Sema3A Overexpression Research Articles

Sema3A Alleviates the Malignant Behaviors of Gastric Cancer Cells by Inhibiting NRP-1.

Semaphorin3A (Sema3a) is lowly expressed in the peripheral blood of gastric cancer patients, suggesting Sema3a may be involved in the progression of gastric cancer. Nevertheless, the specific role and the potential regulatory mechanism of Sema3a in gastric cancer is still obscure. Neuropilin-1 (NRP-1) has been reported to interact with Sema3a; herein, we intended to reveal the role and regulatory mechanism of Sema3a/neuropilin-1 (NRP-1) in gastric cancer progression. Cell transfection was carried out to regulate gene expression. CCK-8 and colony formation assays were applied to estimate cell proliferation. Scratch assay and transwell assay were conducted to assess the cell migration and invasion abilities. Angiogenesis ability was assessed using a tubule-forming assay. The expression of corresponding genes and proteins were detected by RT-qPCR and western blot, respectively. Data showed that Sema3a was downregulated in gastric cancer cells and NRP-1 was upregulated. Sema3a overexpression repressed NRP-1 level in AGS cells. Overexpression of Sema3a inhibited cell proliferation, migration, and invasion abilities as well as epithelial-mesenchymal transition (EMT) of AGS cells. Overexpression of Sema3a inhibited tube formation and reduced the expression of VEGFA/VEGFR2 in AGS cells. However, the effects of Sema3a overexpression on the malignant behaviors in AGS cells were partly reversed by NRP-1 overexpression. Additionally, Sema3a overexpression enhanced the inhibitory effects of Ramucirumab, an anti-VEGFR2 agent, on the proliferative, migratory, and invasive capabilities as well as EMT in AGS cells. In conclusion, Sema3a alleviates the proliferation, migration, invasion, and angiogenesis capabilities of gastric cancer cells via repressing NRP-1. This finding may provide potential targets for gastric cancer therapy.

Reduced expression of semaphorin 3A in osteoclasts causes lymphatic expansion in a Gorham-Stout disease (GSD) mouse model.

Gorham-Stout disease (GSD) is a sporadic chronic disease characterized by progressive bone dissolution, absorption, and disappearance along with lymphatic vessel infiltration in bone-marrow cavities. Although the osteolytic mechanism of GSD has been widely studied, the cause of lymphatic hyperplasia in GSD is rarely investigated. In this study, by comparing the RNA expression profile of osteoclasts (OCs) with that of OC precursors (OCPs) by RNA sequencing, we identified a new factor, semaphorin 3A (Sema3A), which is an osteoprotective factor involved in the lymphatic expansion of GSD. Compared to OCPs, OCs enhanced the growth, migration, and tube formation of lymphatic endothelial cells (LECs), in which the expression of Sema3A is low compared to that in OCPs. In the presence of recombinant Sema3A, the growth, migration, and tube formation of LECs were inhibited, further confirming the inhibitory effect of Sema3A on LECs in vitro. Using an LEC-induced GSD mouse model, the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo. We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss, whereas the injection of lentivirus expressing Sema3A short hairpin RNA (shRNA) into the tibiae caused GSD-like phenotypes. Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment, compared with the control. Based on the above results, we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.

Semaphorin‑3A alleviates cardiac hypertrophy by regulating autophagy.

Cardiac hypertrophy, characterized by cardiomyocyte enlargement, is an adaptive response of the heart to certain hypertrophic stimuli; however, prolonged hypertrophy results in cardiac dysfunction and can ultimately cause heart failure. The present study evaluated the role of semaphorin-3A (Sema3A), a neurochemical inhibitor, in cardiac hypertrophy, utilizing an isoproterenol (ISO) induced H9c2 cell model. Cells were stained with rhodamine-phalloidin to assess the cell surface area and reverse transcription-quantitative PCR was performed to quantify mRNA expression levels of Sema3A, brain natriuretic factor (BNF) and β-myosin heavy chain (β-MHC). The protein expression levels of the autophagy-related proteins light chain 3 (LC3), p62 and Beclin-1, and the Akt/mTOR signaling pathway associated proteins Akt, phosphorylated (p)-Akt, mTOR, p-mTOR, 4E-binding protein 1 (4EBP1) and p-4EBP1 were semi-quantified using western blotting. Rapamycin, a canonical autophagy inducer, was administered to H9c2 cells to elucidate the regulatory mechanism of Sema3A. The results indicated significantly increased cell surface area and elevated BNF and β-MHC mRNA expression levels, increased LC3II/I ratio and Beclin-1 protein expression levels and significantly decreased p62 protein expression levels after treatment of H9c2 cardiomyocytes with ISO for 24 h. Sema3A overexpression improved ISO-induced hypertrophy in H9c2 cells, indicated by decreased cell surface area and reduced BNF and β-MHC mRNA expression levels. Moreover, Sema3A overexpression inhibited ISO-induced autophagy in H9c2 cells, indicated by decreased LC3II/I ratio and Beclin-1 protein expression levels and increased p62 protein expression levels. The autophagy activator rapamycin partially inhibited the protective effect of Sema3A on ISO-induced hypertrophy. Sema3A overexpression suppressed the decrease of the protein expression levels of p-Akt, mTOR and their downstream target 4EBP1, which is induced by ISO. Collectively, these results suggested Sema3A prevented ISO-induced cardiac hypertrophy by inhibiting autophagy via the Akt/mTOR signaling pathway.

