Delbert G. Gillespie

17β-Estradiol, Its Metabolites, and Progesterone Inhibit Cardiac Fibroblast Growth

Postmenopausal women (PMW) have increased incidence of cardiovascular disease, and estrogen substitution therapy has been shown to have cardioprotective effects. Since abnormal growth of cardiac fibroblasts (CFs) is associated with hypertension and myocardial infarction and estrogen inhibits vascular smooth muscle cell (SMC) growth, it is feasible that estrogen may attenuate cardiac remodeling by inhibiting CF growth, and this possibility was investigated by using cultured CFs. 17Beta-estradiol and progesterone, but not 17alpha-estradiol, estrone, or estriol, inhibited 2.5% FCS-induced proliferation (DNA synthesis and cell number) and collagen synthesis (3H-proline incorporation) in a concentration-dependent manner and to a similar extent in male and female CFs. Compared to 17beta-estradiol, its metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent in inhibiting FCS-induced DNA synthesis, collagen synthesis, and cell proliferation. The inhibitory effects of 17beta-estradiol and its metabolites were enhanced in presence of progesterone and 4-hydroxytamoxifen (high-affinity estrogen receptor ligand). Moreover, like estrogens, the dietary phytoestrogens biochanin A and daidzein inhibited FCS-induced growth of CFs. In conclusion, 17beta-estradiol, its metabolites, and progesterone inhibit CF growth in a gender-independent fashion. Moreover, hormone replacement therapy using 17beta-estradiol and progesterone may protect PMW against cardiovascular disease by inhibiting CF growth and cardiac remodeling; whereas estrogens that do not inhibit CF growth may be less effective in protecting PMW against cardiovascular disease. Finally, our studies provide evidence that phytoestrogens inhibit CF growth and may be clinically useful as a substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Exogenous and Endogenous Adenosine Inhibits Fetal Calf Serum–Induced Growth of Rat Cardiac Fibroblasts Role of A2B Receptors

BACKGROUND Because proliferation of cardiac fibroblasts participates in cardiac hypertrophy/remodeling associated with hypertension and myocardial infarction, it is important to elucidate factors regulating cardiac fibroblast proliferation. Adenosine, a nucleoside abundantly produced by cardiac cells, is antimitogenic vis-à-vis vascular smooth muscle cells; however, the effect of adenosine on cardiac fibroblast proliferation is unknown. The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast-derived) adenosine on cardiac fibroblast proliferation. METHODS AND RESULTS Growth-arrested cardiac fibroblasts were stimulated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; adenosine deaminase inhibitor); (2) dipyridamole (adenosine transport blocker); and (3) iodotubericidin (adenosine kinase inhibitor). All of these agents inhibited, in a concentration-dependent manner, FCS-induced cardiac fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation) and cell counting. EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 2.3- to 5.6-fold when added separately to cardiac fibroblasts, and EHNA+iodotubericidin or EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by >690-fold. Both KF17837 (selective A2 antagonist) and DPSPX (nonselective A2 antagonist) but not DPCPX (selective A1 antagonist) blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA synthesis, suggesting the involvement of A2A and/or A2B but excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors and suggested a major role for A2B receptors. This conclusion was confirmed by the rank order potencies of four adenosine analogues. CONCLUSIONS Cardiac fibroblasts synthesize adenosine, and exogenous and cardiac fibroblast-derived adenosine inhibits cardiac fibroblast proliferation via activation of A2B receptors. Cardiac fibroblast-derived adenosine may regulate cardiac hypertrophy and/or remodeling by modulating cardiac fibroblast proliferation.

