Platelet-derived Growth Factor Isoforms Research Articles

Deletion of platelet-derived growth factor receptor β suppresses tumorigenesis in metabolic dysfunction-associated steatohepatitis (MASH) mice with diabetes

The platelet-derived growth factor (PDGF) family contributes to the progression of steatohepatitis; however, changes in and the characteristics of isoform-specific expression remain unclear. Since diabetes is a major driver of metabolic dysfunction-associated steatohepatitis (MASH), we characterized the mouse model of diabetic MASH (dMASH) by focusing on PDGF signaling. Pdgfa-d expression was markedly higher in hepatic stellate cells among flow-sorted cells in control mice and also increased in dMASH. In contrast, a reanalysis of human single-cell RNA-Seq data showed the distinct distribution of each PDGF isoform with disease progression. Furthermore, inflammation and fibrosis in the liver were less severe in diabetic MASH using tamoxifen-induced PDGF receptor β (PDGFRβ)-deficient mice (KO) than in control dMASH using floxed mice (FL) at 12 weeks old. Despite the absence of tumors, the expression of tumor-related genes was lower in KO than in FL. Tumorigenesis was significantly lower in 20-week-old KO. An Ingenuity Pathway Analysis of differentially expressed miRNA between FL and KO identified functional networks associated with hepatotoxicity and cancer. Therefore, PDGFRβ signals play important roles in the progression of steatohepatitis and tumorigenesis in MASH, with the modulation of miRNA expression posited as a potential underlying mechanism.

Comparative Evaluation of Platelet-derived Growth Factor Isoforms between Platelet-rich Fibrin Prepared with Three Different Centrifugation Protocols

Comparative Evaluation of Platelet-derived Growth Factor Isoforms between Platelet-rich Fibrin Prepared with Three Different Centrifugation Protocols

The E3 Ubiquitin Ligase TRIM21 Regulates Basal Levels of PDGFRβ.

Activation of platelet-derived growth factor (PDGF) receptors α and β (PDGFRα and PDGFRβ) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRβ in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRβ. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRβ in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRβ in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRβ, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRβ, thus suggesting that ubiquitination of PDGFRβ by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.

PDGF-AB Reduces Myofibroblast Differentiation Without Increasing Proliferation After Myocardial Infarction

After myocardial infarction (MI), fibroblasts progress from proliferative to myofibroblast states, resulting in fibrosis. Platelet-derived growth factors (PDGFs) are reported to induce fibroblast proliferation, myofibroblast differentiation, and fibrosis. However, we have previously shown that PDGFs improve heart function post-MI without increasing fibrosis. We treated human cardiac fibroblasts with PDGF isoforms then performed RNA sequencing to show that PDGFs reduced cardiac fibroblasts myofibroblast differentiation and downregulated cell cycle pathways. Using mouse/pig MI models, we reveal that PDGF-AB infusion increases cell-cell interactions, reduces myofibroblast differentiation, does not affect proliferation, and accelerates scar formation. RNA sequencing of pig hearts after MI showed that PDGF-AB reduces inflammatory cytokines and alters both transcript variants and long noncoding RNA expression in cell cycle pathways. We propose that PDGF-AB could be used therapeutically to manipulate post-MI scar maturation with subsequent beneficial effects on cardiac function.

CD44 Depletion in Glioblastoma Cells Suppresses Growth and Stemness and Induces Senescence.

Simple SummaryThe hyaluronan receptor CD44 has an important role in glioblastoma multiforme (GBM) progression, but the precise mechanisms have not been elucidated. We have analyzed U251MG glioma cells, expressing CD44 or not, and grown in stem cell-like enriched spheres. Our results revealed that CD44 is important for cell growth and stemness, and for the prevention of senescence. Analysis by RNA sequencing revealed that CD44 is important for the interaction with the hyaluronan-enriched microenvironment. In addition, CD44 depletion impairs certain gene signatures, such as those for platelet-derived growth factor (PDGF) isoforms and PDGF receptors, as well as signatures related to hypoxia, glycolysis, and anti-tumor immune responses.Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the γ-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.