Abstract 12226: Aging Impairs the Neuro-Vascular Interface in the Heart

Aging is a major risk factor for impaired cardiovascular function. The aging heart is characterized by vascular dysfunction, increased hypertrophy, fibrosis and electrophysiological alterations. However, the interaction between the different age-associated changes in cardiac remodelling is poorly define. Since vessels are aligned with nerves, and this interplay is critical for tissue homeostasis, we investigated whether an impairment of the neuro-vascular interface may contribute to age-associated pathologies in the heart. We show that aging reduces sympathetic and sensory nerve fibers, which both align to arteries and arterioles. Aging induced a dysregulation of vascular-derived neuro-regulatory genes, especially the axon repellent factor Sema3A, which was augmented by 2.4±0.1-fold in old heart endothelial cells. Consistently, endothelial Sema3a overexpression reduced axon density in vivo, thus mimicking the observed aged heart phenotype. Since cellular senescence was induced at the same time point when nerve density declined (16 months of age) and senescence endothelial cells showed a higher expression of Sema3a, we hypothesized that endothelial senescence may contribute to cardiac denervation. Indeed, supernatants of senescent endothelial cells reduced neurite outgrowth in vitro in a Sema3A-dependent manner. In vivo studies further confirmed that endothelial-specific induction of senescence by overexpression of Progerin induced left ventricular denervation and induced Sema3a expression. By contrast, the pharmacological removal of senescent cells by the senolytic drugs dasatinib and quercetin rescued age-induced denervation and prevented the age-induced induction of endothelial Sema3a expression. Functional studies additionally demonstrated that endothelial senescence reduced heart rate variability, while senolytics preserved heart rate variability, restored age-associated declines in sympathetic and parasympathetic activity and reduced age-associated increase in arrhythmias. These data suggest that senescence-associated regulation of neuro-regulatory genes in endothelial cells is associated with reduced nerve density and, thereby, contributes to age-associated cardiac dysfunction.

Aging impairs the neurovascular interface in the heart.

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.

CircSEC11A knockdown alleviates oxidative stress and apoptosis and promotes cell proliferation and angiogenesis by regulating miR-29a-3p/SEMA3A axis in OGD-induced human brain microvascular endothelial cells (HBMECs).

Circular RNA (circRNA) has been found to play an important role in the progression of many diseases, including ischemic stroke. However, the regulatory mechanism of circSEC11A in ischemic stroke progression need to further investigation. Human brain microvascular endothelial cells (HBMECs) were stimulated by oxygen glucose deprivation (OGD). CircSEC11A, SEC11A mRNA and miR (microRNA)-29a-3p were quantified by quantitative real-time PCR (qRT-PCR). SEMA3A, BAX and BCL2 protein level was quantified by western blot. Oxidative stress, cell proliferation, angiogenesis and apoptosis abilities were gauged by oxidative stress assay kit, 5-Ethynyl-2'-Deoxyuridine (EdU) staining, tube formation assay and flow cytometry assays, respectively. Direct relationship between miR-29a-3p and circSEC11A or SEMA3A was validated by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. CircSEC11A was upregulated in OGD-induced HBMECs. OGD promoted the oxidative stress and apoptosis and inhibited cell proliferation and angiogenesis, while circSEC11A knockdown relieved the effects. CircSEC11A functioned as the sponge for miR-29a-3p, and miR-29a-3p inhibitor reversed the effects of si-circSEC11A on OGD-induced HBMECs oxidative injuries. Moreover, SEMA3A served as the target gene of miR-29a-3p. MiR-29a-3p inhibition ameliorated OGD-induced HBMECs oxidative injuries, while SEMA3A overexpression rescued the impacts of miR-29a-3p mimic. CircSEC11A promoted the malignant progression in OGD-induced HBMECs through the mediation of miR-29a-3p/SEMA3A axis. This study has provided the new insight into the underlying application of circSEC11A in cell model of ischemic stroke.

