Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDGFAB and PDGFBB), transforming growth factors (TGF-β1and TGF-β2), and vascular endothelial growth factor (VEGF165) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDGFBB, but not PDGFAB, TGF-β1, and TGF-β2, increased with ovariectomy. VEGF165 was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDGFBB in platelets. Higher levels of PDGFBB in platelets in synergy with other platelet-derived products could contribute to increased proliferative arterial response to injury after ovariectomy.
- platelet-derived growth factor
- transforming growth factor
platelets are essential for hemostasis after vascular injury. At sites of vascular injury, aggregating platelets release factors that have vasoactive and mitogenic properties (2, 14, 34). In the absence of intact or functional endothelium, these factors cause contraction of vascular smooth muscle (32, 41), recruitment of more platelets and other blood cells, cholesterol synthesis (13), low-density lipoprotein receptor expression (45), and proliferation and migration of vascular smooth muscle cells (SMCs) from the media to the intima layer of the vessel wall (11, 15). All of these actions also contribute to arterial occlusive disease (22, 36, 38).
Epidemiological studies indicate that the incidence of coronary artery disease (CAD) is lower in premenopausal women compared with age-matched men (7). However, the incidence of CAD increases with menopause, but the risk is decreased in healthy postmenopausal women taking estrogen replacement (28). These observations suggest that ovarian hormones, in particular 17β-estradiol, may be protective against development of CAD in women.
Ovarian hormones may affect platelet function. Ovariectomy increases prostacyclin content in platelets from female pigs (26). Incubating platelets from postmenopausal women with 17β-estradiol inhibits ADP-mediated platelet aggregation and ATP release (4). Platelets release platelet-derived growth factors (PDGFs) and transforming growth factors (TGFs) on platelet activation (1, 11). Both platelet-derived factors are involved in modulating cell migration, proliferation, and extracellular matrix production by SMCs. However, it is unknown whether ovarian hormones affect concentrations of growth factors in platelets. Therefore, experiments were designed to test the hypothesis that ovariectomy (loss of ovarian hormones to mimic menopause) increases content of PDGFAB, PDGFBB, TGF-β1, TGF-β2, and vascular endothelial growth factor (VEGF165) in platelets and, furthermore, as a consequence of such increase, that the proliferative response of vascular SMCs would increase to platelet-lysate from ovariectomized (Ovx) pigs compared with lysate from ovary-intact pigs.
Animal and tissue collection.
Adult (6 mo old, 110–120 kg) gonadally intact and Ovx (for 4 wk) female Yorkshire pigs were used for this study. Ovariectomy was by laparoscopy. Sham laparoscopy surgeries were also performed in age-matched, gonadally intact female pigs to determine the effects of laparoscopy surgery on platelets. Pigs were housed individually (22°C; 12:12-h light-dark cycle) and were fed twice a day (Lean Grow 93, Land O' Lakes Farmland Feed; Fort Dodge, IA). The external genitalia from gonadally intact females showed changes associated with an estrus cycle. Serum levels of estrogen from gonadally intact females range from 10 to 30 pg/ml, and estrogen levels in Ovx females are below the sensitivity level of assay (6, 43). Uterine weight from Ovx females was significantly lower compared with uterine weight from gonadally intact females (51.3 ± 7.8 vs. 86.6 ± 12.3), thus validating efficacy of the surgical intervention. Four week after surgery, Ovx and age-matched gonadally intact pigs were anesthetized intramuscularly with ketamine (8 mg/kg) and xylazine (12 mg/kg). Blood (450 ml) was collected aseptically (22°C) from the right carotid artery directly into collecting bags (Baxter Healthcare) containing 50 ml of acid citrate-dextrose (ACD) solution (pH 6.5, 1:9 vol/vol). Blood (45 ml) was collected (from the femoral artery) before and 4 wk after ovariectomy into polypropylene tubes containing 5 ml ACD solution (pH 6.5, 1:9 vol/vol) for determination of the number of peripheral platelets, red blood cells, white blood cells, hematocrit, and hemoglobin by a Coulter Gen-S hematology analyzer. Bone marrow was biopsied from the ribs using an 8-gauge × 4-in. bone biopsy needle (Medical Device Technologies). Coronary arteries were removed for tissue culture.
