, 40Levine R.L. Chen S.J. Durand J. Chen Y.F. Oparil S. Circulation. 1996; 94: 2221-2227Crossref PubMed Scopus (108) Google Scholar). The finding that progesterone inhibits re-endothelialization of denuded aortae suggests that this hormone could have an opposite effect on estradiol in endothelial repair of denuded atherosclerotic lesions (41Krasinski K. Spyridopoulos I. Asahara T. van der Zee R. Isner J.M. Losordo D.W. Circulation. 1997; 95: 1768-1772Crossref PubMed Scopus (220) Google Scholar).Atherosclerosis is a multifactorial process and the principal contributor to myocardial and cerebral infarction (42Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9929) Google Scholar). Response to injury is one of the favored hypotheses for development of atherogenesis. It postulates that an alteration of the intima by various risk factors (such as mechanical injury, chemically altered low density lipoprotein, viruses, or toxins) initiates a primary endothelial cell dysfunction that leads to subsequent vascular changes, giving rise to the initial atherosclerotic lesion. The plaque progresses by the accumulation of layers of smooth muscle, macrophages, and foam cells; further deposition of extracellular matrix; and possibly neovascularization. Progression or rupture of the plaque is frequently associated with physical disruption of the endothelium. Because of the role of the endothelium in providing an antithrombotic and anticoagulant surface, rapid endothelial repair is of importance to the containment of atherosclerotic lesions. Although progesterone has shown suppressive effects on smooth muscle cell proliferation in vitro (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar), its participation on intimal endothelial repair, according to this study, appears to be equally inhibitory and, by extension, possibly deleterious to the endothelial healing of an exposed lesion.The inhibitory effect of progesterone on endothelial proliferation also provides an explanation for the episodes of vessel breakdown and irregular bleeding associated with progestin-based contraceptives (21Macqueo M. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 138-152Google Scholar). Physiological, but constant, levels of circulating progestins most likely affect the high mitotic rate associated with the endometrial vasculature, 3M. Graubert, M. Lombardo, M. A. Ortega, L. F. Brown, B. Kessel, J. F. Mortola, and M. L. Iruela-Arispe, unpublished observations. leading to capillary rupture. Furthermore, progesterone has been implicated in the suppression of tumor-induced neovascularization (43Rao B.R. Anticancer Res. 1997; 17: 1019-1022PubMed Google Scholar).In this study, we showed that progesterone partially blocks and delays endothelial cell cycle progression through a receptor-mediated mechanism that involves changes in expression of cell cycle proteins, cyclins E and A, and changes in pRb phosphorylation. The participation of PR in the regulation of cell cycle has been controversial, with studies indicating that progesterone stimulates, inhibits, or does not alter cell cycle progression even on the same cell type (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar, 44Groshong S.D. Owen G.I. Grimison B. Schauer I.E. Todd M.C. Langan T.A. Sclafani R.A. Lange C.A. Horwitz K.B. Mol. Endocrinol. 1997; 11: 1593-1607Crossref PubMed Scopus (229) Google Scholar, 45Ishida Y. Heersche J.N. Bone. 1997; 20: 17-25Crossref PubMed Scopus (71) Google Scholar, 46Said T.K. Conneely O.M. Medina D. O'Malley B.W. Lyndon J.P. Endocrinology. 1997; 138: 3933-3939Crossref PubMed Scopus (72) Google Scholar). When inhibitory in mammary epithelial cells, the mechanism by which progesterone mediates cell cycle arrest has been shown to implicate both suppression of Cdk activity (47Musgrove E.A. Swarbrick A. Lee C.S.L. Cornish A.L. Sutherland R.L. Mol. Cell. Biol. 1998; 18: 1812-1825Crossref PubMed Scopus (105) Google Scholar) and up-regulation of p21 (48Owen G.I. Richer J.K. Tung L. Takimoto G. Horwitz K.B. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In endothelial cells, however, it is likely that progesterone acts by regulation of Cdk activity only, because we have not detected changes in either p21 nor p27 expression levels (data not shown). It has now become increasingly clear that the functional contribution of PR as a transcription factor is also dependent on the expression of several binding proteins that modulate PR activity (49Prapapanich V. Chen S. Nair S.C. Rimerman R.A. Smith D.F. Mol. Endocrinol. 