High glucose abolishes the antiproliferative effect of 17beta -estradiol in human vascular smooth muscle cells

Shanhong Ling1, Peter J. Little2, Maro R. I. Williams1, Aozhi Dai1, Kazuhiko Hashimura1, Jun-Ping Liu3, Paul A. Komesaroff1,*, and Krishnankutty Sudhir1,*

1 Hormones and the Vasculature Laboratory, 2 Cell Biology of Diabetes Laboratory, and 3 Molecular Signaling Laboratory, Baker Medical Research Institute, Melbourne, Victoria 8008, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined effects of 17beta -estradiol (E2) on human vascular smooth muscle cell (VSMC) proliferation under normal (5 mmol/l) and high (25 mmol/l) glucose concentrations. Platelet-derived growth factor (PDGF) BB (20 ng/ml)-induced increases in DNA synthesis and proliferation were greater in high than normal glucose concentrations; the difference in DNA synthesis was abolished by a protein kinase C (PKC)-beta inhibitor, LY-379196 (30 nmol/l). Western blotting showed that PKC-beta 1 protein increased in cells exposed to high glucose, whereas PKC-alpha protein and total PKC activity remained unchanged, compared with normal glucose cultures. In normal glucose, E2 (1-100 nmol/l) inhibited PDGF-induced DNA synthesis by 18-37% and cell proliferation by 16-22% in a concentration-dependent manner. The effects of E2 were blocked by the estrogen receptor (ER) antagonist ICI-182780, indicating ER dependence. In high glucose, the inhibitory effect of E2 on VSMC proliferation was abolished but was restored in the presence of the PKC-beta inhibitor LY-379196. Thus high glucose enhances human VSMC proliferation and attenuates the antiproliferative effect of E2 in VSMC via activation of PKC-beta .

high glucose; estrogen; proliferation; smooth muscle cells; protein kinase C-beta


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS CURRENTLY ACCEPTED that premenopausal women have relative protection from cardiovascular disease, compared with men and postmenopausal women, probably due to the protective effects of estrogen (7, 21). However, premenopausal women with diabetes mellitus lose this gender-based cardiovascular protection (3, 16), suggesting that hyperglycemia possibly overcomes some of the beneficial effects of sex steroids. To date, little is known of the cellular and subcellular interactions between the signaling pathways associated with hyperglycemia and sex hormones in the vasculature.

Several lines of evidence suggest that estrogens inhibit vascular smooth muscle cell (VSMC) proliferation (14, 19, 23), whereas hyperglycemia stimulates VSMC growth (24, 26). Because VSMC proliferation is an important cellular mechanism in the development of atherosclerosis (17), an interaction between estrogens and glucose-related signaling pathways in regulating VSMC proliferation is possible. We hypothesized that high glucose concentrations might attenuate the antiproliferative effect of estrogens. In the present study, we examined the antiproliferative effects of 17beta -estradiol (E2) under high (25 mmol/l glucose) and normal (5 mmol/l glucose) glucose concentrations on human internal mammary artery smooth muscle cells. We found that E2 inhibits VSMC proliferation in a dose-dependent manner, but its antiproliferative effect is abolished by high glucose concentrations through a mechanism dependent on the activation of protein kinase C (PKC)-beta .


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. VSMC were prepared from internal mammary arteries of two women with coronary artery disease. The patients had hypertension and hypercholesterolemia, but no diabetes, and one of the patients had had a previous myocardial infarction. The internal mammary artery segments were obtained at the time of coronary artery bypass grafting, and VSMC were harvested by an explant technique (18). In brief, segments of internal mammary artery were cut out and placed into ice-cold Dulbecco's modified Eagle's medium (DMEM). All external fat and connective tissue were cleaned from the vessel under microscope. The outer membrane was torn off carefully, the vessel was cut longitudinally, and the endothelial layer was removed by scraping with forceps. Small strips of media were peeled off, transferred to 60-mm dishes anchored under 9 × 22-mm sterile coverslips, and incubated in normal glucose (5 mmol/l glucose) DMEM in the presence of 10% fetal bovine serum (FBS) in an incubator of 5% CO2 in air at 37°C. Culture media were changed every three days, and after ~1 wk, VSMC were observed migrating from the pieces and growing on the dishes. Cells were grown to near confluence and passaged, and the presence of smooth muscle alpha -actin was confirmed by immunofluorescence staining and Western blot analysis as a marker of VSMC. Cells from the first patient were used at passages 11-15, and cells from the second patient were used at passages 6-7.