Sema3A inactivates the ERK/JNK signalling pathways to alleviate inflammation and oxidative stress in lipopolysaccharide-stimulated rat endothelial cells and lung tissues

Semaphorin 3A (Sema3A) is a secretory member of the semaphorin family of immune response regulators. This research focuses on its effects on inflammation and oxidative stress in acute respiratory distress syndrome (ARDS). By analysing the GEO dataset GSE57011, we obtained Sema3A as the most downregulated gene in ARDS samples. Lipopolysaccharide (LPS) was used to stimulate rat pulmonary microvascular endothelial cells (PMVECs) and rats to induce ARDS-like symptoms in vitro and in vivo, respectively. LPS induced severe damage in rat lung tissues, in which reduced immunohistochemical staining of Sema3A was detected. Sema3A overexpression reduced apoptosis and angiogenesis of LPS-induced PMVECs and alleviated lung injury and pulmonary edoema of rats. Moreover, ELISA results showed that Sema3A overexpression downregulated the levels of inflammatory cytokines and oxidative stress markers both in PMVECs and the rat lung. Activation of ERK/JNK signalling aggravated LPS-induced damage on PMVECs; however, the aggravation was partly blocked by Sema3A, which suppressed phosphorylation of ERK/JNK. Overall, this study demonstrates that Sema3A inactivates the ERK/JNK signalling to ameliorate inflammation and oxidative stress in LPS-induced ARDS models. Sema3A might therefore represent a candidate option for ARDS treatment.

Sema3A alleviates viral myocarditis by modulating SIRT1 to regulate cardiomyocyte mitophagy.

Viral myocarditis (VMC) is a common myocardial inflammatory disease characterized by inflammatory cell infiltration and cardiomyocyte necrosis. Sema3A was reported to reduce cardiac inflammation and improve cardiac function after myocardial infarction, but its role in VMC remains to be explored. Here, a VMC mouse model was established by infection with CVB3, and Sema3A was overexpressed in vivo by intraventricular injection of an adenovirus-mediated Sema3A expression vector (Ad-Sema3A). We found that Sema3A overexpression attenuated CVB3-induced cardiac dysfunction and tissue inflammation. And Sema3A also reduced macrophage accumulation and NLRP3 inflammasome activation in the myocardium of VMC mice. In vitro, LPS was used to stimulate primary splenic macrophages to mimic the macrophage activation state in vivo. Activated macrophages were co-cultured with primary mouse cardiomyocytes to evaluate macrophage infiltration-induced cardiomyocyte damage. Ectopic expression of Sema3A in cardiomyocytes effectively protected cardiomyocytes from activated macrophage-induced inflammation, apoptosis, and ROS accumulation. Mechanistically, cardiomyocyte-expressed Sema3A mitigated macrophage infiltration-caused cardiomyocyte dysfunction by promoting cardiomyocyte mitophagy and hindering NLRP3 inflammasome activation. Furthermore, NAM (a SIRT1 inhibitor) reversed the protective effect of Sema3A against activated macrophage-induced cardiomyocyte dysfunction by suppressing cardiomyocyte mitophagy. In conclusion, Sema3A promoted cardiomyocyte mitophagy and suppressed inflammasome activation by regulating SIRT1, thereby attenuating macrophage infiltration-induced cardiomyocyte injury in VMC.