The Platelet-Derived Growth Factor Pathway in Pulmonary Arterial Hypertension: Still an Interesting Target?

The lack of curative options for pulmonary arterial hypertension drives important research to understand the mechanisms underlying this devastating disease. Among the main identified pathways, the platelet-derived growth factor (PDGF) pathway was established to control vascular remodeling and anti-PDGF receptor (PDGFR) drugs were shown to reverse the disease in experimental models. Four different isoforms of PDGF are produced by various cell types in the lung. PDGFs control vascular cells migration, proliferation and survival through binding to their receptors PDGFRα and β. They elicit multiple intracellular signaling pathways which have been particularly studied in pulmonary smooth muscle cells. Activation of the PDGF pathway has been demonstrated both in patients and in pulmonary hypertension (PH) experimental models. Tyrosine kinase inhibitors (TKI) are numerous but without real specificity and Imatinib, one of the most specific, resulted in beneficial effects. However, adverse events and treatment discontinuation discouraged to pursue this therapy. Novel therapeutic strategies are currently under experimental evaluation. For TKI, they include intratracheal drug administration, low dosage or nanoparticles delivery. Specific anti-PDGF and anti-PDGFR molecules can also be designed such as new TKI, soluble receptors, aptamers or oligonucleotides.

The epigenetic enzyme DOT1L orchestrates vascular smooth muscle cell-monocyte crosstalk and protects against atherosclerosis via the NF-κB pathway.

Histone H3 dimethylation at lysine 79 is a key epigenetic mark uniquely induced by methyltransferase disruptor of telomeric silencing 1-like (DOT1L). We aimed to determine whether DOT1L modulates vascular smooth muscle cell (VSMC) phenotype and how it might affect atherosclerosis in vitro and in vivo, unravelling the related mechanism. Gene expression screening of VSMCs stimulated with the BB isoform of platelet-derived growth factor led us to identify Dot1l as an early up-regulated epigenetic factor. Mouse and human atherosclerotic lesions were assessed for Dot1l expression, which resulted specifically localized in the VSMC compartment. The relevance of Dot1l to atherosclerosis pathogenesis was assessed through deletion of its gene in the VSMCs via an inducible, tissue-specific knock-out mouse model crossed with the ApoE-/- high-fat diet model of atherosclerosis. We found that the inactivation of Dot1l significantly reduced the progression of the disease. By combining RNA- and H3K79me2-chromatin immunoprecipitation-sequencing, we found that DOT1L and its induced H3K79me2 mark directly regulate the transcription of Nf-κB-1 and -2, master modulators of inflammation, which in turn induce the expression of CCL5 and CXCL10, cytokines fundamentally involved in atherosclerosis development. Finally, a correlation between coronary artery disease and genetic variations in the DOT1L gene was found because specific polymorphisms are associated with increased mRNA expression. DOT1L plays a key role in the epigenetic control of VSMC gene expression, leading to atherosclerosis development. Results identify DOT1L as a potential therapeutic target for vascular diseases.

Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants

BackgroundLimited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.MethodsFull-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, −AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.ResultsSuperficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.ConclusionsIn enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFR\u03b2 and affect PDGF-BB-induced STAT3 signalling

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

Atherogenic oxoaldehyde of cholesterol induces innate immune response in monocytes and macrophages.

Cholesterol oxidation product, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol 5,6-secosterol, ChSeco or Atheronal-A), formed at inflammatory sites, has been shown to promote monocyte differentiation into macrophages and cause elevated expression of macrophage scavenger receptors. Since inflammation is a key event at all stages of atherosclerotic plaque formation, the pro-inflammatory actions of ChSeco in human THP-1 monocytes and mouse J774 macrophages were investigated in the present study by employing ELISA, qRT-PCR, and functional assays. An increase in the secretion of interleukin-8 and platelet-derived growth factor (PDGF) isoform AA and, to a limited extent, PDGF isoform BB was observed into the culture medium of THP-1 monocytes exposed to ChSeco. However, no changes were seen in the secretion of tumor necrosis factor-alpha. In J774 macrophages treated with ChSeco, there was an upregulation of gene expression of several pro-inflammatory cytokines and their receptors. Concomitantly, there was down-regulation of gene expression of interleukin-1ß, interleukin-10, and lymphotoxin-beta. An increase in the release of interleukin-18 and chemokine (C-C motif) ligand-20 from J774 macrophages (which corroborated well with their gene expression profiles) and increased binding of THP-1 monocytes to ChSeco-exposed human aortic endothelial cells were observed. The results of the present study, for the first time, demonstrate the pro-inflammatory action of ChSeco and suggest the underlying pro-atherogenic mechanisms. These could be mediated through enhanced monocyte recruitment into the sub-endothelial space and subsequent proliferation of smooth muscle cells under the influence of monocyte-derived PDGF.

