Rosuvastatin Inhibits the Apoptosis of Platelet-Derived Growth Factor-Stimulated Vascular Smooth Muscle Cells by Inhibiting p38 via Autophagy.

The secretion of platelet-derived growth factors (PDGFs) into vascular smooth muscle cells (VSMCs) induced by specific stimuli, such as oxidized low-density lipoprotein (LDL) cholesterol, initially increases the proliferation and migration of VSMCs, and continuous stimulation leads to VSMC apoptosis, resulting in the formation of atheroma. Autophagy suppresses VSMC apoptosis, and statins can activate autophagy. Thus, this study aimed to investigate the mechanism of the autophagy-mediated vasoprotective activity of rosuvastatin, one of the most potent statins, in VSMCs continuously stimulated with PDGF-BB, a PDGF isoform, at a high concentration (100 ng/ml) to induce phenotypic switching of VSMC. Rosuvastatin inhibited apoptosis in a concentration-dependent manner by reducing cleaved caspase-3 and interleukin-1ß (IL-1ß) levels and reduced intracellular reactive oxygen species (ROS) levels in PDGF-stimulated VSMCs. It also inhibited PDGF-induced p38 phosphorylation and increased the expression of microtubule-associated protein light chain 3 (LC3) and the conversion of LC3-I to LC3-II in PDGF-stimulated VSMCs. The ability of rosuvastatin to inhibit apoptosis and p38 phosphorylation was suppressed by treatment with 3-methyladenine (an autophagy inhibitor) but promoted by rapamycin (an autophagy activator) treatment. SB203580, a p38 inhibitor, reduced the PDGF-induced increase in intracellular ROS levels and inhibited the formation of cleaved caspase-3, indicating the suppression of apoptosis. In carotid ligation model mice, rosuvastatin decreased the thickness and area of the intima and increased the area of the lumen. In conclusion, our observations suggest that rosuvastatin inhibits p38 phosphorylation through autophagy and subsequently reduces intracellular ROS levels, leading to its vasoprotective activity. SIGNIFICANCE STATEMENT: This study shows the mechanism responsible for the vasoprotective activity of rosuvastatin in vascular smooth muscle cells under prolonged platelet-derived growth factor stimulation. Rosuvastatin inhibits p38 activation through autophagy, thereby suppressing intracellular reactive oxygen species levels, leading to the inhibition of apoptosis and reductions in the intima thickness and area. Overall, these results suggest that rosuvastatin can be used as a novel treatment to manage chronic vascular diseases such as atherosclerosis.

Methotrexate inhibits effects of platelet-derived growth factor and interleukin-1ß on rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: A key feature of joints in rheumatoid arthritis (RA) is the formation of hyperplastic destructive pannus tissue, which is orchestrated by activated fibroblast-like synoviocytes (FLS). We have demonstrated that the RA risk gene and tumor suppressor Limb bud and heart development (LBH) regulates cell cycle progression in FLS. Methotrexate (MTX) is the first-line treatment for RA, but its mechanisms of action remain incompletely understood. Here, we studied the effects of MTX on mitogen-induced FLS proliferation and expression of cell cycle regulators in vitro. METHODS: Primary FLS from patients with RA or osteoarthritis were stimulated with the mitogen platelet-derived growth factor (PDGF) and the cytokine interleukin-1ß (IL-1ß) in the presence or absence of MTX. Cells were then subjected to qPCR for gene expression and cell cycle analysis by flow cytometry. RESULTS: Stimulation with PDGF and IL-1ß increased the percentage of FLS in the G2/M phase and shifted the cell morphology to a dendritic shape. These effects were inhibited by MTX. Furthermore, PDGF + IL-1ß reduced LBH mRNA expression. However, MTX treatment yielded significantly higher transcript levels of LBH, and of CDKN1A (p21) and TP53 (p53), compared to untreated samples upon mitogen stimulation. The expression of DNA methyltransferase-1 (DNMT1) was also higher in the presence of MTX and there was strong correlation between DNMT1 and LBH expression. CONCLUSIONS: Therapeutic concentrations of MTX abolish the effects of PDGF and IL-1ß on tumor suppressor expression and inhibit mitogen-promoted FLS proliferation. These data demonstrate novel and important effects of MTX on pathogenic effector cells in the joint, which might involve epigenetic mechanisms.

Inhibition of airway remodeling and inflammation by isoforskolin in PDGF-induced rat ASMCs and OVA-induced rat asthma model.