An increase in Semaphorin 3A biases the axonal direction and induces an aberrant dendritic arborization in an in vitro model of human neural progenitor differentiation

BackgroundSemaphorins (Sema) belong to a large family of repellent guidance cues instrumental in guiding axons during development. In particular, Class 3 Sema (Sema 3) is among the best characterized Sema family members and the only produced as secreted proteins in mammals, thereby exerting both autocrine and paracrine functions. Intriguingly, an increasing number of studies supports the crucial role of the Sema 3A in hippocampal and cortical neurodevelopment. This means that alterations in Sema 3A signaling might compromise hippocampal and cortical circuits and predispose to disorders such as autism and schizophrenia. Consistently, increased Sema 3A levels have been detected in brain of patients with schizophrenia and many polymorphisms in Sema 3A or in the Sema 3A receptors, Neuropilins (Npn 1 and 2) and Plexin As (Plxn As), have been associated to autism.ResultsHere we present data indicating that when overexpressed, Sema 3A causes human neural progenitors (NP) axonal retraction and an aberrant dendritic arborization. Similarly, Sema 3A, when overexpressed in human microglia, triggers proinflammatory processes that are highly detrimental to themselves as well as NP. Indeed, NP incubated in microglia overexpressing Sema 3A media retract axons within an hour and then start suffering and finally die. Sema 3A mediated retraction appears to be related to its binding to Npn 1 and Plxn A2 receptors, thus activating the downstream Fyn tyrosine kinase pathway that promotes the threonine-serine kinase cyclin-dependent kinase 5, CDK5, phosphorylation at the Tyr15 residue and the CDK5 processing to generate the active fragment p35.ConclusionsAll together this study identifies Sema 3A as a critical regulator of human NP differentiation. This may imply that an insult due to Sema 3A overexpression during the early phases of neuronal development might compromise neuronal organization and connectivity and make neurons perhaps more vulnerable to other insults across their lifespan.

KCNQ1OT1 inhibition alleviates high glucose-induced podocyte injury by adsorbing miR-23b-3p and regulating Sema3A.

Diabetic nephropathy (DN), a diabetic complication, is the leading cause of end-stage renal disease. KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), a long non-coding RNA, has been unmasked to participate in the pathogenesis of DN. However, the specific mechanism by which KCNQ1OT1 regulates podocyte injury remains unclear. Relative expression of KCNQ1OT1 was measured with quantitative real-time polymerase chain reaction (qRT-PCR). The levels of inflammatory cytokines were analyzed by enzyme linked immunosorbent assay (ELISA). The viability, proliferation, and apoptosis of high glucose (HG)-treated podocyte were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Protein levels were analyzed by western blotting. The regulatory mechanism of KCNQ1OT1 was surveyed by bioinformatics analysis, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. We observed an apparent upregulation in KCNQ1OT1 expression in serums of DN patients and HG-treated podocytes. Furthermore, KCNQ1OT1 downregulation alleviated HG-induced inflammation, proliferation repression, and apoptosis in podocytes. Notably, KCNQ1OT1 was identified as a miR-23b-3p sponge, and miR-23b-3p directly targeted Semaphorin-3A (Sema3A). Moreover, miR-23b-3p silencing reversed KCNQ1OT1 knockdown-mediated effects on inflammation, proliferation, and apoptosis of HG-induced podocytes. Also, Sema3A overexpression reversed the effects of miR-23b-3p mimic on inflammation, proliferation, and apoptosis of HG-induced podocytes. Importantly, KCNQ1OT1 regulated Sema3A expression by sponging miR-23b-3p. HG-induced KCNQ1OT1 promoted inflammation, proliferation repression, and apoptosis of podocytes via increasing Sema3A expression through sponging miR-23b-3p. This study provided evidence to support the involvement of KCNQ1OT1 in the pathogenesis of DN.

Silicified collagen scaffold induces semaphorin 3A secretion by sensory nerves to improve in-situ bone regeneration

Sensory nerves promote osteogenesis through the release of neuropeptides. However, the potential application and mechanism in which sensory nerves promote healing of bone defects in the presence of biomaterials remain elusive. The present study identified that new bone formation was more abundantly produced after implantation of silicified collagen scaffolds into defects created in the distal femur of rats. The wound sites were accompanied by extensive nerve innervation and angiogenesis. Sensory nerve dysfunction by capsaicin injection resulted in significant inhibition of silicon-induced osteogenesis in the aforementioned rodent model. Application of extracellular silicon in vitro induced axon outgrowth and increased expression of semaphorin 3 A (Sema3A) and semaphorin 4D (Sema4D) in the dorsal root ganglion (DRG), as detected by the upregulation of signaling molecules. Culture medium derived from silicon-stimulated DRG cells promoted proliferation and differentiation of bone marrow mesenchymal stem cells and endothelial progenitor cells. These effects were inhibited by the use of Sema3A neutralizing antibodies but not by Sema4D neutralizing antibodies. Knockdown of Sema3A in DRG blocked silicon-induced osteogenesis and angiogenesis almost completely in a femoral defect rat model, whereas overexpression of Sema3A promoted the silicon-induced phenomena. Activation of “mechanistic target of rapamycin” (mTOR) pathway and increase of Sema3A production were identified in the DRG of rats that were implanted with silicified collagen scaffolds. These findings support the role of silicon in inducing Sema3A production by sensory nerves, which, in turn, stimulates osteogenesis and angiogenesis. Taken together, silicon has therapeutic potential in orthopedic rehabilitation.

