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Atherosclerosis is a major cause of cardiovascular disease, in which vascular smooth muscle cells (VSMCs) proliferation and migration play a vital role. Circular RNAs (circRNAs) have been reported to be correlated with the VSMCs function. Therefore, this study is designed to explore the role and mechanism of circRNA lipase maturation factor 1 (circLMF1) in Human aortic VSMCs (HASMCs). The microarray was used for detecting the expression of circLMF1 in proliferative and quiescent HASMCs. Levels of circLMF1, microRNA-125a-3p (miR-125a-3p), vascular endothelial growth factor A (VEGFA), and fibroblast growth factor 1 (FGF1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, cell cycle progression, and migration were assessed by Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and transwell assays, respectively. Western blot assay determined proliferating cell nuclear antigen (PCNA), Cyclin D1, matrix metalloproteinase (MMP2), osteopontin (OPN), VEGFA, and FGF1 protein levels. The possible interactions between miR-125a-3p and circLMF1, and miR-125a-3p and VEGFA or FGF1 were predicted by circbank or targetscan, and then verified by a dual-luciferase reporter, RNA Immunoprecipitation (RIP), RNA pull-down assays. CircLMF1, VEGFA, and FGF1 were increased, and miR-125a-3p was decreased in platelet-derived growth factor-BB (PDGF-BB)-inducted HASMCs. Functionally, circLMF1 knockdown hindered cell viability, cell cycle progression, and migration in PDGF-BB-treated HASMCs. Mechanically, circLMF1 could regulate VEGFA or FGF1 expression through sponging miR-125a-3p. Our findings revealed that circLMF1 deficiency could inhibit cell viability, cell cycle progression, and migration of PDGF-BB stimulated atherosclerosis model partly through the miR-125a-3p/VEGFA or FGF1 axis, suggesting that targeting circLMF1 can be a feasible therapeutic strategy for atherosclerosis.

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibers in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

Vascular smooth muscle cell (VSMC) hyperplasia is closely associated with AS progression. Hence, it is of great significance to elucidate the molecular mechanisms underlying the involvement of VSMCs in AS. SHH antagonist can inhibit the excessive proliferation, migration and phenotypic transformation of PDGF-BB-induced VSMCs. It has been proved that CUL3 can suppress Hedgehog signaling. This current work was designed to identify the biological role of CUL3 in the behaviors of VSMCs in AS and investigate the potential molecular mechanism. VSMCs were treated with PDGF-BB to establish the cell model in vitro. Levels of CUL3, SHH and Gli1 in PDGF-BB-stimulated VSMCs were measured by RT-qPCR analysis. Then, the precise functions of CUL3 in VSMCs were determined from the perspectives of proliferation, migration, apoptosis and phenotype transformation. Besides, the influence of CUL3 on inflammatory response in VSMCs was evaluated. Moreover, the impact of CUL3 on Hedgehog signaling pathway was also investigated. In the present research, it was observed that CUL3 was lowly expressed and SHH and Gli1 were highly expressed in PDGF-BB-stimulated VSMCs. Upregulation of CUL3 suppressed the excessive proliferation, migration and phenotypic transformation and facilitated the apoptosis of PDGF-BB-stimulated VSMCs. In addition, elevation of CUL3 alleviated inflammatory response in PDGF-BB-stimulated VSMCs. Importantly, CUL3 overexpression inactivated Hedgehog signaling pathway. To conclude, CUL3 might regulate the biological behaviors of VSMCs in AS by modulating Hedgehog signaling pathway. These data encourage to further investigate any potential therapeutic role of CUL3 in animal models of AS and explore therapeutic values for AS clinically.

Objective: Vascular smooth muscle cell (SMC) proliferation contributes to neointima formation following vascular injury. Circular RNA—a novel type of noncoding RNA with closed-loop structure—exhibits cell- and tissue-specific expression patterns. However, the role of circular RNA in SMC proliferation and neointima formation is largely unknown. The objective of this study is to investigate the role and mechanism of circSOD2 in SMC proliferation and neointima formation. Approach and Results: Circular RNA profiling of human aortic SMCs revealed that PDGF (platelet-derived growth factor)-BB up- and downregulated numerous circular RNAs. Among them, circSOD2, derived from back-splicing event of SOD2 (superoxide dismutase 2), was significantly enriched. Knockdown of circSOD2 by short hairpin RNA blocked PDGF-BB–induced SMC proliferation. Inversely, circSOD2 ectopic expression promoted SMC proliferation. Mechanistically, circSOD2 acted as a sponge for miR-206, leading to upregulation of NOTCH3 (notch receptor 3) and NOTCH3 signaling, which regulates cyclin D1 and CDK (cyclin-dependent kinase) 4/6. In vivo studies showed that circSOD2 was induced in neointima SMCs in balloon-injured rat carotid arteries. Importantly, knockdown of circSOD2 attenuated injury-induced neointima formation along with decreased neointimal SMC proliferation. Conclusions: CircSOD2 is a novel regulator mediating SMC proliferation and neointima formation following vascular injury. Therefore, circSOD2 could be a potential therapeutic target for inhibiting the development of proliferative vascular diseases.

The accumulation of vascular smooth muscle cells (VSMCs) is considered to play important roles in atherosclerosis (AS) development and progression. Circ_0002984 was found to be increased in oxidized low-density lipoprotein (ox-LDL) human VSMCs (HVSMCs). However, the function and mechanism of circ_0002984 in VSMC dysfunction remain unknown. In this study, the expression of circ_0002984, microRNA (miR)-379-5p, and fibroblast growth factor receptor substrate 2 (FRS2) was detected using quantitative real-time polymerase chain reaction and western blot. Cell proliferation, cell cycle, migration, and invasion were detected using Cell Counting Kit-8, flow cytometry, and transwell assays. The binding interaction between miR-379-5p and circ_0002984 or FRS2 was confirmed by the dual-luciferase reporter assay. Collectively, this study found that circ_0002984 was elevated in platelet-derived growth factor type bb (PDGF-bb)-induced HVSMCs. Circ_0002984 knockdown abrogated PDGF-bb-induced proliferation, migration, and invasion in HVSMCs. Mechanistically, circ_0002984 was confirmed to target miR-379-5p, and miR-379-5p upregulation reversed the protective effects of circ_0002984 knockdown on PDGF-bb-induced HVSMCs. Besides, when FRS2 was a target of miR-379-5p, miR-379-5p restoration abolished PDGF-bb-evoked HVSMC dysfunction, which was attenuated by the overexpression of FRS2. Moreover, circ_0002984 could regulate FRS2 expression through sponging miR-379-5p in HVSMCs. Collectively, these results demonstrated that circ_0002984 promoted PDGF-bb-induced VSMC proliferation, migration, and invasion through the regulation of miR-379-5p/FRS2 axis, suggesting a new insight into the pathogenesis of AS and the potential application of circ_0002984 in AS treatment.

Background Abnormal proliferation and migration of human airway smooth muscle cells (HASMCs) play an important role in the development of childhood asthma. Long non-coding RNAs (lncRNAs) have been demonstrated to participate in HASMC proliferation and migration. We aimed to explore more effects and molecular mechanism of taurine upregulated gene 1 (TUG1) in childhood asthma. Results TUG1 and SMURF2 were overexpressed and miR-216a-3p was downregulated in childhood asthma patients and PDGF-BB-stimulated HASMCs. TUG1 knockdown attenuated PDGF-BB-triggered proliferation and migration of HASMCs. MiR-216a-3p was targeted by TUG1, and miR-216a-3p suppression counteracted the repressive effects of TUG1 interference on proliferation and migration in PDGF-BB-treated HASMCs. SMURF2 was a downstream target of miR-216a-3p, and SMURF2 upregulation abated the inhibiting effects of miR-216a-3p on migration and proliferation in PDGF-BB-exposed HASMCs. TUG1 sponged miR-216a-3p to positively regulate SMURF2 expression. Conclusion TUG1 downregulation inhibited PDGF-BB-induced HASMC proliferation and migration by regulating miR-216a-3p/SMURF2 axis, offering novel insight into the potential application of TUG1 for childhood asthma treatment.

