Platelet-derived Growth Factor Stimulation Research Articles

Yohimbine Inhibits PDGF-Induced Vascular Smooth Muscle Cell Proliferation and Migration via FOXO3a Factor.

Yohimbine (YHB) has been reported to possess anti-inflammatory, anticancer, and cardiac function-enhancing properties. Additionally, it has been reported to inhibit the proliferation, migration, and neointimal formation of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) stimulation by suppressing the phospholipase C-gamma 1 pathway. However, the transcriptional regulatory mechanism of YHB controlling the behavior of VSMCs is not fully understood. In this study, YHB downregulated the expression of cell cycle regulatory proteins, such as proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4), and cyclin E, by modulating the transcription factor FOXO3a in VSMCs induced by PDGF. Furthermore, YHB decreased p-38 and mTOR phosphorylation in a dose-dependent manner. Notably, YHB significantly reduced the phosphorylation at Y397 and Y925 sites of focal adhesion kinase (FAK), and this effect was greater at the Y925 site than Y397. In addition, the expression of paxillin, a FAK-associated protein known to bind to the Y925 site of FAK, was significantly reduced by YHB treatment in a dose-dependent manner. A pronounced reduction in the migration and proliferation of VSMCs was observed following co-treatment of YHB with mTOR or p38 inhibitors. In conclusion, this study shows that YHB inhibits the PDGF-induced proliferation and migration of VSMCs by regulating the transcription factor FOXO3a and the mTOR/p38/FAK signaling pathway. Therefore, YHB may be a potential therapeutic candidate for preventing and treating cardiovascular diseases such as atherosclerosis and vascular restenosis.

Characterization of PDGF-Induced Subcellular Calcium Regulation through Calcium Channels in Airway Smooth Muscle Cells by FRET Biosensors

The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet-derived growth factor (PDGF), inducing the proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulation in the different compartments of ASM cells. A PDGF-induced cytoplasmic calcium [Ca2+]C increase was attributed to both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca2+]ER release, which was partially regulated by the PLC-IP3R pathway. Interestingly, the removal of the extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effects on the calcium-dependent activation of the ER ryanodine receptor. The inhibition of the L-type calcium channel on the plasma membrane or the SERCA pump on the ER resulted in both reduced [Ca2+]C and [Ca2+]ER from PDGF stimulation, while IP3R channel inhibition led to reduced [Ca2+]C only. The inhibited SERCA pump caused an immediate [Ca2+]C increase and [Ca2+]ER decrease, indicating active calcium exchange between the cytosol and ER storage in resting cells. PDGF-induced calcium at the outer mitochondrial membrane sub-region showed a similar regulatory response to cytosolic calcium, not influenced by the inhibition of the mitochondrial calcium uniporter channel. Therefore, our work identifies calcium flow pathways among the extracellular medium, cell cytosol, and ER via regulatory calcium channels. Specifically, extracellular calcium flow has an essential function in fully activating ER calcium release.

Platelet-derived growth factor-stimulated pulmonary artery smooth muscle cells regulate pulmonary artery endothelial cell dysfunction through extracellular vesicle miR-409-5p.

Platelet-derived growth factor (PDGF)-induced changes in vascular smooth muscle cells (VSMCs) stimulate vascular remodeling, resulting in vascular diseases such as pulmonary arterial hypertension. VSMCs communicate with endothelial cells through extracellular vesicles (EVs) carrying cargos, including microRNAs. To understand the molecular mechanisms through which PDGF-stimulated pulmonary artery smooth muscle cells (PASMCs) interact with pulmonary artery endothelial cells (PAECs) under pathological conditions, we investigated the crosstalk between PASMCs and PAECs via extracellular vesicle miR-409-5p under PDGF stimulation. miR-409-5p expression was upregulated in PASMCs upon PDGF signaling, and it was released into EVs. The elevated expression of miR-409-5p was transported to PAECs and led to their impaired function, including reduced NO release, which consequentially resulted in enhanced PASMC proliferation. We propose that the positive regulatory loop of PASMC-extracellular vesicle miR-409-5p-PAEC is a potential mechanism underlying the proliferation of PASMCs under PDGF stimulation. Therefore, miR-409-5p may be a novel therapeutic target for the treatment of vascular diseases, including pulmonary arterial hypertension.

Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes.

Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype. HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice. Cobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN. Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.

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.