MiR-379-5p aggravates experimental autoimmune uveitis in mice via the regulation of SEMA3A

Uveitis is a disease resulting in the inflammation of uveal tracts, but the factors resulting in uveitis is still obscure. Previous studies have shown that miR-379-5p was involved in the pathogenesis of several diseases, however, the role and regulatory mechanism of miR-379-5p in uveitis were unclear. In our study, we established experimental autoimmune uveitis (EAU) mouse models to explore the role of miR-379-5p in uveitis. RT-qPCR identified that miR-379-5p level was increased in serum of EAU mice. In mechanism, SEMA3A 3’UTR was proven to be directly targeted by miR-379-5p and SEMA3A expression was negatively regulated by miR-379-5p in CD4+ T cells. Moreover, ELISA analysis revealed that knockdown of miR-379-5p suppressed the production of inflammation cytokines including IL-17, TNF-α and IL-β in vitro. These results were reversed by SEMA3A overexpression. In addition, the reduction of Th17 cells under miR-379-5p inhibitor was neutralised by SEMA3A knockdown in vitro. Furthermore, we demonstrated that knockdown of miR-379-5p significantly reversed the increased clinical scores and inflammatory response resulting from EAU treatment and this effect was further countervailed by SEMA3A silencing. Our study suggested that miR-379-5p aggravated uveitis in EAU mice via the regulation of SEMA3A, which may provide a novel insight for uveitis treatment.

Semaphorin 3A promotes the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in inflammatory environments by suppressing the Wnt/β-catenin signaling pathway.

After periodontal treatment, the local inflammatory environment surrounding periodontal tissues cannot be entirely eliminated. The means by which alveolar bone repair and regeneration are promoted in inflammatory environments have important clinical significance. As a powerful protein that promotes the differentiation of osteocytes, semaphorin 3A (Sema3A) shows potential for bone regeneration therapy. However, the effect of Sema3A on osteogenic differentiation in an inflammatory environment, as well as the underlying mechanism, have not yet been explored. We used lentivirus to transduce rat bone marrow-derived mesenchymal stem cells (rBMSCs) to stably overexpress Sema3A. Lipopolysaccharide from Escherichia coli (E. coli LPS) was used to stimulate rBMSCs to establish an inflammatory environment. ALP staining, Alizarin red staining, ALP activity tests, quantitative RT-PCR (qRT-PCR), and Western blotting were used to elucidate the effect of Sema3A on the osteogenesis of rBMSCs in inflammatory environments. XAV939 and LiCl were used to determine whether the Wnt/β-catenin signaling pathway was involved in attenuating the inhibition of Sema3A-induced osteogenic differentiation by LPS. The qRT-PCR and Western blot results demonstrated that the lentiviral vector (LV-NC) and lentiviral-Sema3A (LV-Sema3A) were successfully transduced into rBMSCs. An inflammatory environment could be established by stimulating rBMSCs with 1 μg/ml E. coli LPS. After Sema3A overexpression, mineral deposition was exacerbated, and the BSP and Runx2 gene and protein expression levels were increased. Furthermore, E. coli LPS activated the Wnt/β-catenin signaling pathway and decreased rBMSC osteogenesis, but these effects were attenuated by Sema3A. In conclusion, Sema3A could protect BMSCs from LPS-mediated inhibition of osteogenic differentiation in inflammatory environments by suppressing the Wnt/β-catenin pathway.

MiR-203-3p inhibits the oxidative stress, inflammatory responses and apoptosis of mice podocytes induced by high glucose through regulating Sema3A expression.

