Release Of Platelet-derived Growth Factor Research Articles

Hypertrophic pulmonary osteoarthropathy and the vagus nerve: an historical note

Hypertrophic pulmonary osteoarthropathy and the vagus nerve: an historical note

Effectiveness of a Treatment Based on Melatonin in Five Patients with Systemic Sclerosis

Systemic sclerosis (SSc) is a multisystem connective disorder in which endothelial damage leads to fibrotic reactions through platelet hyperaggregability and inappropriate release of platelet-derived growth factors. With other colleagues, I have previously reported that melatonin seems to have a direct favorable effect on endothelium (stopping of bleeding symptoms in thrombocytopenic patients). Furthermore, melatonin has been described as performing an antiaggregating activity, and ACTH and vitamin E have been described as increasing melatonin effects. This provided the rationale to treat 5 SSc patients with melatonin plus ACTH and vitamin E. Patients received the treatment for 1 month. The therapy was continued for 2 additional months in patients with stable or responding disease. After 3 months, the stable or responding patients continued the therapy for 2 months and longer. All patients had a partial response after 1 month. Continuing with the treatment, none of the 5 patients had disease progression (average follow-up time of 16.6 months; range, 7-44 months). Toxicity was lacking, with the only side effect being drowsiness. Our experience suggests that the combination of melatonin-ACTH-vitamin E may be safe and effective in patients with SSc.

Release of platelet‐derived growth factors and proliferation of fibroblasts in the releasates from platelets stored in the liquid state at 22°C after stimulation with agonists

Fresh platelet (PLT)-rich plasma (PRP) treated with thrombin plus calcium chloride (CaCl(2)) is used to prepare a PLT gel to promote hemostasis and wound healing in a variety of surgical procedures. The effects of various agonists on stimulating the release of growth factors from liquid-preserved PLTs and the effects of the PLT releasate on the growth of fibroblasts in tissue culture were investigated. Plateletpheresis PLTs stored at 22 degrees C as high-yield PLTs for 3 to 6 days or outdated PLTs for 9 days were treated with agonists to assess release of platelet-derived growth factor (PDGF) AA, PDGF AB, PDGF BB, transforming growth factor-beta1 (TGF-beta1), and osteocalcin and the proliferation of fibroblasts treated with the PLT releasates in tissue culture. All treatments except for CaCl(2) alone and zeolite-CaCl(2) produced significant increases in PDGF AA compared to PRP. Thrombin-CaCl(2) produced significant increases in PDGF BB. Treatment by all the agonists produced similar increases in PDGF AB. TGF-beta1 and osteocalcin levels after treatment were similar to those in PRP. PRP releasate before and after stimulation with different agonists increased proliferation of fibroblasts in tissue culture. High-yield and outdated liquid-preserved PLTs released PDGF AA, AB, and BB but not TGF-beta1 or osteocalcin. The releasate from untreated PRP stimulated the proliferation of fibroblasts in tissue culture similar to the releasates from PRP treated with the different agonists. Further studies are needed to assess whether or not high-yield and outdated PLTs may be useful in wound healing.

Nicotinic and PDGF‐receptor function are essential for nicotine‐stimulated mitogenesis in human vascular smooth muscle cells

