Platelet-derived Growth Factor Receptor mRNA Research Articles

Inhibited proliferation of human umbilical artery smooth muscle cells by xanthinol nicotinate

Vascular smooth muscle cell proliferation is a key event in the development of hypertension, instant restenosis and other cardiac disorders. Inhibition of this proliferation could lead to better prevention and treatment of these diseases. This study was designed to investigate the effects and mechanisms of different concentrations of xanthinol nicotinate (XN) on human umbilical artery smooth muscle cell (HUASMC) proliferation in vitro. HUASMCs were cultured by the tissue adherent method, passaged three times, and then identified by immunohistochemistry. HUASMCs were then treated with different concentrations of XN (0, 2.76, 27.6 or 276µM), and a 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to detect the inhibition of HUASMC proliferation. The levels of platelet-derived growth factor receptor (PDGFR) mRNA and protein (PDGFR-β) were detected on the cell membrane of these treated HUASMCs using RT-PCR and Western blot analysis, respectively. After culturing and passaging three times, 90% of the cultured cells were identified as HUASMCs by immunohistochemistry. HUASMC proliferation was inhibited by XN in a dose-dependent manner (P<0.05). Furthermore, XN dose-dependently decreased the PDGFR mRNA and PDGFR-β levels on the cell membranes of HUASMCs (P<0.05). Thus, the results suggest that XN could become a potent therapeutic agent for regulating VSMC-associated vascular disease such as cardiovascular disease and restenosis after angioplasty.

Role of platelet-derived growth factor/platelet-derived growth factor receptor axis in the trafficking of circulating fibrocytes in pulmonary fibrosis.

Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4-/- mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor beta in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to alpha4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin alpha4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin alpha4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.

Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension

Rationale Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries. Objectives To analyze the expression and pathogenic role of PDGF in idiopathic PAH. Methods PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription-polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-beta. The effects of imatinib on PDGF-B- induced proliferation and chemotaxis were tested on human PASMCs. Results PDGF-A, PDGF-B, PDGFR-alpha, and PDGFR-beta mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-beta in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR-alpha and PDGFR-beta mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-beta. PDGF- BB-induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis. Conclusions PDGF may play an important role in human PAH Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.

Platelet-derived Growth Factor Expression and Function in Idiopathic Pulmonary Arterial Hypertension

Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries. To analyze the expression and pathogenic role of PDGF in idiopathic PAH. PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription-polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH. Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-beta. The effects of imatinib on PDGF-B-induced proliferation and chemotaxis were tested on human PASMCs. PDGF-A, PDGF-B, PDGFR-alpha, and PDGFR-beta mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-beta in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR-alpha and PDGFR-beta mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-beta. PDGF-BB-induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis. PDGF may play an important role in human PAH. Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.

The Role of Platelet-Derived Growth Factor Receptor in Eotaxin Signaling of Eosinophils

Background: Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) are capable of eliciting kinase activity after ligand binding. In several cells, RTKs are activated via the G-protein-coupled receptor independent of the ligand-RTK interaction. We have previously found that EGFR is transactivated via CC chemokine receptor 3 in bronchial epithelial cells and that this pathway is important for mitogen-activated protein (MAP) kinase activation and cytokine production. It has recently been suggested that hypereosinophilic syndrome results from the fusion tyrosine kinase FIP1L1-PDGFRA. Although it is possible that the PDGFR signal is involved in eosinophil function, the details are still unclear. Methods: Blood eosinophils were purified using Percoll and anti-CD16 antibody-coated magnetic beads. Expression of PDGFR mRNA was examined by RT-PCR. After stimulating eosinophils with eotaxin, the phosphorylation of MAP kinases was examined by Western blotting with the antiphosphospecific MAP kinase antibody. The eotaxin-induced eosinophil chemotaxis was studied using Boyden chambers. Results: Eosinophils expressed PDGFRβ mRNA in 4 out of 8 donors, while PDGFRα mRNA was expressed in only 1 donor. Protein expression of PDGFR was also detectable in eosinophils from some donors. AG1295, a specific inhibitor of PDGFR, showed dose-dependent inhibition of eotaxin-induced MAP kinase phosphorylation in the eosinophils expressing PDGFRβ mRNA. The chemotaxis of these eosinophils was significantly inhibited by AG1295 (n = 3). Conclusions: Our results suggest that PDGFR modifies the CCR3-MAP kinase signaling pathway and chemotactic response in some donors. The pharmacological targeting of PDGFR may be a new strategy to treat eosinophilic disorders.

