Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. We now report that numerous human tumor-derived cell lines simultaneously express the genes for platelet-derived growth factor (PDGF) A and B chains and the PDGF receptor (PDGF-R). Measurement of mRNA transcribed from these genes showed that among 16 malignant glioma cell lines tested, 15 expressed the PDGF A gene, 12 expressed the PDGF B gene, and 13 expressed the PDGF-R gene. Of three osteosarcoma lines, three expressed PDGF A, two expressed PDGF B, and three expressed PDGF-R. For eight malignant melanoma lines, seven expressed PDGF A, five expressed PDGF B, and three expressed PDGF-R genes. Thus, 13 of 16 malignant glioma, 3 of 3 osteosarcomas, and 3 of 8 malignant melanoma cell lines expressed the PDGF receptor gene and either or both PDGF genes. Five cell lines were tested for production of biologically active PDGF and PDGF receptor protein. Media conditioned by each of the five cell lines induced tyrosine phosphorylation of a protein identical in size to the PDGF receptor. These five cell lines also produced PDGF receptor protein as measured by Western blot analysis or metabolic labeling and immunoprecipitation using PDGF-R antibodies. The PDGF receptors of these cell lines were activated by human platelet PDGF or by recombinant AA or BB homodimers. Intracellular interaction of these receptors with the growth factor simultaneously produced may provide continuous stimulation to the proliferation of these cells.

Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) beta-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) alpha as the critical signaling component that regulates the sorting of the PDGF beta-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKCalpha, using myristoylated inhibitory peptides or by knockdown of PKCalpha with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF beta-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF beta-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF beta-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF beta-receptors on early endosomes is regulated by sequential activation of PKCalpha and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.

A novel, p125FAK homologue, CADTK, has been detected in neural, epithelial, or hematopoietic cells but not in fibroblasts. We now demonstrate CADTK expression in a mesenchymal cell, rat aortic smooth muscle cells (RSMC). Angiotensin II (Ang II) or platelet-derived growth factor (PDGF-BB and PDGF-AA) markedly stimulated CADTK tyrosine phosphorylation in RSMC but did not affect p125FAK phosphorylation. The PDGF-depedent CADTK tyrosine phosphorylation was slower and more prolonged than that of Ang II, correlating well with the differential effects of these agonists on cytosolic calcium ([Ca2+]i) signaling. An intracellular calcium chelator inhibited both the rapid and sustained activation of CADTK by Ang II and PDGF. Extracellular calcium chelation inhibited the PDGF-stimulated increase in CADTK tyrosine phosphorylation as well as the sustained (but not the early) activation by Ang II. In contrast, p125FAK tyrosine phosphorylation was maximal in quiescent, adherent RSMC and was not affected by incubation with EGTA. Depletion of protein kinase C activity partially inhibited both the Ang II- and PDGF-induced CADTK tyrosine phosphorylation. Additional results confirm a relation between CADTK and the cytoskeleton. First, the tyrosine phosphorylation of paxilin correlated with activation of CADTK; this increase was inhibited by EGTA. Second, cytochalasin D blocked the PDGF- or Ang II-stimulated tyrosine phosphorylation of CADTK, suggesting a role for the cytoskeleton in agonist-dependent CADTK activation. Third, immunofluorescence analysis of CADTK localization demonstrated actin-like cytoskeleton staining extending into focal contacts. These results suggest that in mesenchymal cells, CADTK is localized to and activated by an actin cytoskeleton-dependent mechanism; a mechanism that is regulated in a calcium and protein kinase C-dependent manner independently of p125FAK.

Migration of medial smooth muscle cells (SMC) into the intima is important in intimal thickening of atherosclerotic tissues. To study the functions of three isoforms of platelet-derived growth factor (PDGF) in atherosclerosis, we investigated their effects on SMC migration by Boyden's chamber method. Although PDGF-AB and PDGF-BB enhanced SMC migration dose-dependently, PDGF-AA did not enhance SMC migration, but instead inhibited SMC migration induced by PDGF-AB or PDGF-BB. PDGF-AA also inhibited SMC migration induced by two other migration factors, fibronectin and SMC-derived migration factor. PDGF-AA is considered to be coexpressed with transforming growth factor (TGF)-beta 1 in atherosclerotic tissues. Treatment of SMC with TGF-beta 1 reduced an autocrine migration activity from SMC. Studies using anti-PDGF antibody revealed that an increased secretion of PDGF-AA by TGF-beta 1 caused the reduced migration activity. cAMP increase by forskolin and dibutyryl cAMP suppressed SMC migration, whereas cAMP decrease by pertussis toxin had no effects on PDGF-AA-suppressed migration. In contrast, staurosporine, an inhibitor of protein kinase C, enhanced SMC migration and neutralized the inhibitory effect of PDGF-AA. These findings suggest that PDGF-AA regulates SMC migration in intimal thickening in atheroma formation and that protein kinase C may play an important role in the inhibitory mechanism of PDGF-AA.

