Association between high levels of growth factors in plasma and progression of coronary atherosclerosis.

Although intimal proliferation of smooth muscle cells (SMC) is recognized as one of the key mechanisms in the development of atherosclerosis, our knowledge of the role of circulating growth factors for SMC in this process is limited. In the present study the plasma levels of platelet-derived growth factor (PDGF), beta-thromboglobulin (beta-TG), platelet factor 4 (PF 4) and total growth factor activity were determined in a group of 30 young postinfarction patients who had participated in an angiographic study of mechanisms associated with progression of coronary atherosclerosis. Significant correlations were found between the total growth factor activity in plasma and progression (r = 0.42, P < 0.05), as well as severity (r = 0.52, P < 0.01), of global coronary atherosclerosis. Attempts to identify the nature of the total growth factor activity indicated that less than 20% could be attributed to PDGF, the major serum mitogen for SMC. PDGF levels determined by radioimmunoassay were not related to progression or severity of global coronary atherosclerosis, but showed a significant association with the number and severity of distinct stenoses (r = 0.40, P < 0.05). Due to the retrospective design of this study, it is not possible to conclude whether there is a causal relationship between circulating growth factors and development of coronary atherosclerosis.     

Growth Factors Released Into the Coronary Circulation After Vascular Injury Promote Proliferation of Human Vascular Smooth Muscle Cells in Culture

Objectives. This study sought to 1) assess in vivo release of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) into the coronary circulation after vascular injury in human subjects; and 2) evaluate mitogenic effects of PDGF and bFGF on the patient’s own vascular smooth muscle cells (VSMCs).

Background. Circumstantial evidence suggests involvement of PDGF and bFGF peptides in the neointimal response to vascular injury. To date, no study has shown biologically active growth factors within the coronary circulation after vascular injury in human subjects.

Methods. In 18 patients, plasma PDGF AB, platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) levels were measured in coronary sinus blood obtained before and up to 30 min after angioplasty. In five patients undergoing atherectomy, coronary sinus serum was added to cultured VSMCs derived from atherectomy tissue to assess the mitogenic potential of the serum. Mitogenicity attributable to PDGF and bFGF was determined using neutralizing antibodies to these factors. PDGF A, PDGF B and bFGF were localized within the atherectomy tissue using immunocytochemical analysis.

Results. Before angioplasty, PDGF AB, PF4 and beta-TG levels were elevated threefold in patients scheduled for angioplasty compared with those in control patients (p < 0.01). Within 5 min of angioplasty, PDGF AB levels increased twofold and returned toward preangioplasty levels at 30 min; PF4 and beta-TG levels remained elevated. Serum obtained at 30 min after atherectomy showed a sixfold increase in mitogenicity compared with preatherectomy serum (p = 0.01). This increase in mitogenicity was reduced by 20%, 40% and 65% in the presence of neutralizing antibodies to PDGF, bFGF and PDGF + bFGF, respectively. PDGF A, PDGF B and bFGF were visualized within the intima of the atherectomy tissue.

Conclusions. The change in plasma PDGF level is consistent with first-phase release of PDGF after vascular injury. The increase in mitogenicity of serum suggests that PDGF and bFGF are biologically active.

(J Am Coll Cardiol 1997;29:1536–41)     

Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions

Platelet-rich plasma is currently promoted to serve as an adjuvant for bone grafts to enhance quantity and quality of newly forming bone; however, the underlying cellular mechanisms are not fully understood. We show here that supernatants of leukocyte-depleted thrombin-activated platelets increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Using neutralizing antibodies raised against platelet-derived growth factor (PDGF), the observed effects of platelet-released supernatants were diminished. The mitogenic response was also decreased when extracellular signal-regulated protein kinase (ERK) signalling was inhibited by PD98059; however, PD98059 did not reverse the effects of platelet-released supernatants on migration and osteogenic differentiation. Consistent with an ERK-mediated mitogenic activity, incubation of serum-starved mesenchymal cell progenitors with platelet-released supernatants increased phosphorylation of the kinase. Together, these observations indicate that PDGF is a key factor released upon platelet activation that can increase migration and proliferation, and decreases osteogenic differentiation of mesenchymal progenitor cells under in vitro conditions. The results further suggest that ERK signalling is required to mediate the mitogenic response to platelet-released supernatants.     