The percentage of reticulated platelets [youngest platelets in circulation containing mRNA (17)] was measured to obtain an indication of platelet turnover before and 4 wk after surgery. A 19-gauge needle was inserted into an ear vein. Blood was allowed to drip, and 20 μl of blood were collected and added to 2 ml of the following dilution buffer (22°C): 20 mol/l HEPES, Hank's balanced salts (Sigma) (pH 7.4), supplemented with 1 mg/ml bovine serum albumin (BSA), 1 μmol/l tick anticoagulant peptide, 25 nM Hirudin, and 1 μg/ml PGE1 (Sigma). All concentrations are final concentrations in the dilution medium. Samples were incubated for 30 min in the dark (22°C) with a porcine GP Ib-biotin-conjugated monoclonal mouse antibody. Streptavidin-phycoerythrin (10 μl)-conjugated antibody was then added for 30 min (in the dark, 22°C). Afterward, control tubes received 1 ml of 1× PBS. The other tubes received 1 ml thiazole orange at 20 ng/ml. Tubes were incubated in the dark for 30 min (22°C). Dilution buffer (1 ml) was added to each tube, and the tubes were centrifuged (3,000 rpm, 15 min, 22°C). Supernatants were removed, and the pellets were resuspended gently in 2 ml of 1× PBS. Platelets were quantified by flow cytometry (FACcalibur, Becton-Dickinson) within 2 h. Log forward scatter (for size characteristic) and log side scatter (for granularity) were used to identify platelets. The platelet cloud was gated electronically to exclude red and white blood cells.
Immunofluorescence and immunohistochemical stainings for estrogen receptor-α and -β in porcine platelets and bone marrow biopsies, respectively.
ACD + 0.1% BSA-washed platelets were smeared on poly-l-lysine-covered slides, air dried, and fixed with buffered formalin-acetone for 30 s. Rib bone marrow biopsies were fixed in 10% formalin, paraffin embedded, sectioned (3 μm), and mounted on glass slides. Slides were incubated for 30 min with 20% normal goat or rabbit serum (Vector Laboratories, diluted in 1× PBS). Excess serum was blotted off, and slides were incubated overnight at 4°C with either affinity-purified primary polyclonal rabbit antibody against estrogen receptor (ER)-α (1:200 in 1× PBS, Santa Cruz Biotechnology) + 20% normal goat serum or with affinity-purified primary polyclonal goat antibody against ER-β (1:200 in 1× PBS, Santa Cruz Biotechnology) + 20% normal rabbit serum. Slides were then washed three times (5 min, 1× PBS). Platelets were incubated for 30 min in the dark with either affinity-purified fluorescein goat anti-rabbit or flourescein rabbit anti-goat antibodies (in 1× PBS + 20% serum). Slides were rinsed with 1× PBS (3 times), and platelets were mounted with ProLong antifade reagent (Molecular Probes), covered with a glass coverslip, and visualized with a confocal microscope. Bone marrow biopsies were incubated (30 min) with biotinylated secondary goat anti-rabbit or rabbit anti-goat IgG antibodies. Peroxidase activity in bone marrow biopsies was visualized with a diaminobenzidine tetrahydrochloride substrate kit (Vector Laboratories) for 2 min. Control stainings were obtained by treating slides similarly with the above procedures with the exception of omitting primary antibodies for either ER-α or ER-β.
Preparation of platelet lysate.
Blood from the right carotid artery collected into sterile collecting bags was centrifuged at 300 g (22°C, 15 min). Platelet-rich plasma was collected into polypropylene tubes and centrifuged at 1,600 g (22°C, 10 min) to precipitate platelets. Platelet-poor plasma (PPP) was removed and stored at −70°C. Precipitated platelet pellets were washed and resuspended with an equal volume of ACD solution + 0.3% BSA. The platelet count was obtained, and afterward platelets were precipitated (1,600g, 10 min, 22°C) and resuspended to adjusted platelet concentration to 1 × 109 platelets/ml in 1× PBS (calcium and magnesium free, 22°C, Sigma) + 0.1% BSA. Aliquots were frozen and stored at −70°C. Platelets were lysed on thawing. Concentrations of PDGFAB, PDGFBB, TGF-β1, TGF-β2, and VEGF165were determined by ELISA in PPP and platelet lysate.
Quantitating growth factors in platelet lysates by ELISA.
Concentrations of PDGFAB, PDGFBB, TGF-β1, TGF-β2, and VEGF165 in platelets and PPP were determined by commercially available human ELISA kits (R&D Systems). All samples were assayed in duplicate. PDGFAB ELISA has 10 and 2% cross-reactivity with PDGFAA and PDGFBB, respectively. PDGFBB ELISA has 0.1% cross-reactivity with PDGFAB. TGF-β1, TGF-β2, and VEGF165 immunoassays have no significant cross-reactivity or interference with other growth factors.