Panax notoginseng saponins have recently been reported to suppress liver fibrosis. Since ginsenoside-Rg1 is the most abundant component of P.notoginseng saponins, we investigated the effect of ginsenoside-Rg1 on experimental liver fibrosis in rats. Histological analysis revealed that ginsenoside-Rg1 significantly improved the extent of liver fibrosis in rats induced by thioacetamide. Ginsenoside-Rg1 markedly suppressed the serum levels of fibrotic markers and hepatic hydroxyproline content in rats treated with thioacetamide. Ginsenoside-Rg1 also reduced the serum levels of alanine transaminase, aspartate transaminase and alkaline phosphatase. Finally, ginsenoside-Rg1 attenuated the levels of thiobarbituric acid reactive substances in livers of rats treated by thioacetamide. In cultured hepatic stellate cells, ginsenoside-Rg1 markedly inhibited cell proliferation, activation and formation of reactive oxygen species stimulated by platelet-derived growth factor-BB (PDGF-BB). Additionally, ginsenoside-Rg1 down-regulated the expression of PDGF receptor-beta by reducing the nuclear factor-kappaB activity, which was required for the gene expression. These results suggest that ginsenoside-Rg1, which exhibits its antioxidant and antifibrotic properties, may be of potential therapeutic value in protecting the liver fibrosis.

We demonstrate that hydrogel functionalization with DNA aptamers can be applied for developing a novel sustained-release system.

The early phase of the biphasic ventilatory response to hypoxia in mammals is critically dependent on NMDA glutamate receptor activation within the nucleus of the solitary tract. However, the mechanisms underlying the subsequent development of the typical ventilatory roll-off are unclear and could underlie important roles in the functional and molecular adaptation to oxygen deprivation. Because the growth factor platelet-derived growth factor (PDGF)-BB can modulate the open channel probability of NMDA receptors by activating PDGF-beta receptors, its contribution to hypoxic ventilatory roll-off was examined. Administration of PDGF-BB, but not PDGF-AA, in the nucleus of the solitary tract was associated with significant attenuations of the early hypoxic ventilatory response in conscious rats. Furthermore, marked reductions in the magnitude of hypoxic ventilatory roll-off occurred in mice heterozygous for a mutation in the PDGF-beta receptor. Administration of a PDGF-beta receptor antagonist to wild-type littermates elicited similar declines in hypoxic ventilatory roll-off. The relative abundance of PDGF-beta receptors was confirmed in the nucleus of the solitary tract and other nuclei implicated in the hypoxic ventilatory response. In nucleus of the solitary tract lysates, PDGF-beta receptor tyrosine phosphorylation was temporally correlated with hypoxic ventilatory roll-off formation. Increased PDGF-B chain mRNA expression was induced by hypoxia in the nucleus of the solitary tract, and PDGF-B chain immunoreactivity colocalized with approximately 40% of nucleus of the solitary tract neurons, demonstrating hypoxia-induced c-Fos enhancements. Thus, PDGF-BB release and PDGF-beta receptor activation in the nucleus of the solitary tract are critical components of hypoxic ventilatory roll-off and may have important functional implications in processes underlying survival and acclimatization to hypoxic environments.

OBJECTIVE

To investigate and compare clinically relevant Platelet-rich plasma (PRP) activation methods.

METHODS

Experimental.

METHODS

PRP was prepared from 6 equine subjects. Activation of the PRP was performed by 4 methods (autologous thrombin, bovine thrombin, calcium chloride (CaCl(2) ), or freeze-thaw). The resultant PDGF-BB (where PDGF is platelet-derived growth factor) and TGFβ1 (where TGFβ is transforming growth factor beta) levels in PRP releasates were quantified by Enzyme-linked immunosorbent assay (ELISA) and compared. Growth factor contents were also compared between platelet-rich clots produced by thrombin or CaCl(2) . The composition and function of equine autologous thrombin were characterized by Western blot analysis and platelet aggregometry.