Platelet-released supernatants increase migration and proliferation,and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions

Platelet-rich plasma is currently promoted to serve as an adjuvant for bone grafts to enhance quantity and quality of newly forming bone; however, the underlying cellular mechanisms are not fully understood. We show here that supernatants of leukocyte-depleted thrombin-activated platelets increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Using neutralizing antibodies raised against platelet-derived growth factor (PDGF), the observed effects of platelet-released supernatants were diminished. The mitogenic response was also decreased when extracellular signal-regulated protein kinase (ERK) signalling was inhibited by PD98059; however, PD98059 did not reverse the effects of platelet-released supernatants on migration and osteogenic differentiation. Consistent with an ERK-mediated mitogenic activity, incubation of serum-starved mesenchymal cell progenitors with platelet-released supernatants increased phosphorylation of the kinase. Together, these observations indicate that PDGF is a key factor released upon platelet activation that can increase migration and proliferation, and decreases osteogenic differentiation of mesenchymal progenitor cells under in vitro conditions. The results further suggest that ERK signalling is required to mediate the mitogenic response to platelet-released supernatants.     

Activity of Platelet-Derived Growth Factor (PDGF) in Platelet Concentrates and Cryopreserved Platelets Determined by PDGF Bioassay

The bioassay of platelet-derived growth factor (PDGF) in platelets was established using the rat fibroblast cell line 3Y1. 3Y1 cells (5 X 10(3)/well) were cultured in 24-well microculture plates with RPMI 1640 medium using 1% of PDGF-free serum and 5% of platelet extracts for 4 days. Cell proliferation was measured by the increase of DNA content. This assay made it possible to measure the biological PDGF activity. PDGF activity of platelet concentrates kept the same level as that of fresh platelet samples during preservation for up to 120 h at 22 degrees C; cryopreserved platelets also retained PDGF activity well.     

The significance of platelet-derived growth factors for proliferation of vascular smooth muscle cells

Platelets are important in acute thrombotic occlusion of injured vessels, e.g., subsequent to angioplasty. In contrast to these acute events of thrombus formation, much less is known about the significance of platelets for the control of smooth muscle cell (SMC) proliferation. A body of experimental and clinical evidence indicates an involvement of platelets in the pathology of atherosclerosis and restenosis. However, the precise role of platelet-derived growth factors for SMC proliferation in atherosclerotic and restenotic vessels is not clear and many questions remain unresolved. Platelet-dependent SMC mitogenesis is determined by a coordinate action of several classes of mitogenic factors which are either released from storage pools or generated upon platelet activation. Although platelet-derived growth factor (PDGF) is considered to be the most important platelet mitogen it is very likely that yet unknown factors and mechanisms are involved. Differential (stimulatory or inhibitory) effects on SMC growth and differentiation have been reported for different platelet-derived growth factors. Thus, for the overall response, complex interactions between multiple factors need to be considered. In addition, multicellular interactions, e.g., between platelets and endothelial cells may modulate the effects of platelet-derived factors on SMC mitogenesis. Taken together, the mechanisms of platelet-dependent SMC proliferation need to be reevaluated. The assessment of the precise role of platelet mitogens in the complex proliferative repair mechanisms of an injured vessel wall clearly requires further studies.     