Phytoestrogens Inhibit Growth and MAP Kinase Activity in Human Aortic Smooth Muscle Cells

-Estrogens are known to induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima formation. However, the use of estrogens as cardioprotective agents is limited by carcinogenic effects in women and feminizing effects in men. If noncarcinogenic and nonfeminizing estrogenlike compounds, such as natural phytoestrogens, afford cardioprotection, this would provide a safe method for prevention of cardiovascular disease in both men and women. Therefore, we evaluated and compared in human aortic SMCs the effects of phytoestrogens (formononetin, genistein, biochanin A, daidzein, and equol) on 2.5% fetal calf serum-induced proliferation (3H-thymidine incorporation and cell number), collagen synthesis (3H-proline incorporation), and total protein synthesis (3H-leucine incorporation) and on PDGF-BB (25 ng/mL)-induced migration (modified Boydens chambers). Moreover, the effects of phytoestrogens on PDGF-BB (25 ng/mL)-induced mitogen-activated protein kinase (MAP kinase) activity in SMCs was also studied. Phytoestrogens inhibited proliferation, collagen and total protein synthesis, migration, and MAP kinase activity in a concentration-dependent manner and in the following order of potency: biochanin A>genistein>equol>daidzein>formononetin. In conclusion, our studies provide the first evidence that in human aortic SMCs phytoestrogens inhibit mitogen-induced proliferation, migration and extracellular matrix synthesis and inhibit/downregulate MAP kinase activity. Thus, phytoestrogens may confer protective effects on the cardiovascular system by inhibiting vascular remodeling and neointima formation and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Clinically Used Estrogens Differentially Inhibit Human Aortic Smooth Muscle Cell Growth and Mitogen-Activated Protein Kinase Activity

Some estrogenic compounds modify vascular smooth muscle cell (SMC) biology; however, whether such effects are mediated in part by estrogen receptors is unknown. The purpose of this study was to evaluate whether the actions of clinically used estrogens on human aortic SMC biology are mediated by estrogen receptors. We examined the effects of various clinically used estrogens in the presence and absence of ICI 182,780, an estrogen receptor antagonist, on cultured human aortic SMC DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell counting), cell migration (modified Boyden chamber), collagen synthesis ([(3)H]proline incorporation), and mitogen-activated protein kinase activity. FCS-induced DNA synthesis, cell proliferation, collagen synthesis, platelet-derived growth factor-induced SMC migration, and mitogen-activated protein kinase activity were significantly inhibited by physiological (10(-9) mol/L) concentrations of 17beta-estradiol and low concentrations (10(-8) to 10(-7) mol/L) of 17beta-estradiol, estradiol valerate, estradiol cypionate, and estradiol benzoate but not by estrone, estriol, 17alpha-estradiol, or estrone sulfate. The inhibitory effects of 17beta-estradiol and other inhibitory estrogens were completely reversed by 100 micromol/L ICI 182,780, and the rank-order potency of various estrogens to inhibit SMC biology matched their rank-order affinity for estrogen receptors. The inhibitory effects of estrogens on SMC biology are in part receptor-mediated. Because the cardioprotective effects of hormone replacement therapy are most likely mediated by modification of SMC biology, whether hormone replacement therapy protects a given postmenopausal woman against cardiovascular disease will depend partially on the affinity of the estrogen for estrogen receptors in vascular SMCs.

Adenosine Inhibits Growth of Human Aortic Smooth Muscle Cells Via A2B Receptors

Adenosine inhibits rat vascular smooth muscle cell (SMC) growth. However, the effects of adenosine on human vascular SMC proliferation and synthesis of extracellular matrix proteins, such as collagen, are unknown. The objective of this study was to characterize the effects of exogenous and endogenous (SMC-derived) adenosine on human aortic SMC proliferation and collagen synthesis. Growth-arrested SMCs were stimulated with 2.5% fetal calf serum (FCS) in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), and with agents that increase endogenous adenosine levels, including erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), dipyridamole, and iodotubericidin. All of these agents inhibited in a concentration-dependent manner FCS-induced SMC proliferation as assessed by DNA synthesis (3H-thymidine incorporation) and cell counting, as well as collagen synthesis (3H-proline incorporation). EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 1.7-fold to 18-fold when added separately to SMCs, and EHNA+iodotubericidin and EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by more than 392-fold. Both KF17837 (selective A2 antagonist) and DPSPX (A1/A2 antagonist), but not DPCPX (selective A1 antagonist), blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA and collagen synthesis, suggesting the involvement of A2A and/or A2B, but excluding the participation of A1, receptors. The lack of effect of CGS21680 (selective A2A agonist), excluded involvement of A2A receptors and suggested a major role for A2B receptors. A comparison of the inhibitory potencies of 2-chloroadenosine, N6-cyclopentyladenosine (selective A1 agonist), NECA (A1/A2 agonist), and MECA (A1/A2 agonist) were consistent with an A2B receptor subtype mediating the inhibitory effects of adenosine on human aortic SMC proliferation. In conclusion, human aortic SMCs synthesize adenosine, and exogenous as well as endogenous (SMC-derived) adenosine inhibits SMC proliferation and collagen synthesis via activation of A2B receptors.