Coronary artery SMC cultures.
SMCs from the right coronary artery were isolated via the explant method (27). SMCs stained positive for α-actin, myosin heavy chain, vimentin, and desmin (Sigma). Forty thousand SMCs per milliliter, third passage, were suspended in phenol red-free M199 (Sigma) with 10% charcoal-stripped fetal bovine serum (CS-FBS, Atlanta Biologicals) and plated in 24-well polystyrene plates (37°C, 95% air-5% CO2 atmosphere). Twenty-four hours later, cells were growth arrested in serum-free medium 199 (M199) for 24 h before the addition of platelet lysate. Platelet lysates prepared from 1 × 109 SMCs/ml diluted with 2% CS-FBS M199 to obtain platelet lysate concentrations ranging from 0.25 to 50% were then added to the cultures. For controls, cells were incubated with 2% CS-FBS (control) or 2% CS-FBS + 1.5% 1× PBS (calcium and magnesium free, 50:50) to control the concentration of 1× PBS in the 50% dilution of platelet lysates. Six hours after the addition of lysate or control solutions, [3H]thymidine (1 μCi/well) was added to the cultures. After an additional 18 h (24-h exposure to platelet lysates or control solutions), SMCs were rinsed twice with cold 1× PBS, incubated with cold trichloroacetic acid for 12 min, digested with 0.1 N NaOH for 5 min, and frozen. The digested cell preparation was thawed once to analyze the [3H]thymidine incorporation by scintillation spectroscopy (Beckman Instruments). Protein concentration was determined by the Bradford assay (Bio-Rad). All experiments were conducted in quadruplicate.
Conditioned cell medium from coronary artery SMC cultures.
Forty thousand SMCs per milliliter, third passage, suspended in phenol red-free M199 with 10% CS-FBS were plated in 24-well plates. After 24 h, cells were growth arrested by incubating them in serum-free M199. Twenty-four hours after growth arrest, 2% CS-FBS-M199 was added to the cultures. After 24 h, the conditioned media was collected and centrifuged to remove any cells, and concentrations of PDGFAB, PDGFBB, TGF-β1, TGF-β2, and VEGF165 were measured by human ELISA kits (R&D Systems). The same growth factors were measured by ELISA in 2% CS-FBS-M199 incubated in the plates but in the absence of SMCs.
All data are expressed as means ± SE; n equals the number of pigs from which platelets were collected. For proliferation experiments, the mean of quadruplicate measures were used for a single experiment, and n equals the number of pigs from which cells were derived for each experiment. Student's t-test (two tailed) for paired or unpaired data was used to assess statistical significance. One-way ANOVA followed by Bonferroni's post hoc analysis was used to compare more than two means. For all statistical tests,P < 0.05 was considered statistically significant.
Peripheral blood cell number and reticulated platelets.
Sham surgery did not significantly change total circulating platelets, percentage of reticulated platelets, or red blood cell and lymphocyte numbers (Table 1). Circulating numbers of platelets and reticulated platelets as percentages of total platelet number were not significantly changed with ovariectomy. However, the number of circulating red blood cells, hemoglobin, and hematocrit was significantly higher in Ovx animals compared with gonadally intact females (Table 1). Ovx females had a significantly lower number of circulating lymphocytes (Table 1). These results indicate that loss of ovarian hormones but not surgical intervention affects blood elements.
Immunohistochemistry in bone marrow biopsies.
Megakaryocytes, the largest and polymorphic nuclear cells in the bone marrow producing platelets from their cytoplasmic fragmentation, stained positive for ER-α (Fig.1 A) and ER-β (Fig. 1 B). Nuclear and cytoplasmic stainings for ERs were observed in megakaryocytes from both groups. Positive staining for ER-α and ER-β was not exclusive to megakaryocytes because other cells in bone marrow also stained positive for both receptors. No staining was observed in the absence of primary antibodies for either ER-α (Fig. 1 C) or ER-β (Fig. 1 D).
Immunofluorescence staining for ERs in female porcine platelets.
Platelets from gonadally intact and Ovx female pigs showed positive immunofluorescent staining for ER-α (Fig.2 A) and ER-β (Fig.2 B). A greater number of platelets stained positive for ER-β in both groups of animals, with only a few platelets staining positive for ER-α. No immunofluorescence was observed in the absence of primary antibody (data not shown, n = 4).
