OBJECTIVE

Systemic sclerosis (scleroderma, SSc) frequently affects the lungs, and interstitial pulmonary fibrosis is one of its major complications. The pathophysiology of SSc lung disease s poorly understood, but recent studies document an inflammatory process resembling that of idiopathic pulmonary fibrosis with increased numbers of activated alveolar macrophages and granulocytes in bronchoalveolar lavage (BAL) fluid). We determined levels of 2 potentially important mediators of fibroproliferative repair in BAL fluid from patients with SSc.

METHODS

Using Western blot and ELISA techniques we measured levels of platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta in BAL fluid from patients with SSc and healthy controls. The mitogenic effect of these cytokines on SSc lung myofibroblasts was determined by [3H]thymidine incorporation.

RESULTS

SSc BAL fluid contains significantly elevated levels of PDGF-AA and PDGF-BB. Where TGF-beta 1 was significantly elevated in SSc lavage fluid, the amount of TGF-beta 2 was significantly less than that observed in normal lavage fluid. Myofibroblasts cultured from SSc lavage fluid exhibited enhanced [3H]thymidine incorporation upon exposure to the growth factors present in SSc BAL fluid: PDGF and TGF-beta 1. SSc lung myofibroblasts pretreated with TGF-beta 1 exhibited an enhanced mitogenic effect upon stimulation by PDGF, due in part to the induction of the PDGF alpha receptor.

CONCLUSIONS

Our studies support a role for PDGF and TGF-beta 1 in the pathogenesis of SSc lung disease.

Platelet-derived growth factor receptor (PDGFR)alpha expression was found in ovarian cancer cells and tumors by microarray hybridization. This led us to test whether ovarian cancers also produce ligands for this receptor, as this would demonstrate that such malignancies support their own growth and spread through autocrine activation. We assayed the expression of ligands for the PDGFR in ovarian tumors, cell lines and peritoneal fluid using RT-PCR, immunohistochemistry (IHC) and ELISA. We detected strong mRNA expression for the PDGFRalpha ligands in most ovarian tumors. Receptor and ligand expressions (PDGFRalpha and PDGF AB) were also detected by IHC in, respectively, 34 and 32 of 47 ovarian tumors. The stainings for PDGFRalpha and PDGF AB were strongly correlated (P-value=0.014), suggesting that an autocrine loop is functional in ovarian cancer. PDGF AA and BB were quantified in peritoneal fluid by ELISA. Both ligands are secreted at higher levels in ovarian cancer ascites specimens (n=54) than in fluid from nonmalignant disorders (n=8). PDGF was detected in media conditioned by ovarian cancer cells. Such conditioned media induced activation of the PDGFR, Akt and MAPK and stimulated cell proliferation. A neutralizing PDGF antibody blocked these effects. Specific PDGFR inhibition by siRNA or a neutralizing antibody to the receptor inhibited PDGF-stimulated receptor activation and cell proliferation, suggesting that receptor targeting has a role in ovarian cancer treatment.

OBJECTIVE

Circulating progenitors and stem cells have been reported to contribute to angiogenesis and arterial repair after injury. In the present study, we investigated whether the arterial wall could host permanently residing progenitor cells under physiological context.

RESULTS

Using the Hoechst-based flow cytometry method, we identified and isolated progenitor cells termed side population (SP) cells at a prevalence of 6.0+/-0.8% in the tunica media of adult mice aortas. Arterial SP cells expressed the ATP-binding cassette transporter subfamily G member 2, frequently present on SP cell surface, and displayed a Sca-1+ c-kit(-/low) Lin- CD34(-/low) profile. They did not form myeloid or lymphoid hematopoietic colonies after plating in methylcellulose-based medium. Importantly, cultured SP cells were able to acquire the phenotype of endothelial cells (CD31, VE-cadherin, and von Willebrand factor expression) or of smooth muscle cells (alpha-smooth muscle actin, calponin, and smooth muscle myosin heavy chain expression), in presence of either vascular endothelial growth factor or transforming growth factor (TGF)-beta1/PDGF-BB, respectively. Moreover, they generated vascular-like branching structures, composed of both VE-cadherin+ cells and alpha-smooth muscle actin+ cells on Matrigel.