Isoforskolin (ISOF) has been reported to play an important role in many illnesses including respiratory, cardiovascular and ophthalmologic diseases. In our study, we aimed to investigate how ISOF regulates airway remodeling and inflammation in asthma. Based on SO2-stimulated mouse cough model, we assessed the role of ISOF in cough and secretion of phlegm. Afterwards, platelet derived growth factor (PDGF)-induced primary rat airway smooth muscle cell (ASMC) model and ovalbumin (OVA)-induced rat asthma model were used to continue our following research. Our results showed that ISOF could prolong the cough latent period, reduce the cough times in two minutes, and increase the excretion of red phenol, which suggested the antitussive and expectorant effects of ISOF. Besides, ISOF pretreatment reversed the hypotonicity and cytoskeleton remodeling in PDGF-induced ASMCs, and reduced mucus hypersecretion and collagen overdeposition in OVA-induced rat asthma model, which indicated its inhibition on airway remodeling in vitro and in vivo. Moreover, ISOF reduced the invasion of inflammatory cells into bronchoalveolar lavage fluid (BALF) and lungs, which revealed its inhibitory role in airway inflammation. The down-regulation of transforming growth factor ß1 (TGF-ß1) and interleukin-1ß (IL-1ß) upon ISOF treatment might be responsible for its anti-remodeling and anti-inflammation roles. In conclusion, ISOF can reduce cough and sputum, as well as inhibit airway remodeling and inflammation by regulating the expression of TGF-ß1 and IL-1ß. These data indicate the potency of ISOF in treating asthma and also provide insights into the development of new anti-asthma agent.

Retroviral delivery of platelet-derived growth factor to spinal cord progenitor cells drives the formation of intramedullary gliomas.

BACKGROUND: High-grade gliomas of the spinal cord are poorly understood tumors that are very commonly associated with bad outcomes. The transforming effects of platelet-derived growth factor (PDGF) on spinal cord glial progenitor cells may play an important role in the development of these tumors. OBJECTIVE: To investigate the possible tumor-initiating effects of PDGF overexpression in the spinal cord, we delivered a PDGF retrovirus directly into the substance of the spinal cord. METHODS: The spinal cords of wild-type adult rats were surgically exposed and injected with 106 colony-forming units of a green fluorescent protein-tagged, PDGF-expressing retrovirus. A control virus was injected to assess the cell types that become infected during retroviral delivery to the spinal cord. RESULTS: It was observed that PDGF overexpression in the spinal cord causes morbidity from high-grade intramedullary glioma formation between 27 and 49 days after PDGF retrovirus injection. Retroviral transduction was highly efficient with 100% of injected animals displaying the tumor phenotype. The tumors produced were highly proliferative, were locally invasive, and displayed the immunophenotype of virus-targeted glial progenitor cells (Olig2+PDGFR+NG2+GFAP-). CONCLUSION: PDGF is capable of driving glial progenitor cells within the adult spinal cord to form high-grade gliomas. Further investigation of PDGF signaling in the spinal cord is needed to better understand and treat these devastating tumors.

Nitidine Chloride inhibits breast cancer cells migration and invasion by suppressing c-Src/FAK associated signaling pathway.

Nitidine is a benzophenanthridine alkaloid, which has been shown to have anti-tumor properties. Here, we demonstrated that Nitidine Chloride (NC) could inhibit breast cancer cells migration and invasion both in vitro and in vivo. Meanwhile, the protrusion formation and partial proteolytic activity of MMP-9 and MMP-2 were attenuated by NC in a dose-dependent manner in MDA-MB-231 cells. Furthermore, addition NC to cells significantly decreases PDGF induced phosphorylation of c-Src, FAK, MAPKs, activation of RhoA, Rac1 and AP-1 transcriptional activity. Taken together, our results indicate that NC could have potential as a novel anti-metastasis drug to breast cancer.

α-Lipoic acid inhibits liver fibrosis through the attenuation of ROS-triggered signaling in hepatic stellate cells activated by PDGF and TGF-ß.

Reactive oxygen species (ROS) have been implicated in hepatic stellate cell activation and liver fibrosis. We previously reported that α-lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) inhibited toxicant-induced inflammation and ROS generation. In the present study, we further examined the effects of LA/DHLA on thioacetamide (TAA)-induced liver fibrosis in rats and the possible underlying mechanisms in hepatic stellate cells in vitro. We found that co-administration of LA to rats chronically treated with TAA inhibited the development of liver cirrhosis, as indicated by reductions in cirrhosis incidence, hepatic fibrosis, and AST/ALT activities. We also found that DHLA inhibited TGF-ß/PDGF-stimulated HSC-T6 activation and ROS generation. These effects could be mediated by the MAPK and PI3K/Akt pathways. According to our current results, LA may have a beneficial role in the treatment of chronic liver diseases caused by ongoing hepatic damage.