Aims The neurotropic growth factor PDGF‐BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF‐BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF‐BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study. Method MPTP‐lesion mice received PDGF‐BB treatment via intracerebroventricular (i.c.v) administration, and the expression of TH in different brain regions was assessed by RT‐PCR, Western blot, and immunohistochemistry staining. The molecular mechanisms of PDGF‐BB‐mediated TH upregulation were examined by RT‐PCR, Western blot, ChIP assay, luciferase reporter assay, and immunocytochemistry. Results We validated a reversal expression of TH in MPTP‐lesion mice upon i.c.v administration of PDGF‐BB for seven days. Similar effects of PDGF‐BB‐mediated TH upregulation were also observed in MPP+‐treated primary neuronal culture and dopaminergic neuronal cell line SH‐SY5Y cells. We next demonstrated that PDGF‐BB rapidly activated the pro‐survival PI3K/Akt and MAPK/ERK signaling pathways, as well as the downstream CREB in SH‐SY5Y cells. We further confirmed the significant induction of p‐CREB in PDGF‐BB‐treated animals in vivo. Using a genetic approach, we demonstrated that the transcription factor CREB is critical for PDGF‐BB‐mediated TH expression. The activation and nucleus translocation of CREB were promoted in PDGF‐BB‐treated SH‐SY5Y cells, and the enrichment of CREB on the promoter region of TH gene was also increased upon PDGF‐BB treatment. Conclusion Our data demonstrated that PDGF‐BB directly regulated the expression of TH via activating the downstream Akt/ERK/CREB signaling pathways. Our finding will further support the therapeutic potential of PDGF‐BB in PD, and provide the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future., The binding of PDGF‐BB to its receptor in DA neurons leads to the activation of downstream PI3K/Akt and MAPK/ERK signaling pathways, which trigger the nucleus translocation of the transcription factor CREB and increased expression of tyrosine hydroxylase.

Background Mucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood. Objective The aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin. Methods The protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER). Results In HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin. Conclusion We found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling.

Osteoarthritis (OA) is a common joint disease that mainly affects the diarthrodial joints. Treatments for OA include non‐pharmacological interventions, topical and oral therapies, intra‐articular therapies and joint surgery. However, all the treatments mentioned above mainly aim to control the symptoms instead of improving or reversing the joint condition. In this research, we observed the effect of recombinant platelet‐derived growth factor (PDGF)‐BB on OA in a monosodium iodoacetate (MIA)–induced rat model and revealed the possible mechanisms. In vitro, the level of inflammation in the chondrocytes was gradually alleviated, and the apoptosis rate was gradually decreased by PDGF‐BB at increasing concentrations. The levels of p‐p38, Bax and caspase‐3 decreased, and the level of p‐Erk increased with increasing PDGF‐BB concentration. In vivo, PDGF‐BB could significantly reverse chondrocyte and matrix loss. Furthermore, high concentrations of PDGF‐BB could alleviate cartilage hyperplasia to remodel the tissue. The level of collagen II was up‐regulated, and the levels of collagen X and apoptosis were down‐regulated by increasing concentrations of PDGF‐BB. In conclusion, recombinant PDGF‐BB alleviated OA by down‐regulating caspase‐3‐dependent apoptosis. The effects of PDGF‐BB on OA mainly include inhibiting chondrocyte loss, reducing cartilage hyperplasia and osteophyte formation, and regulating collagen anabolism.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet‐derived growth factor‐BB (PDGF‐BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF‐BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding‐2 (Id2) and activator protein‐1 (AP‐1) were important factors implicated in harmine‐enhanced preosteoclast PDGF‐BB production. Exposure of RANKL‐induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP‐1. Knockdown of Id2 by Id2‐siRNA reduced the number of preosteoclasts as well as secretion of PDGF‐BB in RANKL‐stimulated BMMs administrated with harmine. Inhibition of c‐Fos or c‐Jun (components of AP‐1) both reversed the stimulatory effect of harmine on preosteoclast PDGF‐BB production. Dual‐luciferase reporter assay analyses determined that PDGF‐BB was the direct target of AP‐1 which was up‐regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF‐BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

We previously suggested an ovariectomy (OVX)-induced osteoporotic rat model showing an impaired alveolar bone defect healing. This study aimed to evaluate and compare the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on alveolar bone defect healing in OVX-induced osteoporotic rats. A total of forty-one female rats were divided into four groups: a collagen group (n=10), a PDGF-BB group (n=11), a BMP-2 group (n=10), and a control group (n=10). Four months after OVX, alveolar bone drill-hole defects were created and grafted with collagen gel, rhPDGF-BB/collagen gel, or rhBMP-2/collagen gel. The defects in the control group were not grafted with any material. Defect healing was evaluated by histological, histomorphometric, and microcomputed tomographic (micro-CT) analyses at 2 and 4 weeks. According to the micro-CT analysis, the BMP-2 group exhibited the greatest bone volume fraction among all groups, while the PDGF-BB group did not show significant differences compared with the collagen group. The histomorphometric analysis showed a significantly larger amount of new bone area in the BMP-2 group than in the control and collagen groups at 4 weeks; however, the PDGF-BB group did not reach significant superiority compared with the other groups. Alveolar bone regeneration was significantly enhanced by the local use of rhBMP-2/collagen gel compared with the use of rhPDGF-BB/collagen gel in OVX-induced osteoporotic rats. A treatment modality using rhBMP-2 may be a promising approach to promote alveolar bone regeneration in patients suffering from postmenopausal osteoporosis.

Previous studies have shown that mesenchymal stem cells (MSCs) derived from various tissue sources can be differentiated into smooth muscle-like cells (SMLCs) in vitro. In this paper, dental pulp-derived mesenchymal stem cells (DPSCs) were evaluated for their differentiation ability towards smooth muscle-like cells (SMLCs) under the effect of widely used cytokines (TGF-β1 and PDGF-BB) with special focus on different culturing environments. For this purpose, both the commercially used culturing plates (Norm-c) and 0.1% gelatin-precoated (Gel-c) plates were used. Isolated cells displayed plastic adherence, pluripotency and cell surface marker profiling, and adipogenic and osteogenic differentiation potential with lineage specific marker expression. Differentiated cells induced under different culturing plates showed successful differentiation into SMLCs by positively expressing smooth muscle cell (SMC) specific markers both at the mRNA and protein levels. Gelatin coating could substantially enhance DPSC differentiation potential than Norm-c-induced cells. However, the absence of mature marker MHY-11 by immunostaining results from all treatment groups further indicated the development of immature and synthetic SMLCs. Finally, it was concluded that DPSC differentiation ability into SMLCs can be enhanced under cytokine treatment as well as by altering the cellular niche by precoating the culturing plates with suitable substrates. However, to get fully functional, contractile, and mature SMLCs, still many different cytokine cocktail combinations and more suitable coating substrates will be needed.

MicroRNA (miRNA) plays a key role in the proliferation and invasion of vascular smooth muscle cells (VSMCs). However, the role and underlying mechanism of miRNAs in VSMCs are not fully understood. Therefore, this study was designed to investigate the role and mechanism of microRNA-1246 (miR-1246) in VSMCs. VSMCs were cultured, and the proliferation of VSMCs was stimulated by platelet-derived growth factor (PDGF-BB) or 15% fetal bovine serum (FBS). The quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of miR-1246 and cystic fibrosis transmembrane conductance regulator (CFTR) in VSMCs. The CCK-8 assay and transwell assay were used to detect the proliferation and invasion of VSMCs. Target gene prediction and screening and luciferase reporter assays were used to verify downstream target genes of miR-1246. Western blotting was used to detect the protein expression levels of PCNA, α-SMA, SM-MHC, Collagen-1, and Cyclin D1 in VSMCs. PDGF-BB and FBS treatment induced VSMCs proliferation and the upregulation of miR-1246 expression. Overexpression of miR-1246 promoted VSMCs proliferation, invasion, and differentiation towards synthetic phenotype, while knockdown of miR-1246 had opposite effects. In addition, CFTR was found to be a direct target for miR-1246, and miR-1246 inhibited the expression of CFTR. Moreover, overexpression of CFTR inhibited VSMC proliferation and synthetic differentiation, while overexpression of miR-1246 partly abolished the effects of CFTR overexpression on VSMCs proliferation and differentiation. Our data suggest that MiR-1246 promotes VSMC proliferation, invasion, and differentiation to synthetic phenotype by regulating CFTR. MiR-1246 may be a potential therapeutic target for atherosclerosis.