CONCLUSIONS

In this study, we provide the first evidence to our knowledge that in the adult mice, the normal arterial wall harbors SP cells with vascular progenitor properties.

MicroRNA (miRNA) are small non-coding RNA molecules that posttranscriptionally effect mRNA stability and translation by targeting the 3'-untranslated region (3'-UTR) of various transcripts. Thus, dysregulation of miRNA affects a wide range of cellular processes such as cell proliferation and differentiation involved in organ remodeling processes. Divergent miRNA patterns were observed during chronic liver diseases of various etiologies. Chronic liver diseases result in uncontrolled scar formation ending up in liver fibrosis or even cirrhosis. Since it has been shown that miR-29 dysregulation is involved in synthesis of extracellular matrix proteins, miR-29 is of special interest. The importance of miR-29 in hepatic collagen homeostasis is underlined by in vivo data showing that experimental severe fibrosis is associated with a prominent miR-29 decrease. The loss of miR-29 is due to the response of hepatic stellate cells to exposure to the profibrogenic mediators TGF-β and PDGF-BB. Several putative binding sites for the Smad proteins and the Ap1 complex are located in the miR-29 promoter, which are suggested to mediate miR-29 decrease in fibrosis. Other miRNA are highly increased after profibrogenic stimulation, such as miR-21. miR-21 is transcriptionally upregulated in response to Smad-3 rather than Smad-2 activation after TGF-β stimulation. In addition, TGF-β promotes miR-21 expression by formation of a microprocessor complex containing Smad proteins. Elevated miR-21 may then act as a profibrogenic miRNA by its repression of the TGF-β inhibitory Smad-7 protein.

The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet-derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet-derived growth factor AA, -BB, and heterodimeric PDGF-AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF-AA, rhPDGF-BB, and rhPDGF-AB. The effect was highest in MPCs and for rhPDGF-BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP-2 as well as rxBMP-4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF-beta1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF-AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone.

Mesangial cell (MC) proliferation and extracellular matrix expansion are involved in the pathogenesis of glomerulosclerosis and renal failure. In vitro, PDGF and basic fibroblast growth factor (bFGF) regulate MC proliferation and/or matrix production. To elucidate the role of PDGF and bFGF in vivo, equimolar concentrations of recombinant PDGF-BB or bFGF or vehicle were infused intravenously into rats over a 7-d period. Rats were either nonmanipulated ("normals") or had received a subnephritogenic dose of anti-MC antibody ("anti-Thy 1.1 rats") before the infusion period. Glomerular cell proliferation (anti-proliferating cell nuclear antigen immunostaining) on days 2, 4, and 7 was unchanged in vehicle-infused normals or anti-Thy 1.1 rats. PDGF infusion increased glomerular cell proliferation 32-fold in anti-Thy 1.1 rats and an 11-fold in normals on day 2. bFGF increased glomerular cell proliferation fourfold in anti-Thy 1.1 rats but was ineffective in normals. Induction of cell proliferation in all kidneys was limited to the glomerulus. The majority of proliferating cells were identified as MC by double immunolabeling. No significant proteinuria, glomerular leukocyte, or platelet influx developed in any group. Glomerular matrix expansion with increased deposition of type IV collagen, laminin, and fibronectin, as well as upregulated laminin and collagen IV mRNA expression was confined to PDGF-infused anti-Thy 1.1 rats. These results show that PDGF and, to a lesser degree, bFGF are selective MC mitogens in vivo and that previous subclinical injury can enhance this MC response. The data thereby support a role of these cytokines in the pathogenesis of glomerulosclerosis.

