Platelet-derived Growth Factor Isoforms Research Articles

Platelet-Derived Growth Factor Receptor β But Not α Mediates Cardiac Fibrosis In Vitro and In Vivo

Background: Platelet-derived growth factor (PDGF) is one of the mediators in cardiac fibrosis but its role has not been fully explored. The aim of this study is to investigate the role of PDGF subtypes (AA, AB, BB, & CC) and their receptors (PDGFRα & β) and signalling pathways involved in cardiac fibrosis both in vitro and in vivo. Methods: Cardiac myocyte (NCM) hypertrophy and fibroblast (NCF) collagen synthesis stimulated by PDGF isoforms (10ng/mL) for 48hours, were determined by [3H]-leucine and [3H]-proline incorporation, respectively. Myocardial infarction (MI) was induced 4 weeks after 5/6th subtotal nephrectomy (STNx), following treatment with imidapril (Imid, 10 mg/kg daily) for 8 weeks. At endpoint, cardiac and renal function and structure were examined. Gene expression and Western Blot analysis were performed. Results: Only PDGF AB and BB stimulated NCM hypertrophy and NCF collagen syntheses, and increased hypertrophic, fibrotic and PDGFR gene expression (Fig). PDGF AB and BB activated p38MAPK and Akt, which were inhibited by their selective inhibitors. Imid treatment significantly improved cardiac and renal function, and reduced hypertrophic and fibrotic gene expression, post-STNx+MI. Imid treatment also reversed PDGFRβ and PDGF BB gene expression post-STNx+MI (Fig). Conclusion: The results demonstrated that only PDGF BB and AB via PDGFRβ subtype activation are involved in cardiac fibrosis in vitro and in vivo, suggesting the therapeutic potential of PDGFRβ subtype specific blockade in cardiac fibrosis.

PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) contribute to normal heart development. Deficient or abnormal expression of Pdgf and Pdgfr genes have a negative impact on cardiac development and function. The cellular effects of PDGFs in the hearts of Pdgf/Pdgfr mutants and the pathogenesis of the resulting abnormalities are poorly understood, but different PDGF isoforms induce varying effects. Here, we generated three new transgenic mouse types which complete a set of studies, where all different PDGF ligands have been expressed under the same heart specific alpha-myosin heavy chain promoter.Transgenic expression of the natural isoforms of Pdgfa and Pdgfb resulted in isoform specific fibrotic reactions and cardiac hypertrophy. Pdgfa overexpression resulted in a severe fibrotic reaction with up to 8-fold increase in cardiac size, leading to lethal cardiac failure within a few weeks after birth. In contrast, Pdgfb overexpression led to focal fibrosis and moderate cardiac hypertrophy. As PDGF-A and PDGF-B have different affinity for the two PDGF receptors, we analyzed the expression of the receptors and the histology of the fibrotic hearts. Our data suggest that the stronger fibrotic effect generated by Pdgfa overexpression was mediated by Pdgfrα in cardiac interstitial mesenchymal cells, i.e. the likely source of extracellular matrix depostion and fibrotic reaction. The apparent sensitivity of the heart to ectopic PDGFRα agonists supports a role for endogenous PDGFRα agonists in the pathogenesis of cardiac fibrosis.

Isoform-Specific Modulation of Inflammation Induced by Adenoviral Mediated Delivery of Platelet-Derived Growth Factors in the Adult Mouse Heart.

Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFRα and PDGFRβ) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRα agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFRα-positive fibroblasts, PDGFRβ agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirus-induced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses.

Platelet-Poor and Platelet-Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells.

Plasma-derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme-linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB, and -AB), insulin-like growth factor binding protein (IGFBP)-2, and IGFBP-6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.

The platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are major players in oncogenesis, drug resistance, and attractive oncologic targets in cancer

The platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) play a key role in signaling pathways in oncogenesis. The overexpression of PDGFs and PDGFRs and the oncogenic alterations of these receptors have been implicated in human cancers and correlated significantly with poor outcomes. This review discusses the biology of the PDGF isoforms and receptors briefly, and their role in oncogenesis. Also, the attractiveness of targeting PDGFs and PDGFRs, based on a wide display of oncologic alterations in cancers, diverse therapeutic strategies, their roles in resistance to cancer treatments with prospects of overcoming drug resistance, and the extent to which validated biomarkers have been developed for effective PDGFs and PDGFRs-based cancer management are discussed.