Large-conductance Ca2 +-activated K+ channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) phenotype switching is very important during the pathogenesis and progression of vascular diseases. However, it is not well understood how normal VSMCs maintain the differentiated state. The large-conductance Ca2+-activated K+ (BKCa) channels are widely expressed in VSMCs and regulate vascular tone. Nevertheless, there is limited understanding of the role of the BKCa channel in modulation of the VSMC phenotype. We assessed BKCa channel expression levels in normal and injured carotid arteries from rats of the balloon-injury model. A strong decrease of BKCa-β1 was seen in the injured carotid arteries, accompanied by a parallel decrease of the VSMC contractile markers. BKCa-β1 in primary rat aortic VSMCs was decreased with the increase of passage numbers and the stimulation of platelet-derived growth factor (PDGF)-BB. Conversely, transforming growth factor β upregulated BKCa-β1. Meanwhile, the BKCa-β1 level was positively associated with the levels of VSMC contractile proteins. Intravenous injection of PDGF-BB induced downregulation of BKCa-β1 expression in the carotid arteries. Knockdown of BKCa-β1 favored VSMC dedifferentiation, characterized by altered morphology, abnormal actin fiber organization, decreased contractile proteins expression and reduced contractile ability. Furthermore, the resultant VSMC dedifferentiated phenotype rendered increased proliferation, migration, enhanced inflammatory factors levels, and matrix metalloproteinases activity. Studies using primary cultured aortic VSMCs from human recapitulated key findings. Finally, protein level of BKCa-β1 was reduced in human atherosclerotic arteries. BKCa-β1 is important in the maintenance of the contractile phenotype of VSMCs. As a novel endogenous defender that prevents pathological VSMC phenotype switching, BKCa-β1 may serve as a potential therapeutic target for treating vascular diseases including post-injury restenosis and atherosclerosis.

Spatial Compartmentation of mTORC1 Signaling at the Mitochondria

The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. The complex is present at the plasma membrane, nucleus, lysosomes, and the outer mitochondrial membrane. Such spatial compartmentation has been suggested to enhance signaling efficiency and specificity. For instance, we recently discovered nuclear mTORC1 activity, which is distinctly regulated from the canonical lysosomal mTORC1 (Zhou et al., 2020). Previous studies have shown that mTOR is present at the outer mitochondrial membrane (OMM), but it is not clear whether mTORC1 is active at this location and what the functional consequences are. To investigate this, we targeted our FRET‐based mTORC1 activity reporter, TORCAR (Zhou et al., 2015), to the OMM and probed the subcellular activity of mTORC1. We found that platelet‐derived growth factor (PDGF) stimulation increases mTORC1 activity at the OMM in addition to at the lysosome and in the nucleus, whereas insulin specifically stimulates mTORC1 activity at the OMM without affecting the lysosomal and nuclear activities. We further dissected the regulation of mitochondrial mTORC1 activity and applied a novel approach of identifying new mTORC1 substrates. Elucidating the signaling events that lead to subcellular mTORC1 activity at mitochondria and its downstream functions will increase our understanding of the roles that mTORC1 may play in diseases associated with altered metabolism or mitochondrial dysfunction, such as diabetes and cancer.

Antifibrotic Effects of Kangxian Ruangan Capsule on Rats with Nonalcoholic Fatty Liver Fibrosis and Hepatic Stellate Cells through Regulation of TGF-β and TLR4 Signaling Pathways

Kangxian ruangan (KXRG) is a traditional Chinese medicine (TCM) formula consisting of 12 herbs. TCM syndrome differentiation proposes that KXRG exerts pharmacological effects against nonalcoholic fatty liver disease (NAFLD) fibrosis. This work investigates the effect of KXRG on NAFLD fibrosis in vivo and in vitro. In vivo, the NAFLD fibrosis model was constructed in Wistar rats using methionine- and choline-deficient (MCD) diet, followed by KXRG (0.92 g/kg/d) treatment for 8 weeks. In vitro, primary hepatic stellate cells (HSCs) were activated using platelet-derived growth factor (PDGF) and treated with KXRG. Molecular mechanisms underlying fibrosis were investigated. After 8 weeks, compared with the control groups, the histological lesions, degree of fibrosis, and inflammatory reaction increased with the MCD diet as demonstrated by histological changes and increased fibrosis-related (α-SMA, TGF-β, COL1A1, and desmin, P < 0.01) and inflammation-related factors (TNF-α, MCP-1, and F4/80, P < 0.01), whereas they decreased with KXRG treatment (P < 0.01). KXRG not only inhibited the proliferation of activated HSCs and promoted their apoptosis but also resulted in G0-G1 arrest. Furthermore, KXRG suppressed HSC activation (P < 0.01), collagen synthesis (P < 0.01), and α-SMA expression (P < 0.01) with PDGF stimulation. In both the MCD diet-induced animal model and PDGF-induced cell model, KXRG inhibited TGF-β and TLR4 signaling (P < 0.01), similar to corresponding small-molecule inhibitors. These results demonstrated that KXRG might exert suppressive effects against NAFLD fibrosis via regulating TGF-β and TLR4 signaling. KXRG may act as a natural and potent therapeutic agent against NAFLD.