RESULTS

CaCl(2) (23 mM) activation of PRP yielded significantly greater PDGF release than did any other method. TGFβ release was comparable after PRP activation by CaCl(2) , bovine thrombin, and freeze thaw. Autologous thrombin was significantly less effective than all other activation methods in eliciting platelet growth factor release and induced significantly less platelet aggregation than bovine thrombin at 5 U/mL. Clots retained substantial concentrations of growth factor, and the amount in the releasate versus the clot differed between activation methods.

CONCLUSIONS

PRP activation methods differ in terms of growth factor output as well as logistical considerations. Autologous thrombin is not recommended for PRP activation. CaCl(2) (23 mM) is an effective and inexpensive method of PRP activation. The PRP releasate derived from CaCl(2) activation contains 80% of the total PDGF content and is easily produced, making it a convenient product for clinical use.

OBJECTIVE

Pulmonary hypertension (PH) is a devastating condition for which no disease-modifying therapies exist. PH is recognized as proliferative disease of the pulmonary artery (PA). In the experimental newborn calf model of hypoxia-induced PH, adventitial fibroblasts in the PA wall exhibit a heightened replication index. Because elevated platelet-derived growth factor β receptor (PDGFβ-R) signalling is associated with PH, we tested the hypothesis that the activation of PDGFβ-R contributes to fibroblast proliferation and adventitial remodelling in PH.

RESULTS

Newborn calves were exposed to either ambient air (P(B) = 640 mmHg) (Neo-C) or high altitude (P(B) = 445 mm Hg) (Neo-PH) for 2 weeks. PDGFβ-R phosphorylation was markedly elevated in PA adventitia of Neo-PH calves as well as in cultured PA fibroblasts isolated from Neo-PH animals. PDGFβ-R activation with PDGF-BB stimulated higher replication in Neo-PH cells compared with that of control fibroblasts. PDGF-BB-induced proliferation was dependent on reactive oxygen species generation and extracellular signal-regulated kinase1/2 activation in both cell populations; however, only Neo-PH cell division via PDGFβ-R activation displayed a unique dependence on c-Jun N-terminal kinase1 (JNK1) stimulation as the blockade of JNK1 with SP600125, a pharmacological antagonist of the JNK pathway, and JNK1-targeted siRNA selectively blunted Neo-PH cell proliferation.

CONCLUSIONS

Our data strongly suggest that hypoxia-induced modified cells engage the PDGFβ-R-JNK1 axis to confer distinctively heightened proliferation and adventitial remodelling in PH.

OBJECTIVE

Green-tea polyphenols are known to have anti-fibrotic properties of the skin and the artery. The proliferation of hepatic stellate cells (HSC) is closely related to the progression of liver fibrosis in chronic liver diseases. We investigated the inhibitory effect of epigallocatechin-3-gallate (EGCG), the major potential inhibitory component of green-tea polyphenols, on the proliferation of HSC. The aim of this study was to clarify the molecular mechanisms of EGCG inhibition of HSC proliferation.

METHODS

A cultured human hepatic stellate cell line LI90 was used for this study. The cells were stimulated by platelet-derived growth factor (PDGF)-BB in the presence or absence of EGCG. Proliferation was determined by bromodeoxy-uridine incorporation. The mRNA expressions of collagen alpha1(I) and (IV) were evaluated by a quantitative reverse transcription-polymerase chain reaction. PDGF receptor tyrosine phosphorylation was detected using anti-phosphotyrosine antibody. PDGF receptor radioligand binding assay was performed by [125I]-PDGF-BB.

RESULTS

EGCG inhibited the PDGF-BB-induced cell-proliferation and collagen alpha1(I) and (IV) mRNA expressions. EGCG reduced the autophosphorylation of the PDGF receptor. EGCG blocked PDGF-BB binding to its receptor in a non-competitive manner.

CONCLUSIONS

EGCG has an inhibitory effect on PDGF-induced proliferation of HSC, and the blocking of PDGF-BB binding to its receptor may be the mechanism behind this effect.