The significance of platelet-derived growth factors for proliferation of vascular smooth muscle cells

Platelets are important in acute thrombotic occlusion of injured vessels, e.g., subsequent to angioplasty. In contrast to these acute events of thrombus formation, much less is known about the significance of platelets for the control of smooth muscle cell (SMC) proliferation. A body of experimental and clinical evidence indicates an involvement of platelets in the pathology of atherosclerosis and restenosis. However, the precise role of platelet-derived growth factors for SMC proliferation in atherosclerotic and restenotic vessels is not clear and many questions remain unresolved. Platelet-dependent SMC mitogenesis is determined by a coordinate action of several classes of mitogenic factors which are either released from storage pools or generated upon platelet activation. Although platelet-derived growth factor (PDGF) is considered to be the most important platelet mitogen it is very likely that yet unknown factors and mechanisms are involved. Differential (stimulatory or inhibitory) effects on SMC growth and differentiation have been reported for different platelet-derived growth factors. Thus, for the overall response, complex interactions between multiple factors need to be considered. In addition, multicellular interactions, e.g., between platelets and endothelial cells may modulate the effects of platelet-derived factors on SMC mitogenesis. Taken together, the mechanisms of platelet-dependent SMC proliferation need to be reevaluated. The assessment of the precise role of platelet mitogens in the complex proliferative repair mechanisms of an injured vessel wall clearly requires further studies.     

Marrow fibroblasts from patients with myeloproliferative disorders show increased sensitivity to human serum mitogens

The growth of marrow fibroblasts from patients with myeloproliferative disorders (MPD) was investigated using platelet derived growth factor (PDGF) and human serum as mitogens in the presence of human plasma derived serum. The proliferation of fibroblasts in MPD patients was increased compared to normal individuals, especially in patients with chronic myelocytic leukaemia and essential thrombocythaemia. This increment of proliferation might be due to higher sensitivity of the fibroblasts to plasma derived serum than to PDGF, because the ratio of proliferation with PDGF to that without PDGF, when compared between patients and normals, remained unchanged. These results suggest that MPD fibroblasts are more sensitive to some factor(s) in plasma, and this fact could partially explain the pathogenesis of myelofibrosis in MPD patients.     

Megakaryocytopoiesis in vitro of patients with essential thrombocythaemia: effect of plasma and serum on megakaryocyte colony formation

To clarify the mechanism of increased numbers of megakaryocytes in patients with essential thrombocythaemia (ET), we studied in vitro megakaryocytopoiesis in ET and other myeloproliferative disorders, using a megakaryocytic colony assay in methylcellulose containing plasma or serum and medium conditioned by phytohaemagglutinin (PHA) stimulated leucocytes (PHA-LCM). Megakaryocytic colony formation was supported well by heparinized or citrated plasma and citrated serum which was harvested after clot formation of citrated plasma. Whole serum was inhibitory for megakaryocytic colony growth. The addition of platelet releasates and partially purified platelet derived growth factor (PDGF) resulted in a decrease in the number of megakaryocytic colonies. These findings suggested that platelet-derived factor(s) in serum was inhibitory to megakaryocytic colony formation. ET plasma supported the megakaryocytic colony formation by normal or ET bone marrow cells better than normal plasma. Moreover, in ET bone marrow cells, spontaneous megakaryocytic colonies were formed in the absence of PHA-LCM. Increased megakaryocytopoiesis in ET may be ascribed to (i) increased megakaryocyte-colony stimulating activity (Meg-CSA) in plasma and (ii) increased sensitivity to Meg-CSA or autonomous proliferation of megakaryocytic progenitor cells.     