The PDGF system and its antagonists in liver fibrosis

Platelet derived growth factor (PDGF) signaling plays an important role in activated hepatic stellate cells and portal fibroblast proliferation, chemotaxis, migration and cell survival. PDGF receptors and ligands are upregulated in experimental liver fibrotic models as well as in human liver fibrotic diseases. Blocking of PDGF signaling ameliorates experimental liver fibrogenesis. The plurality of molecular and cellular activities of PDGF and its involvement in initiation, progression and resolution of hepatic fibrogenesis offers an infinite number of therapeutic possibilities. These include the application of therapeutic antibodies (e.g. AbyD3263, MOR8457) which specifically sequester individual PDGF isoforms or the inhibition of PDGF isoforms by synthetic aptamers. In particular, the isolation of innovative slow off-rate modified aptamers (e.g., SOMAmer SL1 and SL5) that carry functional groups absent in natural nucleic acids by the Systematic Evolution of Ligands by EXponential (SELEX) enrichment technique offers the possibility to design high affinity aptamers that target PDGF isoforms for clinical purposes. Dominant-negative soluble PDGF receptors are also effective in attenuation of hepatic stellate cell proliferation and hepatic fibrogenesis. Moreover, some multikinase inhibitors targeting PDGF signaling have been intensively tested during the last decade and are on the way into advanced preclinical studies and clinical trials. This narrative review aims to gauge the recent progression of research into PDGF systems and liver fibrosis.

The pivotal role of PDGF and its receptor isoforms in adipose-derived stem cells.

Platelet-derived growth factor (PDGF) is one of the growth factors that reportedly regulates cell growth and division of mesenchymal cells. Although PDGF isoforms and their receptors reportedly play a pivotal role in mesenchymal stem cell regulation, there is a paucity of literature reviewing the role of PDGF in adipose-derived stem cells (ASCs). Therefore, we summarized previous reports on the expression and functional roles of PDGF and its receptor isoforms in this review. In addition, we examined findings pertaining to underlying molecular mechanisms and signaling pathways with special focus on PDGF-D/PDGFRβ. ASCs only express PDGF-A, -C, -D, PDGFRα, and PDGFRβ. PDGFRα expression decreases with adipocyte lineage, while PDGFRβ inhibits white adipocyte differentiation. In addition, PDGFRβ induces proliferation, migration, and angiogenesis and up-regulates the expression of paracrine factors in ASCs. Although PDGF-B and -D mediate their functions mainly by PDGFRβ and ROS generation, there are many differences between them in terms of regulating ASCs. PDGF-D is endogenous, generates ROS via the mitochondrial electron transport system, and regulates the autocrine loop of ASCs in vivo. Furthermore, PDGF-D has stronger mitogenic effects than PDGF-B.

Platelet-Rich Gel Supernatants Stimulate the Release of Anti-Inflammatory Proteins on Culture Media of Normal Equine Synovial Membrane Explants.

The aims were as follows: (1) to evaluate the effects at 48 and 96 h of two concentrations (25 and 50%) of leukocyte and platelet-rich gel (L-PRG) and pure PRG (P-PRG) supernatants on the production/degradation in normal equine synovial membrane explants (SME) of platelet derived growth factor isoform BB, transforming growth factor beta-1, tumor necrosis factor alpha, interleukin (IL-) 4 (IL-4), IL-1 receptor antagonist (IL-1ra), and hyaluronan (HA) synthesis and (2) to correlate these molecules with their respective PRG supernatant treatments. SME from 6 horses were cultured for 96 h with L-PRG and P-PRG supernatants at 25 and 50% concentrations, respectively. SME culture media were changed each 48 h and used for determination by ELISA of the molecules, which were also determined in synovial fluid. 25% L-PRG supernatant produced a sustained release over time of IL-1ra and a gradual release of HA, whereas 50% L-PRG supernatant produced a sustained increase over time of IL-4 and HA. 50% P-PRG supernatant produced an increased and sustained production of IL-1ra and IL-4. The cellular composition and the articular concentration (volume) of a platelet-rich plasma preparation could affect the anti-inflammatory and anabolic joint responses in horses with osteoarthritis.