1996; 10: 420-431PubMed Google Scholar, 50Prapapanich V. Chen S. Smith D.F. Mol. Cell. Biol. 1998; 18: 944-952Crossref PubMed Scopus (50) Google Scholar, 51Nair S.C. Rimerman R.A. Toran E.J. Chen S. Prapapanich V. Butts R.N. Smith D.F. Mol. Cell. Biol. 1997; 17: 594-603Crossref PubMed Scopus (165) Google Scholar, 52Segnitz B. Gehring U. J. Biol. Chem. 1997; 272: 18694-18701Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Whether or not these binding proteins play a role in the regulation of endothelial cell cycle or any other cell type is, at this point, not clear and deserves further investigation. Nevertheless, we found that endothelial cell proliferation was suppressed by physiological levels of 17-α-hydroxy-progesterone in every endothelial cell type examined.In conclusion, the presence of progesterone receptor in endothelial cells and the direct effect of progesterone described here can be of physiological relevance to the balance between potentiation of endothelial growth, mediated by estradiol, and suppressive signals, mediated by progesterone. This balance could play an important role in the regulation of angiogenesis in the endometrium and corpus luteus during the menstrual cycle. Nonetheless, constant levels of progestins in HRT could be counter-productive. Although the benefits of progestins, as first or second line therapy in the treatment of breast and endometrial carcinoma, have been widely acknowledged, the long-term use of progestins as contraceptives or for the prophylaxis of menopause should be under closer scrutiny. It is clear from these and other data that progestins may have direct growth-inhibitory actions in nonreproductive sites, apart from their ability to inhibit estrogen-mediated cell proliferation in such classical reproductive targets as the endometrium. Progesterone receptor (PR)1 is a member of a family of nuclear receptors capable of regulating gene expression upon binding to the appropriate hormones (1Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 2Edwards D.P. Altmann M. DeMarzo A. Zhang Y. Weigel N.L. Beck C.A. J. Steroid Biochem. Mol. Biol. 1995; 53: 449-458Crossref PubMed Scopus (69) Google Scholar, 3Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar, 4Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar). As such, PR is considered a transcription factor with known ability to influence development and morphogenesis (5Lydon J.P. DeMayo F.J. Cindee R.F. Mani S.K. Hughes A.R. Montgomery Jr C.A. Shyamala G. Conneely O.M. O'Malley B.W. Genes Dev. 1995; 9: 2266-2278Crossref PubMed Scopus (1495) Google Scholar). Aside from its expression in several cell types of the mammary gland, uterus, and ovary, PR has also been identified in brain and vascular tissue (6Apostolakis E.M. Garai J. Clark J.H. O'Malley B.W. Mol. Endocrinol. 1996; 10: 1595-1604Crossref PubMed Scopus (35) Google Scholar, 7Perrot-Applanat M. Groyer-Picard M.T. Garcia E. Lorenzo F. Milgrom E. Endocrinology. 1988; 123: 1511-1519Crossref PubMed Scopus (185) Google Scholar, 8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar). The presence of PR in blood vessels has relevance because of the increase use of progestins in hormone replacement therapy (HRT). HRT with estrogen and progestins has become a well accepted treatment regimen for postmenopausal women, particularly those with an absence of familial breast cancer history. A large body of literature has demonstrated that the benefits of HRT extend beyond the amelioration of symptoms associated with menopause. HRT also aids in the prevention of osteoporosis and in the reduction of cardiovascular disease (9Lobo R.A. Obstet. Gynecol. 1990; 75: 18S-25SCrossref PubMed Scopus (129) Google Scholar, 10Sullivan J.M. Circulation. 1996; 94: 2699-2702Crossref PubMed Scopus (21) Google Scholar, 11Rossouw J.E. Circulation. 1996; 94: 2982-2985Crossref PubMed Scopus (51) Google Scholar). Most of these effects have been attributed to estradiol, as demonstrated by several experimental and epidemiological studies. In fact, estradiol has been shown to diminish the incidence of cardiovascular disease up to 45% (12Stampfer M.J. Colditz G.A. Prev. Med. 1991; 20: 47-63Crossref PubMed Scopus (1496) Google Scholar, 13Sullivan J.M. Prog. Cardiovasc. Dis. 1995; 38: 211-222Crossref PubMed Scopus (17) Google Scholar, 14Sullivan J.M. Fowlkes L.P. Cardiol. Clin. 1996; 14: 105-116Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The contribution of progesterone to the overall effect of HRT is less clear. Historically, progestins were included to counteract endometrial dysplasia caused by estradiol (11Rossouw J.E. Circulation. 