Pericytes and vascular smooth muscle cells (VSMCs), which are recruited to developing blood vessels by platelet-derived growth factor BB, support endothelial cell survival and vascular stability. Here, we report that imatinib, a tyrosine kinase inhibitor of platelet-derived growth factor receptor β (PDGFRβ), impaired growth of lymphoma in both human xenograft and murine allograft models. Lymphoma cells themselves neither expressed PDGFRβ nor were growth inhibited by imatinib. Tumor growth inhibition was associated with decreased microvascular density and increased vascular leakage. In vivo, imatinib induced apoptosis of tumor-associated PDGFRβ(+) pericytes and loss of perivascular integrity. In vitro, imatinib inhibited PDGFRβ(+) VSMC proliferation and PDGF-BB signaling, whereas small interfering RNA knockdown of PDGFRβ in pericytes protected them against imatinib-mediated growth inhibition. Fluorescence-activated cell sorter analysis of tumor tissue revealed depletion of pericytes, endothelial cells, and their progenitors following imatinib treatment. Compared with imatinib, treatment with an anti-PDGFRβ monoclonal antibody partially inhibited lymphoma growth. Last, microarray analysis (Gene Expression Omnibus database accession number GSE30752) of PDGFRβ(+) VSMCs following imatinib treatment showed down-regulation of genes implicated in vascular cell proliferation, survival, and assembly, including those representing multiple pathways downstream of PDGFRβ. Taken together, these data indicate that PDGFRβ(+) pericytes may represent a novel, nonendothelial, antiangiogenic target for lymphoma therapy.

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum-starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF-AA, venous SMCs showed no proliferative response to PDGF-AA, but instead demonstrated a significantly greater proliferative response to PDGF-BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5-fold higher (P < 0.05) density of PDGF receptor-α (PDGF-Rα) and a 6.6-fold lower (P < 0.05) density of PDGF-Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF-AA in arterial SMCs was a marked PDGF-Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c-Jun NH2-terminal kinase (JNK), and a significant reduction in expression of the cell-cycle inhibitor p27(kip1). This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF-Rα was detected after venous SMC exposure to PDGF-AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF-BB stimulation of venous SMC resulted in PDGF-Rβ activation as well as transactivation of epidermal growth factor receptor (EGF-R); transactivation of EGF-R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins.

OBJECTIVE

Excessive orbital fibroblast proliferation and hyaluronan production are characteristic of Graves' ophthalmopathy (GO) and are driven by local mediators. Imatinib mesylate and AMN107 are tyrosine kinase inhibitors that inhibit fibroblast proliferation and collagen production in lungs and skin. This study was conducted to determine whether imatinib mesylate and AMN107 inhibit orbital fibroblast proliferation and hyaluronan production induced by PDGF-BB and TGF-beta(1) and whether expression of the genes PDGF-B and TGF-B(1) (growth factors suggested to play a role in GO) are increased in GO orbital tissues.

METHODS

PDGF-B and TGF-B(1) mRNA levels were determined in orbital tissues of 13 patients with GO and 5 control patients. Orbital fibroblasts were cultured from eight patients with GO and three control patients and the effect of imatinib mesylate and AMN107 on PDGF-BB and TGF-beta(1)-induced orbital fibroblast proliferation, signaling cascades, hyaluronan synthase (HAS) gene expression and hyaluronan production were determined.

RESULTS

PDGF-B and TGF-B(1) mRNA levels were significantly increased in GO orbital tissues. Imatinib mesylate and AMN107 inhibited PDGF-BB-induced orbital fibroblast proliferation, HAS induction and hyaluronan production by blocking PDGF-receptor phosphorylation. TGF-beta(1) induced HAS expression and hyaluronan production. This induction was not inhibited by imatinib mesylate or AMN107, due to the inability of TGF-beta(1) to activate c-Abl kinase activity in orbital fibroblasts.

CONCLUSIONS

Imatinib mesylate and AMN107 inhibit orbital fibroblast proliferation and hyaluronan production induced by PDGF-BB; a factor highly expressed in orbital tissue from patients with GO. The drugs, however, had no effect on TGF-beta(1)-induced HAS expression and hyaluronan production. Nevertheless, imatinib mesylate and AMN107 should be considered as treatment candidates for GO.