A review of nonclinical toxicology studies of becaplermin (rhPDGF-BB).

Becaplermin (recombinant human platelet-derived growth factor-BB [BB homodimer, rhPDGF-BB]) has demonstrated a favorable safety profile in a series of nonclinical studies designed to assess its systemic toxicity, sensitization, local irritation, and genotoxic potential. No significant local or systemic toxicity directly attributable to becaplermin was observed following single and multiple intravenous or subcutaneous administration at doses up to 3 mg/kg in monkeys. Administration of single large intravenous doses (up to 100 mg/kg) and repeated dosing at 1 or 3 mg/kg in mice resulted in rapidly reversible vasodilation and central nervous system depression. In a bone-toxicity study, becaplermin produced histomorphologic changes suggestive of accelerated bone remodeling, which were judged to be potentially reversible. Similar findings have not been observed in humans. Although becaplermin was not considered a dermal or ocular irritant, some skin-sensitizing effects were observed in animals; this finding was not unexpected for a recombinant human-derived protein. Becaplermin was not genotoxic in a variety of in vitro assays and in one in vivo assay.

Platelet-derived growth factor delays oligodendrocyte differentiation and axonal myelination in vivo in the anterior medullary velum of the developing rat.

The AA dimeric form of platelet-derived growth factor (PDGF-AA) is implicated in the differentiation of cells of the oligodendrocyte lineage, which express PDGF receptors of the alpha subunit type (PDGF-alphaR). In the present study, we show that a single injection of PDGF-AA into the cerebrospinal fluid of neonatal rats delays oligodendrocyte differentiation and interrupts the progress of myelination in the anterior medullary velum (AMV), a white matter tract roofing the IVth ventricle of the brain. PDGF-AA or saline was injected intrathecally in postnatal day (P) 7 rats, and the AMV was subsequently removed and immunolabelled with the oligodendrocyte-specific antibody Rip, at P9, P12, and P21, corresponding to postinjection days (PID) 2, 5, and 14. At P9 (PID2), myelination was retarded in PDGF-AA-treated rats as opposed to saline-treated controls but progressed rapidly after P12 (PID5). Quantification supported the qualitative observations that PDGF-AA mediated an acute decrease in the number of Rip+ oligodendrocytes at P9-12, which largely recovered by P21, suggesting that PDGF-AA may have delayed recruitment of myelinating oligodendrocytes. However, the definitive number of Rip+ oligodendrocytes in the AMV was not increased, suggesting that its action as a promoter of early oligodendrocyte survival may not ultimately affect the definitive number of myelinating oliogdendrocytes in vivo. We discuss the possibilities that excess PDGF-AA may have acted on early oligodendrocytes (precursors or preoligodendrocytes) to either (1) delay their differentiation by maintaining them in the cell cycle or (2) accelerate their differentiation, which may result in premature cell death in the absence of synchronised survival signals. This study supports a role for PDGF-AA in the timing of oligodendrocyte differentiation in vivo, as has been shown in vitro.

Tyrosine kinase inhibitor markedly suppresses the development of coronary lesions induced by long-term treatment with platelet-derived growth factor in pigs in vivo.

Platelet-derived growth factor (PDGF) plays an important role in the development of coronary atherosclerosis. However, it remains to be examined what morphologic and functional changes are induced in vivo by the long-term treatment with PDGF itself or what pharmacologic interventions could suppress those changes in vivo. Our study was designed to address these points. We examined the effects of long-term treatment with PDGF on the porcine coronary artery in vivo. Under aseptic conditions, the proximal segments of the left porcine coronary artery were gently wrapped with cotton mesh absorbing sepharose beads either with or without recombinant human PDGF-AA or -BB. Two weeks after the operation, coronary hyperconstrictions to intracoronary serotonin or histamine were noted at the sites treated with PDGF-AA or -BB. Histologically, neointimal formation and geometric remodeling (reduction of the total vessel area) were noted at the PDGF-treated sites. These functional and histologic changes of the coronary artery induced by PDGF were markedly inhibited by cotreatment with ST 638, a specific inhibitor of tyrosine kinases. A Western blot analysis showed that ST 638 markedly suppressed the PDGF-induced tyrosine phosphorylations in the coronary segment. These results indicate that long-term treatment with PDGF induces neointimal formation, geometric remodeling, and vasospastic responses in vivo, for all of which, activation of tyrosine kinases is substantially involved.