Activated pancreatic stellate cells (PSCs) play an important role in pancreatic fibrosis and inflammation, where oxidative stress is implicated in the pathogenesis. NADPH oxidase might be a source of reactive oxygen species (ROS) in the injured pancreas. This study aimed to clarify the expression and regulation of cell functions by NADPH oxidase in PSCs. PSCs were isolated from rat and human pancreas tissues. Expression of NADPH oxidase was assessed by reverse transcription-PCR and immunostaining. Intracellular ROS production was assessed using 2',7'-dichlorofluorescin diacetate. The effects of diphenylene iodonium (DPI) and apocynin, inhibitors of NADPH oxidase, on key parameters of PSC activation were evaluated in vitro. In vivo, DPI (at 1 mg.kg body wt(-1).day(-1)) was administered in drinking water to 10-wk-old male Wistar Bonn/Kobori rats for 10 wk and to rats with chronic pancreatitis induced by dibutyltin dichloride (DBTC). PSCs expressed key components of NADPH oxidase (p22(phox), p47(phox), NOX1, gp91(phox)/NOX2, NOX4, and NOX activator 1). PDGF-BB, IL-1beta, and angiotensin II induced ROS production, which was abolished by DPI and apocynin. DPI inhibited PDGF-induced proliferation, IL-1beta-induced chemokine production, and expression of alpha-smooth muscle actin and collagen. DPI inhibited transformation of freshly isolated cells to a myofibroblast-like phenotype. In addition, DPI inhibited the development of pancreatic fibrosis in Wistar Bonn/Kobori rats and in rats with DBTC-induced chronic pancreatitis. In conclusion, PSCs express NADPH oxidase to generate ROS, which mediates key cell functions and activation of PSCs. NADPH oxidase might be a potential target for the treatment of pancreatic fibrosis.

Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.

Increased matrix metalloproteinase-9 (MMP-9) expression is associated with intimal hyperplasia in saphenous vein (SV) bypass grafts. Recent evidence suggests that HMG-CoA reductase inhibitors (statins) can prevent the progression of vein graft failure. Here we investigated whether statins inhibited MMP-9 secretion from cultured human SV smooth muscle cells (SMC) and examined the underlying mechanisms. SV-SMC from different patients were exposed to phorbol ester (TPA) or PDGF-BB plus interleukin-1alpha (IL-1). MMP-9 secretion and mRNA expression were analyzed using gelatin zymography and RT-PCR, respectively. Specific signal transduction pathways were investigated by immunoblotting and pharmacological inhibition. Simvastatin reduced TPA- and PDGF/IL-1-induced MMP-9 secretion and mRNA levels, effects reversed by geranylgeranyl pyrophosphate and mimicked by inhibiting Rho geranylgeranylation or Rho-kinase (ROCK). MMP-9 secretion induced by PDGF/IL-1 was mediated via the ERK, p38 MAPK, and NFkappaB pathways, whereas that induced by TPA was mediated specifically via the ERK pathway. Simvastatin failed to inhibit activation of these signaling pathways. Moreover, simvastatin did not affect MMP-9 mRNA stability. Together these data suggest that simvastatin reduces MMP-9 secretion from human SV-SMC by inhibiting the RhoA/ROCK pathway and decreasing MMP-9 mRNA levels independently of effects on signaling pathways required for MMP-9 gene expression.

OBJECTIVE

Periostin is dramatically upregulated in rat carotid arteries after balloon injury. The objective of the present study was to understand mechanisms underlying periostin upregulation in balloon-injured rat carotid arteries and in cultured vascular smooth muscle cells (VSMCs).

RESULTS

Periostin protein was strongly expressed at 3 days (in the medial SMCs) and 7 days (in the neointima) after injury. It was also abundantly expressed in the neointima in the late phase (at 14 and 28 days) after injury. Periostin upregulation was mediated through PI-3-kinase-dependent signaling pathway. In vivo, wortmannin, a PI-3-kinase inhibitor, inhibited balloon injury-induced Akt phosphorylation and periostin mRNA expression. In vitro, periostin mRNA expression in cultured VSMCs was stimulated by growth factors (transforming growth factor-beta1 (TGF-beta1), fibroblast growth factors (FGFs), PDGF-BB, and angiotensin II). This stimulatory effect was inhibited by the PI-3-kinase inhibitor LY294002. Further, periostin protein was mostly located in the cytoplasma of VSMCs in culture and abundantly secreted into the culture medium (CM) after stimulation with FGF-2, which significantly promoted VSMC migration in vitro. Immunodepletion of periostin from the VSMC-CM or blockade of periostin function with an anti-periostin antibody significantly reduced VSMC migration.

CONCLUSIONS

Upregulation of periostin expression in rat carotid arteries following balloon injury and in cultured VSMCs after stimulation by growth factors is mediated through PI-3-kinase-dependent signaling pathway. Periostin protein secreted by VSMCs plays a significant role in regulating VSMC migration in vitro.