MicroRNAs are crucial regulators in the phenotype switch of vascular smooth muscle cells (VSMCs). Nonetheless, the role of miR-146b-3p in VSMCs remains unclear. In the present study, platelet-derived growth factor-BB (PDGF-BB) at different concentrations was employed to stimulate VSMCs for different times, to establish the model of VSMC dysfunction. The relative expression of miR-146b-3p was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation of VSMCs was measured by BrdU assay. Flow cytometry analysis was employed for the analysis of cell cycle. VSMC migration was detected by Transwell assay. Phosphoinositide-3 kinase catalytic subunit-gamma (PIK3CG) and markers of VSMC differentiation, including α-SMA, SM-22α, SMMHC, and Calponin were examined employing Western blot. The targeting relationship between miR-146b-3p and PIK3CG 3ʹ-UTR was affirmed by dual-luciferase gene assay. We report that the reduction of miR-146b-3p expression was induced by PDGF-BB in a time-dependent and dose-dependent manner (P, Graphical Abstract

As one of the significant serum cytokines, platelet-derived growth factor-BB (PDGF-BB) is a crucial protein biomarker overexpressed in human life-threatening tumors, the sensitive identification and quantification of which are urgently desired but challenging. Herein we report a novel core–shell nanoarchitecture consisting of Cu-based metal–organic frameworks (Cu-MOFs) and covalent organic frameworks (denoted as TpBD-COFs), which was used to prepare an aptasensor for the detection of platelet-derived growth factor-BB (PDGF-BB). The central Cu-MOFs function as signal labels with no need for extra redox media, whereas the porous TpBD serves as the shell to immobilize the PDGF-BB-targeted aptamer strands in abundance via strong interactions involving π–π stacking, electrostatic, and hydrogen bonding interactions. The proposed aptasensor based on Cu-MOF@TpBD can achieve a detection limit as low as 0.034 pg mL−1 within the dynamic detection range from 0.0001 to 60 ng mL−1. The hybridization of MOFs and COFs, together with the immobilization with the specific analyte targeted aptamer, provides a promising and propagable approach to prepare an aptasensor for the simple, sensitive, and selective detection of a specific biomarker in clinical diagnosis.

It is of great importance to develop biosensing methods for the sensitive and selective analysis of biomarkers at very low levels in biological samples. Using a new target-induced activation of the DNA polymerase activity for recycling amplification cascades, we describe an aptamer-based method for highly sensitive detection of platelet-derived growth factor BB (PDGF-BB) in human serums. The polymerase activity is initially inhibited by the binding of the polymerase to the enzyme aptamer sequence. PDGF-BB associates with and switches a PDGF-BB binding aptamer to trigger the release of an active polymerase, which further initiates the simultaneous recycling of the target PDGF-BB molecules and the enzyme aptamer sequence for the subsequent displacement of the fluorescently quenched probes to recover the fluorescence. Due to two recycling cascades, substantial fluorescence magnification is obtained for the highly sensitive detection of PDGF-BB with a low detection limit of 5.1 pM. Moreover, the potential applicability of this method for real samples was verified by determining PDGF-BB in diluted human serums, relying on the excellent specificity and selectivity of the aptamer. The demonstration of the PDGF-BB assay method here thus can be expanded for the construction of diverse sensing platforms for detecting different trace biomarkers with the integration of an elaborate design of the aptamer probes.

Introduction: Thrombocytopenia has been identified as an independent risk factor for retinopathy of prematurity (ROP), although underlying mechanisms are unknown. In this study, the association of platelet count and serum platelet-derived factors with ROP was investigated. Methods: Data for 78 infants born at gestational age (GA) Results: Postnatal platelet counts and serum concentrations of the 3 growth factors followed the same postnatal pattern, with lower levels in infants developing severe ROP at PMA 32 and 36 weeks (p < 0.05–0.001). With adjustment for GA, low platelet counts and low serum concentrations of all 3 factors at PMA 32 weeks were significantly associated with severe ROP. Serum concentrations of all 3 factors also strongly correlated with platelet count (p < 0.001). Conclusion: In this article, we show that ROP, platelet counts, and specific pro-angiogenic factors correlate. These data suggest that platelet-released factors might be involved in the regulation of retinal and systemic angiogenesis after extremely preterm birth. Further investigations are needed.

Objective: This study evaluated the use of novel peptides derived from platelet-derived growth factor (PDGF-BB) as potential wound healing stimulants. One of the compounds (named PDGF2) was subjected for further research after cytotoxicity and proliferation assays on human skin cells. Further investigation included evaluation of: migration and chemotaxis of skin cells, immunological and allergic safety, the transcriptional analyses of adipose-derived stem cells (ASCs) and dermal fibroblasts stimulated with PDGF2, and the use of dorsal skin wound injury model to evaluate the effect of wound healing in mice. Approach: Colorimetric lactate dehydrogenase and tetrazolium assays were used to evaluate the cytotoxicity and the effect on proliferation. PDGF2 effect on migration and chemotaxis was also checked. Immunological safety and allergic potential were evaluated with a lymphocyte activation and basophil activation test. Transcriptional profiles of ASCs and primary fibroblasts were assessed after stimulation with PDGF2. Eight-week-old BALB/c female mice were used for dorsal skin wound injury model. Results: PDGF2 showed low cytotoxicity, pro-proliferative effects on human skin cells, high immunological safety, and accelerated wound healing in mouse model. Furthermore, transcriptomic analysis of ASCs and fibroblasts revealed the activation of processes involved in wound healing and indicated its safety. Innovation: A novel peptide derived from PDGF-BB was proved to be safe drug candidate in wound healing. We also present a multifaceted in vitro model for the initial screening of new compounds that may be potentially useful in wound healing stimulation. Conclusion: The results show that peptide derived from PDGF-BB is a promising drug candidate for wound treatment.

Aberrant proliferation and migration of vascular smooth muscle cells contributes to cardiovascular diseases (CVDs), including atherosclerosis. MicroRNA-223 (miR-223) protects against atherosclerotic CVDs. We investigated the contribution of miR-223 to platelet-derived growth factor-BB (PDGF-BB)-induced proliferation and migration of human aortic smooth muscle cells (HASMCs). We found that miR-223 was downregulated in PDGF-BB-treated HASMCs in a dose- and time-dependent manner, while nuclear factor of activated T cells 5 (NFAT5) was upregulated. Gain- and loss-of-function studies demonstrated that miR-223 treatment reduced PDGF-BB-induced HASMC proliferation and motility, whereas miR-223 inhibitor enhanced these processes. Moreover, NFAT5 was identified as a direct target of miR-223 in HASMC. The inhibitory effects of miR-223 on HASMC proliferation and migration were partly rescued by NFAT5 restoration. Overall, these findings suggest that miR-223 inhibits the PDGF-BB-induced proliferation and motility of HASMCs by targeting NFAT5 and that miR-223 and NFAT5 may be potential therapeutic targets for atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory disease that mainly involves the large and middle arteries, but the specific mechanism is not precise. Chemokine ligand 19 (CCL19) has been reported highly expressed in peripheral blood of patients with atherosclerosis, but its role lacks explicit data. By ELISA assay and immunohistochemical (IHC) analysis, we found that the CCL19 was significantly up-regulated in AS. Therefore, we tried to clarify whether CCL19 expression was related to the progression of AS. QRT-PCR and western blot demonstrated that overexpression of CCL19 promoted the secretion of inflammatory factors and the deposition of the extracellular matrix, and facilitated the proliferation and migration of VSMCS. Besides, knockdown of CCL19 reduced the inflammation, collagen secretion, proliferation and migration of VSMCS induced by PGDF-BB. The results of database analysis, chromatin immunoprecipitation (ChIP) and luciferase assay showed that interferon regulatory factor 1 (IRF-1) activated the expression of CCL19 at the transcriptional level. Importantly, silencing IRF-1 inhibited atherosclerosis in high-fat-fed mice, inhibited the proliferation and migration of VSMCS, and down-regulated the expression of CCL19. Summing up, the results demonstrated that IRF-1 contributed to the pathological phenotype of VSMCs during atherogenesis by increasing CCL19 transcription.

Platelet Derived Growth Factor Receptor (PDGFR) signaling is a central mitogenic pathway in development, as well as tissue repair and homeostasis. The rules governing the binding of PDGF ligand to the receptor to produce activation and downstream signaling have been well defined over the last several decades. In cultured cells after a period of serum deprivation, treatment with PDGF leads to the rapid formation of dramatic, actin-rich Circular Dorsal Ruffles (CDRs). Using CDRs as a robust visual readout of early PDGFR signaling, we have identified several contradictory elements in the widely accepted model of PDGF activity. Employing CRISPR/Cas9 gene editing to disrupt the Pdgfra gene in two different murine cell lines, we show that in addition to the widely accepted function for PDGFR-beta in CDR formation, PDGFR-alpha is also clearly capable of eliciting CDRs. Moreover, we demonstrate activity for heterodimeric PDGF-AB ligand in the vigorous activation of PDGFR-beta homodimers to produce CDRs. These findings are key to a more complete understanding of PDGF ligand-receptor interactions and their downstream signaling consequences. This knowledge will allow for more rigorous experimental design in future studies of PDGFR signaling and its contributions to development and disease.