Concentration of growth factors in platelets and PPP.
Concentrations of PDGFAB were similar in lysates from 1 × 109 platelets/ml from gonadally intact (1,676 ± 183.6 pg/ml, n = 12) and Ovx (1,220 ± 134.7 pg/ml, n = 12) female pigs. However, concentrations of PDGFBB increased significantly in platelet lysates with ovariectomy (Fig.3).
Concentrations of TGF-β1 and TGF-β2 in platelet lysates did not change with ovariectomy (Fig.4); concentrations of VEGF165were below the sensitivity level of the assay (9.0 pg/ml).
Concentrations of PDGFAB, PDGFBB, and TGF-β1 in PPP from Ovx female pigs were greater than those in PPP from intact females (Table2). However, VEGF165 was detected only in PPP from gonadally intact female pigs. Therefore, ovariectomy differentially affects the concentration of growth factors in platelets and plasma.
Proliferation of coronary artery SMCs in response to platelet lysate.
Proliferation of right coronary arterial SMCs from gonadally intact and Ovx female pigs treated with 2% CS-FBS-M199 only (control) were similar (Fig. 5). Treating SMCs from both groups with 1× PBS + 2% CS-FBS-M199 inhibited their proliferation. However, adding 2% CS-FBS-M199 + platelet lysates (containing 1× PBS) restored SMC proliferation in both groups. Treating SMCs from both groups with 0.25–1.5% of autologous platelet lysates increased their proliferation by ∼1.5-fold (data not shown). Incubating SMCs from Ovx females with 6.5%, 12.5%, and 25% of autologous platelet lysates (from 1 × 109platelets/ml) significantly increased SMC proliferation 1.5- to 2.5-fold compared with controls (Fig. 5 B). A 1.5- to 2-fold increase was observed in proliferation of SMCs from intact females (Fig. 5 A) when treated with 1.5–12.5% of autologous platelet lysates. However, treating SMCs from intact females with 25% of autologous platelet lysates decreased their proliferation back to control levels, and treatment with 50% of autologous platelet lysates completely inhibited their proliferation. Incubating SMCs from Ovx females with 25–50% of autologous platelet lysates produced a 2.5- and 1.5-fold SMC growth, respectively, in their proliferation (Fig. 5 B). These results suggest that mitogenic factors in platelet lysates from Ovx females increase proliferation of SMCs to a greater extent than lysates from intact females. However, to determine whether responsiveness of SMCs affected responses to platelet lysates, SMCs from gonadally intact females were treated with lysate from platelets of Ovx females, and SMCs from Ovx females were treated with lysate from platelets of intact females. Variable cell proliferation at different platelet lysate concentrations was observed (Fig.6). However, unlike proliferation to autologous platelet lysate, treating SMCs of intact females with the highest concentrations (25% and 50%) of platelet lysates from Ovx females increased proliferation. Treating SMCs from Ovx females with either autologous platelet lysate or lysate from gonadally intact female platelets increased proliferation twofold (Fig. 6). These results suggest that both platelet content of growth factors and SMCs are affected by ovariectomy.
Concentrations of growth factors in media conditioned by SMCs.
Concentrations of TGF-β1 and VEGF165, but not PDGFAB, were significantly higher in media conditioned by SMCs from Ovx female pigs than in media conditioned by SMCs from intact female pigs (Table 3). Levels of PDGFBB and TGF-β2 were not detected in media conditioned by either groups of cells (n = 3, data not shown). These results indicate that SMCs release growth factors that could alter proliferation as autocrine hormones.
Results from the present study indicate that removal of ovarian hormones by ovariectomy increases content of growth factors, particularly PDGFBB, in platelets without altering the total circulating platelet number and percentage of reticulated platelets.
Because platelets do not have a nucleus, only residual amounts of mRNA, and limited protein synthesis (9), the presence of ER-α and ER-β in megakaryocytes (in the nucleus and cytoplasm) from both groups indicates that ovarian hormones, including 17β-estradiol, may be involved in modulating content of growth factors genomically in megakaryocytes. ER-β may be the main ER in porcine platelets because platelets from both groups had more extensive positive immunofluorescence staining to ER-β than to ER-α. These results are consistent with results from studies on human platelets because only ER-β, but not ER-α, has been detected (19). With the use of Western blotting as a more quantitative measure of ER distribution, recent studies from our laboratory have extended these immunofluorescent data and found that both ER-α and ER-β are upregulated with ovariectomy (18). Therefore, in addition to regulation of growth factors in platelets by ovarian hormones, ERs themselves are regulated.