Estrogen receptor studies. Estrogen receptor (ER) density in these VSMC was determined by radioligand binding assay. Confluent monolayers of cells on 6-well plates (~106 cells/well) were incubated with [3H]estradiol (0.31-5 nmol/l) and nonradioactive diethylstilbestrol (1 µmol/l) for 90 min. Cells were washed twice with Dulbecco's PBS (D-PBS), scraped in the presence of 1.5 ml/well of 0.1% Triton-acetic acid, and put into scintillation vials with 5 ml of Scintillation Liquid Instagel (Bio-Rad, Sydney, NSW, Australia) for counting (5 min/vial) in a beta -counter. Functional ER studies were carried out using the pure ER antagonist ICI-182780 (Tocris Cookson, Bristol, UK).

Experimental protocol. Cells were seeded in culture plates, grown to ~90% confluence, and growth-arrested in serum-free DMEM with normal-glucose medium (5 mmol/l glucose, with 20 mmol/l mannose for control of osmolarity) or high- glucose medium (25 mmol/l glucose) for 48 h. Cell proliferation was induced with PDGF-BB (20 ng/ml), and effects of E2 were detected by preincubation with the hormone for 4 h before PDGF-BB stimulation.

Assays of cell proliferation. DNA synthesis was determined by a [3H]thymidine incorporation assay. Cells in 24-well plates were incubated with 1 µCi/well of [3H]thymidine during the last 3 h of PDGF-BB treatment, washed three times with ice-cold D-PBS, incubated with ice-cold 0.2 N HClO4 (1 ml/well) on ice for 30 min, washed (0.5 ml/well, 3 times) with 0.2 N HClO4, incubated with 0.5 ml/well of 0.2 N NaOH at 37°C for 1 h, and neutralized with 0.2 ml/well of 6% acetic acid. Contents of the well were transferred into scintillation vials with 3 ml of Instagel and counted for 2 min per vial in a beta -counter. Cell number was measured by an automatic cell counter (S.ST.II/ZM, Coulter Electronics, London, UK) before and after PDGF-BB treatment for 48 h.

Analysis of PKC activity. Cellular PKC activity was measured using a previously published method (1). Briefly, cells in 24-well plates were stimulated with PDGF-BB for 15 min and, after the medium was removed, incubated at 37°C for 10 min with 120 µl/well of assay buffer containing, in mmol/l: 137 NaCl, 5.4 KCl, 0.3 Na2HPO4, 0.4 KH2PO4, 20 HEPES, 10 MgCl2, 5 EGTA, 25 beta -glycerophosphate, and 2.5 CaCl2 and 1 g/l glucose, pH 7.2-7.4, 50 mg/l digitonin, 0.05 mg/ml myristolated alanine-rich PKC kinase substrate (MARCKS) peptide, and 100 µmol/l [gamma -32P]ATP (added freshly before use). To terminate the reaction, 30 µl/well of 25% trichloroacetic acid were added for 5 min, and 135 µl of the cell lysate were transferred into 1.5-ml tubes containing 15 µl of 3.75% BSA (0.375 mg/ml final concentration) and incubated on ice for 30 min. After being centrifuged for 5 min at 12,000 g, 100 µl of supernatant were dotted onto Whatman P-81 cation exchange paper (3 × 3 cm). After being washed in 75 mmol/l phosphoric acid twice for 1 min, once for 5 min, and three times for 10 min, the paper was dried and put into scintillation vials containing Instagel (3 ml/vial) for counting (5 min) in a beta -counter. Nonspecific background, defined as the amount of radioactivity retained in the absence of PKC substrate MARCKS peptide, was subtracted from all values. The PKC inhibitor LY-379196 was a gift from Eli Lilly (Sydney, Australia). Similar to the compound LY-333531 (5, 25), LY-379196 at concentrations of 10-30 nmol/l selectively inhibits PKC-beta activity and at 600 nmol/l induces nonselective PKC inhibition and can thus be used for analysis of total and specific PKC activity.