Platelet‑derived growth factor (PDGF) is a potent mitogen and chemoattractant that serves a role in the development of several types of solid cancer, and abnormal PDGF activity has been reported in numerous human tumors. Tumor‑derived PDGF ligands are considered to act in either a paracrine or autocrine manner, serving roles in the phosphorylation of receptors on tumor and stromal cells in the tumor microenvironment. Despite the well‑established association between PDGF and tumor progression, the precise mechanisms of autocrine PDGF signaling in pancreatic tumor cells remain elusive. Therefore, the present study aimed to analyze the influence of PDGF‑BB in pancreatic cancer. Pancreatic adenocarcinoma BxPC‑3 cells were cultured and treated with recombinant human PDGF‑BB in vitro. Cell proliferation was tested using an MTT assay. Cell apoptosis was measured using flow cytometry. Tumor cell migration and invasion were examined via wound‑healing and Transwell assays, respectively. The expression and subcellular localization of Yes‑associated protein (YAP) was determined using western blotting and immunofluorescence. The transcriptional activity of target genes was tested using a luciferase assay and reverse transcription‑quantitative PCR. The present study revealed that PDGF‑BB significantly promoted cell proliferation in pancreatic adenocarcinoma BxPC‑3 cells and enhanced the aggressiveness of this cell line, as demonstrated by Transwell and wound‑healing assays. Anoikis resistance is an important mechanism by which metastatic cells avoid apoptosis when detaching from adjacent cells or the extracellular matrix. PDGF‑BB treatment inhibited anoikis under anchorage‑independent conditions. Mechanistic experiments revealed that PDGF‑BB promoted the upregulation and activation of the transcriptional coactivator YAP, an effector of the Hippo signaling pathway. RhoA or protein phosphatase‑1 (PP‑1) inhibition partially abolished the accumulation and activation of YAP, suggesting PDGF‑BB‑mediated YAP dephosphorylation and transactivation via the RhoA/PP‑1 cascade. Pharmacologic inhibition of the PDGF receptor directly downregulated YAP activity and the expression levels of downstream genes. Furthermore, verteporfin, a small molecular inhibitor of the Hippo/YAP signaling pathway, partially reversed the effects of PDGF‑BB on cell proliferation, anoikis resistance and cell migration. In conclusion, the present study revealed that the Hippo/YAP signaling pathway may be involved in the tumor‑promoting activity of PDGF‑BB in pancreatic cancer.

Objective: We sought to identify and investigate the functional role of the major endothelial cell (EC)-derived factors that control pericyte recruitment to EC tubes and pericyte-induced tube maturation during capillary network formation. Approach and Results: We identify PDGF (platelet-derived growth factor)-BB, PDGF-DD, ET (endothelin)-1, TGF (transforming growth factor)-β, and HB-EGF (heparin-binding epidermal growth factor), as the key individual and combined regulators of pericyte assembly around EC tubes. Using novel pericyte only assays, we demonstrate that PDGF-BB, PDGF-DD, and ET-1 are the primary direct drivers of pericyte invasion. Their addition to pericytes induces invasion as if ECs were present. In contrast, TGF-β and HB-EGF have minimal ability to directly stimulate pericyte invasion. In contrast, TGF-β1 can act as an upstream pericyte primer to stimulate invasion in response to PDGFs and ET-1. HB-EGF stimulates pericyte proliferation along with PDGFs and ET-1. Using EC-pericyte cocultures, individual, or combined blockade of these EC-derived factors, or their pericyte receptors, using neutralizing antibodies or chemical inhibitors, respectively, interferes with pericyte recruitment and proliferation. As individual factors, PDGF-BB and ET-1 have the strongest impact on these events. However, when the blocking reagents are combined to interfere with each of the above factors or their receptors, more dramatic and profound blockade of pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition occurs. Under these conditions, ECs form tubes that become much wider and less elongated as if pericytes were absent. Conclusions: Overall, these new studies define and characterize a functional role for key EC-derived factors controlling pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition during capillary network assembly.

Platelet α-granules release growth factors (GFs) that promote healing and tissue regeneration. Platelet-rich plasma (PRP) is shown to be beneficial in treating alopecia, and however, clinical response can be inconsistent. Due to several fold enrichment of platelets secreting large quantities of GFs following PRP injections, heterogeneity in amounts of GFs secreted by platelets may contribute to inconsistent clinical responses. Herein, we evaluated factors that could potentially contribute to heterogeneous secretion of GFs by platelets. We measured platelet secretion of transforming growth factor beta1 (TGFβ1), platelet-derived growth factor (PDGF-BB), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF2) in aliquots of de-identified PRP samples from female patients undergoing therapy in the hair disease clinic. Although secretion of GFs by platelets was comparable in PRP samples of patients with non-cicatricial and cicatricial alopecia, a Shapiro-Wilk test for normal distribution indicated significant variability across all patient samples. The amount of GF secreted by platelets was comparable when PRP prepared from two FDA-cleared devices with distinct techniques were compared. We provide evidence of platelets secreting heterogeneous amounts of GFs within each sample as high and low secretion of random factors could be simultaneously detected. These results suggest inherent heterogeneity in secretion of GFs by platelets in patient samples that are not influenced by the device used to prepare PRP. Since some GFs could have antagonistic effects on hair growth, a balance between amounts of growth promoting and inhibiting factors may be crucial in determining clinical response to PRP therapy.

Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing l-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Although magnesium and its alloys are candidates for orthopedic implants, they can effectively promote osteogenesis and angiogenesis. However, due to the degradability of magnesium, different concentrations of magnesium ions have different effects on cells, which affects the safety of magnesium implants. The cellular and molecular mechanisms by which magnesium promotes osteogenesis and angiogenesis are still unclear, which further affects its clinical use. In this study, HUVECs were treated with different concentrations of magnesium ions, and the concentration between 1 and 5 mM, especially 5 mM, was most suitable for cultivation of HUVECs. Using gene sequencing, RT-PCR, ELISA and Western blot analysis, we found that magnesium can promote MC3T3-E1 expression and secretion of PDGF-BB. Then, osteogenesis-related tests and angiogenesis-related tests found that magnesium promotes the secretion of PDGF-BB by MC3T3-E1 not only to improve the osteogenic differentiation ability of osteoblasts but also to effectively promote the angiogenic ability of HUVECs.

As a receptor for transforming growth factor-β, nodal and activin, activin receptor-like kinase 7 (ALK7) previously acts as a suppressor of tumorigenesis and metastasis, which has emerged to play a key role in cardiovascular diseases. However, the potential effect and molecular mechanism of ALK7 on vascular smooth muscle cells' (VSMCs) phenotypic modulation have not been investigated. Using cultured mouse VSMCs with platelet-derived growth factor-BB administration, we observed that ALK7 showed a significantly increased expression in VSMCs accompanied by decreased VSMCs differentiation marker genes. Loss-of-function study demonstrated that ALK7 knockdown inhibited platelet-derived growth factor-BB-induced VSMCs phenotypic modulation characterized by increased VSMCs differentiation markers, reduced proliferation, and migration of VSMCs. Such above effects were reversed by ALK7 overexpression. Notably, we noticed that ALK7 silencing dramatically enhanced PPARγ expression, which was required for the attenuated effect of ALK7 knockdown on VSMCs phenotypic modulation. Collected, we identified that ALK7 acted as a novel and positive regulator for VSMCs phenotypic modulation partially through inactivation of PPARγ, which suggested that neutralization of ALK7 might act as a promising therapeutic strategy of intimal hyperplasia.