OBJECTIVE

The use of platelet concentrates has gained increasing awareness in recent years for regenerative procedures in modern dentistry. The aim of the present study was to compare growth factor release over time from platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and a modernized protocol for PRF, advanced-PRF (A-PRF).

METHODS

Eighteen blood samples were collected from six donors (3 samples each for PRP, PRF, and A-PRF). Following preparation, samples were incubated in a plate shaker and assessed for growth factor release at 15 min, 60 min, 8 h, 1 day, 3 days, and 10 days. Thereafter, growth factor release of PDGF-AA, PDGF-AB, PDGF-BB, TGFB1, VEGF, EGF, and IGF was quantified using ELISA.

RESULTS

The highest reported growth factor released from platelet concentrates was PDGF-AA followed by PDGF-BB, TGFB1, VEGF, and PDGF-AB. In general, following 15-60 min incubation, PRP released significantly higher growth factors when compared to PRF and A-PRF. At later time points up to 10 days, it was routinely found that A-PRF released the highest total growth factors. Furthermore, A-PRF released significantly higher total protein accumulated over a 10-day period when compared to PRP or PRF.

CONCLUSIONS

The results from the present study indicate that the various platelet concentrates have quite different release kinetics. The advantage of PRP is the release of significantly higher proteins at earlier time points whereas PRF displayed a continual and steady release of growth factors over a 10-day period. Furthermore, in general, it was observed that the new formulation of PRF (A-PRF) released significantly higher total quantities of growth factors when compared to traditional PRF.

CONCLUSIONS

Based on these findings, PRP can be recommended for fast delivery of growth factors whereas A-PRF is better-suited for long-term release.

The chemotactic activities of three different isoforms of platelet-derived growth factor (PDGF) on fibroblasts, monocytes, and granulocytes of human origin were investigated. PDGF-AB and PDGF-BB induced strong, dose-dependent responses in both fibroblasts and monocytes, whereas PDGF-AA did not stimulate chemotaxis of these cell types. Instead, PDGF-AA inhibited the chemotactic activity of PDGF-AB and PDGF-BB on fibroblasts and monocytes. However, PDGF-AA was not able to block monocyte chemotaxis induced by FMLP. In contrast, in granulocytes, dose-dependent chemotactic responses were obtained with all three isoforms of PDGF. All isoforms gave maximal responses at concentrations between 5 and 20 ng/ml. At higher concentrations the migration was reduced. Reduction and alkylation of the PDGF molecule, which leads to loss of the mitogenic activity, also caused a loss of the chemotactic activities for all three cell types. The data suggest that the various isoforms of PDGF stimulate and inhibit chemotaxis in an isoform- and cell type-specific manner.

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta1 or AA. TGF-beta1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22alpha was significantly up-regulated by 30 microM AA. We also investigated the additive effect of TGF-beta1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.

BACKGROUND

Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known.

OBJECTIVE

Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis.

METHODS

Controlled laboratory study.

METHODS

Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration.

RESULTS

Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons.

CONCLUSIONS

Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that minimizing leukocytes in PRP is more important than maximizing platelet numbers with respect to decreasing inflammation and enhancing matrix gene synthesis.

CONCLUSIONS

This study suggests that reducing leukocytes to minimize catabolic signaling appears to be more important than increasing platelets in an effort to maximize anabolic signaling. Further, a maximum biological threshold of benefit was demonstrated with regard to the number of platelets beyond which further increases in platelet concentration did not result in further anabolic upregulation. In vivo investigations documenting the use of platelets for the treatment of tendinopathy are justified as well as further in vitro characterization of the ideal PRP product for the treatment of tendinopathy and other musculoskeletal applications.