1996; 94: 2982-2985Crossref PubMed Scopus (51) Google Scholar, 15Whitehead M. Lobo R.A. Lancet. 1988; 2: 1243-1244Abstract PubMed Google Scholar). Although the inclusion of progestins to HRT has recognized value, dosage levels and frequency have been the object of much controversy, mostly due to collateral effects (16Fraser I.S. Diczfalusy E. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 384-413Google Scholar, 17Sivin I. Stud. Fam. Plan. 1988; 19: 81-94Crossref PubMed Scopus (224) Google Scholar, 18Shoupe D. Mishell D.R.J. Bopp B.L. Fielding M. Obstet. Gynecol. 1991; 77: 256-260Crossref PubMed Scopus (71) Google Scholar). In blood vessels, PR has been localized in smooth muscle cells of the tunica media (7Perrot-Applanat M. Groyer-Picard M.T. Garcia E. Lorenzo F. Milgrom E. Endocrinology. 1988; 123: 1511-1519Crossref PubMed Scopus (185) Google Scholar, 19Colburn P. Buonassisi V. Science. 1978; 201: 817-819Crossref PubMed Scopus (219) Google Scholar, 20Ingegno M.D. Money S.M. Thelmo W. Greene G.L. Davidian M. Jaffe B.M. Pertschuk L.P. Lab. Invest. 1988; 59: 353-356PubMed Google Scholar) and has been shown to suppress smooth muscle cell proliferation in vitro (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar). Nevertheless, neither the presence of functional PR nor the effect of progesterone have been addressed on endothelial cells. Because constant levels of exogenous progestins can be associated with the breakdown of vessels and irregular endometrial bleedings (21Macqueo M. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 138-152Google Scholar), we hypothesized that this hormone might play a direct role in endothelial function. Because progesterone mediates signals through its receptor (2Edwards D.P. Altmann M. DeMarzo A. Zhang Y. Weigel N.L. Beck C.A. J. Steroid Biochem. Mol. Biol. 1995; 53: 449-458Crossref PubMed Scopus (69) Google Scholar, 3Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar, 4Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar), we investigated the presence of PR in endothelial cells from several organs and addressed the effect of the ligand on several aspects of endothelial function. DISCUSSIONIn this study, we have provided functional support to the presence of progesterone receptor on vascular endothelium. Although expression of progesterone receptor has been previously reported in the intima of blood vessels using biochemical and immunocytochemical approaches (19Colburn P. Buonassisi V. Science. 1978; 201: 817-819Crossref PubMed Scopus (219) Google Scholar,20Ingegno M.D. Money S.M. Thelmo W. Greene G.L. Davidian M. Jaffe B.M. Pertschuk L.P. Lab. Invest. 1988; 59: 353-356PubMed Google Scholar), to our knowledge, this is the first study that performs a broad-based analysis on several endothelial types and that provides a functional relevance to this receptor on endothelial biology.The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. However, the contribution of progesterone to the replacement regime has been controversial. Although experimental studies evaluating intimal thickness (37Hanke H. Hanke S. Bruck B. Brehme U. Gugel N. Finking G. Muck A.O. Schmahl F.W. Hombach V. Haasis R. Atherosclerosis. 1996; 121: 129-138Abstract Full Text PDF PubMed Scopus (86) Google Scholar) and bromodeoxyuridine incorporation (38Hanke H. Hanke S. Finking G. Muhic-Lohrer A. Muck A.O. Schmahl F.W. Haasis R. Hombach V. Circulation. 1996; 94: 175-181Crossref PubMed Scopus (78) Google Scholar) conclude that progesterone directly inhibits the atheroprotective effect of estrogen, several epidemiological and in vivo studies demonstrate otherwise (39Grodstein F. Stampfer M.J. Manson J.E. Colditz G.A. Willett W.C. Rosner B. Speizer F.E. Hennekens C.H. N. Engl. J. Med. 1996; 335: 453-461Crossref PubMed Scopus (1285) Google Scholar, 40Levine R.L. Chen S.J. Durand J. Chen Y.F. Oparil S. Circulation. 1996; 94: 2221-2227Crossref PubMed Scopus (108) Google Scholar). The finding that progesterone inhibits re-endothelialization of denuded aortae suggests that this hormone could have an opposite effect on estradiol in endothelial repair of denuded atherosclerotic lesions (41Krasinski K. Spyridopoulos I. Asahara T. van der Zee R. Isner J.M. Losordo D.W. Circulation. 