Smooth muscle cells (SMCs) are the major type of cells constituting arterial walls and play a role to maintain stiffness via producing extracellular matrix. Here, the loss and degenerative changes of SMCs become the major histopathological features of an intracranial aneurysm (IA), a major cause of subarachnoid hemorrhage. Considering the important role of SMCs and the loss of this type of cells in IA lesions, we in the present study subjected rats to IA models and examined how SMCs behave during disease progression. We found that, at the neck portion of IAs, SMCs accumulated underneath the internal elastic lamina according to disease progression and formed the intimal hyperplasia. As these SMCs were positive for a dedifferentiation marker, myosin heavy chain 10, and contained abundant mitochondria and rough endoplasmic reticulum, SMCs at the intimal hyperplasia were dedifferentiated and activated. Furthermore, dedifferentiated SMCs expressed some pro-inflammatory factors, suggesting the role in the formation of inflammatory microenvironment to promote the disease. Intriguingly, some SMCs at the intimal hyperplasia were positive for CD68 and contained lipid depositions, indicating similarity with atherosclerosis. We next examined a potential factor mediating dedifferentiation and recruitment of SMCs. Platelet derived growth factor (PDGF)-BB was expressed in endothelial cells at the neck portion of lesions where high wall shear stress (WSS) was loaded. PDGF-BB facilitated migration of SMCs across matrigel-coated pores in a transwell system, promoted dedifferentiation of SMCs and induced expression of pro-inflammatory genes in these cells in vitro. Because, in a stenosis model of rats, PDGF-BB expression was expressed in endothelial cells loaded in high WSS regions, and SMCs present nearby were dedifferentiated, hence a correlation existed between high WSS, PDGFB and dedifferentiation in vivo. In conclusion, dedifferentiated SMCs presumably by PDGF-BB produced from high WSS-loaded endothelial cells accumulate in the intimal hyperplasia to form inflammatory microenvironment leading to the progression of the disease.

Increasing evidence suggests that T‑cell immunoglobulin and mucin domain 3 (TIM‑3) displays anti‑atherosclerotic effects, but its role in vascular smooth muscle cells (VSMCs) has not been reported. The present study aimed to investigate the function of TIM‑3 and its roles in human artery VSMCs (HASMCs). A protein array was used to investigate the TIM‑3 protein expression profile, which indicated that TIM‑3 expression was increased in the serum of patients with lower extremity arteriosclerosis obliterans disease (LEAOD) compared with healthy individuals. Immunohistochemistry and western blotting of arterial tissue further revealed that TIM‑3 expression was increased in LEAOD artery tissue compared with normal artery tissue. Additionally, platelet‑derived growth factor‑BB (PDGF‑BB) displayed a positive correlation with TIM‑3 expression in HASMCs. TIM‑3 decreased the migration and proliferation of PDGF‑BB‑induced HASMCs, and anti‑TIM‑3 blocked the effects of TIM‑3. The effect of TIM‑3 on the proliferation and migration of HASMCs was further investigated using LV‑TIM‑3‑transduced cells. The results revealed that TIM‑3 also inhibited PDGF‑BB‑induced expression of the inflammatory factors interleukin‑6 and tumor necrosis factor‑α by suppressing NF‑κB activation. In summary, the present study revealed that TIM‑3 displayed a regulatory role during the PDGF‑BB‑induced inflammatory reaction in HASMCs, which indicated that TIM‑3 may display anti‑atherosclerotic effects.

In this study we investigated the regulatory role of cell-migration-inducing and hyaluronan-binding protein (CEMIP) in the proliferation and migration of vascular smooth muscle cells (VSMCs). The mRNA and protein levels of CEMIP were upregulated in the plasma samples from patients with atherosclerosis, and in VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB), compared with plasma from healthy subjects and untreated VSMCs. Silencing CEMIP suppressed PDGF-BB-induced cell migration and proliferation in VSMCs, as determined using a Cell Counting Kit-8 assays, 5-ethynyl-2′-deocyuridine (EDU) assays, flow cytometry, wound healing assays, and Transwell assays. Overexpression of CEMIP promoted the proliferation and migration of VSMCs via activation of the Wnt–β-catenin signaling pathway and the upregulation of its target genes, including matrix metalloproteinase-2, matrix metalloproteinase-7, cyclin D1, and c-myc, whereas CEMIP deficiency showed the opposite effects. The knockdown of CEMIP in ApoE−/− mice by intravenous injection of lentiviral vector expressing si-CEMIP protected against high-fat-diet-induced atherosclerosis, as shown by the reduced aortic lesion areas, aortic sinus lesion areas, and the concentration of blood lipids compared with mice normally expressing CEMIP. These results demonstrated that CEMIP regulates the proliferation and migration of VSMCs in atherosclerosis by activating the WNT–β-catenin signaling pathway, which suggests the therapeutic potential of CEMIP for the management of atherosclerosis.

Dental implants are very successful medical devices, yet implant failures do occur due to biological and mechanical complications. Peri-implantitis is one such biological complication that is primarily caused by bacteria and their products at the implant-soft tissue interface. Bacterial infiltration can be prevented by the formation of a reliable soft tissue seal encircling dental implants. Platelet derived growth factor-BB (PDGF-BB) has significant chemotactic and proliferative effects on various mesenchymal cell types, including fibroblasts, and therefore can be an effective molecule to enhance the peri-implant soft tissue seal. To overcome the limitations of the recombinant protein form of PDGF-BB, such as cost and the need for supraphysiological doses, we have developed and characterized a titanium surface that is rendered bioactive by coating it with polyethylenimine (PEI)-plasmid DNA (pDNA) nanoplexes in the presence of sucrose. Human embryonic kidney 293T (HEK293T) cells and human primary gingival fibroblasts (GFs) were successfully transfected in culture with enhanced green fluorescent protein (EGFP)-encoding pDNA or platelet derived growth factor subunit B (PDGFB)-encoding pDNA loaded into nanoplexes and coated onto titanium discs in a dose-dependent manner. GFs were shown to secrete PDGF-BB for at least 7 days after transfection, and displayed both minimal viability loss and increased integrin-α2 expression 4 days post-transfection.

Vascular smooth muscle cell (SMC) from arterial stenotic-occlusive diseases is featured with deficiency in mitochondrial respiration and loss of cell contractility. However, the regulatory mechanism of mitochondrial genes and mitochondrial energy metabolism in SMC remains elusive. Here, we described that DNA methyltransferase 1 (DNMT1) translocated to the mitochondria and catalyzed D-loop methylation of mitochondrial DNA in vascular SMCs in response to platelet-derived growth factor-BB (PDGF-BB). Mitochondrial-specific expression of DNMT1 repressed mitochondrial gene expression, caused functional damage, and reduced SMC contractility. Hypermethylation of mitochondrial D-loop regions were detected in the intima-media layer of mouse carotid arteries subjected to either cessation of blood flow or mechanical endothelial injury, and also in vessel specimens from patients with carotid occlusive diseases. Likewise, the ligated mouse arteries exhibited an enhanced mitochondrial binding of DNMT1, repressed mitochondrial gene expression, defects in mitochondrial respiration, and impaired contractility. The impaired contractility of a ligated vessel could be restored by ex vivo transplantation of DNMT1-deleted mitochondria. In summary, we discovered the function of DNMT1-mediated mitochondrial D-loop methylation in the regulation of mitochondrial gene transcription. Methylation of mitochondrial D-loop in vascular SMCs contributes to impaired mitochondrial function and loss of contractile phenotype in vascular occlusive disease.

SIRT1 and STAT3 are key to human aortic vascular smooth muscle cells (HAVSMCs) proliferation, migration and phenotypic transformation, but the regulatory mechanism of SIRT1-STAT3 in this process is still unclear. Septin4 is a cytoskeleton-related protein that regulates oxidative stress-vascular endothelial injury. However, the role and underlying mechanism of Septin4 in atherosclerosis remains unknown. Here, we revealed the role and mechanism of Septin4 in regulating SIRT1-STAT3 in atherosclerosis. We determined that the expression of Septin4 were markedly increased in Apoe-/- atherosclerosis mice and PDGF-BB-induced HAVSMCs. Knockdown of Septin4 significantly increased PDGF-BB-induced HAVSMCs proliferation, migration and phenotypic transformation, while overexpression of Septin4 had the opposite effects. Mechanically, co-immunoprecipitation results demonstrated that Septin4 was a novel interacting protein of STAT3 and SIRT1. Septin4 formed a complex with SIRT1-STAT3, enhancing the interaction between SIRT1 and STAT3, ensuing promoting SIRT1-regulated STAT3-K685 deacetylation and STAT3-Y705 dephosphorylation, which inhibited PDGF-BB-induced HAVSMCs proliferation, migration and phenotype transformation. Therefore, our findings provide novel insights into the prevention and treatment of atherosclerosis.