Potential role of lysophosphatidic acid in hypertension and atherosclerosis

BACKGROUND: Lysophosphatidic acid (LPA) is present in both serum and cytosol. Serum LPA is mainly released from platelets whereas cytosolic LPA is the metabolite of phosphatidic acid due to the action of phopholipase A2. Because platelet function and phospholipase A2 activity are upregulated in hypertensive and coronary heart disease patients, respectively, plasma and cytosolic LPA levels are expected to be higher in these pathological conditions. OBSERVATIONS: LPA is known to cause platelet aggregation and thus release more LPA as well as platelet-derived growth factor; this positive feedback circuit leads to the continuous growth of vascular smooth muscle cells (VSMCs). LPA also increases the intracellular concentration of free calcium in VSMCs and elevates the blood pressure. LPA content in the atherosclerotic plaque is elevated about 13 times in comparison with normal tissues because oxidized low-density lipoproteins promote the production of LPA. On the other hand, LPA has been shown to protect the heart from ischemia and reperfusion-induced damage due to its antiapoptosis effect. Because LPA has been reported to stimulate mitogen-activated protein kinase, phosphatidylinositide-3-kinase and protein kinase C, this bioactive phospholipid may be involved in the signal transduction mechanisms during the process of cardiac hypertrophy. CONCLUSIONS: Due to its ability to increase intracellular Ca2+ and proliferation of VSMCs, LPA may play an important role in the development of hypertension and atherosclerosis. It is therefore suggested that LPA antagonists may prove useful in the treatment of both hypertension and atherosclerosis.     

急性冠脉综合征血小板源性生长因子与血管紧张素Ⅱ的相关性及其临床意义

目的 探讨急性冠脉综合征（ACS）患者中血清血小板源性生长因子（PDGF）与血管紧张素（Ang）II的相关性及其意义。方法 ACS组132例,按临床类型分为不稳定型心绞痛组（n=40）、非ST段抬高型心肌梗死组(n=54)和ST段抬高型心肌梗死组（n=38）,采用酶联免疫吸附法检测PDGF的水平、放射免疫法检测血清AngⅡ的水平,并进行相关统计学分析。结果 ACS各组患者外周血中PDGF及AngⅡ水平均有显著差异（均P<0.05）,相关性分析显示ACS患者外周血中PDGF与AngⅡ水平呈正相关（r=0.413,P<0.05）,且与患者的GRACE评分存在显著正相关（r=0.527,P<0.05）。结论 随着ACS病情加重,PDGF表达水平升高,PDGF与ACS的GRACE积分和AngⅡ水平有相关性,可为评估ACS的危险分层及预后提供新的指标。     

Locally acting growth factors for vascular smooth muscle cells: endogenous synthesis and release from platelets

Release of platelet-derived growth factor (PDGF) from platelets has been postulated to stimulate at least some of the cell proliferation seen at sites of tissue damage, both beneficially (wound healing) and perniciously (during formation of atherosclerotic lesions). Two other growth factors have been localized to the platelet: epidermal growth factor and transforming growth factor. These factors may function synergistically with PDGF in promoting smooth muscle cell proliferation in the injured vessel wall. PDGF-like molecules (PDGF-c) that bind to the PDGF receptor and are at least partially recognized by antiserum against PDGF may also be synthesized by vessel wall cells themselves under certain circumstances. Arterial endothelial cells secrete several mitogens, one of which is a PDGF-c. Release is greatly stimulated by exposure of the cells to physiologic concentrations of thrombin. Also, aortic smooth muscle cells from 2-week-old rats secrete mitogenic levels of PDGF-c. In this case, PDGF-c accounts for all the mitogenic activity in conditioned medium (when assayed on 3T3 cells). Smooth muscle cells obtained from adult rat aortae secrete 150-fold less PDGF-c. In a third example, when adult rat carotid arteries are damaged with a balloon catheter, smooth muscle cells migrate into the intima of the artery and proliferate. By 2 weeks, the number of smooth muscle cells in the artery has doubled. When these intimal smooth muscle cells are cultured, they are found to secrete PDGF-c. These findings suggest that activation of endogenous synthesis of PDGF-c may contribute to the smooth muscle cell proliferation seen in response to vascular injury.     