Cigarette smoking is implicated in the formation of occlusive vascular diseases. Nicotine's role in this process is incompletely understood. Nicotine's effect on human aortic vascular smooth muscle cells (HaVSMC) and the role of the nicotinic receptor (nAChR), platelet-derived growth factor (PDGF), and the PDGF-receptor (PDGF-R) in this response were studied. Nicotine's mitogenic effect was characterized by three methods: thymidine incorporation, a viability/proliferation assay based on metabolic conversion of tetrazolium salt to formazan dye and cell counting. Nicotine administration (10(-6) M) stimulated cell cycle entry marked by increased DNA synthesis, PCNA and cyclin D1 production, and increased cell division. Nicotinic receptor blockade with d-tubocurarine, a nicotinic AchR blocker, decreased nicotine-induced DNA synthesis, and cell division (0.33 +/- 0.04, 0.77 +/- 0.31-fold decrease, respectively). Nicotine increased cellular PDGF-BB transcript levels and protein release (ELISA: 1.6 +/- 0.5-fold increase) but not PDGF-AA or PDGF-AB release. Nicotine increased PDGFbeta-receptor protein content. PDGF inactivation with anti-PDGF antibody abolished nicotine-induced DNA synthesis (1.9 +/- 0.08-fold decrease). PDGF-R blockade with the PDGF-R antagonist tyrphostin AG 1295 decreased nicotine-induced DNA synthesis and cell division (0.25 +/- 0.01, 0.44 +/- 0.2-fold decrease, respectively). PDGF-R blockade reversed nicotine-stimulated increases in PDGF release, PDGF-BB transcripts, and PDGF-receptor levels (0.68 +/- 0.34; 0.46 +/- 0.01; 0.28 +/- 0.01-fold decrease, respectively). In conclusion, nicotine-mediated activation of nAChRs increases PDGF-BB transcription and protein production as well as PDGF beta-receptor levels. PDGF-BB/PDGF-R interaction is vital in nicotine's mitogenic actions on human aortic smooth muscle cells.

Differential growth factor retention by platelet-rich plasma composites

This study evaluates the temporal sequence and growth factor release from platelet-rich plasma (PRP) combined with different bone substitutes (BS), to identify an optimal substrate for extended growth factor retention. PRP was clotted with bovine thrombin or thrombin receptor activator peptide-6 (TRAP). In addition, PRP was clotted using Allogro (Ceramed, Lakewood, CO), BioGlass (Mo-Sci, Rolla, MN), or BioOss (Osteohealth, Shirley, NY). The effects of media exchange and BS on platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF beta) release were quantified via enzyme-linked immunosorbent assay. At day 1, the thrombin group released 36% more PDGF than the TRAP group and 80% more than the BS groups. At 7 days, PDGF release was the greatest for the TRAP group. PDGF release was minimal for all groups at day 14, with BS groups retaining 60% more PDGF than thrombin clots. Similarly, the thrombin group released the greatest amount of TGF beta (81.4% of the total), whereas TRAP and BS groups released significantly less TGF beta at day 1. Compared with thrombin, TRAP retained 39.2% more TGF beta, whereas BS groups retained even greater levels (Allogro, 54.3%; BioOss, 45.8%; BioGlass, 67.0%). No significant difference in TGF beta release was observed among the substitutes after day 1. The BS groups continued to retain TGF beta after 14 days, whereas all TGF beta in the thrombin clots was depleted. PRP preparation with thrombin results in a large, immediate release of growth factors that could be lost into the interstitium in vivo. TRAP-BS may prove more efficacious than thrombin in sustaining growth factor levels critical for the cascade of events leading to bone formation.

The Role of the Platelet in the Pathogenesis of Atherothrombosis

Platelet adhesion, activation, and aggregation at sites of vascular endothelial disruption caused by atherosclerosis are key events in arterial thrombus formation. Platelet tethering and adhesion to the arterial wall, particularly under high shear forces, are achieved through multiple high-affinity interactions between platelet membrane receptors (integrins) and ligands within the exposed subendothelium, most notably collagen and von Willebrand factor (vWF). Platelet adhesion to collagen occurs both indirectly, via binding of the platelet glycoprotein (GP) Ib-V-IX receptor to circulating vWF, which binds to exposed collagen, and directly, via interaction with the platelet receptors GP VI and GP Ia/IIb. Platelet activation, initiated by exposed collagen and locally generated soluble platelet agonists (primarily thrombin, ADP, and thromboxane A2), provides the stimulus for the release of platelet-derived growth factors, adhesion molecules and coagulation factors, activation of adjacent platelets, and conformational changes in the platelet alpha(IIb)beta3 integrin (GP IIb/IIIa receptor). Platelet aggregation, mediated primarily by interaction between the activated platelet GP IIb/IIIa receptor and its ligands, fibrinogen and vWF, results in the formation of a platelet-rich thrombus. Currently available antiplatelet drugs (aspirin [acetylsalicylic acid], dipyridamole, clopidogrel, ticlopidine, abciximab, eptifibatide, tirofiban) act on specific targets to inhibit platelet activation and aggregation. Elucidation of the multiple mechanisms involved in platelet thrombus formation provides opportunities for selectively inhibiting the pathways most relevant to the pathophysiology of atherothrombosis.