BACKGROUND

Platelet-rich plasma (PRP) has more concentrated platelets than normal plasma (approximately 150-400×10(3) cell/dL). Platelets excrete several growth factors and cytokines that are associated with the healing and regeneration process. However, even though PRP is widely used, the mechanism or actual effect is presently unclear. Therefore, this study was performed to investigate the levels of growth factors and platelet concentration rate.

METHODS

Autologous blood for preparing PRP was obtained from healthy subjects aged 25 to 35 years. The samples were divided into 4 experimental groups (inactivated whole blood, inactivated PRP, activated whole blood with thrombin and calcium chloride, and activated PRP). The platelet counts in the blood were analyzed and the growth factors were quantitatively measured. A statistical analysis was performed by using Dunn's multiple comparison test.

RESULTS

In the blood cell analysis, the platelet count of the PRP group was approximately 4.25 times higher than that of the whole blood group. In the quantitative analysis of growth factors, the platelet-derived growth factor (PDGF)-AB, PDGF-BB, and transforming growth factor-β of the inactivated and activated PRP groups were higher than those of the inactivated and activated whole blood groups (P<0.05).

CONCLUSIONS

In this study, the platelet count and the levels of PDGF-AB and PDGF-BB in the PRP were determined. Further, more research is required on the bioactivity level of the growth factors secreted during the process of PRP preparation and the potency of growth factors that can be exerted physiologically in vivo.

Matrix metalloproteinase- (MMP-) 9 is one of the main metalloproteinases reported to be involved in extracellular matrix degradation and recently also in triggering of angiogenic switch in the course of inflammatory bowel diseases (IBD). The goal of our studies was to estimate in one experimental setting the levels of MMP-9 in sera of Crohn's Disease (CD) and ulcerative colitis (UC) patients and to evaluate its possible diagnostic potential in comparison with other biochemical markers and selected proinflammatory and angiogenic factors. The study group included 176 subjects (CD = 64, UC = 85, control = 27). Concentrations of serum MMP-9 were significantly higher in active than inactive forms of IBD, being higher in active UC than in active CD. Both in the case of CD and UC serum MMP-9 positively correlated with disease activity, IL-6 levels, platelet and leukocyte count, midkine, and PDGF-BB, as well as in UC with ESR and in CD with CRP, IL-1, and VEGF-A. Diagnostic accuracy of MMP-9 in distinguishing active UC from active CD was 66%, and displayed higher specificity than CRP (79.0% versus 61.6%, resp.). Evaluation of serum MMP-9 concentrations could aid in differentiation of active UC from active CD. MMP-9 correlated better with inflammatory and angiogenic parameters in CD than in UC.

OBJECTIVE

Locally-active growth factors have been implicated in the pathogenesis of many diseases in which organ fibrosis is a characteristic feature. In the setting of chronic kidney disease (CKD), two such pro-fibrotic factors, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) have emerged as lead potential targets for intervention. Given the incomplete organ protection afforded by blocking the actions of TGF-β or PDGF individually, we sought to determine whether an agent that inhibited the actions of both may have broader effects in ameliorating the key structural and functional abnormalities of CKD.

METHODS

Accordingly, we studied the effects of a recently described, small molecule anti-fibrotic drug, 3-methoxy-4-propargyloxycinnamoyl anthranilate (FT011, Fibrotech Therapeutics, Australia), which should have these effects.

RESULTS

In the in vitro setting, FT011 inhibited both TGF-β1 and PDGF-BB induced collagen production as well as PDGF-BB-mediated mesangial proliferation. Consistent with these in vitro actions, when studied in a robust model of non-diabetic kidney disease, the 5/6 nephrectomised rat, FT011 attenuated the decline in GFR, proteinuria and glomerulosclerosis (p<0.05 for all). Similarly, in the streptozotocin-diabetic Ren-2 rat, a model of advanced diabetic nephropathy, FT011 reduced albuminuria, glomerulosclerosis and tubulointerstitial fibrosis.

CONCLUSIONS

Together these studies suggest that broadly antagonising growth factor actions, including those of TGF-β1 and PDGF-BB, has the potential to protect the kidney from progressive injury in both the diabetic and non-diabetic settings.