1997; 95: 1768-1772Crossref PubMed Scopus (220) Google Scholar).Atherosclerosis is a multifactorial process and the principal contributor to myocardial and cerebral infarction (42Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9929) Google Scholar). Response to injury is one of the favored hypotheses for development of atherogenesis. It postulates that an alteration of the intima by various risk factors (such as mechanical injury, chemically altered low density lipoprotein, viruses, or toxins) initiates a primary endothelial cell dysfunction that leads to subsequent vascular changes, giving rise to the initial atherosclerotic lesion. The plaque progresses by the accumulation of layers of smooth muscle, macrophages, and foam cells; further deposition of extracellular matrix; and possibly neovascularization. Progression or rupture of the plaque is frequently associated with physical disruption of the endothelium. Because of the role of the endothelium in providing an antithrombotic and anticoagulant surface, rapid endothelial repair is of importance to the containment of atherosclerotic lesions. Although progesterone has shown suppressive effects on smooth muscle cell proliferation in vitro (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar), its participation on intimal endothelial repair, according to this study, appears to be equally inhibitory and, by extension, possibly deleterious to the endothelial healing of an exposed lesion.The inhibitory effect of progesterone on endothelial proliferation also provides an explanation for the episodes of vessel breakdown and irregular bleeding associated with progestin-based contraceptives (21Macqueo M. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 138-152Google Scholar). Physiological, but constant, levels of circulating progestins most likely affect the high mitotic rate associated with the endometrial vasculature, 3M. Graubert, M. Lombardo, M. A. Ortega, L. F. Brown, B. Kessel, J. F. Mortola, and M. L. Iruela-Arispe, unpublished observations. leading to capillary rupture. Furthermore, progesterone has been implicated in the suppression of tumor-induced neovascularization (43Rao B.R. Anticancer Res. 1997; 17: 1019-1022PubMed Google Scholar).In this study, we showed that progesterone partially blocks and delays endothelial cell cycle progression through a receptor-mediated mechanism that involves changes in expression of cell cycle proteins, cyclins E and A, and changes in pRb phosphorylation. The participation of PR in the regulation of cell cycle has been controversial, with studies indicating that progesterone stimulates, inhibits, or does not alter cell cycle progression even on the same cell type (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar, 44Groshong S.D. Owen G.I. Grimison B. Schauer I.E. Todd M.C. Langan T.A. Sclafani R.A. Lange C.A. Horwitz K.B. Mol. Endocrinol. 1997; 11: 1593-1607Crossref PubMed Scopus (229) Google Scholar, 45Ishida Y. Heersche J.N. Bone. 1997; 20: 17-25Crossref PubMed Scopus (71) Google Scholar, 46Said T.K. Conneely O.M. Medina D. O'Malley B.W. Lyndon J.P. Endocrinology. 1997; 138: 3933-3939Crossref PubMed Scopus (72) Google Scholar). When inhibitory in mammary epithelial cells, the mechanism by which progesterone mediates cell cycle arrest has been shown to implicate both suppression of Cdk activity (47Musgrove E.A. Swarbrick A. Lee C.S.L. Cornish A.L. Sutherland R.L. Mol. Cell. Biol. 1998; 18: 1812-1825Crossref PubMed Scopus (105) Google Scholar) and up-regulation of p21 (48Owen G.I. Richer J.K. Tung L. Takimoto G. Horwitz K.B. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In endothelial cells, however, it is likely that progesterone acts by regulation of Cdk activity only, because we have not detected changes in either p21 nor p27 expression levels (data not shown). It has now become increasingly clear that the functional contribution of PR as a transcription factor is also dependent on the expression of several binding proteins that modulate PR activity (49Prapapanich V. Chen S. Nair S.C. Rimerman R.A. Smith D.F. Mol. Endocrinol. 