Glomerular expression of platelet-derived growth factor (PDGF)-A, -B chain and PDGF receptor-alpha, -beta in human diabetic nephropathy.

Mesangial expansion is thought to be a major cause of diabetic nephropathy (DN). Platelet-derived growth factor (PDGF) plays an important role in the production of extracellular matrix proteins in renal diseases. The present study was designed to determine the expression of PDGF and PDF receptor (PDGFR) mRNA in the renal tissues of type 2 diabetic patients with DN. We examined open renal biopsies of 20 type 2 diabetic patients with DN, and 10 normal human kidneys (NHK). Histopathologically, the severity of DN was classified as grade I (DN I, n = 10; mild mesangial expansion) or grade II (DN II, n = 10; moderate mesangial expansion). We evaluated the expression and localization of PDGF-A, -B, and PDGFR-Alpha, -Beta using in situ hybridization, and quantified PDGF and PDGFR mRNA expression by counting all nuclei, and nuclei surrounded by PDGF-positive cytoplasm and PDGFR-positive cytoplasm, in at least ten randomly selected cross-sections of nonsclerotic glomeruli. In all glomeruli, PDGF and PDGFR mRNAs were expressed mainly in glomerular resident cells, predominantly glomerular mesangial and epithelial cells. The percentages of cells positive for PDGF-A and PDGFR-Alpha mRNA in DN were similar to those in NHK. In contrast, the percentages of PDGF-B and PDGFR-Beta mRNA-positive cells in DN were significantly higher than those in NHK, and were significantly higher in DN I than in DN II: The percentages of cells positive for PDGF-B correlated with the PDGFR-Beta mRNA level. Our results suggest that the expression of PDGF-B and PDGFR-Beta is an important factor in histologically early glomerular lesions of DN. Mesangial expansion is thought to be a major cause of diabetic nephropathy (DN). Platelet-derived growth factor (PDGF) plays an important role in the production of extracellular matrix proteins in renal diseases. The present study was designed to determine the expression of PDGF and PDGF receptor (PDGFR) mRNA in the renal tissues of type 2 diabetic patients with DN.

Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling.

Angiogenesis and vascular cell proliferation are pivotal in physiological and pathological processes including atherogenesis, restenosis, wound healing, and cancer development. Here we show that mammalian target of rapamycin (mTOR) signaling plays a key role in hypoxia-triggered smooth muscle and endothelial proliferation and angiogenesis in vitro. Hypoxia significantly increased DNA synthesis and proliferative responses to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) in rat and human smooth muscle and endothelial cells. In an in vitro 3-dimensional model of angiogenesis, hypoxia increased PDGF- and FGF-stimulated sprout formation from rat and mouse aortas. Hypoxia did not modulate PDGF receptor mRNA, protein, or phosphorylation. PI3K activity was essential for cell proliferation under normoxic and hypoxic conditions. Activities of PI3K-downstream target PKB under hypoxia and normoxia were comparable. However, mTOR inhibition by rapamycin specifically abrogated hypoxia-mediated amplification of proliferation and angiogenesis, but was without effect on proliferation under normoxia. Accordingly, hypoxia-mediated amplification of proliferation was further augmented in mTOR-overexpressing endothelial cells. Thus, signaling via mTOR may represent a novel mechanism whereby hypoxia augments mitogen-stimulated vascular cell proliferation and angiogenesis.

Direct effects of simvastatin on proliferation and matrix accumulation in cultured murine mesangial cells

Background. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been demonstrated to suppress glomerular injuries in various renal diseases. However, it is not fully clear whether HMG-CoA reductase inhibitors directly regulate matrix protein accumulation in mesangial cells. Methods. We investigated the effect of simvastatin (SIM), an HMG-CoA reductase inhibitor, on DNA synthesis in cultured murine mesangial cells stimulated by fetal calf serum (FCS). We then determined whether SIM affected the expression of regulatory factors of cell growth and matrix protein accumulation, using Northern analysis. Results. SIM dose-dependently inhibited FCS-induced DNA synthesis after 24 h of incubation. SIM treatment for 24 h suppressed the mRNA expression of platelet-derived growth factor (PDGF)-B chain, PDGF receptor β-subunit and c-myc, while the expression of transforming growth factor β (TGF-β) was not affected. Concerning matrix protein synthesis, the mRNA expression of type IV collagen was suppressed, whereas that of type III collagen was markedly upregulated. As for matrix turnover proteins, SIM had a markedly suppressive effect on the mRNA expression of plasminogen activator inhibitor-1 (PAI-1), with a constant expression of tissue-type plasminogen activator (tPA). Conclusions. These results indicate that SIM may sup-press mesangial cell proliferation in part through the downregulation of PDGF, PDGF-receptor, and c-myc mRNA expressions. In addition, the suppression of the synthesis of collagen IV and PAI-1 appears to be a direct inhibitory effect of SIM on glomerular matrix accumulation.

Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

Determination of cytokine mRNA-expression in term human placenta of patients with gestational hypertension, intrauterine growth retardation and gestational diabetes mellitus using polymerase chain reaction.

Our objective was to test the hypothesis, that pregnancy-related diseases are going along with changes in cytokine mRNA-expression at the placental site, either as a part of a pathological process or in connection with regulatory mechanisms induced by disturbances at the feto-maternal interface resulting from previous pathological changes--in the sense of counterregulation. The cytokines chosen for this investigation are known to 1.) be expressed in the human placental tissue, 2.) to be involved in immunological processes and 3.) the regulation of growth and differentiation processes of different cell types of the placenta or decidua, 4.) to play a role in the angiogenesis at the feto-placental interface and 5.) to be involved in pathological processes in other human diseases. 32 samples derived from term human placentas were examined for messenger RNA levels of interleukin 1 alpha (II-1 alpha), tumor necrosis factor-alpha (TNF-alpha), platelet derived growth factor-A chain (PDGF-A), platelet derived growth factor-B chain (PDGF-B), and platelet derived growth factor receptor (PDGF-R) using a semiquantitative reverse transcriptase (RT) polymerase chain reaction (PCR) protocol. To calibrate samples in our procedure, beta-actin mRNA (messenger ribonucleic acid) known as a "house keeping" gene was proven to be constantly expressed. The sample-groups consisted of normal pregnancies (n = 8), gestational hypertension (GH, n = 7), intrauterine growth retardation (IUGR, n = 6), gestational diabetes mellitus (GDM, n = 5), and gemini (n = 3 x 2). Throughout the 32 samples, a significant correlation between PDGF-A and PDGF-R expression, PDGF-A and TNF-alpha expression was stated (p = 0.007). Compared with the pattern of expression in normal placentas, placentas of growth retarded pregnancies had higher Il-1 alpha mRNA (p = 0.016), PDGF-A (p = 0.029) and PDGF-B (p = 0.001) levels. The samples of the gestational hypertension group and placentas of patients with gestational diabetes displayed a significantly stronger PDGF-R mRNA signal (p = 0.0029 and p = 0.008). Though these marked differences in cytokine mRNA levels between clinical groups were statistically proven, clear correlation of these differences with clinical data was not found.

Kawasaki disease on PDGF expression and VSMC proliferation.

The effect of serum of patients with Kawasaki disease (KD) on expression of platelet-derived growth factor (PDGF) B chain protein in vascular endothelial cells (VEC) was studied by immunocytochemical method. Meanwhile, the effects of the endothelial cell conditioned media (ECM) on expression of PDGF receptor mRNA in vascular smooth muscle cells (VSMC) and on cell cycle of VSMC were investigated by the methods of nucleic acid hybridization and flow cytometry (FCM). The results showed that the serum of patients with KD induced the expression of PDGF-B chain protein significantly. ECM significantly promoted the expression of PDGF receptor mRNA and induced the proliferation of VSMC. These data suggest that the activation of PDGF-PDGF receptor may play a role in the pathogenesis of coronary complication of KD.

Study on platelet-derived growth factor mRNA and copper-zinc superoxide dismutase mRNA expression changes of aortic artery endothelium with restenosis after aortic artery dilatation treated with Buyang Huanwu Decoction

To study on dynamic mechanism of restenosis after PTCA treated with Buyang Huanwu Decoction (BYHWD). Twenty-eight male New Zealand rabbits with mimic restenosis after aortic artery dilatation were detected by prime in situ labeling in aortic endothelium. The items included gene expression of copper zinc superoxide dismutase (SOD-1) and platelet-derived growth factor receptor (PDGFR) mRNA to analyse dynamic mechanism of restenosis. Seven days after aortic artery dilatation, PDGFR mRNA of aortic endothelium decreased clearly. The comparison between experimental and the control group showed significant difference (P < 0.05). By the same condition, the gene expression of SOD-1 was increasing, which showed a significant difference with the control group (P < 0.05). BYHWD increased SOD-1 gene expression of artery endothelium, to improve oxygen feed for ischemia reperfusion in the same way, BYHWD suppressed PDGFR mRNA expression to control cellular pathological proliferation.