Western blotting for protein expression of PKC subtypes. Cells in 60-mm dishes were cultured under both normal- and high-glucose conditions in the presence or absence of E2 (10 nmol/l) and LY-379196 (30 nmol/l). Cells were lysed by incubation on ice for 30 min with lysis buffer [in mmol/l: 20 Tris base, pH 7.7, 250 NaCl, 2 EDTA, 2 EGTA, 20 beta -glycerophosphate, and 1 Na-vanadate and 0.5% NP-40 and 10% glycerol; 10 µl/ml leupeptin, 5 µl/ml aprotinin, 1 µmol/l pepstatin, 1 mmol/l 4-(2-aminoethyl)benzenesulfonyl fluoride, and 10 mmol/l dithiothreitol were added before use]. Plasma membrane proteins were isolated by centrifugation at 14,000 rpm for 15 min, and 30 µg of protein were electrophoresed on 10% SDS-polyacrylamide gels and transferred to Hybond enhanced chemiluminescence (ECL) filters (Sigma). The filters were blocked with 5% nonfat dry milk in TBST (20 mmol/l Tris, pH7.5, 50 mmol/l NaCl, and 0.1% Tween-20) overnight and then washed and incubated with primary antibodies against PKC-alpha or PKC-beta 1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. After being washed (3 × 10 min), blots were incubated with horseradish peroxidase-conjugated secondary antibody (DAKO) for 1 h, washed (3 × 10 min), incubated for 1 min with ECL reagents (Amersham), and exposed to X-ray films. For protein loading controls, the blots were washed again and probed with an anti-human smooth alpha -actin antibody (DAKO) by use of the same method as described. For quantification, bands were scanned in a PowerLook Scanner.

Statistical analysis. All data are presented as means ± SE. Comparisons between two means were made using Student's t-test and multiple comparisons using ANOVA. Differences of P < 0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

E2 inhibits PDGF-BB-induced VSMC proliferation by a mechanism dependent on ER and negatively regulated by high glucose concentrations. In normal-glucose medium, preincubation of VSMC with E2 (1-100 nmol/l) for 4 h resulted in a decrease of PDGF-BB-induced DNA synthesis in a dose-dependent manner (Fig. 1), with inhibition reaching ~40% of control at 10 and 100 nmol/l. In contrast, no inhibition was observed for E2 in VSMC treated with PDGF-BB in high-glucose culture cells (Fig. 1). Consistent with the effect on DNA synthesis, direct cell counting showed a significant inhibition in PDGF-BB-stimulated increase in cell number by E2 (1-10 nmol/l), an inhibition observed for cells cultured in normal- but not in high-glucose medium (Fig. 2). High glucose itself induced an increase in cell proliferation, and the [3H]thymidine incorporation and cell number in high glucose concentrations were higher than in normal glucose concentrations (see controls in Figs. 1 and 2). Similar results were also observed in VSMC from the aorta of Wistar Kyoto rats (data not shown).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Vascular smooth muscle cells (VSMC) in 24-well plates at near confluence were growth-arrested in serum-free DMEM with glucose at normal [5 mmol/l glucose + 20 mmol/l mannose, normal glucose (NG)] or high [25 mmol/l glucose, high glucose (HG)] concentrations for 48 h. The cells were treated with platelet-derived growth factor (PDGF)-BB (20 ng/ml) for 20 h in the presence of 17beta -estradiol (E2) or vehicle (ethanol at final concentration of 0.1%) as control, and during the last 3 h, [3H]thymidine incorporation assay was performed (4 wells per treatment). Data are shown as means ± SE of a representative experiment from 3 similar ones. *P < 0.05 and **P < 0.01 vs. control (C); #P < 0.01 between controls (C).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   VSMC were grown to 60% confluence in 24-well plates and then cultured in serum-free DMEM with glucose concentrations at 5 mmol/l (NG) or 25 mmol/l (HG) for 48 h and treated with PDGF-BB (20 ng/ml) in the presence of E2 for 2 days. Cells were harvested and counted with an automatic cell counter. Data are shown as means ± SE of 4 wells per treatment. *P < 0.05 vs. control; #P < 0.01 between controls.