Proteomic and biomarker studies and neurological complications of pediatric sickle cell disease.

Biomarker analysis and proteomic discovery in pediatric sickle cell disease has the potential to lead to important discoveries and improve care. The aim of this review article is to describe proteomic and biomarker articles involving neurological and developmental complications in this population. A systematic review was conducted to identify relevant research publications. Articles were selected for children under the age of 21 years with the most common subtypes of sickle cell disease. Included articles focused on growth factors (platelet-derived growth factor), intra and extracellular brain proteins (glial fibrillary acidic protein, brain-derived neurotrophic factor), and inflammatory and coagulation markers (interleukin-1β, l-selectin, thrombospondin-1, erythrocyte, and platelet-derived microparticles). Positive findings include increases in plasma brain-derived neurotrophic factor and platelet-derived growth factor with elevated transcranial Dopplers velocities, increases in platelet-derived growth factor isoform AA with overt stroke, and increases in glial fibrillary acidic protein with acute brain injury. These promising potential neuro-biomarkers provide insight into pathophysiologic processes and clinical events, but their clinical utility is yet to be established. Additional proteomics research is needed, including broad-based proteomic discovery of plasma constituents and blood cell proteins, as well as urine and cerebrospinal fluid components, before, during and after neurological and developmental complications.

Platelet-derived growth factors (PDGFs) in glomerular and tubulointerstitial fibrosis

Renal fibrosis is the hallmark of chronic kidney disease progression and is characterized by an exaggerated wound-healing process with the production of renal scar tissue. It comprises both the glomerular and the tubulointerstitial compartments. Among the factors that contribute to kidney fibrosis, the members of the platelet-derived growth factor (PDGF) family are among the best characterized ones. They appear to be the key factors in driving renal fibrosis, independent of the underlying kidney disease. The PDGF family consists of four isoforms (PDGF-A, -B, -C, and -D) and two receptor chains (PDGFR-α and -β), which are constitutively or inducibly expressed in most renal cells. These components have an irreplaceable role in kidney development by recruitment of mesenchymal cells to the glomerular and tubulointerstitial compartments. They further regulate multiple pathophysiologic processes including cell proliferation, cell migration, expression and accumulation of extracellular matrix, production and secretion of pro- and anti-inflammatory mediators, vascular permeability, and hemodynamics. This review provides a brief update on the role of different PDGF isoforms in the development of glomerulosclerosis and tubulointerstitial fibrosis, newly identified endogeneous PDGF antagonists, and resulting potential therapies.

Differential gene expression and immunolocalization of platelet-derived growth factors and their receptors in caprine ovaries

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-β were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-β (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-β were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats.

Platelet-derived growth factor BB and DD and angiopoietin1 are altered in follicular fluid from polycystic ovary syndrome patients.

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet-derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin-5 was significantly increased in endothelial cells incubated for 24 hr in the presence of FF from PCOS versus from control patients, while vascular-endothelial cadherin, β-catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF-induced vascular permeability in the PCOS ovary by regulating endothelial-cell-junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women.

Aptamer-functionalized hybrid carbon nanofiber FET-type electrode for a highly sensitive and selective platelet-derived growth factor biosensor.

Precise selectivity and rapid responses to target biomolecules are important in the development of biosensors. In particular, highly sensitive and selective biosensors have been used in clinical treatment to detect factors such as cancer oncoproteins and endocrine disruptors. Herein, highly sensitive liquid electrolyte field-effect transistor (FET) system biosensors were fabricated to detect platelet-derived growth factor (PDGF) using a PDGF-B binding aptamer conjugated with carboxylic polypyrrole-coated metal oxide-decorated carbon nanofibers (CPMCNFs) as the signal transducer. First, CPMCNFs were fabricated using vapor deposition polymerization (VDP) of the carboxylic pryrrole monomer (CPy) on metal oxide-decorated carbon nanofiber (MCNF) surfaces with no treatment for carbon surface functionalization. Furthermore, a 3 nm thick uniformly coated carboxylic polypyrrole (CPPy) layer was formed without aggregation. The CPMCNFs were integrated with the PDGF-B binding aptamer and immobilized on the interdigitated array substrate by covalent anchoring to produce a FET-type biosensor transducer. The PDGF-B binding aptamer conjugated CPMCNF (CPB-Apt) FET sensor was highly sensitive (5 fM) and extremely selective for isoforms of PDGFs. Additionally, the CPB-Apt FET sensor could be reused over a few weeks.