Regulation of PDGF and its receptors in fibrotic diseases

Platelet-derived growth factor (PDGF) isoforms play a major role in stimulating the replication, survival, and migration of myofibroblasts during the pathogenesis of fibrotic diseases. During fibrogenesis, PDGF is secreted by a variety of cell types as a response to injury, and many pro-inflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF. PDGF action is determined by the relative expression of PDGF α-receptors (PDGFRα) and β-receptors (PDGFRβ) on the surface of myofibroblasts. These receptors are induced during fibrogenesis, thereby amplifying biological responses to PDGF isoforms. PDGF action is also modulated by extracellular binding proteins and matrix molecules. This review summarizes the literature on the role of PDGF and its receptors in the development of fibrosis in a variety of organ systems, including lung, liver, kidney, and skin.

A photo-crosslinked poly(vinyl alcohol) hydrogel growth factor release vehicle for wound healing applications.

The objective of this study was to develop and evaluate a hydrogel vehicle for sustained release of growth factors for wound healing applications. Hydrogels were fabricated using ultraviolet photo-crosslinking of acrylamide-functionalized nondegradable poly(vinyl alcohol) (PVA). Protein permeability was initially assessed using trypsin inhibitor (TI), a 21 000 MW model protein drug. TI permeability was altered by changing the solids content of the gel and by adding hydrophilic PVA fillers. As the PVA content increased from 10% to 20%, protein flux decreased, with no TI permeating through 20% PVA hydrogels. Further increase in model drug release was achieved by incorporating hydrophilic PVA fillers into the hydrogel. As filler molecular weight increased, TI flux increased. The mechanism for this is most likely an alteration in protein/gel interactions and transient variations in water content. The percent protein released was also altered by varying protein loading concentration. Release studies conducted using growth factor in vehicles with hydrophilic filler showed sustained release of platelet-derived growth factor (PDGF-beta,beta) for up to 3 days compared with less than 24 hours in the controls. In vitro bioactivity was demonstrated by doubling of normal human dermal fibroblast numbers when exposed to growth factor-loaded vehicle compared to control. The release vehicle developed in this study uses a rapid and simple fabrication method, and protein release can be tailored by modifying solid content, incorporating biocompatible hydrophilic fillers, and varying protein loading concentration.

Factor Xa Induces Mitogenesis of Vascular Smooth Muscle Cells via Autocrine Production of Epiregulin ,

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell.

Transient leukaemia--a benign form of leukaemia in newborn infants with trisomy 21.

Transient leukaemia--a benign form of leukaemia in newborn infants with trisomy 21.

Overexpression of von Willebrand factor is an independent risk factor for pathogenesis of intimal hyperplasia: Preliminary studies