Inhibition of platelet-derived growth factor C and their receptors additionally increases doxorubicin effects in triple-negative breast cancer cells

Complex of platelet-derived growth factor (PDGF) isoforms and PDGF receptors have important functions in the regulation of growth and survival of various cell types. Herein, it was found that aberrant PDGFC expression is closely associated with survival rates in triple-negative breast cancer (TNBC) patients. In addition, PDGFC expression was identified to be significantly increased in TNBC cells unlike other subtypes such as PDGFA, PDGFB, and PDGFD. Apparently, the effects of specific PDGF receptor (PDGFR) inhibitors such as sunitinib and ponatinib on HCC1806 and Hs578T TNBC cells were investigated. Both inhibitors decreased cell viability in a dose-dependent manner. In addition, the inhibitors completely inhibited cell growth in both the cell lines and decreased the expression of matrix metalloproteinase-1 (MMP-1), one of the metastasis-related genes. Cell migration was also decreased by the inhibitors. Finally, the combined effects of the inhibitors with doxorubicin (DOX) were investigated. The results showed that the combination of two PDGFR inhibitors with DOX inhibited the growth of cells and enhanced the apoptotic cell death more uniformly than DOX. Consequently, it is demonstrated that PDGFR inhibitors, sunitinib and ponatinib hold the potential for effective treatment of TNBC.

Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study.

Simple SummaryThere are few publications on the subject of canine platelet concentrates and further studies are required to characterize these for clinical applications. The aim of this study determined platelet, erythrocyte, and leukocyte counts in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy). Whole blood (WB) from 30 healthy dogs was used. In 10 L-PRP bovine thrombin activated samples platelet-derived growth factor isoform BB (PDGF-BB) concentration was also measured. The CPUNT 20 produced clinically useful quantities of sterile canine L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Activated L-PRP has an average of 3442 ± 2061 pg/mL of PDGF-BB.Background: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. Methods: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) according to the manufacturer’s instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. Results: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Conclusion: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.

PDGF regulated migration of mesenchymal stem cells towards malignancy acts via the PI3K signaling pathway.

Mesenchymal stem cells (MSCs) have been described in breast cancer models to migrate towards carcinoma and integrate into tumor associated stroma supporting tumor growth, increasing their metastatic potency and contributing to tumor-angiogenesis. Platelet-derived growth factor (PDGF) isoforms (AA, BB, CC) stimulate growth, survival and motility of MSCs and certain other cell types. Noteworthy, breast carcinomas are known to express PDGF. We aim to further shed light on i) the relevance of the different PDGF isoforms on adipose tissue derived stem cells (ASCs) migration and ii) the underlying pathway dependent on PDGF stimulation. Breast cancer cell lines were purchased and ASC's were isolated from murine subcutaneous adipose tissue. The transmigration of ASC's towards the PDGF-isoforms was assessed by using recombinant human PDGF-AA, PDGF-BB and PDGF-CC in a trans-well culture dish system. Transmigrated ASC's were quantified in 5 randomly selected fields per condition using fluorescence microscopy after calcein-staining. PDGF-BB depended transmigration of ASC's was verified by downregulation and overexpression of PDGF-BB in breast cancer cell line using lentiviral vectors. In addition, a PI3-kinase inhibitor (LY294002) and a MAP-kinase inhibitor (PD98059) were used to identify the pathway involved in the PDGF-BB mediated migration of ASC's towards tumor. ASC's transmigration significantly increased towards PDGF AA at 50 ng and only showed further increase by 500 ng which was similar to cell behavior when exposed to PDGF CC. In comparison, PDGF-BB significantly increased ASC's transmigration already at a low level of 5 ng with further significant increase for 20 ng and 40 ng. Cell transmigration was blocked with PDGFR-α antibodies but only for PDGF-AA and PDGF-CC whereas PDGFR-β blockage showed a significant effect on transmigration for PDGF-BB and PDGF-CC but not for PDGF-AA. Neutralizing antibodies in combination with PDGF receptor blockage confirmed findings. In addition, only PI3-kinase inhibitor but not the MEK-1 selective inhibitor caused a significant decrease of transmigration for ASCs towards breast cancer cells. The transmigration of ASC's is most significantly enhanced by PDGF-BB via the PI3-kinase pathway. This data support that PI3-kinase is an important key player for MSC migration towards malignancy which need further research to prevent tumor progression in early disease stage.