Adenosine Inhibits Growth of Rat Aortic Smooth Muscle Cells: Possible Role of A2b Receptor

Abnormal growth of vascular smooth muscle cells (SMC) is frequently associated with hypertension and atherosclerosis, and homeostasis within a normal vessel is maintained by the balanced generation of both vasoconstrictors and vasodilators. Moreover, several endogenous vasoconstricting factors induce SMC growth, whereas several vasodilators inhibit SMC growth. Inasmuch as adenosine is a potent vasodilator, it is possible that it too could inhibit SMC growth. Hence, the effects of adenosine (10(-8) to 10(-3) mol/L), 2-chloroadenosine (a stable analogue of adenosine; 10(-8) to 10(-3) mol/L), and 8-bromo-cAMP (10(-8) to 10(-3) mol/L) on fetal calf serum (FCS; 2.5%)-induced growth of rat aortic SMC were evaluated. Growth was analyzed by assaying DNA synthesis (thymidine incorporation in SMC pulsed for 4 hours with 1 microCi/mL [3H]thymidine) and cell proliferation (change in cell number). Growth-arrested SMC were treated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine, or 8-bromo-cAMP for 24 hours for DNA synthesis or 4 days for cell proliferation. All three substances inhibited DNA synthesis and cell proliferation in a concentration-dependent manner. Compared with adenosine, 2-chloroadenosine was more potent in inhibiting growth. The inhibitory effects of 2-chloroadenosine were reversed by KF17837 (a specific A2 receptor antagonist) but not by DPCPX (a specific A1 receptor antagonist). Furthermore, the inhibitory effects of 2-chloroadenosine were not mimicked by CGS21680 (an A2a receptor agonist), and the effects of N6-cyclopentyladenosine (CPA; an A1 receptor agonist) were not markedly more potent than those of 2-chloroadenosine, suggesting that the inhibitory effects of adenosine are possibly mediated via A2b receptors. These studies provide evidence that adenosine inhibits SMC growth and suggest that a decrease in local levels of adenosine may initiate SMC growth and contribute to the vascular remodeling process observed in hypertension and atherosclerosis.

A2B Receptors Mediate the Antimitogenic Effects of Adenosine in Cardiac Fibroblasts

Adenosine inhibits growth of cardiac fibroblasts; however, the adenosine receptor subtype that mediates this antimitogenic effect remains undefined. Therefore, the goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat left ventricular cardiac fibroblasts, PDGF-BB (25 ng/mL) stimulated DNA synthesis (3H-thymidine incorporation), cellular proliferation (cell number), collagen synthesis (3H-proline incorporation), and MAP kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, but not N6-cyclopentyladenosine, 4-aminobenzyl-5′-N-methylcarboxamidoadenosine, or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A2B receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A2 receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A2B receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5′-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A2B receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of CF growth by activating A2B receptors coupled to inhibition of MAP kinase activity. Thus, A2B receptors may play a critical role in regulating cardiac remodeling associated with CF proliferation. Pharmacologic or molecular biological activation of A2B receptors may prevent cardiac remodeling associated with hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.

2-Methoxyestradiol, an Estradiol Metabolite, Inhibits Neointima Formation and Smooth Muscle Cell Growth via Double Blockade of the Cell Cycle

2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by ≥50% blocked cell-cycle progression in G0/G1 and in G2/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D1 and cyclin B1 expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D1, cyclin B1, phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME–treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME–induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.