Diabetic nephropathy (DN) is the most serious long-term microvascular complication of diabetes, which mainly causes podocyte injury. Many studies have shown that microRNAs play a vital role in the development of DN. Studies have shown that miR-203-3p is involved in mesangial cell proliferation and apoptosis of DN mice. Therefore, we speculated that miR-203-3p might be related to the development of DN, but our study does not provide any evidence. In animal experiments, diabetic mice (db/db) were transfected with iR-203-3p overexpression lentiviral vectors (LV-miR-203-3p) and their control (LV-miR-con), with normal mice (db/m) being used as the control. High glucose (HG)-induced podocytes were used to construct a DN cell model in vitro. The expression levels of miR-203-3p, Semaphorin 3A (Sema3A) and inflammatory cytokines were detected by quantitative real-time polymerase chain reaction. Also, serum creatinine and blood urea nitrogen levels were used to evaluate the degree of renal injury in DN mice. Sema3A and apoptosis-related protein levels were assessed by the western blot analysis. Enzyme-linked immunosorbent assay was used to determine the different oxidative stress-related indicators and inflammatory cytokines. Flow cytometry and caspase-3 activity detection were used to analyze the degree of podocyte apoptosis. Our results suggested that the expression of miR-203-3p was lower in DN mice and in HG-induced podocytes. Overexpression of miR-203-3p reduced the body weight, blood glucose and renal injury of DN mice in vivo, as well as relieve the oxidative stress, inflammatory response and apoptosis of HG-induced podocytes in vitro. Functionally, Sema3A was a target of miR-203-3p, and Sema3A overexpression reversed the inhibitory effect of miR-203-3p on HG-induced podocyte injury. Our findings revealed that miR-203-3p alleviated the podocyte injury induced by HG via regulating Sema3A expression, suggesting that miR-203-3p might be a new therapeutic target to improve the progression of DN.

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model.

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.

Semaphorin 3A-hypoxia inducible factor 1 subunit alpha co-overexpression enhances the osteogenic differentiation of induced pluripotent stem cells-derived mesenchymal stem cells in vitro.

Background:Mesenchymal stem or stromal cells (MSCs) derived from the induced pluripotent stem cells (iPSCs) have uniform biological activity, which makes the clinical application of MSCs in bone repair possible. Culturing the iPSC-MSCs onto osteoconductive materials is a promising tissue engineering-based strategy in bone regeneration. The aim of this work was to evaluate the effects of semaphorin 3A (Sema3A) and hypoxia inducible factor 1 subunit alpha (HIF1α) co-overexpression on the survival and osteogenic differentiation of iPSC-MSCs.Methods:Sema3A and HIF1α were linked together with the three (GGGGS; G, glycine; S, serine) peptide fragment, and their co-expression in iPSC-MSCs was mediated by a lentiviral vector. The fusion protein retained the immune reactivity for both Sema3A and HIF1α as determined with Western blotting. iPSC-MSCs were infected with overexpression lentivirus (oeLenti) as negative control, oeLenti-Sema3A, oeLenti-HIF1α or oeLenti-Sema3A-HIF1α lentiviruses.Results:Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs (the activity and/or expression of osteoblast markers, such as alkaline phosphatase, osteopontin, and osteocalcin, were upregulated), and suppressed cell survival. The Sema3A-HIF1α fusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival. We further seeded iPSC-MSCs modified by SemaA-HIF1α overexpression onto hydroxyapatite (HA) scaffolds, and evaluated their growth and differentiation on this three-dimensional material. Additional data indicated that, as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes, cells cultured in HA scaffolds grew (blank vs. HA scaffolds: 0.83 vs. 1.39 for survival) and differentiated better (blank vs. HA scaffolds: 11.29 vs. 16.62 for alkaline phosphatase activity).Conclusion:Modifying iPSC-MSCs with pro-osteogenic (Sema3A) and pro-survival (HIF1α) factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.

Sema3a-Nrp1 Signaling Mediates Fast-Twitch Myofiber Specificity of Tw2+ Cells.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