Crocetin inhibits PDGF-BB-induced proliferation and migration of retinal pigment epithelial cells

In proliferative vitreoretinopathy (PVR), the proliferation and migration of retinal pigment epithelial (RPE) cells are important to pathogenesis. Platelet-derived growth factor (PDGF) is an important factor in the underlying mechanism. Several studies have shown that PDGF induced the proliferation and migration effects on RPE cells in PVR. Crocetin—anantioxidant carotenoid that is abundant in saffron—has been shown to suppress the migration and proliferation of many cell types, but studies of the effects on RPE cell migration and proliferation are incomplete. Therefore, we investigated the inhibitory effect of crocetin on the proliferation and migration of ARPE-19 cells induced by PDGF-BB, an isoform of PDGF. The proliferation of cells was assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays. The apoptosis of cells was assessed by flow cytometric analysis. The migration of RPE cells was detected by a Transwell migration assay and an in vitro scratch assay. The levels of main regulatory proteins for apoptosis and the PDGF-BB-induced signaling pathway were determined by western blot analysis. The proliferation and migration of ARPE-19 cells treated with crocetin (100–400 μM) and PDGF-BB (20 ng/ml) were significantly inhibited in a concentration- and time-dependent manner. Crocetin exhibited potent inducing effects on the apoptosis of PDGF-BB-induced ARPE-19 cells via the modulation of Bcl-2 family regulators in a concentration-dependent manner. The inhibitory effects of crocetin on PDGF-BB-induced platelet-derived growth factor receptor β (PDGFRβ) and the underlying pathways of PI3K/Akt and ERK, p38, JNK activation were identified. The results showed that crocetin is an effective inhibitor of PDGF-BB-induced proliferation and migration of ARPE-19 cell through the downregulation of regulatory signaling pathways.

Equine suspensory ligament and tendon explants cultured with platelet-rich gel supernatants release different anti-inflammatory and anabolic mediators.

The aim of this study was to evaluate the release of pro- and anti-inflammatory as well as anabolic mediators stimulated by a leukocyte-reduced platelet-rich gel supernatant (Lr-PRGS) and a leukocyte-reduced plasma supernatant (Lr-PL) at two concentrations (25 and 50%) on normal equine suspensory ligament explants (SLEs) and tendon explants (TEs). SLEs and TEs from six horses were independently incubated for 48 h with Lr-PRGS and Lr-PL at concentrations of 25 and 50%, respectively. Samples were collected from the incubated tissues at 1 h and 48 h, which were employed for ELISA determination of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), IL-4, IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor isoform BB (PDGF-BB), transforming growth factor beta-1 (TGF-β1, and hyaluronic acid (HA). Overall, 50% Lr-PRGS induced significantly less IL-1β release than the other hemoderivatives in both tissues. At 48 h, both Lr-PRGS and 25% Lr-PL induced significantly higher TNF-α concentrations in SLEs when compared to TEs, whereas both Lr-PRGS concentrations induced significantly higher IL-4 concentrations in SLEs in comparison to TEs. IL-1ra release was not different between tissues. However, this cytokine was significantly higher in tissue explants cultured with both Lr-PRGS concentrations. HA concentration was lower in tissue explants cultured with all hemoderivatives at two concentrations when compared to the control group. The positive effects observed for ligaments and tendons treated with Lr-PRGS may be mediated by the inhibition of IL-1β release of and increased release of IL-4 and IL-1ra. Furthermore, PDGF-BB could be a polypeptide responsible for mediating the release of anti-inflammatory cytokines in SLEs and TEs incubated with Lr-PRGS.