Synergistic action of purified alpha-fetoprotein and growth factors on the proliferation of porcine granulosa cells in monolayer culture.

alpha-Fetoprotein (AFP) is present in high levels in fetal fluids, certain neoplasias, and regenerating liver. Although AFP's physiological role remains an enigma, we have recently demonstrated mitogenic activity for AFP. Using a primary monolayer culture system, we have further investigated the proliferative activity of purified AFP. Porcine granulosa cells from small ovarian follicles were attached for 2 days in Ham's F-12-Dulbecco's Modified Eagle's Medium (1:1) and 5% fetal calf serum, followed by 6 days of culture in medium containing 0.25% plasma-derived serum plus 25 micrograms/ml low density lipoprotein with or without growth factors and/or purified human AFP. In this system AFP alone does not stimulate proliferation. However, when combined with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I; 10 ng/ml each), AFP (5 micrograms/ml) significantly (P less than 0.01) enhanced growth factor-mediated proliferation 4.5-fold over that of medium controls. Equivalent doses of purified human serum albumin or transferrin demonstrated no effect. The effects of AFP were dose dependent, with significant (P less than 0.05) enhancement of proliferation (2.7-fold over controls) observed with as little as 0.313 micrograms/ml AFP. Increased proliferation was noticed as early as 24 h after the addition of AFP and by 48 h AFP, EGF, and IGF-I had significantly (P less than 0.05) increased proliferation over that seen in medium controls, cells treated with EGF plus IGF-I, or cells treated with 10% fetal calf serum plus EGF, and this trend continued linearly over 5 days of culture. AFP (5 micrograms/ml) significantly increased the proliferative response observed with increasing doses of EGF, IGF-I, or EGF plus IGF-I, but did not appear to alter the dose-response curves. AFP dose-dependently (1.25-5 micrograms/ml) and significantly (P less than 0.05) increased proliferation of porcine granulosa cells in response to platelet-derived growth factor (PDGF) and EGF (25 and 10 ng/ml, respectively), but not to PDGF alone. In contrast, AFP produced no further proliferation of porcine thecal cells in response to PDGF plus EGF. Binding of EGF, IGF-I, or PDGF to purified AFP could not be demonstrated. These results demonstrate that physiological levels of AFP, although not mitogenic alone, can significantly enhance the mitogenic activity of EGF plus IGF-I/PDGF and may function to modulate growth factor-mediated cell proliferation during development and neoplasia.     

The measurement of arterial smooth muscle cell mitogens in the blood of oral contraceptive users

We have previously shown that serum from young women receiving the same combined mestranol-norethindrone containing oral contraceptive (OC) preparation accelerated the proliferation of arterial smooth muscle cells (SMC) in tissue culture, and this in vitro effect was not a direct action of either of its estrogenic or progestogenic constituents. To identify the substance(s) which might contribute to this potentially atherogenic action, blood was obtained from 20 OC users and control women for the measurement of growth hormone, insulin, somatomedins (insulin-like growth factor IGF-I and IGF-II), and the platelet alpha-granule constituents platelet-derived growth factor (PDGF), beta-thromboglobulin, and platelet factor 4 (PF4). No difference was demonstrable between OC users and controls in the levels of any of these growth-promoting hormones, nor in plasma concentrations of any of the platelet alpha-granule proteins. These studies indicate that the enhanced mitogenicity found in OC sera is probably not attributable directly to these hormones or PDGF, and may instead result from an in vivo OC-induced alteration in other as yet unidentified mediators of cellular growth.     