The effects of platelet-derived growth factor-BB and insulin-like growth factor-1 on epithelial dysplasia.

Growth factors are multi-functional and multi-targeted proteins which play a significant role in wound healing. Platelet-derived growth factor B-chain homodimer (PDGF-BB) and insulin-like growth factor-1 (IGF-1) have demonstrated efficacy for periodontal regeneration in animal models. Although primarily associated with wound healing, PDGF-BB and IGF-1 also facilitate growth of a number of malignant neoplasms. Of particular concern to periodontists is epithelial dysplasia, a necessary precursor to squamous cell carcinoma, the most common oral malignancy. Certain risk factors for oral cancer, such as tobacco, age, and alcohol, are also associated with an increased incidence of periodontal disease. The effects of the combination of PDGF-BB and IGF-1 on epithelial dysplasia have not previously been reported. The purpose of this study was to examine the effects of the combination of PDGF-BB and IGF-1 on epithelial dysplasia induced in the buccal cheek pouch of the Syrian golden hamster. A total of 66 hamsters received 18 applications of 0.5% dimethylbenzanthracene (DMBA), a topical carcinogen, over a 6-week period for the induction of dysplasia. The hamsters were subsequently divided into a baseline and 3 experimental groups (growth factors, saline vehicle, untreated control). Following the final DMBA application (day 0), the baseline group (N = 6) was sacrificed, the growth factor group (N = 21) received a single injection in the cheek pouch containing 4 micrograms of PDGF-BB and 4 micrograms of IGF-1 in saline, the saline group (N = 19) received an injection in the cheek pouch containing the saline vehicle only, and the untreated control group (N = 20) received no injection. Animals in experimental groups were sacrificed on days 3, 6, and 10. The cheek pouches were harvested for histologic and histochemical evaluation. Dysplasia was histologically graded from 0 to 4. Statistical analysis of the histologic data revealed no significant differences either by sacrifice date or by group. Histochemical evaluation, via staining for gamma-glutamyl transpeptidase (GGT), a marker for dysplastic cell colonies, revealed that the density of GGT-positive cells in experimental groups differed significantly from baseline levels. No significant differences were detected between experimental groups. There was poor correlation between the density of GGT-positive cells and the histologic grading of dysplasia. It is concluded that exposure to PDGF-BB and IGF-1 had no demonstrable effect on epithelial dysplasia in this hamster model.

Platelet-derived growth factor causes pulmonary cell proliferation and collagen deposition in vivo.

Platelet-derived growth factor (PDGF) is postulated to play a role in the pathophysiology of pulmonary fibrosis. Recombinant human PDGF-BB administered as a single intratracheal injection in rats causes an increase in peribronchial and perivascular stromal cells on days 2 and 3 after injection as evaluated by hematoxylin and eosin histology and 5-bromodeoxyuridine incorporation. Proliferation of bronchial epithelial cells and arterial smooth muscle cells, although not evident by routine histological examination alone, is detected on days 2 and 3 by increased 5-bromodeoxyuridine incorporation. A mild increase in 5-bromodeoxyuridine labeling is observed in peripheral alveolar parenchyma after injection of PDGF. The proliferative peribronchial and perivascular mesenchymal cells appear by light microscopic and ultrastructural criteria to be fibroblasts that are immunoreactive for vimentin but negative for alpha-smooth muscle actin and desmin. Daily intratracheal injection of PDGF-BB for 3 days causes a slightly more pronounced peribronchial and perivascular spindle cell proliferation accompanied by collagen deposition as evaluated by Masson's trichrome stain. PDGF-induced increases in cellularity and collagen resolve within 5 days after the last PDGF injection. In conclusion, intratracheal injection of PDGF-BB causes transient proliferation of pulmonary mesenchymal and epithelial cells accompanied by collagen deposition.

Tranilast suppresses intimal hyperplasia in the balloon injury model and cuff treatment model in rabbits.