1996; 10: 420-431PubMed Google Scholar, 50Prapapanich V. Chen S. Smith D.F. Mol. Cell. Biol. 1998; 18: 944-952Crossref PubMed Scopus (50) Google Scholar, 51Nair S.C. Rimerman R.A. Toran E.J. Chen S. Prapapanich V. Butts R.N. Smith D.F. Mol. Cell. Biol. 1997; 17: 594-603Crossref PubMed Scopus (165) Google Scholar, 52Segnitz B. Gehring U. J. Biol. Chem. 1997; 272: 18694-18701Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Whether or not these binding proteins play a role in the regulation of endothelial cell cycle or any other cell type is, at this point, not clear and deserves further investigation. Nevertheless, we found that endothelial cell proliferation was suppressed by physiological levels of 17-α-hydroxy-progesterone in every endothelial cell type examined.In conclusion, the presence of progesterone receptor in endothelial cells and the direct effect of progesterone described here can be of physiological relevance to the balance between potentiation of endothelial growth, mediated by estradiol, and suppressive signals, mediated by progesterone. This balance could play an important role in the regulation of angiogenesis in the endometrium and corpus luteus during the menstrual cycle. Nonetheless, constant levels of progestins in HRT could be counter-productive. Although the benefits of progestins, as first or second line therapy in the treatment of breast and endometrial carcinoma, have been widely acknowledged, the long-term use of progestins as contraceptives or for the prophylaxis of menopause should be under closer scrutiny. It is clear from these and other data that progestins may have direct growth-inhibitory actions in nonreproductive sites, apart from their ability to inhibit estrogen-mediated cell proliferation in such classical reproductive targets as the endometrium. In this study, we have provided functional support to the presence of progesterone receptor on vascular endothelium. Although expression of progesterone receptor has been previously reported in the intima of blood vessels using biochemical and immunocytochemical approaches (19Colburn P. Buonassisi V. Science. 1978; 201: 817-819Crossref PubMed Scopus (219) Google Scholar,20Ingegno M.D. Money S.M. Thelmo W. Greene G.L. Davidian M. Jaffe B.M. Pertschuk L.P. Lab. Invest. 1988; 59: 353-356PubMed Google Scholar), to our knowledge, this is the first study that performs a broad-based analysis on several endothelial types and that provides a functional relevance to this receptor on endothelial biology. The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. However, the contribution of progesterone to the replacement regime has been controversial. Although experimental studies evaluating intimal thickness (37Hanke H. Hanke S. Bruck B. Brehme U. Gugel N. Finking G. Muck A.O. Schmahl F.W. Hombach V. Haasis R. Atherosclerosis. 1996; 121: 129-138Abstract Full Text PDF PubMed Scopus (86) Google Scholar) and bromodeoxyuridine incorporation (38Hanke H. Hanke S. Finking G. Muhic-Lohrer A. Muck A.O. Schmahl F.W. Haasis R. Hombach V. 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Atherosclerosis is a multifactorial process and the principal contributor to myocardial and cerebral infarction (42Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9929) Google Scholar). Response to injury is one of the favored hypotheses for development of atherogenesis. It postulates that an alteration of the intima by various risk factors (such as mechanical injury, chemically altered low density lipoprotein, viruses, or toxins) initiates a primary endothelial cell dysfunction that leads to subsequent vascular changes, giving rise to the initial atherosclerotic lesion. The plaque progresses by the accumulation of layers of smooth muscle, macrophages, and foam cells; further deposition of extracellular matrix; and possibly neovascularization. Progression or rupture of the plaque is frequently associated with physical disruption of the endothelium. Because of the role of the endothelium in providing an antithrombotic and anticoagulant surface, rapid endothelial repair is of importance to the containment of atherosclerotic lesions. Although progesterone has shown suppressive effects on smooth muscle cell proliferation in vitro (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar), its participation on intimal endothelial repair, according to this study, appears to be equally inhibitory and, by extension, possibly deleterious to the endothelial healing of an exposed lesion. The inhibitory effect of progesterone on endothelial proliferation also provides an explanation for the episodes of vessel breakdown and irregular bleeding associated with progestin-based contraceptives (21Macqueo M. Diczfalusy E. Fraser I.S. Webb F.T.G. Endometrial Bleeding and Steroidal Contraception. Lakartidmingen, Geneva1980: 