Radioactive ligand binding assay showed that these human VSMC contained estradiol-specific binding sites (ER) at a relatively high density (24,000 ± 2,000 sites/cell; dissociation constant = 0.5 nmol/l, maximum binding capacity = 41 fmol), with no change in ER density after 48-h culture in high-glucose medium (Fig. 3). In addition, the ER antagonist ICI-182780 (100 nmol/l) completely abolished the inhibitory effect of E2 (10 nmol/l) on cell DNA synthesis and proliferation (Fig. 4), consistent with an ER-mediated effect.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Confluent monolayers of VSMC in 6-well plates (~106 cells/well) were incubated with [3H]estradiol (0.31-5 nmol/l) and nonradioactive diethylstilbestrol (1 µmol/l) in normal glucose medium for 90 min at 37°C. Specific binding of [3H]estradiol was observed in the cells with dissociation constant = 0.5 nmol/l, maximum binding capacity = 41 fmol, and estrogen receptor (ER) number = 23,741 ± 1,976 sites/cell (n = 3).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   VSMC in 24-well plates at near confluence were growth-arrested in serum-free DMEM with NG or HG for 48 h. Cells were treated with PDGF-BB (PDGF; 20 ng/ml) for 20 h (for DNA synthesis) or 48 h (for cell proliferation) in the presence of vehicles (control), E2 (10 nmol/l) alone, or E2 (10 nmol/l) + ICI-182780 (ICI; 100 nmol/l). Data are shown as means ± SE of 4 wells per treatment. *P < 0.05 vs. control and PDGF+E2+ICI.

Involvement of PKC-beta in the stimulatory effect of high glucose on VSMC proliferation. Because PKC is a key regulatory element in signal transduction and PKC-beta has been implicated in diabetes-associated vascular complications (6, 12), we assessed the potential role of PKC in the effect of elevated glucose on E2 signaling by determining the effects on PKC activity and protein expression and of the PKC antagonist LY-379196.

PDGF-BB (20 ng/ml) induced a 1.3- to 1.6-fold increase in total PKC activity in these VSMC; this elevation was not further enhanced by high-glucose culture. The PDGF-BB-induced increase in total PKC activity was reduced ~30% by 600 nmol/l LY-379196, whereas neither 30 nmol/l LY-379196 nor E2 affected this increase in total PKC activity (Fig. 5).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5.   Confluent VSMC in 24-well plates were cultured in NG or HG serum-free DMEM for 48 h, pretreated with E2 and LY-379196 (LY) for 4 h, and treated with PDGF-BB (20 ng/ml) for 15 min, and cellular total protein kinase C (PKC) activity was determined. Data are shown as means ± SE of 4 wells per treatment. *P < 0.05 vs. PDGF-BB alone.

Western blotting analysis (Fig. 6) showed that protein expression of PKC-beta 1 increased significantly (~3-fold) in high-glucose cultures of 24 h or longer, whereas PKC-alpha , a protein expressed at relative high levels, was not changed in high glucose. The expression of PKC-alpha and -beta 1 was not affected by either E2 or LY-379196 (data not shown).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 6.   Confluent VSMC in 60-mm dishes were cultured in NG or HG serum-free DMEM for 0, 6, 24 and 48 h. Membrane proteins were isolated, and specific expression of PKC-alpha and PKC-beta 1 protein was detected by Western blot. Photograph shows the protein bands on a blot, by use of smooth muscle alpha -actin (SM actin) protein as loading control. Bar graph shows means ± SE of the relative protein levels from 3 similar Western blots. *P < 0.01 vs. NG at same time points.

The difference in DNA synthesis between normal and high-glucose cultures was completely abolished by LY-379196 at 30 nmol/l (Fig. 7), the concentration for selective inhibition of PKC-beta , indicating a key role of the PKC-beta subtype in the action of glucose on cell growth. Under high-glucose conditions, E2 (10 nmol/l) alone did not significantly affect PDGF-BB-induced DNA synthesis but did so when added together with 30 nmo/l LY-379196 (Fig. 7), indicating that the antiproliferative effect of estradiol on VSMC is restored under high glucose conditions by selective inhibition of PKC-beta .


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   VSMC in 24-well plates at near confluence were continually cultured in NG or HG serum-free DMEM for 48 h, pretreated with LY-379196 (30 nmol/l) and E2 (10 nmol/l) for 4 h, and treated with PDGF-BB (20 ng/ml) for 20 h. [3H]thymidine incorporation was measured during the last 3 h of PDGF-BB treatment. Data are shown as means ± SE of two similar experiments with 6-8 wells per treatment. *P < 0.05 vs. control; #P < 0.05, comparisons of E2+LY with E2 alone and to LY alone in cells under HG conditions.