Reduced platelet-derived growth factor receptor expression is a primary feature of human bronchopulmonary dysplasia.

Animal studies have shown that platelet-derived growth factor (PDGF) signaling is required for normal alveolarization. Changes in PDGF receptor (PDGFR) expression in infants with bronchopulmonary dysplasia (BPD), a disease of hypoalveolarization, have not been examined. We hypothesized that PDGFR expression is reduced in neonatal lung mesenchymal stromal cells (MSCs) from infants who develop BPD. MSCs from tracheal aspirates of premature infants requiring mechanical ventilation in the first week of life were studied. MSC migration was assessed in a Boyden chamber. Human lung tissue was obtained from the University of Rochester Neonatal Lung Biorepository. Neonatal mice were exposed to air or 75% oxygen for 14 days. PDGFR expression was quantified by qPCR, immunoblotting, and stereology. MSCs were isolated from 25 neonates (mean gestational age 27.7 wk); 13 developed BPD and 12 did not. MSCs from infants who develop BPD showed lower PDGFR-α and PDGFR-β mRNA and protein expression and decreased migration to PDGF isoforms. Lungs from infants dying with BPD show thickened alveolar walls and paucity of PDGFR-α-positive cells in the dysmorphic alveolar septa. Similarly, lungs from hyperoxia-exposed neonatal mice showed lower expression of PDGFR-α and PDGFR-β, with significant reductions in the volume of PDGFR-α-positive alveolar tips. In conclusion, MSCs from infants who develop BPD hold stable alterations in PDGFR gene expression that favor hypoalveolarization. These data demonstrate that defective PDGFR signaling is a primary feature of human BPD.

Analysis of growth factors in serum and induced sputum from patients with asthma.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), the AA and BB isoforms of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) are involved in the pathogenesis of airway inflammation in asthma. In the present study, the associations between asthmatic phenotypes and the expression levels of these mediators in induced sputum and serum were investigated. A total of 62 asthmatic patients were divided into eosinophilic or neutrophilic phenotypes by cytological classification of the induced sputum. In addition, patients were classified according to lung function (FEV1/FVC >70% or FEV1/FVC <70%) and asthma severity (mild, moderate or severe). The concentrations of EGF, bFGF, PDGF-AA, PDGF-BB and VEGF in the serum and induced sputum were measured using sandwich enzyme immunoassays. VEGF levels in the serum and induced sputum were higher in patients with an eosinophilic phenotype compared with those with a neutrophilic phenotype. In addition, VEGF expression was higher in patients with an FEV1/FVC value of <70% as compared with patients with an FEV1/FVC value of >70%. Furthermore, the levels of VEGF were higher in patients with severe asthma compared with the patients with mild and moderate asthma. There were no statistically significant differences observed with regard to EGF, bFGF, PDGF-AA and PDGF-BB levels among the various phenotypes. Therefore, the observations of the present study indicated that increased VEGF expression in the serum and induced sputum of patients may be associated with eosinophilic airway inflammation, severe airflow limitation and the severity of asthma.

The roles of platelet-derived growth factors and their receptors in brain radiation necrosis