Effects of Estradiol and Its Metabolites on Glomerular Endothelial Nitric Oxide Synthesis and Mesangial Cell Growth

Reduced nitric oxide synthesis by glomerular endothelial cells and increased proliferation of glomerular mesangial cells is associated with glomerular remodeling that leads to accelerated glomerulosclerosis. Estradiol induces nitric oxide synthesis and slows the progression of renal disease. Because the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol are more potent than estradiol in inhibiting growth of vascular smooth muscle cells, which are phenotypically similar to mesangial cells, we compared the effects of estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol on growth of glomerular mesangial cells and on basal nitric oxide synthesis by glomerular endothelial cells. In human glomerular mesangial cells, estradiol and its metabolites concentration-dependently (1 nmol/L to 10 &mgr;mol/L) inhibited serum (2.5%)-induced DNA synthesis, cell proliferation, and collagen synthesis with the order of potency being 2-methoxyestradiol > 2-hydroxyestradiol > estradiol. ICI182780 (100 &mgr;mol/L, an estrogen receptor antagonist) blocked the growth inhibitory effects of estradiol but not 2-hydroxyestradiol or 2-methoxyestradiol. Treatment with estradiol, but not 2-hydroxyestradiol and 2-methoxyestradiol, induced nitric oxide synthesis (P <0.05, assayed by the formation of 3H-l-citrulline from 3H-l-arginine) in human glomerular endothelial cells, and these effects were blocked by ICI182780 and L-NMA (a nitric oxide synthesis inhibitor). In conclusion, estradiol may attenuate glomerulosclerosis by inducing nitric oxide synthesis via an estrogen receptor–dependent mechanism and by conversion to 2-hydroxyestradiol and 2-methoxyestradiol, which inhibit glomerular mesangial cell proliferation independent of estrogen receptors.

Cyclic AMP-Adenosine Pathway Induces Nitric Oxide Synthesis in Aortic Smooth Muscle Cells

The main purpose of this investigation was to evaluate whether the cyclic AMP-adenosine pathway, ie, the conversion of cAMP to AMP and, hence, to adenosine, is involved in the regulation of nitric oxide (NO) synthesis by vascular smooth muscle cells (SMCs). Treatment of confluent monolayers of SMCs with adenosine, 2-chloroadenosine (stable analog of adenosine), and agents that elevate endogenous (SMC-derived) adenosine (EHNA and iodotubericidin) increased nitrite/nitrate (stable metabolites of NO) levels in the medium and enhanced the conversion of 3H-L-arginine to 3H-L-citrulline by cytosolic extracts obtained from the pretreated SMCs. The stimulatory effects of adenosine were not mimicked by low (1 to 100 nmol/L) concentrations of CGS21680, an A2A receptor agonist, or CPA, a selective A1 receptor agonist. The stimulatory effects of 2-chloroadenosine and EHNA plus iodotubericidin were significantly inhibited by KF17837, a selective A2 receptor antagonist, and by DPSPX, an A1/A2 receptor antagonist, but not by DPCPX, a selective A1 receptor antagonist. DDA (adenylyl cyclase inhibitor) and Rp-cyclic AMP (protein kinase A inhibitor) did not block the effects of adenosine on NO synthesis. Incubation of SMCs with exogenous cyclic AMP, at concentrations previously shown to elevate levels ofadenosine in the medium, also increased nitrite/nitrate levels and 3H-L-citrulline formation, and the effects of cyclic AMP on NO synthesis were blocked by DPSPX and KF17837, but not by DPCPX. These findings provide evidence that exogenous and SMC-derived adenosine induce NO synthesis via A2B receptors linked to a pathway not involving adenylyl cyclase/protein kinase A. Moreover, extracellular cyclic AMP induces NO synthesis via conversion to adenosine and activation of A2B adenosine receptors. The cyclic AMP-adenosine pathway may be importantly involved in the vascular production of NO.