Deposition of von Willebrand factor (vWF) is increased in hyperplastic intima of grafts, vWF levels are elevated in patients with cardiovascular diseases, and there is resistance to progression of atherosclerosis in pigs with von Willebrand disease. We hypothesize that increased expression of endothelial vWF has mitogenic effects on smooth muscle cell (SMC) proliferation. In an in vitro study, mouse aortic smooth muscle cells (SMC) were exposed to vWF in various concentrations (0, 5, 20, 100, 500, and 1000 ng/mL). DNA synthesis of SMC was measured with (3)H-thymidine incorporation. In an in vivo study, 108 mice from inbred strains of C57BL/6J (control) and RIIIS/J (characteristic of low plasma vWF) underwent carotid artery ligation (flow cessation model) and were divided into three groups: C57BL/6J, RIIIS/J, and RIIIS/J treated with desmopressin (DDAVP; intraperitoneal injection at 3 micro g/kg/d). At 2 and 4 weeks, carotid arteries were harvested for analysis with immunohistochemical analysis, morphometric studies, and reverse transcriptase polymerase chain reaction; plasma vWF was measured with an enzyme-linked immunosorbent assay. In vitro SMC proliferation showed a positive dose-response curve with vWF stimulation. Intimal hyperplasia (IH) in carotid arteries was prominent in C57BL/6J mice, absent in RIIIS/J mice, and moderate in RIIIS/J treated with DDAVP (intima-media ratio, 71% +/- 18%, 0, and 32% +/- 12%, respectively; P <.01). vWF deposition occurred in all hyperplastic intima subjacent to intact endothelium. Plasma vWF correlated with degree of IH (110% +/- 10%, 21% +/- 7%, and 45% +/- 8%, respectively; P <.05). vWF-messenger RNA was 9 times higher in carotid arteries of C57BL/6J mice and 4 times higher in RIIIS/J with DDAVP, compared with RIIIS/J. vWF directly stimulates SMC proliferation in vitro via a direct dose-response effect. In vivo low shear stress accelerates IH proportional to vWF expression. This could occur under intact endothelium without platelet activation and platelet-derived growth factor release. In effect, control of IH may entail modulation of vWF expression.

Time- and pH-dependent release of PDGF and TGF-ß from platelets in vitro

We studied the spontaneous and thrombin-induced activation of platelets and their release of platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) at different pH values. Platelet activation was assayed with anti-bodies against P-selectin and performed in serum-free media. The release of PDGF and TGF-β was determined by ELISA after 15 min and 12 h. There was no activation at pH 5.0, while a time-dependent release of growth factors occurred at neutral and alkaline pH. The results suggest that release of growth factors is not only dependent on platelet activation but also on incubation time and pH. Although the used serum-free experimental situation is different from normal conditions for platelets in vivo, the findings of a late release of growth factors may, nevertheless, be relevant to wound healing.

Tumor infiltrating lymphocytes and macrophages have a potential dual role in lung cancer by supporting both host-defense and tumor progression

A prerequisite to the developement of an efficient cell and/or gene therapy for lung cancer is a precise characterization of the inflammatory cell populations spontaneously present in the tumor stroma associated with this cancer. This study was designed to define the cytotoxic potential and the relationship with stroma development of tumor infiltrating lymphocytes (TIL) and tumor associated macrophages (TAM). Tumor samples from 48 patients undergoing surgery for non-small cell lung cancer (NSCLC) were analyzed, by immunohistochemistry and in situ hybridization, with a panel of antibodies and probes specific for cell proteins linked to cytotoxicity, cytokines, and growth factors, and the replication status of TIL and TAM was evaluated by in vivo 5-bromodeoxyuridine incorporation. It was shown that, in NSCLC: (1) tumor stroma inflammatory cells are mainly TIL (~2/3) (among them, 80 % are T-cells) and TAM (~1/3), with almost no natural killer (NK) cells, and a few dentritic cells; (2) TAM and TIL are poorly replicating, but mainly recruited to the tumor stroma; (3) more than half TAM show an antibody-dependent cytotoxic potential, and one third of T-cells are TIA-1 positive CD8 activated cytotoxic lymphocytes; (4) cancer cells from only a few tumor express HLA class I and II antigens; (5) TAM production of cytotoxic cytokines [interleukin-1α (IL-1α), IL-1β, IL-6, tumor necrosis factor-α (TNF-α)] and of transforming growth factor-β1 (TGF-β1) is low, in contrast to their strong release of platelet-derived growth factor (PDGF). We concluded that, in NSCLC, TIL cytotoxicity is likely to be low because of a poor class I MHC expression by tumor cells, and TAM low production of cytotoxic cytokines is a major limit to their possible cytotoxic activity. In contrast, TAM may favor tumor progression by contributing to tumor stroma formation and angiogenesis through their release of PDGF, in conjunction with TGF-β1 production by tumor cells. (J Lab Clin Med 2002;140:320-8)

Factors in the fracture microenvironment induce primary osteoblast angiogenic cytokine production.

Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (<1% oxygen) conditions for 0, 3, 6, 12, and 24 hours, and VEGF gene expression was analyzed by Northern blot analysis. To determine whether rhPDGF-BB-induced VEGF messenger RNA (mRNA) expression was transcriptionally mediated or required de novo protein synthesis, transcription, and translation, studies were performed using actinomycin D and cycloheximide, respectively. Treatment with 50 ng/ml rhPDGF-BB resulted in a 2.4-fold increase in VEGF mRNA expression after 3 hours. Interestingly, rhPDGF-BB and hypoxia seemed to have an additive effect, resulting in a 3.7-fold increase in VEGF mRNA expression after 6 hours in primary neonatal rat calvarial osteoblasts. Furthermore, by using actinomycin D and cycloheximide, the authors demonstrated that the rhPDGF-BB-induced VEGF mRNA expression was transcriptionally mediate and not dependent on de novo protein synthesis. These data demonstrate that rhPDGF-BB transcriptionally increases osteoblasts VEGF mRNA expression in vitro. Furthermore, the semiquantitative results suggest that rhPDGF-BB and hypoxia act additively to increase VEGF mRNA expression. It is postulated that similar mechanisms may occur in vivo, at a site of skeletal injury, to induce neoangiogenesis and promote fracture repair.

Platelet α-granule secretion and its modification by SC-57101A, a GPIIb/IIIa antagonist

Platelet–agonist interaction results in aggregatory and secretory responses. While the activation of glycoprotein (GP) IIb/IIIa plays an essential role in platelet aggregation, its role in granule secretion is not clear. The present study was performed to examine the effect of 3-[[[[1-[4-(aminoiminomethyl) phenyl]-2-oxo-3 S-pyrrolidinyl]amino]carbonyl]amino]-propanoate monohydrochloride salt (SC-57101A), a GPIIb/IIIa antagonist, on platelet α-granule secretion responses to collagen, ADP, and thrombin receptor activating peptide (TRAP). Both SC-57101A and prostaglandin E 1 (PGE 1) inhibited collagen-, ADP-, and TRAP-induced platelet aggregation in a concentration-dependent manner. SC-57101A inhibited the collagen- and ADP-induced release of platelet-derived growth factor (PDGF) and β-thromboglobulin (β-TG) from platelets, but not TRAP-induced secretion of these granule contents. On the other hand, PGE 1 inhibited the release of PDGF and β-TG from platelets activated with all the agonists used. ADP and TRAP elicited P-selectin expression in the absence of platelet aggregation, while collagen produced no such reaction. SC-57101A only moderately inhibited P-selectin expression induced by ADP and had no inhibitory effect on that induced by TRAP. The inhibition of ADP-induced secretion of α-granule contents by SC-57101A was abolished when platelets were pretreated with aspirin. These results suggest that GPIIb/IIIa activation plays a minor role, if any, in α-granule secretion in human platelets.

Close relationship between the platelet activation marker CD62 and the granular release of platelet-derived growth factor.

The expression of CD62 on the surface of platelets is considered to be an indicator of platelet degranulation and secretion. We characterized the relationship between CD62 expression and platelet-derived growth factor (PDGF)(AB) and PDGF(BB) secretion in response to thrombin-receptor activating peptide (TRAP). The principal findings were 1) expression of CD62 as a constituent of platelet alpha-granule membrane and secretion of PDGF, an important ingredient of alpha-granules, can be stimulated by TRAP-induced activation in a dose-dependent fashion; 2) the activation marker and secretion product are closely correlated with each other; and 3) changes in the CD62 expression induced by a drug, namely clopidogrel, or by a disease, namely diabetes, are paralleled by changes in PDGF secretion. Although CD62 is perceived as an activation marker of platelets indicating enhanced aggregability and secretion of alpha-granular content, the proof that the CD62 status and its modifications reflect directly the actual secretion of the most important platelet mitogen, PDGF, has so far not been given. This ex vivo-in vitro study shows that at least for the activation pathway provided by the PAR-1 receptor for which TRAP is the selective agonist, CD62 expression on platelets could be a surrogate for their secretory activity.