Expression of PDGF and PDGF receptor mRNA in glomeruli in IgA nephropathy.

This study investigated the mRNA expression of the platelet-derived growth factor (PDGF) A-chain and B-chain and PDGF-beta receptor in glomeruli of 15 immunoglobulin A (IgA) nephropathy kidneys and those with minimal-change lesion (N = 7), membranous nephropathy (N = 3), and focal segmental glomerulonephritis (N = 5), by using competitive RT-PCR methods. The level of PDGF B-chain and beta receptor mRNA expression in IgA nephropathy was significantly higher than in the other forms of glomerulonephritis, but mRNA expressions of PDGF A-chain were not significantly different. Significant correlations were observed between the urinary protein level and the mRNA level of PDGF-beta receptor expression and PDGF B-chain expression, and between the serum creatinine level and the mRNA level of PDGF-beta receptor expression. The PDGF B-chain and beta-receptor may be upregulated and accelerate cell proliferation in a paracrine or autocrine manner and may play a role in the pathogenesis of IgA nephropathy.

Temporal expression of PDGF receptors and PDGF regulatory effects on osteoblastic cells in mineralizing cultures.

Platelet-derived growth factor (PDGF) is mitogenic and chemotactic for osteoblastic cells in vitro. It is expressed during osseous wound healing and stimulates formation of new bone in vivo. PDGF stimulates cells by binding to specific cell surface receptors. The purpose of this study was to examine the effects of PDGF on osteoblastic proliferation and differentiation in long-term mineralizing cultures. Utilizing Northern blot analysis, we found that continuous PDGF treatment increased histone expression, indicative of enhanced proliferation, but suppressed osteoblast differentiation, demonstrated by inhibition of alkaline phosphatase, type I collagen, and osteocalcin expression. The inhibitory effect of PDGF on the differentiated function of osteoblasts was further established by findings that PDGF significantly inhibited nodule formation. The expression of PDGF receptors varied at different stages of culture. PDGF receptor mRNA expression increased when the cells had achieved a mature phenotype, during the stage of matrix maturation, and then decreased. However, as demonstrated by thymidine incorporation assays, the capacity of PDGF to stimulate DNA synthesis actually decreased during osteoblast maturation, as receptor expression increased. To investigate this apparent contradiction, tyrosyl phosphorylation and immunoblot assays were performed to assess changes in PDGF activation of their cognate receptors. The pattern of PDGF-induced tyrosyl phosphorylation remained relatively constant. This suggests that the diminished mitogenic activity of PDGF that occurs after osteoblast differentiation is regulated at a postreceptor level.

Different roles for PDGF-alpha and -beta receptors in embryonic lung development.

Platelet-derived growth factor (PDGF) is implicated in the process of normal lung development. We have previously shown the presence of PDGF-AA and BB homodimers in embryonic rat lung. Also, we reported that PDGF-AA is involved in embryonic lung branching, whereas PDGF-BB influences embryonic lung growth. PDGF isoforms bind with different affinities to two related receptors, denoted the PDGF alpha- and beta-receptors, respectively. The alpha-receptor binds both PDGF isoforms, whereas the beta-receptor binds only PDGF-BB. In the present study, we investigated the role of both receptors in early embryonic rat lung development. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that both PDGF alpha- and beta-receptor mRNAs are mainly expressed in the mesenchyme. Phosphorothioate antisense receptor oligonucleotides decreased PDGF receptor mRNA expression in early lung explants. PDGF-induced receptor tyrosine phosphorylation was also reduced by the antisense oligonucleotides. Incubation of embryonic lung explants with antisense beta-receptor oligonucleotides inhibited lung growth but not early lung branching. Neither growth nor branching were affected by sense beta-receptor oligonucleotides. The inhibitory effect of antisense beta-receptor oligonucleotides on embryonic lung growth was reversed by the addition of PDGF-BB or PDGF-AA, suggesting that the alpha-receptor can transduce similar mitogenic signals as the beta-receptor in early lung development. Antisense alpha-receptor oligonucleotides reduced both embryonic lung growth and branching. Sense alpha-receptor treatment had no effect on lung growth and branching. PDGF-BB but not PDGF-AA partially attenuated the inhibitory effect of antisense alpha-receptor oligonucleotides on lung growth. In contrast, PDGF-BB did not overcome the inhibitory effect on early lung branching, indicating that the beta-receptor cannot replace this biologic role of the alpha-receptor in early lung development. These data suggest that PDGF-BB stimulation of both receptors leads to lung growth, whereas PDGF-AA stimulation of the alpha-receptor induces transduction pathways that lead lung branching.