Dipyridamole and Platelet Release of Platelet-derived Growth Factor

Platelet-derived growth factor (PDGF) and β-thromboglobulin (β-TG) are released from platelet alpha-granules during platelet activation. PDGF is a potent chemoattractant and mitogen for human vascular smooth muscle cells, and may be important in the development of late restenosis following angioplasty and in atherogenesis. In recent studies, where PDGF release into serum was evaluated indirectly by measuring (3)H-thymidine incorporation into fibroblasts, it was reported that the antiplatelet drug dipyridamole (DPM) decreased serum levels of PDGF. Such selective inhibition of the PDGF-release would have potential important implications for patients with atherosclerosis and for patients undergoing angioplasty. We therefore measured platelet content of PDGF and β-TG as well as platelet release of PDGF using a newly developed radioimmunoassay in healthy volunteers before and immediately after ingestion of DPM 100 mg t.i.d. for 3 days. We found no significant differences in platelet content of PDGF or β-TG before and after DPM. PDGF release from platelets isolated from plasma by gel filtration and stimulated with thrombin as well as platelet release of PDGF into serum was also unaffected by DPM. In conclusion, treatment with DPM does not affect platelet content of PDGF or β-TG. The treatment did not inhibit the platelet-release of PDGF as previously reported, neither via direct effects on platelets nor on inhibitory plasma components. DPM may, however, inhibit (3)H-thymidine incorporation into fibroblasts.     

高胆固醇血症患者循环血小板衍化生长因子的变化

采用生物活性法测定血循环中血小板衍化生长因子（ＰＤＧＦ）活性。结果表明高胆固醇血症患者循环ＰＤＧＦ活性明显增高。提示高胆固醇血症患者ＰＤＧＦ的改变可能与内皮细胞损害及原癌基因异常表达有关，高活性的ＰＤＧＦ又与动脉粥样硬化平滑肌细胞增殖有密切的关系。     

Increased proliferation of bone marrow-derived fibroblasts in primitive hypertrophic osteoarthropathy with severe myelofibrosis

Pachydermoperiostosis or primary hypertrophic osteoarthropathy (HOA) is a rare congenital growth disorder of connective tissue. We report a case of severe myelofibrosis in a patient with HOA. When cultured in vitro, patient bone marrow-derived fibroblasts displayed a high proliferative potential with a shortened doubling time (24 hours v 36 to 48 hours for normal fibroblasts). The role of platelet-derived growth factor (PDGF), previously implicated in the pathogenesis of secondary acquired myelofibrosis, was studied. HOA fibroblasts expressed an increased number of PDGF-BB binding sites (300,000 sites/cell v 200,000 sites/cell for normal fibroblasts) without any modification of affinity. The increased expression of PDGF-R beta appeared to result from an accelerated rate of PDGF-R beta resynthesis with normal kinetics of endocytosis. As a consequence, a several-fold increase of PDGF-R beta tyrosine kinase activity was observed. No autocrine mechanism of growth was suspected as neither spontaneous PDGF- R beta autophosphorylation nor mitogenic activity in HOA fibroblast- conditioned medium was detected. Patient serum and platelet lysate were less potent than controls in inducing [3H]thymidine incorporation into HOA fibroblasts. This was inconsistent with a paracrine mechanism of growth. In vitro, human serum or PDGF-BB were not more mitogenic for HOA than normal fibroblasts. High levels of cyclin D1, a putative oncogene, were detected in serum-deprived HOA fibroblasts. Cyclin D1 overexpression could be implicated in the accelerated growth of these cells. Our results suggest that the mechanism of fibroblastic proliferation observed in this case of myelofibrosis might differ from those reported in other acquired myeloproliferative syndromes and could be associated with an intrinsic abnormality of HOA fibroblast growth.     

Interaction of two GPIIb/IIIa monoclonal antibodies with platelet Fc receptor (FcγRII)