Differential actions of naftopidil, doxazosin and nifedipine on platelet thromboxane generation and platelet-derived growth factor efflux in vitro

We examined the effects of the f 1 -adrenoreceptor blockers naftopidil and doxazosin and the Ca 2+ antagonist nifedipine on platelet function with reference to stimulus-induced thromboxane (TxB 2 ) generation and platelet-derived growth factor (PDGF) efflux. Collagen (5 w g/ml) caused a 12.5-fold increase in TxB 2 generation, from a basal level of 7.69 - 1.28 ng/10 8 platelets to 96.34 - 13.37 ng/10 8 platelets ( P <0.001). Adrenaline (16 w M) increased TxB 2 production 3-fold from 2.44 - 0.61 to 8.02 - 1.08 ng/10 8 platelets ( P <0.01). Adrenaline-induced TxB 2 generation was inhibited 42.5 - 10.3% ( P <0.05) and 81.8 - 7.5% ( P <0.05) by 10 and 40 w M naftopidil, respectively. Collagen-stimulated TxB 2 generation was inhibited 59.5 - 9.2% ( P <0.01) by 40 w M naftopidil and 53.7 - 11.3% ( P <0.01) by 28 w M nifedipine. Doxazosin (7.5 and 30 w M) did not influence adrenaline- or collagen-induced TxB 2 synthesis. Collagen increased PDGF efflux from 1.17 - 0.39 to 4.25 - 0.51 ng/10 8 platelets ( P <0.01), whilst adrenaline raised concentrations from 1.08 - 0.19 to 5.37 + 1.02 ng/10 8 platelets ( P <0.01). Naftopidil had no effect on collagen-induced PDGF release. Adrenaline-stimulated PDGF efflux was, however, inhibited 82.9 - 13.7% ( P <0.001) and 125.7 - 16.3% ( P <0.001) by 10 and 40 w M naftopidil, respectively. Doxazosin (30 w M) inhibited adrenaline-induced PDGF release by 70.3 - 31.5% ( P <0.05), whilst nifedipine (28 w M) had no effect on collagen-stimulated release. We conclude that naftopidil, like nifedipine, may block stimulated TxB 2 generation via inhibition of phospholipase A 2 , the Ca 2+ -dependent, rate-limiting enzyme in thromboxane synthesis. Although adrenaline-induced PDGF release was inhibited by naftopidil and doxazosin, collagen-induced release was unaffected by either f 1 -adrenoreceptor blocker or nifedipine, indicating that platelet f -granular release is not dependent on Ca 2+ mobilisation or thromboxane generation. Thus, the effects of these drugs on PDGF release may be mediated through alternative cellular signalling mechanisms.

Mechanical strain stimulates a mitogenic response in coronary vascular smooth muscle cells via release of basic fibroblast growth factor

Mechanical strain has been shown to induce mitogenesis in a rat neonatal vascular smooth muscle (VSM) cell line in a response mediated predominantly by transcription, expression, and release of platelet-derived growth factor (PDGF). We examined the effect of cyclic mechanical strain and growth factor production on mitogenic response in ovine coronary artery smooth muscle cells. Vascular smooth muscle cells were cultured from explants of left anterior descending (LAD) coronary arteries from young sheep. Cells for experiments were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Tritiated thymidine incorporation was used as a measure of DNA synthesis. Cell membrane damage was assessed with differentially permeable nuclear staining dyes. We observed an increase in tritiated thymidine incorporation in response to strain with a temporal response identical to that observed in response to exogenous growth factors (PDGF-BB and basic fibroblast growth factor [bFGF]). Supernatant medium obtained from stretched cells induced a twofold increase in DNA synthesis in unstretched cells. The mitogenic response was abolished by monoclonal antibodies to bFGF, but not by antibodies to PDGF-AB. Studies of fluorescent dye exclusion indicated the stretching protocol caused no cell membrane damage. Thus, mechanical strain is an important stimulus for growth factor release in coronary VSM cells. The mitogenic response is mediated by release of bFGF.