Studies were also carried out in VSMC from the second patient by direct counting of cell numbers. The inhibitory effect of E2 (1-100 nmol/l) on cell proliferation was evident in VSMC in normal glucose culture in a dose-dependent manner but was minimal in high-glucose cultures, with only 11% inhibition by E2 at 100 nmol/l (Fig. 8A). Selective inhibition of PKC-beta activity by LY-379196 (30 nmol/l) reversed the antiproliferative effect of E2 at 10 nmol/l, and the ER antagonist ICI-182780 abolished E2 effects in these VSMC (Fig. 8B). These results were consistent with those in the first patient and indicate a general phenomenon in human VSMC.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   Human VSMC from a second patient at passage 6-7 were cultured in NG or HG DMEM for 2 days in the presence or absence of E2 with or without LY-379196 (LY; 30 nmol/l) and ICI-182780 (100 nmol/l). Cells were treated with PDGF-BB (20 ng/ml) for 48 h and then counted with an automatic counter. Data are shown as means ± SE of 4 wells per treatment. *P < 0.05 and **P < 0.01 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have shown that E2 inhibits PDGF-BB-induced VSMC proliferation in a concentration-dependent manner but that this inhibition is lost in high-glucose cultures. We have further shown that high-glucose medium induces an approximately threefold increase in PKC-beta 1 protein expression after 24-h culture and that selective inhibition of PKC-beta activity with LY-379196 at 30 nmol/l inhibits high-glucose-induced cell proliferation and restores the antiproliferative effect of E2 in VSMC cultured in high-glucose medium.

The effects of estrogens on the vasculature include nongenomic vasodilatation, reduction of circulating lipids (9, 22), and genomic actions on vascular cells (15). Some studies show that estrogens inhibit growth factor-induced cell proliferation in cultured VSMC via ER-mediated reduction of mitogen-activated protein (MAP) kinase (14), c-myc, and other early-response genes (15). It has also been shown, however, that cultured smooth muscle cells from human uterine artery or aortic smooth muscle cells from pregnant rats have higher proliferative responses to serum and PDGF and that E2 stimulates proliferation in these VSMC via activation of PKC (8). These studies indicate that estrogens have variable actions on VSMC, possibly dependent on the hormonal milieu of the cells, as well as their phenotype (20).

The present study shows that, in normal glucose, E2 attenuates PDGF-BB-induced proliferation in VSMC from human internal mammary artery, similar to our previous observations showing that E2 at physiological levels inhibits mechanical strain-induced mitosis in human aortic smooth muscle cells (11) and consistent with studies of cultured VSMC from different sources (14, 19, 23). A salient finding of the present study is that the antiproliferative effect of E2 was abolished under high glucose conditions via activation of PKC-beta . Such an interpretation is supported by our observations that high-glucose medium increased PKC-beta 1 protein expression and selective inhibition of PKC-beta (LY-379196 at 30 nmol/l) restored this antiproliferative effect of E2 in high-glucose conditions.

PDGF is an important factor in atherosclerosis. PDGF, mainly as PDGF-BB, is produced and secreted by vascular endothelial cells and contributes, via the PDGF-beta receptor, to VSMC migration and proliferation (17). These PDGF actions on VSMC are believed to be mediated through a complex array of intracellular signaling pathways including MAP kinase, PKC, early growth response genes, and intracellular calcium (4). The antiproliferative effects of estrogens are reportedly mediated via the MAP kinase pathway (2, 14). In the present study, total PKC activity (Fig. 5) or PKC-beta 1 protein expression was not affected by E2, indicating that the inhibitory effect of E2 on PDGF-BB-induced proliferation is not via the PKC pathway. The increased activation of PKC by high-glucose medium counteracts the antiproliferative effect of E2 in VSMC. Our results show that the antiproliferative effects of estrogen are abolished by high-glucose medium but that this potentially beneficial effect of estrogen can be restored, even in a high-glucose milieu, by a PKC-beta inhibitor.