Platelet-rich plasma (PRP) therapy has been widely applied in various medical fields including humans and horses. This study aimed to establish an optimal activation method to stably and reproducibly maximize the concentrations of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) contained in equine PRP. Autologous PRP was prepared from 11 Thoroughbreds. For the activation test, PRP was activated by either a single freeze-thaw cycle (Fr) or adding calcium and autologous serum containing thrombin (Ca). PDGF-BB and TGF-β1 concentrations in Fr, Ca, nonactivated (No), and platelet-poor plasma (PPP) samples were determined using ELISA and compared. For repetitive freeze-thaw test, PRP was subjected to single (Fr1), double (Fr2), triple (Fr3), or quadruple (Fr4) freeze-thaw cycles and the concentrations of both growth factors in samples were compared similarly. The PDGF-BB concentration in Ca was significantly higher than that in other preparations. The TGF-β1 concentrations in Fr and Ca were significantly higher than those in PPP and No, with no significant differences between Fr and Ca. The concentrations of both factors were significantly increased in PRP treated with multiple cycles of freeze-thaw compared with that in PRP treated with a single cycle. No significant differences were noted among Fr2, Fr3, and Fr4. Our findings suggest that activation by adding calcium and autologous serum is optimal for instant use of PRP and that double freeze-thawing is an easier and optimal activation method for cryopreserved PRP.

Biomaterials play a pivotal role in cell-free cartilage repair approaches, where cells must migrate through the scaffold, fill the defect, and then proliferate and differentiate facilitating tissue remodeling. Here we used multiple assays to test the influence of chemokines and growth factors on cell migration and cartilage repair in two different hyaluronan (HA)-based hydrogels. We first investigated bone marrow Mesenchymal Stromal Cells (BMSC) migration in vitro, in response to different concentrations of platelet-derived growth factor-BB (PDGF-BB), chemokine ligand 5 (CCL5/RANTES) and stromal cell-derived factor 1 (SDF-1), using a 3D spheroid-based assay. PDGF-BB was selected as most favourable chemotactic agent, and MSC migration was assessed in the context of physical impediment to cell recruitment by testing Fibrin-HA and HA-Tyramine hydrogels of different cross-linking densities. Supplementation of PDGF-BB stimulated progressive migration of MSC through the gels over time. We then investigated in situ cell migration into the hydrogels with and without PDGF-BB, using a cartilage-bone explant model implanted subcutaneously in athymic mice. In vivo studies show that when placed into an osteochondral defect, both hydrogels supported endogenous cell infiltration and provided an amenable microenvironment for cartilage production. These processes were best supported in Fibrin-HA hydrogel in the absence of PDGF-BB. This study used an advanced preclinical testing platform to select an appropriate microenvironment provided by implanted hydrogels, demonstrating that HA-based hydrogels can promote the initial and critical step of endogenous cell recruitment and circumvent some of the clinical challenges in cartilage tissue repair. STATEMENT OF SIGNIFICANCE: The challenge of articular cartilage repair arises from its complex structure and architecture, which confers the unique mechanical behavior of the extracellular matrix. The aim of our research is to identify biomaterials for implants that can support migration of endogenous stem and progenitor cell populations from cartilage and bone tissue, in order to permanently replace damaged cartilage with the original hyaline structure. Here, we present an in vitro 3D spheroid-based migration assay and an osteochondral defect model, which provide the opportunity to assess biomaterials and biomolecules, and to get stronger experimental evidence of the not well-characterized dynamic process of endogenous cells colonization in an osteochondral defect. Furthermore, the delicate step of early cell migration into biomaterials towards functional tissue engineering is reproduced. These tests can be used for pre-clinical testing of newly developed material designs in the field of scaffold engineering.

Biological research and clinical testing frequently require simultaneous quantification of multiple biomarkers for accurate disease diagnosis. In particular, the application of nanopore technology to real-sample analysis is limited in terms of discriminating multiple analytes in a mixture. In this study, we designed a series of highly specific double-stranded DNA-based probes and used them for synchronous detection of and discrimination among three protein biomarkers associated with lung cancer in a single nanopore. The probe could specifically identify and bind to the target, releasing an output DNA hybrid that generated signature current signals upon translocation through the nanopore. DNA hybrids with different hybridization stability and structure produced blockade currents clearly distinguishable in nanopore recordings, which could be unequivocally assigned to each analyte, thereby enabling accurate differentiation among multiple biomarkers. This assay based on the excellent discrimination ability of the probes facilitates unambiguous identification and quantification of multiple analytes on a natural serum background, thus presenting a powerful tool for biomedical research and clinical practice.

Hemodynamic forces have an important role in venous intimal hyperplasia, which is the main cause of arteriovenous fistula dysfunction. Endothelial cells (ECs) constantly exposed to the shear stress of blood flow, converted the mechanical stimuli into intracellular signals, and interacted with the underlying vascular smooth muscle cells (VSMCs). Caveolin-1 is one of the important mechanoreceptors on cytomembrane, which is related to vascular abnormalities. Extracellular signal-regulated kinase1/2 (ERK1/2) pathway is involved in the process of VSMCs proliferation and migration. In the present study, we explore the effects of Caveolin-1-ERK1/2 pathway and uremia toxins on the endothelial cells and VSMCs following shear stress application. Different shear stress was simulated with a ECs/VSMCs cocultured parallel-plate flow chamber system. Low shear stress and oscillating shear stress up-regulated the expression of fibroblast growth factor-4, platelet-derived growth factor-BB, vascular endothelial growth factor-A, ERK1/2 phosphorylation in endothelial cells, and proliferation and migration of VSMCs but down-regulated the Caveolin-1 expression in endothelial cells. Uremia toxin induces the proliferation and migration of VSMCs but not in a Caveolin-1-dependent manner in the static environment. Low shear stress-induced proliferation and migration of VSMCs is inhibited by Caveolin-1 overexpression and ERK1/2 suppression. Shear stress-regulated VSMC proliferation and migration is an endothelial cells-dependent process. Low shear stress and oscillating shear stress exert atherosclerotic influences on endothelial cells and VSMCs. Low shear stress modulated proliferation and migration of VSMCs through Caveolin-1-ERK1/2 pathway, which suggested that Caveolin-1 and ERK1/2 can be used as a new therapeutic target for the treatment of arteriovenous fistula dysfunction.Impact statementVenous intimal hyperplasia is the leading cause of arteriovenous fistula (AVF) dysfunction. This article reports that shear stress-regulated vascular smooth muscle cells (VSMCs) proliferation and migration is an endothelial cell (EC)-dependent process. Low shear stress (LSS) and oscillating shear stress (OSS) exert atherosclerotic influences on the ECs and VSMCs. LSS-induced proliferation and migration of VSMCs is inhibited by Caveolin-1 overexpression and extracellular signal-regulated kinase1/2 (ERK1/2) suppression, which suggested that Caveolin-1 and ERK1/2 can be used as a new therapeutic target for the treatment of AVF dysfunction.

Evidence links osteoporosis and cardiovascular disease but the cellular and molecular mechanisms are unclear. Here we identify skeleton-secreted platelet-derived growth factor-BB (PDGF-BB) as a key mediator of arterial stiffening in response to aging and metabolic stress. Aged mice and those fed high-fat diet (HFD), relative to young mice and those fed normal chow food diet, respectively, had higher serum PDGF-BB and developed bone loss and arterial stiffening. Bone/bone marrow preosteoclasts in aged mice and HFD mice secrete an excessive amount of PDGF-BB, contributing to the elevated PDGF-BB in blood circulation. Conditioned medium prepared from preosteoclasts stimulated proliferation and migration of the vascular smooth muscle cells. Conditional transgenic mice, in which PDGF-BB is overexpressed in preosteoclasts, had 3-fold higher serum PDGF-BB concentration and developed simultaneous bone loss and arterial stiffening spontaneously at a young age. Conversely, in conditional knockout mice, in which PDGF-BB is deleted selectively in preosteoclasts, HFD did not affect serum PDGF-BB concentration; as a result, HFD-induced bone loss and arterial stiffening were attenuated. These studies confirm that preosteoclasts are a main source of excessive PDGF-BB in blood circulation during aging and metabolic stress and establish the role of skeleton-derived PDGF-BB as an important mediator of vascular stiffening.

Vascular intimal hyperplasia is a hallmark event in vascular restenosis. The excessive proliferation, migration and phenotypic transformation of vascular smooth muscle cells (VSMCs) play important roles in the pathological mechanism of vascular intimal hyperplasia. Physalin B is an alcoholate isolated from Physalis (Solanaceae) that has a wide range of biological activities. However, the effect of physalin B on VSMCs is currently unclear. In this study, we demonstrated that physalin B significantly inhibited the proliferation, migration and phenotypic transformation of VSMCs induced by PDGF-BB. Physalin B also reduced inflammation and oxidative stress in VSMCs induced by PDGF-BB. Mechanistic studies showed that physalin B plays a role mainly by activating Nrf2. After Nrf2 activation, physalin B mitigates oxidative stress by enhancing the expression of the antioxidant gene HO-1; on the other hand, physalin B inhibits the NF-κB pathway to alleviate the inflammatory response. These two effects ultimately reduce the proliferation, migration and phenotypic transformation of VSMCs induced by PDGF-BB. In addition, in the mouse carotid artery ligation model, physalin B prevented intimal hyperplasia and inhibited the proliferation, migration and phenotypic transformation of cells in the hyperplastic intima. In conclusion, we provided significant evidence that physalin B abrogates PDGF-BB-induced VSMC proliferation, migration, phenotypic transformation and intimal hyperplasia by activating Nrf2-mediated signal transduction. Therefore, physalin B may be a potential therapeutic agent for preventing or treating restenosis.