Neuroinflammation associated with advanced HIV-1 infection is often exacerbated in cocaine-abusing, HIV-infected patients. The underlying mechanisms could, in part, be attributed to the increased impairment of blood brain barrier integrity in the presence of cocaine. Platelet-derived growth factor (PDGF) has been implicated in several pathologic conditions, specifically attributable to its potent mitogenic effects. Its modulation by drug abuse, however, has received very little attention. In the present study, we demonstrated cocaine-mediated induction of PDGF-BB in human brain microvascular endothelial cells through the binding to its cognate σ receptor. Furthermore, this effect was mediated, with subsequent activation of mitogen-activated protein kinases and Egr-1 pathways, culminating ultimately into increased expression of PDGF-BB. Cocaine exposure resulted in increased permeability of the endothelial barrier, and this effect was abrogated in mice exposed to PDGF-BB neutralizing antibody, thus underscoring its role as a vascular permeant. In vivo relevance of these findings was further corroborated in cocaine-treated mice that were administered neutralizing antibody specific for PDGF-BB as well as in Egr-1(-/-) mice. Understanding the regulation of PDGF-BB expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with HIV infection and drug abuse.

Regenerating liver was evaluated for the spatiotemporal expression of angiogenic growth factor receptors on endothelial cell (EC) membranes during revascularization resulting from 70% partial hepatectomy (PHx). Fractions enriched in EC membranes were examined by Western blot for angiogenic growth factor receptor expression from 1 to 14 days after PHx. Increases in vascular endothelial growth factor (VEGF) receptors Flt-1 and Flk-1/KDR, angiopoietin receptors Tie-1, Tie-2, and platelet-derived growth factor receptor beta (PDGF-Rbeta), modest increases in epidermal growth factor receptor (EGF-R), and no increase in hepatocyte growth factor receptor (c-Met) or fibroblast growth factor receptors (FGF-R) were observed in isolated membranes during EC proliferation. All receptors were tyrosine phosphorylated, and therefore activated, during peak expression. Immunofluorescence staining of regenerating liver identified populations with increased receptor expression, indicating cells receptive to ligand signaling. EGF-R was upregulated evenly throughout the sinusoidal membrane, whereas c-Met was observed on hepatocyte canaliculae, bile duct epithelium, and large vessel EC. Tie-2 and PDGF-Rbeta were increased on sinusoidal and large vessel EC, whereas Tie-1 was expressed in EC surrounding avascular hepatic islands. Flk-1/KDR was increased on large vessels with slight increases on sinusoidal EC, whereas Flt-1 was increased in arterioles, sinusoidal EC as well as in hepatocytes. Although Flt-1 was phosphorylated on isolated hepatocytes, vascular endothelial growth factor(165) (VEGF(165)) did not induce a proliferative or motogenic response. Proliferation assays on isolated EC indicated responsiveness to VEGF(165), but synergism among several growth factors including PDGF-BB was also observed. The data identify novel autocrine and paracrine interactions and indicate that each growth factor acts on a specific set of EC at specific times during revascularization of regenerating liver.

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [(3)H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.

OBJECTIVE

This phase II trial assessed the activity and tolerability of an oral dose of imatinib mesylate 400 mg twice daily in patients with recurrent or persistent epithelial ovarian or primary peritoneal carcinoma. The association between the expression of certain markers and clinical outcome was investigated.

METHODS

Primary measure of clinical efficacy was progression-free survival (PFS) at 6 months. Mutational analysis of KIT, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay for markers (KIT, platelet-derived growth factor [PDGF] receptor [-R], AKT2, phosphorylated AKT [p-AKT], stem cell factor [SCF], and PDGF) were performed.

RESULTS

Fifty-six eligible patients were evaluated. Nine patients were progression free for at least 6 months including one complete responder. The median PFS and survival were 2 and 16 months, respectively. The most common grade 3 and 4 toxicities were neutropenia, GI, dermatologic effects, pain, and electrolyte disturbances. At least one target of imatinib (KIT, PDGFR-alpha, or PDGFR-beta) was expressed in all tumors, and most tumors expressed all three receptors. Higher expression of p-AKT and PDGFR-beta were associated with shorter PFS, and higher IHC scores (% immunopositive cells x staining intensity) of SCF and p-AKT were associated with decreased overall survival. No sequence mutations were detected in the KIT gene. Higher pretreatment plasma concentrations of PDGF-AB, PDGF-BB, and vascular endothelial growth factor (VEGF) were individually associated with shorter PFS and survival.