Caracterização estrutural e papel do fator de crescimento derivado de plaquetas (PDGF) na foliculogênese ovariana

Background: : : : The platelet-derived growth factor (PDGF) is expressed in a wide variety of cell types, exerts a potent mitogenic role and acts on the growth, differentiation and cell chemotaxis. Studies have shown that during folliculogenesis, PDGF and their receptors are expressed in oocytes, granulosa cells and thecal cells of ovarian follicles at different developmental stages in several species. Although exist many information about its expression sites, as well as about its action in different cells types, the role of PDGF on ovarian folliculogenesis remains understudied. Thus, this article aims to review issues related to PDGF, suggesting the involvement of this mitogenic factor during follicular development. Review: Along this work, it was shown aspects related to structural characterization of PDGF and its receptors, as well as PDGF expression in different cells types, emphasizing its importance to follicular development. PDGF family is composed by four polypeptide chains (each encoded by a different gene), which are synthesized in the form of inactive pro-proteins. After a proteolytic processing, these chains undergo homo or heterodimerization, resulting in five isoforms (PDGF-AA, -BB, -AB, -CC e -DD). The cellular effects of these different PDGF isoforms are mediated by binding, with different specificities, to three transmembrane receptors isoforms of type tyrosine kinase generated by the association of subunits ? e ? (PDGFR- ??, - ??, - ??). PDGF was initially purified from platelets, being later verified its production by many varieties of cell types. It acts as an important mitogenic factor, especially to cells of mesenchymal origin as fibroblasts and smooth muscle cells, and also exerts a role on cellular migration and survival. Studies with PDGF and its receptors have demonstrated its importance as a paracrine growth factor for normal embryo development and angiogenesis. Although the action of this growth factor has been shown in several cellular responses, its role in ovarian folliculogenesis of mammals is understudied. Folliculogenesis, event initiated at pre-natal life in most species, can be defined as the process of follicular formation, growth and maturation, beggining with the primordial follicle formation and resulting in preovulatory stage. Until the present moment, the different PDGF types and their receptors were detected in ovarian follicles of murines, rodents, swines and humans according to developmental stage. Evidences suggest that this growth factor may acts as a potent regulator of ovarian function, increasing DNA synthesis in granulosa cells and stimulating theca cells growth in swines. In addition, PDGF has shown benefic effects in in vitro maturation of bovine oocytes when associated with follicle stimulating hormone (FSH). Conclusion: Nevertheless, the specific actions of PDGF on ovarian follicles development have not been described due to lack of data available in the literature about this subject. Therefore, this review was performed to clarify the involvement of this growth factor in the regulation of ovarian function in mammals. However, it is necessary to perform additional studies that may provide a greater understanding about the importance of PDGF during folicullogenesis.

Involvement of platelet-derived growth factor ligands and receptors in tumorigenesis.

Platelet-derived growth factor (PDGF) isoforms and their receptors have important roles during embryogenesis, particularly in the development of various mesenchymal cell types in different organs. In the adult, PDGF stimulates wound healing and regulates tissue homeostasis. However, overactivity of PDGF signalling is associated with malignancies and other diseases characterized by excessive cell proliferation, such as fibrotic conditions and atherosclerosis. In certain tumours, genetic or epigenetic alterations of the genes for PDGF ligands and receptors drive tumour cell proliferation and survival. Examples include the rare skin tumour dermatofibrosarcoma protuberance, which is driven by autocrine PDGF stimulation due to translocation of a PDGF gene, and certain gastrointestinal stromal tumours and leukaemias, which are driven by constitute activation of PDGF receptors due to point mutations and formation of fusion proteins ofthe receptors, respectively. Moreover, PDGF stimulates cells in tumour stroma and promotes angiogenesis as well as the development of cancer-associated fibroblasts, both of which promote tumour progression. Inhibitors of PDGF signalling may thus be of clinical usefulness in the treatment of certain tumours.