Platelet-derived growth factor (PDGF) is thought to play some role in the genesis of fibrosis associated with myeloproliferative disorders. In addition, transforming growth factor-beta (TGF-beta) has been confirmed to promote fibrotic process. Both PDGF and TGF-beta have been shown to cooperate with epidermal growth factor (EGF) in regulating the growth of human marrow fibroblasts. All three are contained in platelet alpha-granules. We report the results of a study in patients with myelofibrosis with myeloid metaplasia (MMM). We evaluated PDGF, TGF-beta and EGF-like activities in circulating platelets from patients compared to healthy subjects. In contrast to EGF-like intraplatelet levels which were similar in patients and in normal donors (1-4 ng/10(9) platelets), we found constantly higher values for both PDGF and TGF-beta in MMM patients. In both radioimmunoassay (RIA) and assay for mitogenic activity on human bone marrow fibroblasts, PDGF levels were increased on the average 2-3.5-fold over the levels found in normal donors (P less than 0.01 and P less than 0.001, respectively). PDGF serum levels in patients were consistent with those found in platelets. In platelet-poor plasma (PPP), PDGF concentrations were undetectable or congruent to 2 ng/ml in patients and in control donors as well. The total TGF-beta activity in platelet lysates, determined using a competitive radioreceptor binding assay on Swiss 3T3 mouse cells and an inhibition growth assay on CCL64 cells, was found 2-3-fold increased in patients with MMM as compared to control subjects (P less than 0.003). These results emphasize that, not only PDGF, but also TGF-beta are implicated in the myelofibrosis with myeloid metaplasia.     

The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells

BACKGROUND: It has been reported that matrix metalloproteinase (MMP) was expressed in coronary arterial atherosclerotic lesions. However, not much is known about the relationship between the production of MMP and the progression of atherosclerosis. PURPOSE AND METHOD: To demonstrate the association between the protein-tyrosine phosphorylation (PTP) and the activation of extracellular MMP in the proliferation and migration of vascular smooth muscle cells (VSMCs), the effect of platelet-derived growth factor (PDGF) and vanadate (an inhibitor of protein-tyrosine phosphatase and an activator of certain protein-tyrosine kinases) on mitogenesis ([3H]thymidine incorporation after 24 hours), migration, PTP (Western blot analysis using anti-phosphotyrosine antibodies), and production of MMP (gelatin zymography) was examined in cultured VSMCs. RESULTS: Both vanadate (1-5 micromol/l) and PDGF (1-10 ng/ml) caused a dose-dependent increase in thymidine incorporation and migration and produced 72-kDa type IV gelatinase (MMP-2) in VSMCs. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation and MMP-2 production. Western blot analysis revealed that PDGF caused an increase in PTP, extracellular signal-regulated kinases (ERK1, ERK2) and PDGF receptor in VSMCs. Vanadate given together with PDGF induced a marked increase in the intensity of tyrosine phosphorylation in these proteins. Tyrosine kinase inhibitors (genistein and herbimycin A) and a synthetic inhibitor of MMP (1,10-phenanthroline) and an anti-MMP-2 neutralizing antibody inhibited the mitogenic effect induced by vanadate and/or PDGF. CONCLUSIONS: The data suggest that the proliferation and migration of cultured VSMCs was closely related to the stimulation of MMP-2 production that was induced through activation of PTK.     

Effect of platelet-derived growth factor and bone marrow-conditioned medium on the proliferation of human bone marrow-derived fibroblastoid colony-forming cells

The effects of various human sera, platelet lysates and platelet-derived growth factor (PDGF) on the proliferation of human bone marrow-derived fibroblastoid colony-forming cells (CFU-F) were examined. We obtained nearly identical growth curves of fibroblastoid colonies with sera, platelet lysates and PDGF as stimulants and concluded that PDGF was a main growth factor for CFU-F in human serum. In contrast to colony size, CFU-F number was irrelevant to the concentration of PDGF. Removal of culture medium containing hemopoietic cells after short-term incubation of bone marrow cells reduced both colony number and size in CFU-F cultures. When each of bone marrow-conditioned medium (BMCM), peripheral blood mononuclear cells (MNC) and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) was added to the cultures, CFU-F number and colony size recovered. The role of PDGF and the factors present in BMCM, MNC and PHA-LCM in the growth of CFU-F and their precursor cells were discussed.