Because platelets first respond to vascular injury (37) and are involved in the development of CAD (35, 42), these results have implications to explain differences in presentation of CAD after menopause. 17β-Estradiol, the main ovarian hormone in premenopausal women, may protect against risks for CAD by reducing content of mitogenic factors in platelets (10). For example, platelets aggregating in response to arterial injury or endothelial dysfunction would release increased amounts of PDGFBB and in combination with other factors synergistically increase proliferation of SMCs in Ovx or estrogen-depleted animals. SMC proliferation may not be arrested by TGF-β (25, 33), because similar levels were observed in platelet lysates from ovary-intact and Ovx animals. TGF-β inhibit growth of vascular SMCs. It has been hypothesized that atherosclerosis may result from a failure in endogenous inhibitory systems that normally limit wound repair (23).
Growth factors released from SMCs may also contribute to the response to injury because autocrine and paracrine factors from media conditioned with SMCs from Ovx females had higher TGF-β1and VEGF165 levels than media conditioned by cells from ovary-intact animals. VEGF165 may act as a paracrine hormone to stimulate migration and proliferation of endothelial cells (21). However, TGF-β1 may induce VEGF165 expression (8) and synergistically with TGF-β1 may increase vascular SMC proliferation (44). Modulation and release of other growth factors [insulin-like growth factor (IGF)-I] in platelets and SMCs from Ovx female porcines cannot be excluded. However, proliferation of vascular SMCs treated with combinations of PDGFBB, IGF-I, epidermal growth factor, and TGF-β was not further increased compared with SMCs treated with PDGFBB only (16). In addition, higher plasma concentrations of circulating PDGF activity are observed in patients with severe CAD compared with age- and sex-matched controls (29, 30).
17β-Estradiol inhibits migration/proliferation of vascular SMCs (20, 24, 39). Ovx female pigs have significantly lower levels of 17β-estradiol compared with gonadally intact female pigs (6). The high concentrations of PDGFAB, PDGFBB, and TGF-β1 and lower levels of VEGF165 in plasma from Ovx females and higher concentrations of PDGFBB that could be released from platelets may synergistically contribute to an increased response to injury (34) and stimulate growth of SMCs at the vascular wall in the absence or low levels of ovarian hormones.
Ovariectomy increased circulating numbers of red blood cells. These cells traveling in the axis of the artery force platelets toward the endothelial lining, increasing platelet-platelet and platelet-endothelial contact (12) as well as increasing blood viscosity. ADP released from red blood cells could activate platelets (3, 40) and contribute to platelet recruitment at the vessel wall.
Differences in platelet irritability between gonadally intact and Ovx female pigs were not determined in the present study. Platelet aggregation and ATP release in vitro are higher in platelets from postmenopausal women than in platelets from premenopausal women (4, 5). Furthermore, in addition to ERs being upregulated by ovariectomy, chaperone proteins of the heat shock family known to regulate nitric oxide and nitric oxide synthase are also upregulated by depletion of ovarian hormones (18). Therefore, interaction between platelet-derived nitric oxide and endothelium-derived nitric oxide will also affect platelet aggregation and secretion at sites of injury and endothelial dysfunction.
In conclusion, removal of ovarian hormones does not affect the number of platelets but increases the number of red blood cells and decreases the number of circulating lymphocytes that could contribute to development of increased arterial response to injury by increasing blood viscosity, platelet-platelet activation, and platelet-endothelial cell interaction. In addition, levels of mitogenic growth factors in plasma are increased with ovariectomy, as is the content of PDGFBB in platelets. Therefore, release of platelet PDGFBB and blood-derived growth factors as well as growth factors produced by SMCs could contribute to migration, proliferation, and synthesis of extracellular matrix proteins by SMCs that ultimately participate in the development of intimal thickening associated with response to injury and development of CAD, as observed in Ovx animals and postmenopausal women, respectively (31). The presence of ER-α and ER-β in platelets and bone marrow megakaryocytes suggests that 17β-estradiol may modulate PDGFBB levels genomically in megakaryocytes. The ERs on circulating platelets could affect nongenomic release of factors or other platelet functions when hormones are placed into ovarian hormone-deficient animals or humans.
Address for reprint requests and other correspondence: V. M. Miller, Dept. of Surgery, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail:).
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.
May 2, 2002;10.1152/ajpheart.00201.2002
- Copyright © 2002 the American Physiological Society