Difference in release kinetics of unwashed and washed platelet-released supernatants from bone substitute materials: the impact of platelet preparation modalities.

In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and β-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS. Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes. Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile.

JAK-STAT signalling and the atrial fibrillation promoting fibrotic substrate.

Left-atrial (LA) fibrosis is an important feature of many atrial fibrillation (AF) substrates. The JAK-STAT system contributes to cardiac remodelling, but its role in AF is unknown. Here we investigated JAK-STAT changes in an AF-model and their potential contributions to LA-fibrosis. LA-remodelling was studied in dogs with heart failure (HF) induced by ventricular tachypacing (VTP, 240 bpm), and in mice with left-ventricular (LV) dysfunction due to myocardial infarction (MI). The selective STAT-3 inhibitor S3I-201 was administered to fibroblasts in vitro or mice in vivo (10 mg/kg/d, osmotic mini-pump). HF-dogs developed LA-selective fibrosis and AF-susceptibility at 1-week VTP. The mRNA-expression of platelet-derived growth factor (PDGF, a JAK-STAT activator) isoforms A, C and D, as well as JAK2, increased in LA fibroblasts from 1-week VTP. HF upregulated protein-expression of PDGF-receptor-β and phosphorylated (activated) signal transducer and activator of transcription 3 (STAT3) in LA. PDGF-AB stimulation of LA fibroblasts increased PDGFR-α, STAT3 and phosphorylated-STAT3 expression, as well as collagen-1 and fibronectin-1 protein secretion (by 1.6- to 20-fold), with smaller changes in LV fibroblasts. Phosphorylated-STAT3 and collagen upregulation were suppressed by the JAK2 inhibitor AG-490, PDGF receptor inhibitor AG1296 and STAT3-inhibitor SI3-201. In vivo S3I-201 treatment of MI-mice attenuated LA-fibrosis, LA-dilation and P-wave duration changes versus vehicle-control. HF activates the LA JAK-STAT system and enhances PDGF-signalling. JAK-STAT inhibition reduces the profibrotic effects of PDGF stimulation on canine fibroblasts in vitro while attenuating in vivo LA-fibrosis and remodelling in post-MI mice, suggesting that the JAK/STAT pathway contributes to LA-fibrogenesis and might be a potential target for LA-fibrosis prevention.

Atorvastatin Calcium Inhibits PDGF-ββ-Induced Proliferation and Migration of VSMCs Through the G0/G1 Cell Cycle Arrest and Suppression of Activated PDGFRβ-PI3K-Akt Signaling Cascade

Background/Aims: Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular lesions, such as atherosclerosis and restenosis. PDGF-ββ, an isoform of PDGF (platelet-derived growth factor), has been demonstrated to induce proliferation and migration of VSMCs. Atorvastatin calcium, a selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, has favorable protective effects on VSMCs. This study examined the effects of atorvastatin calcium on the proliferation and migration of PDGF-ββ-treated VSMCs, as well as its underlying mechanisms. Methods: MTT assays, Edu imaging, cell cycle analysis, wound healing assays, transwell migration assays, and western blot analysis were performed. Results: Atorvastatin calcium significantly inhibited cell proliferation, DNA synthesis and cell migration of PDGF-ββ-treated VSMCs. We demonstrated that atorvastatin calcium induced cell cycle arrest in the G0/G1 phase in response to PDGF-ββ stimulation and decreased the expression of G0/G1-specific regulatory proteins, including proliferating cell nuclear antigen (PCNA), CDK2, cyclin D1, cyclin E and CDK4 in PDGF-ββ-treated VSMCs. Moreover, pretreatment with atorvastatin calcium inhibited the PDGF-ββ-treated phosphorylation of PDGFRβ and Akt, whereas atorvastatin calcium did not affect the phosphorylation of PLC-γ1 or (ERK) 1/2. Conclusion: Our data suggested that atorvastatin calcium inhibited abnormal proliferation and migration of VSMCs through G0/G1 cell cycle arrest and suppression of the PDGFRβ-Akt signaling cascade.