CONCLUSIONS

Imatinib mesylate was well tolerated but had minimal single-agent activity in patients with recurrent ovarian or primary peritoneal carcinoma. No marker was identified that would predict activity of imatinib; however, tumor p-AKT and plasma VEGF levels were associated with poor outcome.

Vascular endothelial growth factor (VEGF) is a potent peptide growth factor specific for vascular endothelial cells, which promotes neovascularization and increases vascular permeability in vivo. Enhanced microvascular permeability and edema are common characteristics of inflammatory and neoplastic disorders. Two proinflammatory mediators, platelet-activating factor (PAF) and platelet-derived growth factor (PDGF), are known to contribute to cellular damage and tissue remodeling in a number of lung diseases. To determine whether PAF or PDGF induce VEGF gene expression in primary cultures of human pulmonary fibroblasts and pulmonary vascular smooth-muscle cells (VSMCs), we performed Northern-blot analysis and enzyme-linked immunosorbent assays (EIA). PAF and all three isoforms of PDGF (PDGF-AA, -AB, and -BB) increased VEGF mRNA in a time- and dose-dependent manner. While PAF was shown to increase VEGF mRNA at picomolar concentrations, all PDGF isoforms were effective in inducing VEGF mRNA at nanomolar concentrations. The transcriptional activation was accompanied by increased levels of VEGF protein as determined by EIA in culture medium. These results indicate that VEGF gene expression in VSMCs and fibroblasts is mediated by PAF and/or PDGF isoforms. In a paracrine mode of action, secreted VEGF may then lead to altered endothelial cell functions and vascular hyperpermeability. In the presence of the corticosteroids cortisone, hydrocortisone, dexamethasone, or prednisolone at nanomolar concentrations, this stimulus-dependent transcription of VEGF was abolished. The inhibitory effect of corticosteroids on VEGF expression could explain the clinically well-known antiedematous potency of corticosteroids on a molecular level.

Platelet-derived growth factor (PDGF) is encoded by separate genes for two possible subunit chains (A-chain and B-chain) which can form three possible dimers (AA, AB, and BB). We have recently presented evidence that multiple forms of PDGF receptor exist which distinguish between these isoforms (Hart, C. H., Forstrom, J. W., Kelley, J. D., Smith, R. A., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1988) Science 240, 1529-1531). We used this specificity to determine the amount of PDGF from different sources which is able to bind to each class of receptor and found that each source had a characteristic isoform composition. Levels of total PDGF activity in sera from different species ranged more than 15-fold, from less than 1 ng/ml in dog, chicken, pig, and calf, to greater than 13 ng/ml in mouse and human. Despite these differences in PDGF content, the total mitogenic activities of the sera were comparable indicating that the relative importance of PDGF as a serum mitogen may vary considerably between species. Analysis of the total PDGF into the amounts of each isoform revealed great differences in composition. PDGF-BB constitutes only about 15% of the total binding activity in human PDGF purified by the method of Raines and Ross (Raines, E. W., and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160) but is the predominant isoform in whole blood serum from all other species. In contrast to serum, medium conditioned by cultured PDGF-secreting cell types contained no detectable PDGF-BB except in two cases: medium conditioned by vascular endothelial cells and by cells transformed by simian sarcoma virus. The existence of isoform-specific PDGF receptors and the large variation in PDGF isoform composition dependent upon source may provide an important mechanism through which the effects of PDGF can be targeted to different cell types and/or toward eliciting different cell responses.

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta-receptors in human diploid foreskin AG 1518 fibroblasts. A transient tyrosine phosphorylation of PDGF beta-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not on poly-L-lysine. In contrast EGF or PDGF alpha-receptors were not phosphorylated on tyrosine residues under these conditions. Tyrosine phosphorylation of PDGF beta-receptors induced by plating on collagen type I was inhibited by cytochalasin D and herbimycin A, unaffected by cycloheximide and enhanced by orthovanadate. Furthermore, a transient phosphorylation of PDGF beta-receptors occurred when AG 518 fibroblasts were cultured in three-dimensional collagen lattices or exposed to external strain exerted through centrifugation. The latter effect was evident already after two minutes. Clustering of cell surface beta1 integrins led to PDGF beta-receptor phosphorylation both in suspended and firmly attached AG 1518 fibroblasts. Plating of cells on collagen type I, fibronectin, and anti-beta1-integrin IgG resulted in the formation of PDGF beta-receptor aggregates as detected by immunofluorescence. Suramin or anti-PDGF-BB IgG had no effect on the plating-induced tyrosine phosphorylation of PDGF beta-receptors. PDGF-B chain mRNA, or protein, were not detected in AG 1518 fibroblasts. Our data suggest that a ligand-independent PDGF beta-receptor activation during cell adhesion and early phases of cell spreading is involved in integrin-mediated signaling in fibroblasts, and constitutes parts of a mechanism for cells to respond during the dynamic phases of externally applied tension as well as fibroblast-mediated tension during cell adhesion and collagen gel contraction.