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-1β (IL-1β) 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.

Platelet-Derived Growth Factor Stimulated Migration of Bone Marrow Mesenchymal Stem Cells into an Injectable Gelatin-Hydroxyphenyl Propionic Acid Matrix.

Bone marrow mesenchymal stem cells (bMSCs) are responsible in the repair of injured tissue through differentiation into multiple cell types and secretion of paracrine factors, and thus have a broad application profile in tissue engineering/regenerative medicine, especially for the musculoskeletal system. The lesion due to injury or disease may be a closed irregular-shaped cavity deep within tissue necessitating an injectable biomaterial permissive of host (endogenous) cell migration, proliferation and differentiation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) is a natural biopolymer hydrogel which is covalently cross-linked by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in situ and can be delivered to the lesion by needle injection. Growth factors and cytokines can be directly incorporated into the gel or into nano- and micro-particles, which can be employed for sustained release of biomolecules while maintaining their bioactivity. In this study, we selected polyelectrolyte complex nanoparticles (PCNs) prepared with dextran sulfate and chitosan as the carrier for platelet-derived growth factor (PDGF)-BB and stromal cell-derived factor (SDF)-1α, which have been tested effectively in recruiting stem cells. Our in vitro results showed a high degree of viability of bMSCs through the process of Gtn-HPA covalent cross-linking gelation. The Gtn-HPA matrix was highly permissive of bMSC migration, proliferation, and differentiation. PDGF-BB (20 ng/mL) directly incorporated into the gel and, alternatively, released from PCNs stimulated bMSC migration and proliferation. There were only small differences in the results for the direct incorporation of PDGF into the gel compared with its release from PCNs, and for increased doses of the growth factor (200 ng/mL and 2 µg/mL). In contrast, SDF-1α elicited an increase in migration and proliferation only when released from PCNs; its effect on migration was notably less than PDGF-BB. The in vitro results demonstrate that PDGF-BB substantially increases migration of bMSCs into Gtn-HPA and their proliferation in the gel, and that these benefits can be derived from incorporation of a relatively low dose of the growth factor directly into the gel. These findings commend the use of Gtn-HPA/PDGF-BB as an injectable therapeutic agent to treat defects in musculoskeletal tissues.

MicroRNA-520c-3p suppresses vascular endothelium dysfunction by targeting RELA and regulating the AKT and NF-κB signaling pathways.

Endothelial injury, which can cause endothelial inflammation and dysfunction, is an important mechanism for the development of atherosclerotic plaque. This study aims to investigate the functional role of miR-520c-3p in vascular endothelium during inflammatory diseases such as atherosclerosis. Quantitative real-time PCR was used to detect miR-520c-3p expression in in human umbilical vein endothelial cells (HUVECs) after treatment with platelet-derived growth factor (PDGF). Furthermore, the effects of miR-520c-3p overexpression and silencing on cell proliferation, adhesion, and apoptosis were assessed. Bioinformatics analysis and Biotin-labeled miRNA pull-down assay were used to confirm the targets of miR-520-3p. Then, the effects of miR-520c-3p on AKT and NF-κB signaling pathways were detected by western blot. Herein, we observed that the expression level of miR-520c-3p was downregulated in HUVECs under PDGF stimulation. Overexpression of miR-520c-3p not only decreased cell adhesion but also promoted proliferation and inhibited apoptosis to protect the viability of endothelial cells. It was confirmed that RELA is the target of miR-520c-3p. MiR-520c-3p inhibited the protein phosphorylation of AKT and RELA, and si-RELA reversed the promotion of AKT and RELA protein phosphorylation by anti-miR-520c-3p. In summary, our study suggested that miRNA-520c-3p targeting RELA through AKT and NF-κB signaling pathways regulated the proliferation, apoptosis, and adhesion of vascular endothelial cells. We conclude that miR-520c-3p may play an important role in the suppression of endothelial injury, which could serve as a biomarker and therapeutic target for atherosclerosis.

Modulation of Vascular Smooth Muscle Cell Phenotype by High Mobility Group AT-Hook 1.