Intimal hyperplasia is a serious problem after percutaneous transluminal coronary angioplasty (PTCA). In this study, we investigated the effects of tranilast on intimal hyperplasia in both in vivo and in vitro experiments. For the in vivo experiments, we used the balloon injury model and the cuff treatment model of rabbits fed regular chow. In the balloon injury model, tranilast decreased intimal area, intima/media ratio, stenosis ratio and vascular DNA content after endothelial injury. Also in the cuff treatment model, tranilast suppressed the intimal hyperplasia. In the in vitro experiments, we assessed the effects of tranilast on platelet-derived growth factor-induced rabbit vascular smooth muscle cell (VSMC) migration and proliferation and on collagen synthesis by VSMCs. Tranilast inhibited VSMC migration, proliferation and collagen synthesis. These results suggest that tranilast has a suppressive effect on intimal hyperplasia after a vascular injury such as PTCA.

Chemotaxis of human osteoblasts. Effects of osteotropic growth factors.

The in vitro chemotactic response of human osteoblasts was investigated towards the following growth factors: TGF-beta, PDGFs, FGFs and IGFs. Human osteoblasts grown from trabecular bone after enzymatic digestion were studied. TGF-beta stimulated the migration of human osteoblasts in a dose-dependent manner with a four-fold increase in migrated cells at 100 pg/ml, which was the optimum concentration. PDGF-BB also stimulated migration four-fold in a dose-dependent manner with a maximum response at 10 ng/ml. PDGF-AA, IGF-I and IGF-II stimulated migration two-fold at 100 ng/ml. The results show that TGF-beta and PDGF-BB are important regulators of human osteoblast migration, but other growth factors IGF-I, IGF-II and PDGF-AA may also stimulate osteoblast migration. Our results additionally suggest that TGF-beta and PDGF-BB may participate in the recruitment of osteoblasts during bone remodeling since both TGF-beta and PDGF-BB are found in bone matrix and could be released during osteoclastic bone resorption. They furthermore support a possible use of TGF-beta and PDGF-BB in growth factor-induced osteogenesis.

Alanine mutagenesis of conserved residues in the platelet-derived growth factor family: identification of residues necessary for dimerization and transformation.

Platelet-derived growth factor (PDGF) and vascular endothelial growth factor define a family of dimeric proteins characterized by eight conserved cysteine residues involved in disulfide bonds. Thirteen non-cysteine residues conserved among the platelet-derived/vascular endothelial growth factors were individually mutated to alanine in v-sis/PDGF-B. In addition, five other residues flanking F148 were also mutated to alanine. The resulting mutants were assayed for transformation of NIH3T3 cells, and the mutant proteins were assayed for their ability to dimerize. Four residues were found to be crucial for disulfide-linked dimer formation: P152 and G162 were mandatory, while R159 and H205 also contributed to efficient dimerization. Four of the mutant proteins (at residues N147, F148, L149 and K185) dimerized efficiently yet exhibited less than 50% transforming activity compared with wild-type v-sis. Two mutants (at residues D142 and F148) were located in a region important for PDGF receptor interaction and were further studied with regard to secretion and PDGF receptor autophosphorylation. A series of substitutions at residue F148 revealed a strong preference for aromatic amino acids. One mutant from this series (F148G) dimerized but was completely inactive for transformation. This study thus identifies four residues in v-sis/PDGF-B important for dimerization and also identifies additional residues critical for full activation of PDGF receptors. The E5 oncoprotein encoded by bovine papillomavirus type I exhibits two short regions of amino acid similarity when compared with the minimal transforming region of v-sis/PDGF-B. Several of the v-sis mutants discussed in this work affect residues that are also present in the E5 oncoprotein, including F148, L149 and H205.

Cellular transformation by coordinated action of three peptide growth factors from human platelets.

Platelet-derived growth factor (PDGF) is known to be involved in regulating the mitosis of connective tissue cells, and recent studies have also shown that it may function in mediating cellular transformation. The oncogene carried by simian sarcoma virus, sis, is homologous to one chain of PDGF, and treatment of non-neoplastic cells with this growth factor results in increased transcription of another oncogene, myc (ref. 9). PDGF also stimulates the synthesis of proteins that are characteristic of transformed cells. However, phenotypic transformation does not appear to result from the action of PDGF alone. For example, expression of myc does not transform cells in the absence of other oncogene expression. We have recently shown that platelets contain another peptide growth factor, transforming growth factor-beta (TGF-beta)12,13, in addition to PDGF. We report here that extracts of human platelets can induce anchorage-independent growth of nonneoplastic rat kidney (NRK) fibroblasts, but that purified PDGF alone does not elicit this effect. Rather, the transforming activity of the platelet extract is due to a concerted action of three distinct peptides: PDGF, TGF-ss and a newly identified analogue of epidermal growth factor (EGF).