BackgroundBrain radiation necrosis (RN) occurring after radiotherapy is a serious complication. We and others have performed several treatments for RN, using anticoagulants, corticosteroids, surgical resection and bevacizumab. However, the mechanisms underlying RN have not yet been completely elucidated. For more than a decade, platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) have been extensively studied in many biological processes. These proteins influence a wide range of biological responses and participate in many normal and pathological conditions. In this study, we demonstrated that PDGF isoforms (PDGF-A, B, C, and D) and PDGFRs (PDGFR-α and β) are involved in the pathogenesis of human brain RN. We speculated on their roles, with a focus on their potential involvement in angiogenesis and inflammation in RN.MethodsSeven surgical specimens of RN, obtained from 2006 to 2013 at our department, were subjected to histopathological analyses and stained with hematoxylin and eosin. We qualitatively analyzed the protein expression of each isoform of PDGF by immunohistochemistry. We also examined their expression with double immunofluorescence.ResultsAll PDGFs were expressed in macrophages, microglia, and endothelial cells in the boundary of the core of RN, namely, the perinecrotic area (PN), as well as in undamaged brain tissue (UB). PDGF-C, D and PDGFR-α were also expressed in reactive astrocytes in PN. PDGFs and PDGFR-α were scarcely detected in UB, but PDGFR-β was specifically expressed in endothelial cells not only in PN but also in UB.ConclusionsPDGFs/PDGFRs play critical roles in angiogenesis and possibly in inflammation, and they contribute to the pathogenesis of RN, irrespective of the original tumor pathology and applied radiation modality. Treatments for the inhibition of PDGF-C, PDGF-D, and PDGFR-α may provide new approaches for the treatment of RN induced by common radiation therapies.

Bacteriostatic effect of equine pure platelet-rich plasma and other blood products against methicillin-sensitive Staphylococcus aureus. An in vitro study.

1) To evaluate the bacteriostatic in vitro effect of pure platelet-rich plasma (P-PRP), pure platelet-rich gel (P-PRG), leukocyte-poor gel (LPG), platelet-poor plasma (PPP), and heat inactivated plasma (IP) against methicillin-sensitive Staphylococcus aureus (MSSA) over a period of 24 hours. 2) To determine the degradation of platelet factor-4 (PF-4), transforming growth factor beta 1 (TGF-β1), and platelet-derived growth factor isoform BB (PDGF-BB) in these equine blood components. 3) To establish correlations between platelet and leukocyte counts, PF-4 concentrations, and MSSA growth. Fourteen horses were used. Blood components were obtained by a manual protocol. Every blood component was mixed with MSSA and Müller-Hinton Broth and cultured at 37°C for 24 hours. Samples for the determination of bacterial growth (colony-forming units) and PF-4, TGF-β1 and PDGF-BB concentrations were taken at one, four, eight, 12 and 24 hours. The bacterial growth was significantly (p = 0.01) inhibited for P-PRP, P-PRG, LPG and PPP in comparison with IP and, the positive control group during the first 12 hours. The P-PRG had higher and sustained TGF-β1 and PDGF-BB concentrations over time in comparison with the other blood components. The plasma complement could be one of the most responsible components of the in vitro bacteriostatic effect of P-PRP, P-PRG, LPG and PPP against MSSA. Additionally, P-PRG was the better biomaterial because it had an acceptable bacteriostatic effect and the highest concentration of growth factors.

Transforming Growth Factor β Inhibits Platelet Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation via Akt-Independent, Smad-Mediated Cyclin D1 Downregulation

In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.

Analysis of Mice Lacking the Heparin-Binding Splice Isoform of Platelet-Derived Growth Factor A

Platelet-derived growth factor A-chain (PDGF-A) exists in two evolutionarily conserved isoforms, PDGF-Along and PDGF-Ashort, generated by alternative RNA splicing. They differ by the presence (in PDGF-Along) or absence (in PDGF-Ashort) of a carboxy-terminal heparin/heparan sulfate proteoglycan-binding motif. In mice, similar motifs present in other members of the PDGF and vascular endothelial growth factor (VEGF) families have been functionally analyzed in vivo, but the specific physiological importance of PDGF-Along has not been explored previously. Here, we analyzed the absolute and relative expression of the two PDGF-A splice isoforms during early postnatal organ development in the mouse and report on the generation of a Pdgfa allele (Pdgfa(Δex6)) incapable of producing PDGF-Along due to a deletion of the exon 6 splice acceptor site. In situations of limiting PDGF-A signaling through PDGF receptor alpha (PDGFRα), or in mice lacking PDGF-C, homozygous carriers of Pdgfa(Δex6) showed abnormal development of the lung, intestine, and vertebral column, pinpointing developmental processes where PDGF-Along may play a physiological role.

Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs

This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function.