Background The vascular pathology of peripheral artery disease (PAD) encompasses abnormal microvascular architecture and fibrosis in response to ischemia-reperfusion (I/R) cycles. We aimed to investigate the mechanisms by which pathological changes in the microvasculature direct fibrosis in the context of I/R. Methods Primary human aortic endothelial cells (ECs) were cultured under cycles of normoxia-hypoxia (NH) or normoxia-hypoxia-hyperoxia (NHH) to mimic I/R. Primary human aortic smooth muscle cells (SMCs) were cultured and treated with media from the ECs. Findings The mRNA and protein expression of the pro-fibrotic factors platelet derived growth factor (PDGF)-BB and connective tissue growth factor (CTGF) were significantly upregulated in ECs undergoing NH or NHH cycles. Treatment of SMCs with media from ECs undergoing NH or NHH cycles led to significant increases in TGF-β1, TGF-β pathway signaling intermediates, and collagen expression. Addition of neutralizing antibodies against PDGF-BB and CTGF to the media blunted the increases in TGF-β1 and collagen expression. Treatment of SMCs with PAD patient-derived serum also led to increased TGF-β1 levels. Interpretation In an in-vitro model of I/R, which recapitulates the pathophysiology of PAD, increased secretion of PDGF-BB and CTGF by ECs was shown to be predominantly driving TGF-β1-mediated expression by SMCs. These cell culture experiments help elucidate the mechanism and interaction between ECs and SMCs in microvascular fibrosis associated with I/R. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I/R. Funding National Institute on Aging at the National Institutes of Health grant number R01AG064420. Research in context Evidence before this study: Previous studies in gastrocnemius biopsies from peripheral artery disease (PAD) patients showed that transforming growth factor beta 1 (TGF-β1), the most potent inducer of pathological fibrosis, is increased in the vasculature of PAD patients and correlated with collagen deposition. However, the exact cellular source of TGF-β1 remained unclear. Added value of this study: Exposing cells to cycles of normoxia-hypoxia-hyperoxia (NHH) resulted in pathological changes that are consistent with human PAD. This supports the idea that the use of NHH may be a reliable, novel in vitro model of PAD useful for studying associated pathophysiological mechanisms. Furthermore, pro-fibrotic factors (PDGF-BB and CTGF) released from endothelial cells were shown to induce a fibrotic phenotype in smooth muscle cells. This suggests a potential interaction between these cell types in the microvasculature that drives increased TGF-β1 expression and collagen deposition. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of ischemia-reperfusion.

Background Childhood asthma is a common respiratory disease characterized by airway inflammation. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) has been found to be involved in the progression of asthma. This study aimed to explore the role of TIPE2 in the regulation of airway smooth muscle cells (ASMCs), which are one of the main effector cells in the development of asthma. Materials and methods ASMCs were transfected with pcDNA3.0-TIPE2 or si-TIPE2 for 48 h and then treated with platelet-derived growth factor (PDGF)-BB. Cell proliferation of ASMCs was measured using the MTT assay. Cell migration of ASMCs was determined by a transwell assay. The mRNA expression levels of calponin and smooth muscle protein 22α (SM22α) were measured using qRT-PCR. The levels of TIPE2, calponin, SM22α, PI3K, p-PI3K, Akt, and p-Akt were detected by Western blotting. Results Our results showed that PDGF-BB treatment significantly reduced TIPE2 expression at both the mRNA and protein levels in ASMCs. Overexpression of TIPE2 inhibited PDGF-BB-induced ASMC proliferation and migration. In addition, overexpression of TIPE2 increased the expression of calponin and SM22α in PDGF-BB-stimulated ASMCs. However, an opposite effect was observed with TIPE2 knockdown. Furthermore, TIPE2 overexpression blocked PDGF-BB-induced phosphorylation of PI3K and Akt, whereas the expression of p-PI3K and p-Akt were aggravated by TIPE2 knockdown. Additionally, the effects of TIPE2 overexpression and TIPE2 knockdown were altered by IGF-1 and LY294002 treatments, respectively. Conclusions Our findings demonstrate that TIPE2 inhibits PDGF-BB-induced ASMC proliferation, migration, and phenotype switching via the PI3K/Akt signaling pathway. Thus, TIPE2 may be a potential therapeutic target for the treatment of asthma.

Cancer treatment commonly relies on chemotherapy. This treatment faces many challenges, including treatment specificity and undesired side effects. To address these, a Dox-loaded Chol-aptamer molecular hybrid (Dox-CAH) was developed. This multivalent interaction system combines the key function of each integrated species: doxorubicin, cholesterol, and two aptamers binding to nucleolin and platelet-derived growth factor BB (PDGF-BB). The study has four stages: preparation of CAH via oligonucleotide hybridization, intercalation of doxorubicin into CAH, verification of CAH binding on SW480 by fluorescence microscopy and flow cytometry, and investigation of effect of Dox-CAH on SW480 proliferation. CAH was successfully prepared, as confirmed by electrophoresis. Flow cytometry and fluorescence microscopy demonstrated CAH binding to SW480, due to the presence of the AS1411 aptamer. This molecular hybrid exhibited specific binding because it did not bind to CCD 841 CoN. CAH binding to PDGF-BB compromises its function, as shown by enzyme-linked immunosorbent assay (ELISA) and cell assay. The DNA duplex in this molecular hybrid reduces the cytotoxicity of the Dox-CAH. Binding and the reduction of Dox-CAH toxicity may improve treatment specificity and minimize side effects. Dox-CAH is a model for more effective anticancer therapy, allowing incorporation of chemotherapeutic drugs and recognition elements.

The purpose of this study was to explore the effect of miR-34c on PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway, so as to find a new method for early diagnosis and treatment of cardiovascular disease. HA-VSMCs were treated with platelet-derived growth factor-BB (PDGF-BB) at 0 h, 12 h, 24 h, 48 h or 36 h to explore the optimal time for phenotypic transformation of VSMCs. And then, PDGF-BB-induced HA-VSMCs were transfected with miR-34c mimics/mimics NC and pcDNA3.1-PDGFR-β/pcDNA3.1-NC to observe cell biological behaviour. CCK8 was used to detect cell proliferation activity. Transwell chamber assay was used to detect cell invasion. Early apoptosis was analyzed by flow cytometry. The expression of α-SMA and Smemb was detected by immunofluorescence staining. The expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 were analyzed by Western blot analysis. The expression of miR-34a, miR-34b and miR-34c was detected by RT-PCR, and the targeting relationship between miR-34c and PDGFR-β was detected by luciferase reporting assay. The results indicated the proliferation and migration of PDGF-BB-induced HA-VSMCs significantly increased, and apoptosis significantly decreased. Besides, α-SMA decreased significantly, while Smemb increased significantly. Furthermore, expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 increased significantly, and SIRT1 decreased significantly. Experimental results showed that, miR-34c mimics significantly inhibited cell proliferation and migration, and promoted cell apoptosis, and miR-34c inhibitor had the opposite effects. MiR-34c mimics significantly increased α-SMA expression and decreased Smemb expression, while the opposite effects were reflected after transfection with miR-34c inhibitor. Moreover, miR-34c mimics significantly decreased the expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1, and significantly increased the expression of SIRT1, while miR-34c inhibitor had the opposite effects. Luciferase assay confirmed that PDGFR-β was a potential target of miR-34c. Subsequently, PDGF-BB-induced HA-VSMCs were co-transfected with miR-34c mimics and pcDNA3.1-PDGFR-β. The results indicated that PDGFR-β reversed the biological function of miR-34c mimic. The results revealed the potential application value of miR-34c as a marker molecule of phenotypic transformation, providing a potential target for improving phenotypic transformation. Supplementary Information The online version contains supplementary material available at 10.1007/s11033-021-06427-5.