138-152Google Scholar). Physiological, but constant, levels of circulating progestins most likely affect the high mitotic rate associated with the endometrial vasculature, 3M. Graubert, M. Lombardo, M. A. Ortega, L. F. Brown, B. Kessel, J. F. Mortola, and M. L. Iruela-Arispe, unpublished observations. leading to capillary rupture. Furthermore, progesterone has been implicated in the suppression of tumor-induced neovascularization (43Rao B.R. Anticancer Res. 1997; 17: 1019-1022PubMed Google Scholar). In this study, we showed that progesterone partially blocks and delays endothelial cell cycle progression through a receptor-mediated mechanism that involves changes in expression of cell cycle proteins, cyclins E and A, and changes in pRb phosphorylation. The participation of PR in the regulation of cell cycle has been controversial, with studies indicating that progesterone stimulates, inhibits, or does not alter cell cycle progression even on the same cell type (8Lee W. Harder J.A. Yoshizumi M. Lee M. Haber E. Nat. Med. 1997; 3: 1005-1008Crossref PubMed Scopus (109) Google Scholar, 44Groshong S.D. Owen G.I. Grimison B. Schauer I.E. Todd M.C. Langan T.A. Sclafani R.A. Lange C.A. Horwitz K.B. Mol. Endocrinol. 1997; 11: 1593-1607Crossref PubMed Scopus (229) Google Scholar, 45Ishida Y. Heersche J.N. Bone. 1997; 20: 17-25Crossref PubMed Scopus (71) Google Scholar, 46Said T.K. Conneely O.M. Medina D. O'Malley B.W. Lyndon J.P. Endocrinology. 1997; 138: 3933-3939Crossref PubMed Scopus (72) Google Scholar). When inhibitory in mammary epithelial cells, the mechanism by which progesterone mediates cell cycle arrest has been shown to implicate both suppression of Cdk activity (47Musgrove E.A. Swarbrick A. Lee C.S.L. Cornish A.L. Sutherland R.L. Mol. Cell. Biol. 1998; 18: 1812-1825Crossref PubMed Scopus (105) Google Scholar) and up-regulation of p21 (48Owen G.I. Richer J.K. Tung L. Takimoto G. Horwitz K.B. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In endothelial cells, however, it is likely that progesterone acts by regulation of Cdk activity only, because we have not detected changes in either p21 nor p27 expression levels (data not shown). It has now become increasingly clear that the functional contribution of PR as a transcription factor is also dependent on the expression of several binding proteins that modulate PR activity (49Prapapanich V. Chen S. Nair S.C. Rimerman R.A. Smith D.F. Mol. Endocrinol. 1996; 10: 420-431PubMed Google Scholar, 50Prapapanich V. Chen S. Smith D.F. Mol. Cell. Biol. 1998; 18: 944-952Crossref PubMed Scopus (50) Google Scholar, 51Nair S.C. Rimerman R.A. Toran E.J. Chen S. Prapapanich V. Butts R.N. Smith D.F. Mol. Cell. Biol. 1997; 17: 594-603Crossref PubMed Scopus (165) Google Scholar, 52Segnitz B. Gehring U. J. Biol. Chem. 1997; 272: 18694-18701Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Whether or not these binding proteins play a role in the regulation of endothelial cell cycle or any other cell type is, at this point, not clear and deserves further investigation. Nevertheless, we found that endothelial cell proliferation was suppressed by physiological levels of 17-α-hydroxy-progesterone in every endothelial cell type examined. In conclusion, the presence of progesterone receptor in endothelial cells and the direct effect of progesterone described here can be of physiological relevance to the balance between potentiation of endothelial growth, mediated by estradiol, and suppressive signals, mediated by progesterone. This balance could play an important role in the regulation of angiogenesis in the endometrium and corpus luteus during the menstrual cycle. Nonetheless, constant levels of progestins in HRT could be counter-productive. Although the benefits of progestins, as first or second line therapy in the treatment of breast and endometrial carcinoma, have been widely acknowledged, the long-term use of progestins as contraceptives or for the prophylaxis of menopause should be under closer scrutiny. It is clear from these and other data that progestins may have direct growth-inhibitory actions in nonreproductive sites, apart from their ability to inhibit estrogen-mediated cell proliferation in such classical reproductive targets as the endometrium. We thank Katie Davies for excellent technical help on the isolation of endothelial cells and Dr. Tim Lane for discussions and comments during the progression of this project.
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