Bimodal Concentration-Dependent Effect of Thrombin on Endothelial Cell Proliferation and Growth Factor Release in Culture

Background. The role of thrombin in the stimulation of endothelial cell (EC) proliferation is controversial. The aim of this study was to investigate if thrombin regulates cell proliferation and production of platelet-derived growth factor (PDGF), bovine fibroblast growth factor (bFGF), and transforming growth factor β1 (TGF-β1) by bovine aortic ECs.Methods. ECs, obtained from thoracic aortas of calves, were stimulated with thrombin at various concentrations (from 0.05 to 1.0 IU/ml) in serum free culture. Mitogenic activity of thrombin on ECs was determined by tritiated thymidine uptake. The release of PDGF, bFGF, and TGF-β1 was assessed by ELISA. PDGF release was confirmed by Western blot and bFGF and TGF-β1 mRNA expression was determined by polymerase chain reaction (PCR).Results. Thrombin at high concentrations did not cause any increase in EC proliferation after 72 h of culture and induced inhibition of EC proliferation after 96 h and 8 days of culture. It induced a decrease in PDGF release and an increase in TGF-β1 release. Thrombin at low concentrations induced a significant increase in EC proliferation at 72 h, 96 h, and 8 days of culture. It induced an increase in PDGF release and a decrease in TGF-β1 release. bFGF release was higher than control at all thrombin concentrations. These data were confirmed by Western blot and PCR studies.Conclusions. Thrombin regulates EC growth through the inhibition of EC proliferation at high concentrations and through the stimulation of EC proliferation at low physiological concentrations. EC proliferation is partially mediated by autocrine production of PDGF, bFGF, and TGF-β1.

Sphingosine-1-Phosphate and the Leading Edg-1 of Vascular Smooth Muscle Cells

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima in concert with their subsequent massive proliferation within the intima characterizes restenosis after angioplasty, occlusion of saphenous vein grafts, transplant organ failure, and the progression of atherosclerosis. The identification of molecules and mechanisms that regulate VSMC migration and proliferation is therefore of considerable clinical relevance. A key discovery that led Ross and Glomset1 to propose the “response to injury” hypothesis of atherosclerosis was the release of platelet-derived growth factor (PDGF), a potent chemoattractant and mitogen for VSMC upon platelet activation. Subsequent studies showed that PDGF is also produced by endothelial cells, macrophages, and SMCs themselves. A role for PDGF signaling in migration and proliferation of intima VSMCs and in the pathogenesis of plaque development and restenosis is now well established.2 Studies disrupting the PDGF-B gene or the PDGF-β receptor show that the PDGF-B pathway is required for normal blood vessel formation during embryonic development. Very recently, another class of mediator and another receptor essential for VSMC migration and vascular maturation has been described that involve the lipid sphingosine-1-phosphate (S1P) and its receptor Edg-1.3,4 S1P and the structurally related sister molecule lysophosphatidic acid (LPA) are potent bioactive lipids that have multiple biologic actions on vascular cells and blood cells.5 As is seen with PDGF, S1P is released in large quantities from activated platelets (through a mechanism, which has yet to be described).6 It is also constitutively produced and secreted by other blood cells (mononuclear cells, neutrophils, and red blood cells) albeit in small quantities.6,7 S1P binds with high affinity to the receptor Edg-1, which couples to the Gi protein pathway.8 This receptor, the first of S1P and LPA receptors characterized, was identified as a gene induced during human endothelial …