Biomaterial designs are increasingly incorporating multiple instructive signals to induce a desired cell response. However, many approaches do not allow orthogonal manipulation of immobilized growth factor signals and matrix stiffness. Further, few methods support patterning of biomolecular signals across a biomaterial in a spatially-selective manner. Here, we report a sequential approach employing carbodiimide crosslinking and benzophenone photoimmobilization chemistries to orthogonally modify the stiffness and immobilized growth factor content of a model collagen-GAG (CG) biomaterial. We subsequently examined the singular and combined effects of bone morphogenetic protein (BMP-2), platelet derived growth factor (PDGF-BB), and CG membrane stiffness on the bioactivity and osteogenic/adipogenic lineage-specific gene expression of adipose derived stem cells, an increasingly popular cell source for regenerative medicine studies. We found that the stiffest substrates direct osteogenic lineage commitment of ASCs regardless of the presence or absence of growth factors, while softer substrates require biochemical cues to direct cell fate. We subsequently describe the use of this approach to create overlapping patterns of growth factors across a single substrate. These results highlight the need for versatile approaches to selectively manipulate the biomaterial microenvironment to identify synergies between biochemical and mechanical cues for a range of regenerative medicine applications.

OBJECTIVE

Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease.

METHODS

A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied.

RESULTS

Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation.

CONCLUSIONS

Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.

OBJECTIVE

Secondary failure due to late restenosis continues to occur in 30-50% of individuals after PTCA. beta-Blockers play an important role in the treatment of CAD. The aim of this study was to investigate the effects of the new beta-blocker nebivolol on cell proliferation of human coronary smooth muscle cells (haCSMCs) and endothelial cells (haECs) in comparison to traditional beta-blockers.

METHODS

The effect of nebivolol and other beta-blockers on proliferation of HaECs and HaCSMCs was analyzed by bromodeoxyuridine incorporation. Apoptosis was measured by determination of hypodiploid DNA in both cell types. Additionally, in HaECs NO formation, endothelin-1 transcription and secretion were determined.

RESULTS

Incubation for 1, 2, 4, 7 or 14 days resulted in a concentration- and time-dependent reduction of proliferation up to 80% in HaECs and HaCSMCs. beta-Blockers such as propranolol, metoprolol or bisoprolol did not exert this effect. Nebivolol inhibited accelerated haCSMC proliferation even in the presence of growth factors such as TGFbeta(1) and PDGF-BB. Nebivolol concentration-dependently induced a moderate apoptosis (10(-5) mol/l: 23%) and a decrease of haCSMCs in the S-phase by 66%. HaECs showed comparable results. During nebivolol incubation NO formation of HaCEs increased, while endothelin-1 transcription and secretion were suppressed.

CONCLUSIONS

Whereas classical beta-blockers do not affect cell growth, only nebivolol inhibits haCSMC or haEC proliferation and induces a moderate rate of apoptosis. Furthermore, in HaCEs NO formation increases and endothelin-1 secretion decreases suggesting that nebivolol may represent a beta-blocker with great promises in CAD therapy.

OBJECTIVE

The therapeutic benefits of platelet-rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation.

METHODS

The levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium- and thrombin-activated PRP samples from five donors were quantified by enzyme-linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody-coated beads on HUVEC proliferation was also tested.

RESULTS

Average concentrations of VEGF, PDGF-BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody-coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose-dependent manner.

CONCLUSIONS

This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.

OBJECTIVE

Migration of smooth muscle cells (SMCs) from the media to the intima of arteries is involved in intimal thickening. The platelet-derived growth factor (PDGF) BB is recognized as a major migratory factor for arterial SMCs both in vitro and during neointima formation. Since PDGF acts in synergy with the matrix protein osteopontin (OPN) and also induces its expression, the present study was conceived to explore the role of the OPN produced in an autocrine fashion by PDGF-stimulated SMCs in the migration process and to define regulatory mechanisms of OPN expression.

RESULTS

PDGF stimulation of quiescent rat aortic SMCs induced their migration (transfilter assays) and the increase of OPN expression (mRNA and protein assays). Blockade of either OPN expression by a specific short interference RNA (siRNA) or of its function by a blocking antibody decreased the PDGF-stimulated migration by about 70%, demonstrating that autocrine production and excretion of OPN are integral to the PDGF-induced SMC migration. In parallel, SMC stimulation by PDGF also activated the transcription factor CREB essentially through mitogen-activated protein kinase (MAPK) 1/2 and protein kinase A (PKA) pathways. Inhibition of either CREB expression (via siRNA) or function (via dominant-negative CREB) decreased both PDGF-induced SMC migration and OPN expression. SMC transfection with OPN promoter reporter constructs demonstrated that PDGF-induced OPN transcription is mediated by CREB binding to two functional sites of the OPN promoter: a CRE site located at -1403 and an AP-1 site located at -76.