ObjectiveThe purpose of this study is to examine the effect of high mobility group AT-hook 1 (HMGA1) on the phenotyptic change of vascular smooth muscle cells (VSMCs).MethodsGene silencing and overexpression of HMGA1 were introduced to evaluate the effect of HMGA1 expression on the phenotypic change of VSMCs. Marker gene expression of VSMCs was measured by promoter assay, quantitative polymerase chain reaction, and western blot analysis. Common left carotid artery ligation model was used to establish in vivo neointima formation.ResultsHMGA1 was expressed strongly in the synthetic type of VSMCs and significantly downregulated during the differentiation of VSMCs. Silencing of HMGA1 in the synthetic type of VSMCs enhanced the expression of contractile marker genes thereby enhanced angiotensin II (Ang II)-dependent contraction, however, significantly suppressed proliferation and migration. Stimulation of contractile VSMCs with platelet-derived growth factor (PDGF) enhanced HMGA1 expression concomitant with the downregulation of marker gene expression which was blocked significantly by the silencing of HMGA1. Silencing of HMGA1 retained the Ang II-dependent contractile function, which was curtailed by PDGF stimulation, however, overexpression of HMGA1 in the contractile type of VSMCs suppressed marker gene expression. Proliferation and migration were enhanced significantly by the overexpression of HMGA1. Furthermore, the Ang II-dependent contraction was reduced significantly by the overexpression of HMGA1. Finally, the expression of HMGA1 was enhanced significantly in the ligated artery, especially in the neointima area.ConclusionHMGA1 plays an essential role in the phenotypic modulation of VSMCs. Therefore, paracrine factors such as PDGF may affect vascular remodeling through the regulation of HMGA1.

High Throughput Screen Identifies the DNMT1 (DNA Methyltransferase-1) Inhibitor, 5-Azacytidine, as a Potent Inducer of PTEN (Phosphatase and Tensin Homolog): Central Role for PTEN in 5-Azacytidine Protection Against Pathological Vascular Remodeling.

Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.

Vascular Smooth Muscle Cell-Derived Exosomal MicroRNAs Regulate Endothelial Cell Migration Under PDGF Stimulation.

Intercellular communication between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is essential for the maintenance of vascular homeostasis. The presence of exosomes, a recently discovered player in vascular cell communication, has been associated with vascular disease progression. However, the detailed mechanism of how the signal mediated by exosomes affects the function of vascular cells during vascular pathogenesis is yet to be further understood. In this study, we investigated the expression of exosomal microRNAs (miRNAs) secreted by VSMCs and their functional relevance to ECs in pathogenesis, including their role in processes such as platelet-derived growth factor (PDGF) stimulation. We observed that PDGF stimulation contributes to a change in exosomal miRNA release from VSMCs; specifically, miR-1246, miR-182, and miR-486 were deficient in exosomes derived from PDGF-stimulated VSMCs. The reduced miRNA expression in these exosomes is associated with an increase in EC migration. These findings increase our understanding of exosome-mediated crosstalk between vascular cells under a pathological condition.

Gab1 mediates PDGF signaling and is essential to oligodendrocyte differentiation and CNS myelination.

Oligodendrocytes (OLs) myelinate axons and provide electrical insulation and trophic support for neurons in the central nervous system (CNS). Platelet-derived growth factor (PDGF) is critical for steady-state number and differentiation of oligodendrocyte precursor cells (OPCs), but its downstream targets are unclear. Here, we show for the first time that Gab1, an adaptor protein of receptor tyrosine kinase, is specifically expressed in OL lineage cells and is an essential effector of PDGF signaling in OPCs in mice. Gab1 is downregulated by PDGF stimulation and upregulated during OPC differentiation. Conditional deletions of Gab1 in OLs cause CNS hypomyelination by affecting OPC differentiation. Moreover, Gab1 binds to downstream GSK3β and regulated its activity, and thereby affects the nuclear accumulation of β-catenin and the expression of a number of transcription factors critical to myelination. Our work uncovers a novel downstream target of PDGF signaling, which is essential to OPC differentiation and CNS myelination.

Autocrine Production of PDGF Stimulated by the Tenascin-C-Derived Peptide TNIIIA2 Induces Hyper-Proliferation in Glioblastoma Cells.