Regulation of differential expression of platelet-derived growth factor α- and β-receptor mRNA in normal and malignant human mesothelial cell lines

In earlier studies we showed that the expression patterns of platelet-derived growth factor (PDGF) aand /3-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF a-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF/3-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF/3-receptor in mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF fl-receptor transcription but of a-receptor transcription as well, as evidenced from run off analysis and RT-PCR using a-receptor specific primers. However, the fact that PDGF a-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF a-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF a-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF /3-receptor mRNA expression was seen. The half-life of the PDGF fl-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF/3-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in fl-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF fl-receptor transcripts in these cells. Transforming growth factor/3-1 (TGF-/31), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-fl 1 revealed decreased PDGF a-receptor mRNA expression in normal mesothelial cells. The effect on PDGF 0-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-fl I effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-/31 may nevertheless play a role in downregulation of an (already) low PDGF a-receptor mRNA level in malignant mesothelioma cell lines.

Regulation of differential expression of platelet-derived growth factor α- and β-receptor mRNA in normal and malignant human mesothelial cell lines

In earlier studies we showed that the expression patterns of platelet-derived growth factor (PDGF) α- and β-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF α-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF β-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF β-receptor in mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF β-receptor transcription but of α-receptor transcription as well, as evidenced from run off analysis and RT-PCR using α-receptor specific primers. However, the fact that PDGF α-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF α-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF α-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF β-receptor mRNA expression was seen. The half-life of the PDGF β-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF β-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in β-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF β-receptor transcripts in these cells. Transforming growth factor β-1 (TGF-β1), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-β1 revealed decreased PDGF α-receptor mRNA expression in normal mesothelial cells. The effect on PDGF β-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-β1 effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-β1 may nevertheless play a role in downregulation of an (already) low PDGF α-receptor mRNA level in malignant mesothelioma cell lines.

Reduction in platelet-derived growth factor receptor mRNA in v- src-transformed fibroblasts

The status of the platelet-derived growth factor (PDGF) receptor in normal rat kidney (NRK) fibroblasts and in NRK fibroblasts transformed by the v- src oncogene or the polyoma middle T (pmt) antigen has been compared. v- src-NRK cells have 7-fold fewer surface binding sites for PDGF than NRK cells, but the affinity of the residual receptors for PDGF is reduced only 2-fold. Levels of the PDGF receptor measured by Western blotting or in an autophosphorylation assay in vitro are 8- and 4-fold lower respectively in v- src-NRK cells than in NRK cells. No PDGF-induced phosphorylation of the PDGF receptor is apparent after 32P-labelling of intact v- src-NRK cells, implying that the reduction in PDGF receptor levels is not a consequence of production of autocrine PDGF. A 10-fold reduction in the amount of mRNA for the PDGF receptor is also observed in v- src-NRK cells. No decrease in PDGF receptor protein or mRNA levels is observed in pmt-NRK cells. We conclude that levels of the PDGF receptor in v- src-transformed NRK fibroblasts are modulated by reduction in the level of PDGF receptor mRNA.

Enhancement of Growth of Rabbit Corneal Endothelial Cells by PDGF

The effect of platelet-derived growth factor (PDGF) on the growth of rabbit corneal endothelial cells and the expression of PDGF receptor mRNA in these cells were examined in the present study. When rabbit corneal endothelial cells were cultured in the presence of natural PDGF, recombinant PDGF-BB, and PDGF-AA for 1, 3, and 5 days, natural PDGF and recombinant PDGF-BB enhanced the growth of the endothelial cells, but PDGF-AA did not. This growth promotion was suppressed by anti-PDGF antibody. PDGF enhanced also the incorporation of 3H-thymidine into the cells. When PDGF was combined with epidermal growth factor (EGF), the additive effect was recognized. The expression of the PDGF receptor mRNA was found in the endothelial cells by the polymerase chain reaction (PCR) method. It was concluded from these results that PDGF enhanced the growth of the corneal endothelial cells and that the PDGF receptor mRNA was expressed in these cells. These results suggest that PDGF may regulate corneal endothelial cell growth.