Commonly used VSMC culture media such as DMEM and Waymouth's medium contain a high level of glucose (25 mmol/l), much higher than physiological levels (3-6 mmol/l). It has been shown, in the present study and other studies (24, 26), that high glucose itself induces VSMC growth, suggesting that glucose concentrations in culture media possibly influence the results of studies examining VSMC proliferation. The PKC pathway is regarded as a major mechanism underlying the vascular effects of hyperglycemia. Normalization of PKC by vitamin D (10) and of PKC-beta by the selective inhibitor LY-333531 (1, 5) prevents the vascular damage of hyperglycemia in experimental diabetes. Direct effects of high glucose on cultured VSMC, as shown in the present study and other studies (10, 24, 26), are also mainly via PKC activation, especially the PKC-beta isoform, which, possibly via phospholipase D, induces vascular proliferation and hypertrophy, which in turn likely contribute to diabetic vascular complications.

Our study provides a possible mechanism underlying the loss of gender-based protection against vascular disease in diabetic women. Vascular proliferation is a key element in diabetic macrovascular disease and is a significant determinant of morbidity and mortality in this condition (13). Estrogen does not appear to inhibit growth factor-induced proliferation in the presence of high glucose concentrations. Nevertheless, our findings suggest that the antiproliferative action of estrogen is restored by inhibition of PKC-beta and may indicate a potential role for PKC-beta inhibitors in diabetic macrovascular disease in women.


    ACKNOWLEDGEMENTS

We are thankful to Dr. He Li for assistance in the PKC analysis, and Prof. J. Funder for critically reviewing the manuscript.


    FOOTNOTES

* K. Sudhir and P. A. Komesaroff have contributed equally to this study.

The study was supported by a block grant from the National Health and Medical Research Council (NH&MRC) of Australia to the Baker Institute. K. Sudhir is funded as a Senior Research Fellow of the NH&MRC. S. Ling is funded by Dora Lush Scholarship from the NH&MRC as a Ph.D. candidate in Monash University at Melbourne.

Address for reprint requests and other correspondence: Address for reprint requests and other correspondence: K. Sudhir, Pharmacyclics, 995 E. Arques Ave., Sunnyvale, CA 94085-4521 (E-mail: ksudhir{at}pcyc.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00111.2001

Received 9 March 2001; accepted in final form 14 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Assender, JW, Irenius E, and Fredholm BB. 5-Hydroxytryptamine, angiotensin and bradykinin transiently increase intracellular calcium concentrations and PKC-alpha activity, but do not induce mitogenesis in human vascular smooth muscle cells. Acta Physiol Scand 160: 207-217, 1997[ISI][Medline].

2.   Dubey, RK, Jackson EK, Gillespie DG, Zacharia LC, Imthurn B, and Keller PJ. Clinically used estrogens differentially inhibit human aortic smooth muscle cell growth and mitogen-activated protein kinase activity. Arterioscler Thromb Vasc Biol 20: 964-972, 2000[Abstract/Free Full Text].

3.   Haffner, SM, Miettinen H, and Stern MP. Relatively more atherogenic coronary heart disease risk factors in prediabetic women than in prediabetic men. Diabetologia 40: 711-717, 1997[ISI][Medline].

4.   Hughes, AD, Clunn GF, Refson J, and Demoliou-Mason C. Platelet-derived growth factor (PDGF): actions and mechanisms in vascular smooth muscle. Gen Pharmacol 27: 1079-1089, 1996[Medline].

5.   Ishii, H, Jirousek MR, Ballas LM, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Heath WF, Stramm LE, Feener EP, and King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKCbeta inhibitor. Science 272: 728-731, 1996[Abstract].

6.   Ishii, H, Koya D, and King GL. Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. J Mol Med 76: 21-31, 1998[ISI][Medline].

7.   Isles, CG, Hole DJ, Hawthrone VM, and Lever AF. Relation between coronary risk and coronary mortality in women of the Renfrew and Paisley survey: comparison with men. Lancet 339: 702-706, 1992[ISI][Medline].

8.   Keyes, LE, Moore LG, Walchak SJ, and Dempsey EC. Pregnancy-stimulated growth of vascular smooth muscle cells: importance of protein kinase C-dependent synergy between estrogen and platelet-derived growth factor. J Cell Physiol 166: 22-32, 1996[ISI][Medline].

9.   Komesaroff, PA, Black CVS, and Westerman RA. A novel, nongenomic action of estrogen on the cardiovascular system. J Clin Endocrinol Metab 83: 2313-2316, 1998[Abstract/Free Full Text].