CONCLUSIONS

The present study demonstrates that the autocrine expression of OPN plays a major role in PDGF-induced SMC migration. It further shows that the transcription factor CREB, activated in PDGF-stimulated SMCs, plays a key role in PDGF-induced SMC migration, probably by regulating OPN expression.

During wound healing, biologically active molecules are released from platelets. The rationale of using platelet-rich plasma (PRP) relies on the concentration of bioactive molecules and subsequent delivery to healing sites. These bioactive molecules have been seldom simultaneously quantified within the same PRP preparation. In the present study, the flexible Bio-Plex system was employed to assess the concentration of a large range of cytokines, chemokines, and growth factors in 16 healthy volunteers so as to determine whether significant baseline differences may be found. Besides IL-1b, IL-1ra, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17, INF-γ, TNF-α, MCP-1, MIP-1a, RANTES, bFGF, PDGF, and VEGF that were already quantified elsewhere, the authors reported also on the presence of IL-2, IL-5, IL-7, IL-9, IL-10, IL-15 G-CSF, GM-CSF, Eotaxin, CXCL10 chemokine (IP-10), and MIP 1b. Among the most interesting results, it is convenient to mention the high concentrations of the HIV-suppressive and inflammatory cytokine RANTES and a statistically significant difference between males and females in the content of PDGF-BB. These data are consistent with previous reports pointing out that gender, diet, and test system affect the results of platelet function in healthy subjects, but seem contradictory when compared to other quantification assays in serum and plasma. The inconsistencies affecting the experimental results found in literature, along with the variability found in the content of bioactive molecules, urge further research, hopefully in form of randomized controlled clinical trials, in order to find definitive evidence of the efficacy of PRP treatment in various pathologic and regenerative conditions.

The periosteum plays a pivotal role during bone development and repair contributing to bone vascularization and osteoprogenitor cells source. We propose a periosteal substitute engineered using a platelet-rich plasma (PRP) membrane incorporating autologous bone marrow-derived mesenchymal stem cells (PRP/BMSC gel membrane) to be wrapped around an osteoconductive scaffold for regeneration of compromised bone defects. The PRP/BMSC gel membrane was optimized using different compositions for optimal release of vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB). Survival and proliferation of cells in the PRP gel membrane with time were confirmed in addition to their osteogenic capacity. Furthermore, to evaluate the possible effects of the PRP/BMSC gel membrane on surrounding progenitor cells in the injury area, we found that the PRP gel membrane products could significantly induce the migration of human endothelial cells in vitro, and increased the expression of bone morphogenetic protein 2 in cultured BMSC. These cells also secreted significant amounts of soluble proangiogenic factors, such as PDGF-BB, VEGF, and interleukin-8 (IL-8). Finally, the functionality of the PRP/BMSC gel membrane periosteal substitute for bone regeneration was tested in vivo both in an ectopic mouse model as well as in a rabbit segmental bone defect model providing evidence of its capacity to biomimic a periosteal response enhancing bone regeneration.

alpha(2)-Macroglobulin (alpha(2)M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-beta (NGF-beta) to a linear sequence in the 180-kDa human alpha(2)M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-beta in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-beta binding to immobilized FP3 were 300 +/- 40 and 180 +/- 30 nM, respectively; these values were comparable with those determined using methylamine-modified alpha(2)M, suggesting that higher-order alpha(2)M structure is not necessary for PDGF-BB and NGF-beta binding. PDGF-BB and NGF-beta blocked the binding of transforming growth factor-beta1 (TGF-beta1) to FP3. Furthermore, murinoglobulin, which is the only known member of the alpha-macroglobulin family that does not bind TGF-beta, also failed to bind PDGF-BB and NGF-beta. These results support the hypothesis that either a single linear sequence in human alpha(2)M or overlapping sequences are responsible for the binding of TGF-beta, PDGF-BB, and NGF-beta, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF beta receptor was fused to the IgG(1) Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.