OBJECTIVE

Angiogenesis is a process stimulated in inflamed synovium of patients with osteoarthritis (OA), and contributes to the progression of the disease. Synovial fibroblasts secrete angiogenic factors, such as vascular endothelial growth factor (VEGF), an up-regulator of angiogenesis, and this ability is increased by interleukin (IL)-1beta. The purpose of this study was to verify whether IL-17 contributes and/or synergizes with IL-1beta and tumor necrosis factor (TNF)-alpha in vessel development in articular tissues by stimulating the secretion of proangiogenic factors by synovial fibroblasts.

METHODS

We stimulated in vitro synovial fibroblasts isolated from OA, rheumatoid arthritis (RA) and fractured patients (FP) with IL-17 and IL-1beta and from OA patients with IL-17, IL-1beta and TNF-alpha. In the supernatants from the cultures, we assayed the amount of VEGF by immunoassay and other angiogenic factors (keratinocyte growth factor, KGF; hepatocyte growth factor, HGF; heparin-binding endothelial growth factor, HB-EGF; angiopoietin-2, Ang-2; platelet-derived growth factor B, PDGF-BB; thrombopoietin, TPO) by chemiluminescence; semiquantitative RT-PCR was used to state mRNA expression of nonreleased angiogenic factors (Ang-2 and PDGF-BB) and tissue inhibitors of metalloproteinase (TIMP)-1.

RESULTS

IL-17, TNF-alpha and IL-1beta increased VEGF secretion by synovial fibroblasts from OA patients. IL-17 and IL-1beta also increased VEGF secretion in RA and FP. Besides, IL-17 increased KGF and HGF secretions in OA, RA and FP; in OA and RA, IL-17 also increased the HB-EGF secretion and the expression of TIMP-1 as protein and mRNA. In OA patients IL-17 had an additive effect on TNF-alpha-stimulated VEGF secretion.

CONCLUSIONS

These results suggest that IL-17 is an in vitro stimulator of angiogenic factor release, both by its own action and by cooperating with TNF-alpha.

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.

OBJECTIVE

To investigate osteoarthritic cartilage in comparison to normal cartilage in humans for the presence of the most relevant cytokines/growth factors known to be important for degradation and formation of new cartilage.

METHODS

Cartilage from knee or hip joints was obtained from 10 patients with osteoarthritis (OA) and from 7 age matched control patients with intact cartilage. Additionally, normal cartilage from 2 young patients (12 and 17 years old) was obtained after knee traumas. Immunohistological staining of cartilage sections was performed using antibodies for the following cytokines/growth factors: tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), IL-1beta, interferon-gamma, IL-6, IL-4, IL-10, transforming growth factor beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), IGF-II, platelet derived growth factor AA (PDGF-AA), and PDGF-BB.

RESULTS

Immunohistochemical stainings were positive for all cytokines in OA cartilage, while only a faint or no staining was found in healthy cartilage. Activated chondrocytes expressing most of the cytokines were located in the middle and partly in the lower layer of cartilage, with the exception of IGF-I, which was expressed exclusively in the upper cartilage layer close to the surface. More chondrocytes stained positive for TNF-alpha than for IL-1, and expression of the degrading cytokine TNF-alpha was inversely correlated to the expression of the regulatory cytokines IL-4, IL-10, and TGF-beta.

CONCLUSIONS

The most relevant cytokines known to be involved in cartilage metabolism are produced by chondrocytes themselves. They are upregulated in OA cartilage, suggesting that they serve some regulatory function and could be a target for future treatment.