10.   Kunisaki, M, Bursell SE, Umeda F, Nawata H, and King GL. Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes 43: 1372-1377, 1994[Abstract].

11.   Ling, S, Deng G, Ives HE, Chatterjee K, Rubanyi G, Komesaroff PA, and Sudhir K. Estrogen inhibits mechanical strain-induced mitogenesis in human vascular smooth muscle cells via down-regulation of Sp-1. Cardiovasc Res 50: 108-114, 2001[ISI][Medline].

12.   Liu, JP. Protein kinase C and its substrates. Mol Cell Endocrinol 116: 1-29, 1996[ISI][Medline].

13.   Massi-Benedetti, M, and Federici MO. Cardiovascular risk factors in type 2 diabetes: the role of hyperglycemia. Exp Clin Endocrinol Diabetes 107, Suppl4: S120-S123, 1999[ISI][Medline].

14.   Morey, AK, Pedram A, Razandi M, Prins BA, Hu RM, Biesiada E, and Levin ER. Estrogen and progesterone inhibit vascular smooth muscle proliferation. Endocrinology 138: 3330-3339, 1997[Abstract/Free Full Text].

15.   Oparil, S. Hormones and vasoprotection. Hypertension 33: 170-176, 1999[Abstract/Free Full Text].

16.   Orchard, TJ. The impact of gender and general risk factors on the occurrence of atherosclerotic vascular disease in non-insulin-dependent diabetes mellitus. Ann Med 28: 323-333, 1996[ISI][Medline].

17.   Ross, R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801-809, 1993[ISI][Medline].

18.   Smirnov, NV, and Orekhov AN. Smooth muscle cells from adult human aorta. In: Cell Culture Techniques in Heart and Vessel Research, edited by Piper HM.. Berlin, Germany: Springer-Verlag, 1990, p. 280-281.

19.   Somjen, D, Kohen F, Jaffe A, Amir-Zaltsman Y, Knoll E, and Stern N. Effects of gonadal steroids and their antagonists on DNA synthesis in human vascular cells. Hypertension 32: 39-45, 1998[Abstract/Free Full Text].

20.   Song, J, Wan Y, Rolfe BE, Campbell JH, and Campbell GR. Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype. Atherosclerosis 140: 97-104, 1998[ISI][Medline].

21.   Stampfer, MJ, and Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiological evidence. Prev Med 20: 47-63, 1991[ISI][Medline].

22.   Sudhir, K, Ko E, Zellner C, Wong HE, Hutchison SJ, Chou TM, and Chatterjee K. Physiological concentrations of estradiol attenuate endothelial 1-induced coronary vasconstriction in vivo. Circulation 96: 3626-3632, 1997[Abstract/Free Full Text].

23.   Suzuki, A, Mizuno K, Ino Y, Okada M, Kikkawa F, Mizutani S, and Tomoda Y. Effect of 17beta -estradiol and progesterone on growth-factor-induced proliferation and migration in human aortic smooth muscle cells in vitro. Cardiovasc Res 32: 516-523, 1996[ISI][Medline].

24.   Williams, B, Gallacher B, Patel H, and Orme C. Glucose-induced protein kinase C activation regulates vascular permeability factor mRNA expression and peptide production by human vascular smooth muscle cells in vitro. Diabetes 46: 1497-1503, 1997[Abstract].

25.   Xia, P, Aiello LP, Ishii H, Jiang ZY, Park DJ, Robinson GS, Newsome WP, Jirousek MR, and King GL. Characterization of vascular endothelial growth factor's (VEGF) effect on the activation of phospholipase protein kinase C (PKC) and its isoforms: pathways and endothelial cell growth. J Clin Invest 98: 2018-2025, 1996[Abstract/Free Full Text].

26.   Yasunari, K, Kohno M, Kano H, Yokokawa K, Horio T, and Yoshikawa J. Possible involvement of phospholipase D and protein kinase C in vascular growth induced by elevated glucose concentration. Hypertension 28: 159-168, 1996[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 282(4):E746-E751
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
282/4/E746    most recent
00111.2001v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Google Scholar
Articles by Ling, S.
Articles by Sudhir, K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Ling, S.
Articles by Sudhir, K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online