Semaphorin-3A protects against neointimal hyperplasia after vascular injury

BackgroundNeointimal hyperplasia is a prominent pathological event during in-stent restenosis. Phenotype switching of vascular smooth muscle cells (VSMCs) from a differentiated/contractile to a dedifferentiated/synthetic phenotype, accompanied by migration and proliferation of VSMCs play an important role in neointimal hyperplasia. However, the molecular mechanisms underlying phenotype switching of VSMCs have yet to be fully understood.MethodsThe mouse carotid artery ligation model was established to evaluate Sema3A expression and its role during neointimal hyperplasia in vivo. Bioinformatics analysis, chromatin immunoprecipitation (ChIP) assays and promoter-luciferase reporter assays were used to examine regulatory mechanism of Sema3A expression. SiRNA transfection and lentivirus infection were performed to regulate Sema3A expression. EdU assays, Wound-healing scratch experiments and Transwell migration assays were used to assess VSMC proliferation and migration.FindingsIn this study, we found that semaphorin-3A (Sema3A) was significantly downregulated in VSMCs during neointimal hyperplasia after vascular injury in mice and in human atherosclerotic plaques. Meanwhile, Sema3A was transcriptionally downregulated by PDGF-BB via p53 in VSMCs. Furthermore, we found that overexpression of Sema3A inhibited VSMC proliferation and migration, as well as increasing differentiated gene expression. Mechanistically, Sema3A increased the NRP1-plexin-A1 complex and decreased the NRP1-PDGFRβ complex, thus inhibiting phosphorylation of PDGFRβ. Moreover, we found that overexpression of Sema3A suppressed neointimal hyperplasia after vascular injury in vivo.InterpretationThese results suggest that local delivery of Sema3A may act as a novel therapeutic option to prevent in-stent restenosis.

A bone-targeting drug-delivery system based on Semaphorin 3A gene therapy ameliorates bone loss in osteoporotic ovariectomized mice.

Osteoporosis is a serious health problem worldwide. Semaphorins (Sema) have been described as key molecules involved in the cross-talk between bone cells (osteoblasts/osteoclasts). In this study, we investigated whether plasmid containing Sema3a could ameliorate bone loss in an ovariectomized (OVX) mouse model via (AspSerSer)6, a selectively bone-targeting moiety. Plasmid pcDNA3.1(+)-Sema3a-GFP was fabricated and transfected cells with the plasmid demonstrated statistically higher levels of Sema3A in vitro (p < 0.001). Mice were ovariectomized and injected twice weekly with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP for four weeks. The aim of the study was twofold: firstly to design an effective bone-targeting drug-delivery system (AspSerSer)6. Secondly, the effects of Sem3A gene therapy on bone loss was investigated. Here, the targeting selectivity of pcDNA3.1(+)-Sema3a-GFP via (AspSerSer)6 to the trabecular bone surface was firstly verified by histological observation of frozen sections and immunofluorescence staining. Then, bone microstructure analysis by Micro-CT indicated significantly less bone loss in mice treated with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP compared to the control group (p < 0.05). Furthermore,H&E staining and Safranin O staining of the decalcified sections demonstrated statistically significantly higher bone area/total area in the mice that were injected with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP (p < 0.001, p < 0.01,respectively). TRAP staining and immunohistochemistry staining of COL I demonstrated lower numbers of osteoclasts and significantly increased numbers of osteoblasts in the bone-targeting moiety delivering pcDNA3.1(+)-Sema3a-GFP group, when compared to the control group (p < 0.01, p < 0.001,respectively). Together, our findings have identified that, (AspSerSer)6, a bone-targeting drug-delivery system based on semaphorin3A gene therapy, ameliorated bone loss in osteoporotic ovariectomized mice, by suppressing osteoclastic bone resorption and simultaneously increasing osteoblastic bone formation. Gene therapy by local site-specific Sema3A overexpression might be a potential new strategy for treating osteoporosis and bone defects.

Bidirectional regulation of bone formation by exogenous and osteosarcoma-derived Sema3A

Semaphorin 3A (Sema3A), a secreted member of the Semaphorin family, increases osteoblast differentiation, stimulates bone formation and enhances fracture healing. Here, we report a previously unknown role of Sema3A in the regulation of ectopic bone formation and osteolysis related to osteosarcoma. Human recombinant (exogenous) Sema3A promoted the expression of osteoblastic phenotype in a panel of human osteosarcoma cell lines and inhibited the ability of these cells to migrate and enhance osteoclastogenesis in vitro. In vivo, administration of exogenous Sema3A in mice after paratibial inoculation of KHOS cells increased bone volume in non-inoculated and tumour-bearing legs. In contrast, Sema3A overexpression reduced the ability of KHOS cells to cause ectopic bone formation in mice and to increase bone nodule formation by engaging DKK1/β-catenin signalling. Thus, Sema3A is of potential therapeutic efficacy in osteosarcoma. However, inhibition of bone formation associated with continuous exposure to Sema3A may limit its long-term usefulness as therapeutic agent.