Expression level of tenascin-C is closely correlated to poor prognosis in glioblastoma patients, while the substantial role of tenascin-C responsible for aggressive progression in glioblastoma cells has not been clarified. We previously found that peptide TNIIIA2, which is derived from the tumor-associated tenascin-C variants, has the ability to promote cell adhesion by activating β1-integrins. Our recent study demonstrated that potentiated activation of integrin α5β1 by TNIIIA2 causes not only a dysregulated proliferation in a platelet-derived growth factor (PDGF)-dependent manner, but also disseminative migration in glioblastoma cells. Here, we show that TNIIIA2 enhances the proliferation in glioblastoma cells expressing PDGF-receptorβ, even without exogenous PDGF. Mechanistically, TNIIIA2 induced upregulated expression of PDGF, which in turn stimulated the expression of tenascin-C, a parental molecule of TNIIIA2. Moreover, in glioblastoma cells and rat brain-derived fibroblasts, tenascin-C upregulated matrix metalloproteinase-2, which has the potential to release TNIIIA2 from tenascin-C. Thus, it was shown that autocrine production of PDGF triggered by TNIIIA2 functions to continuously generate a functional amount of PDGF through a positive spiral loop, which might contribute to hyper-proliferation in glioblastoma cells. TNIIIA2 also enhanced in vitro disseminative migration of glioblastoma cells via the PKCα signaling. Collectively, the tenascin-C/TNIIIA2 could be a potential therapeutic target for glioblastoma.

OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway.

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.

Salidroside inhibits platelet-derived growth factor-induced proliferation and migration of airway smooth muscle cells.

Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) have been found to be important for the airway remodeling during the pathogenesis of asthma. Salidroside a bioactive glucoside that exerts antitumor activity via inhibiting the cell proliferation and migration of cancer cells. The aim of the current study was to evaluate the effects of salidroside on the proliferation and migration of ASMCs. Our results showed that salidroside inhibited the proliferation and migration of ASMCs in response to platelet-derived growth factor (PDGF) stimulation. Salidroside markedly attenuated the PDGF-induced production of matrix metalloproteinase 2 (MMP-2) and MMP-9 in ASMCs. The levels of contractile phenotype markers including smooth muscle α-actin and calponin were reduced in response to PDGF stimulation, which was attenuated by salidroside pretreatment. Salidroside diminished the increase in the expression levels of type I collagen and fibronectin in PDGF-stimulated ASMCs. Furthermore, salidroside blocked the PDGF-induced activation of the nuclear factor-κB (NF-κB) pathway in ASMCs. The results suggested that salidroside functionally regulated the proliferation, migration, phenotype plasticity, and extracellular matrix deposition in PDGF-induced ASMCs and the NF-κB pathway might be implicated in the effects of salidroside on ASMCs induced by PDGF.

Copper transporter ATP7A interacts with IQGAP1, a Rac1 binding scaffolding protein: role in PDGF-induced VSMC migration and vascular remodeling.

Vascular smooth muscle cell (VSMC) migration contributes to neointimal formation after vascular injury. We previously demonstrated that copper (Cu) transporter ATP7A is involved in platelet-derived growth factor (PDGF)-induced VSMC migration in a Cu- and Rac1-dependent manner. The underlying mechanism is still unknown. Here we show that ATP7A interacts with IQGAP1, a Rac1 and receptor tyrosine kinase binding scaffolding proteins, which mediates PDGF-induced VSMC migration and vascular remodeling. In cultured rat aortic SMCs, PDGF stimulation rapidly promoted ATP7A association with IQGAP1 and Rac1 and their translocation to the lipid rafts and leading edge. Cotransfection assay revealed that ATP7A directly bound to NH2-terminal domain of IQGAP1. Functionally, either ATP7A or IQGAP1 depletion using siRNA significantly inhibited PDGF-induced VSMC migration without additive effects, suggesting that IQGAP1 and ATP7A are in the same axis to promote migration. Furthermore, IQGAP1 siRNA blocked PDGF-induced ATP7A association with Rac1 as well as its translocation to leading edge, while PDGF-induced IQGAP1 translocation was not affected by ATP7A siRNA or Cu chelator. Overexpression of mutant IQGAP1 lacking a Rac1 binding site prevented PDGF-induced translocation of Rac1, but not ATP7A, to the leading edge, thereby inhibiting lamellipodia formation and VSMC migration. In vivo, ATP7A colocalized with IQGAP1 at neointimal VSMCs in a mice wire injury model, while neointimal formation and extracellular matrix deposition induced by vascular injury were inhibited in ATP7A mutant mice with reduced Cu transporter function. In summary, IQGAP1 functions as ATP7A and Rac1 binding scaffolding protein to organize PDGF-dependent ATP7A translocation to the lamellipodial leading edge, thereby promoting VSMC migration and vascular remodeling.