Growth factors can stimulate tissue regeneration, but the side effects and low effectiveness associated with suboptimal delivery systems have impeded their use in translational regenerative medicine. Physiologically, growth factor interactions with the extracellular matrix control their bioavailability and spatiotemporal cellular signalling. Growth factor signalling is also controlled at the cell surface level via binding to heparan sulfate proteoglycans, such as syndecans. Here we show that vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) that were engineered to have a syndecan-binding sequence trigger sustained low-intensity signalling (tonic signalling) and reduce the desensitization of growth factor receptors. We also show in mouse models that tonic signalling leads to superior morphogenetic activity, with syndecan-binding growth factors inducing greater bone regeneration and wound repair than wild-type growth factors, as well as reduced tumour growth (associated with PDGF-BB delivery) and vascular permeability (triggered by VEGF-A). Tonic signalling via syndecan binding may also enhance the regenerative capacity of other growth factors. Attaching a syndecan-binding domain to vascular endothelial and platelet-derived growth factor variants enhances their binding to syndecans and triggers tonic signalling for enhanced bone regeneration and wound repair in mice.

BACKGROUND The long noncoding RNA LIPCAR is a type of transcription product (>200 nucleotides long). Recent studies demonstrated that LIPCAR is a potential biomarker in cardiovascular disease and can predict survival in patients with cardiovascular disease. Therefore, the present study explored the role of LIPCAR in the regulation of proliferation, migration, and change in phenotype of vascular smooth muscle cells. MATERIAL AND METHODS Human vascular smooth muscle cells (VSMCs) were treated with 20 g/mL oxidatively modified low-density lipoprotein (ox-LDL) or 20 ng/ml platelet-derived growth factor BB (PDGF-BB) for 24 h, then the expression levels of LIPCAR were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. LIPCAR-overexpressing plasmids were transfected into VSMCs. After transfection, cell proliferation and migration were measured using the Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The levels of a-smooth muscle actin (a-SMA) a molecular marker of the contractile VSMC phenotype, were measured using Western blot and immunofluorescence assays. Protein levels of cyclin-dependent kinase-2 (CDK2), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor A (VEGF-A), and angiopoietin-2 (Ang-2) were assessed by Western blot. The level of tissue factor (TF) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS Treatment with PDGF-BB or ox-LDL significantly increased levels of LIPCAR in VSMCs. Overexpression of LIPCAR markedly promoted cell proliferation and migration. Further, upregulation of LIPCAR increased CDK2, p21, PCNA, MMP2, MMP9, VEGF-A, Ang-2, and TF expression and decreased p21 expression. In addition, LIPCAR significantly decreased a-SAM expression. CONCLUSIONS Together, our data suggest that overexpression of LIPCAR promotes cell proliferation, migration, and phenotypic switch of vascular smooth muscle cells.

The relationship between aging and restenosis are unclear. The purposes of this study were to investigate the possible pathological role and mechanism of aging on formation of restenosis. Our data indicated that cell proliferation and migration of the oxidative stress-induced senescent vascular smooth muscle cells were obviously desensitized to stimulation by platelet-derived growth factor (PDGF)-BB, which may have been caused by suppression of promoter activity, transcription, translation, and activation levels of PDGF receptor (PDGFR)-β. The analyzed data obtained from the binding array of transcription factors (TFs) showed that binding levels of eighteen TFs on the PDGFR-β promoter region (-523 to -1) were significantly lower in senescent cells compared to those of non-senescent cells. Among these TFs, the bioinformatics prediction suggested that the putative binding sites of ten TFs were found in this promoter region. Of these, transcriptional levels of seven TFs were markedly reduced in senescent cells. The clinical data showed that the proportion of restenosis was relatively lower in the older group than that in the younger group. Our study results suggested that a PDGFR-β-mediated pathway was suppressed in aging cells, and our clinical data showed that age and the vascular status were slightly negatively correlated in overall participants.

Endothelial barrier dysfunction is a critical pathophysiological process of sepsis. Impaired endothelial cell migration is one of the main reasons for endothelial dysfunction. Statins may have a protective effect on endothelial barrier function. However, the effect and mechanism of statins on lipopolysaccharide (LPS)‐induced endothelial barrier dysfunction remain unclear. Simvastatin (SV) was loaded in nanostructured lipid carriers to produce SV nanoparticles (SV‐NPs). Normal SV and SV‐NPs were used to treat human umbilical vein vascular endothelial cells (HUVECs) injured by LPS. Barrier function was evaluated by monitoring cell monolayer permeability and transendothelial electrical resistance, and cell migration ability was measured by a wound healing assay. LY294002 and imatinib were used to inhibit the activity of PI3K/Akt and platelet‐derived growth factor receptor (PDGFR) β. IQ‐GTPase‐activating protein 1 (IQGAP1) siRNA was used to knockdown endogenous IQGAP1, which was used to verify the role of the PDGFRβ/PI3K/Akt/IQGAP1 pathway in SV/SV‐NPs‐mediated barrier protection in HUVECs injured by LPS. The results show that SV/SV‐NPs promoted the migration and decreased the permeability of HUVECs treated with LPS, and the efficacy of the SV‐NPs exceeded that of SV significantly. LY294002, imatinib and IQGAP1 siRNA all suppressed the barrier protection of SV/SV‐NPs. SV/SV‐NPs promoted the secretion of platelet‐derived growth factor‐BB (PDGF‐BB) and activated the PDGFRβ/PI3K/Akt/IQGAP1 pathway. SV preparations restored endothelial barrier function by restoring endothelial cell migration, which is involved in the regulation of the PDGFRβ/PI3K/Akt/IQGAP1 pathway and PDGF‐BB secretion. As an appropriate formulation for restoring endothelial dysfunction, SV‐NPs may be more effective than SV.

In this study, the expression changes and the potential effects of CD200 and its receptors during the process of retinal neovascularization (RNV) development had been detected, using a classic oxygen-induced retinopathy (OIR) mice model and CD200Fc (a CD200R1 agonist) intravitreal injection. 7 day postnatal (P7) C57BL/6J mice were raised in hyperoxia incubators with 75±2% oxygen for 5 days, and returned to room air at P12. All animals were subdivided into three groups: normoxia control, OIR, and OIR+CD200Fc group. The mice of OIR+CD200Fc group were intravitreal injected with CD200Fc (2μg/μL, 0.5μL) at P12. Retinas and vitreous samples were harvested at P17. The expression and localization of CD200 and its receptors were analyzed by Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and retinal whole-mount immunofluorescence. To investigate the effects of CD200Fc treatment, vascular endothelial growth factor (VEGF)-A, platelet-derived growth factor (PDGF)-BB, pro-inflammatory cytokines, NV area, and microglial activation were detected respectively. In OIR group, both protein and RNA levels of CD200 and CD200R1 were significantly up-regulated. The increased CD200 and CD200R1 were co-localized with Alex594-labeled Griffonia simplicifolia isolectin B4 (IB4) on vascular endothelial cells in NV area of OIR samples, and CD200R1 was co-expressed with ionized calcium-bind adapter molecule 1 (iba1) on microglia in OIR samples at the same time. CD200Fc intravitreal injection could significantly reduce the release of VEGF-A, PDGF-BB, and pro-inflammatory cytokines; shrink the NV area; and inhibit the activation of microglia in OIR mice. These findings suggested that the up-regulation of CD200 and CD200R1 was closely related to RNV development, and the antiangiogenic effects of CD200Fc in OIR model might be realized by inhibition of inflammatory response and microglia activation. The results may provide a new therapeutic target for RNV diseases.

Detection of biomarkers in body fluids is critical to both diagnosing the life-threatening diseases and optimizing therapeutic interventions. We herein report use of liquid crystals (LCs) to detect biomarkers in blood with high sensitivity and specificity by employing in situ rolling circle amplification (RCA) on magnetic beads (MBs). Specific recognition of cancer biomarkers, such as platelet derived growth factor BB (PDGF-BB) and adenosine, by aptamers leads to formation of a nucleic acid circle on MBs preassembled with ligation DNA, linear padlock DNA, and aptamers, thereby triggering in situ RCA. LCs change from dark to bright appearance after the in situ RCA products being transferred onto the LC interface decorated with octadecy trimethylammonium bromide (OTAB), which is particularly sensitive to the amplified DNA on MBs. Overall, this label-free approach takes advantages of high specificity of aptamer-based assay, efficient enrichment of signaling molecules on MBs, remarkable DNA elongation performance of the RCA reaction, and high sensitivity of LC-based assay. It successfully eliminates the matrix interference on the LC-based sensors and thus achieves at least 4 orders of magnitude improvement in sensitivity for detection of biomarkers compared to other LC-based sensors. In addition, performance of the developed sensor is comparable to that of the commercial ones. Thus, this study provides a simple, powerful, and promising approach to facilitate highly sensitive, specific, and label-free detection of biomarkers in body fluids.