Semaporin3A inhibitor ameliorates renal fibrosis through the regulation of JNK signaling.

Renal fibrosis is the common pathological pathway in progressive renal diseases. In the present study, we analyzed the roles of semaphorin 3 A (SEMA3A) on renal fibrosis and the effect of SEMA3A inhibitor (SEMA3A-I) using a unilateral ureteral obstruction (UUO) mouse model. Expression of SEMA3A in the proximal tubulus and neuropilin-1, a recepor of SEMA3A, in fibloblast and tubular cells were increased in UUO kidneys. The expression of myofibroblast marker tenascin-C and fibronection as well as renal fibrosis were increased in UUO kidneys, all of which were ameliorated by SEMA3A-I. In addition, the JNK signaling pathway, known as the target of SEMA3A signaling, was activated in proximal tubular cells and fibroblast cells after UUO surgery, and SEMA3A-I significantly attenuated the activation. In vitro, treatments with SEMA3A as well as transforming growth factor-ß1 (TGF-ß1) in human proximal tubular cells lost epithelial cell characteristics, and SEMA3A-I significantly ameliorated this transformation. The JNK inhibitor SP600125 partially reversed SEMA3A and TGF-ß1-induced cell transformation, indicating that JNK signaling is involved in SEMA3A-induced renal fibrosis. In addition, treatment with SEMA3A in fibroblast cells activated expression of tenascin-C, collagen type I, and fibronection, indicating that SEMA3A may accelerate renal fibrosis through the activation of fibroblast cells. Analysis of human data revealed the positive correlation between urinary SEMA3A and urinary N-acetyl-ß-d-glucosaminidase, indicating the association between SEMA3A and tubular injury. In conclusion, SEMA3A signaling is involved in renal fibrosis through the JNK signaling pathway and SEMA3A-I might be a therapeutic option for protecting from renal fibrosis.NEW & NOTEWORTHY Renal fibrosis is the common pathological pathway in the progression of renal diseases. This study, using a unilateral ureteral obstruction (UUO) mouse model, indicated increased semaphorin3A (SEMA3A) signaling in renal tubular cells as well as fibroblast cells under UUO surgery, and SEMA3A inhibitor ameliorated UUO-induced renal fibrosis through the regulation of JNK signaling. The study proposes the potential therapeutic option of SEMA3A inhibitor to treat renal fibrosis.

Semaphorin3A-Inhibitor Ameliorates Doxorubicin-Induced Podocyte Injury.

Podocyte injury is an independent risk factor for the progression of renal diseases. Semaphorin3A (SEMA3A), expressed in podocytes and tubular cells in the mammalian adult kidneys, has been reported to regulate diverse biological functions and be associated with renal diseases. Here, we investigated pathological roles of SEMA3A signaling on podocyte injury using a doxorubicin (Dox)-induced mouse model and examined the therapeutic effect of SEMA3A-inhibitor (SEMA3A-I). We demonstrated that Dox caused massive albuminuria and podocyte apoptosis as well as an increase of SEMA3A expression in podocytes, all of which were ameliorated with SEMA3A-I treatment. In addition, c-Jun N-terminal kinase (JNK), known as a downstream of SEMA3A signaling, was activated in Dox-injected mouse podocytes while SEMA3A-I treatment partially blocked the activation. In vitro, SEMA3A-I protected against Dox-induced podocyte apoptosis and recombinant SEMA3A caused podocyte apoptosis with activation of JNK signaling. JNK inhibitor, SP600125, attenuated SEMA3A-induced podocyte apoptosis, indicating that the JNK pathway would be involved in SEMA3A-induced podocyte apoptosis. Furthermore, the analysis of human data revealed a positive correlation between levels of urinary SEMA3A and protein, suggesting that SEMA3A is associated with podocyte injury. In conclusion, SEMA3A has essential roles in podocyte injury and it would be the therapeutic target for protecting from podocyte injury.

MicroRNA-30b regulates the polarity of retinal ganglion cells by inhibiting semaphorin-3A.

Purpose: Retinal ganglion cell (RGC) polarity plays an important role in optic nerve regeneration. This study was designed to investigate whether semaphorin-3A (Sema3A) is involved in the regulation of RGC polarity and Sema3A protein expression. Methods: Cultured primary RGCs were treated with Fc-Sema3A or Sema3A siRNA or transfected with purified miR-30b recombinant adenoassociated virus (rAAV). The polarity of the RGCs was observed with immunofluorescence. A western blot analysis of phosphorylated protein kinase A (p-PKA), the downstream effector molecule phosphorylated glycogen synthase kinase 3 beta (GSK-3ß), and collapsing response mediator protein 2 (CRMP2) was performed. Results: We found that Sema3A could statistically significantly promote dendritic branching while inhibiting the growth of axons in RGCs. miR-30b overexpression and Sema3A siRNA could statistically significantly promote the growth of axons while inhibiting the growth of dendrites from RGCs. Additionally, miR-30b could restrain the expression of Sema3A protein and its downstream PKA/GSK-3ß/CRMP2 signaling pathways. Conclusions: The results indicate that Sema3A promotes dendritic growth and inhibits axonal growth, which is not conducive to the early repair of optic nerve injury. The overexpression of miR-30b can overcome this problem, and may represent a new target for the treatment of nerve injury and regeneration in the future.

Astrocyte-Derived Exosomes Treated With a Semaphorin 3A Inhibitor Enhance Stroke Recovery via Prostaglandin D2 Synthase.

Background and Purpose- Exosomes play a pivotal role in neurogenesis. In the peri-infarct area after stroke, axons begin to regenerate but are inhibited by astrocyte scar formation. The direct effect and underlying molecular mechanisms of astrocyte-derived exosomes on axonal outgrowth after ischemia are not known. Methods- Using a semaphorin 3A (Sema3A) inhibitor, we explored neuronal signaling during axonal outgrowth after ischemia in rats subjected to middle cerebral artery occlusion and in cultured cortical neurons challenged with oxygen-glucose deprivation. Furthermore, we assessed whether this inhibitor suppressed astrocyte activation and regulated astrocyte-derived exosomes to enhance axonal outgrowth after ischemia. Results- In rats subjected to middle cerebral artery occlusion, we administered a Sema3A inhibitor into the peri-infarct area from 7 to 21 days after occlusion. We found that phosphorylated high-molecular weight neurofilament-immunoreactive axons were increased, glial fibrillary acidic protein-immunoreactive astrocytes were decreased, and functional recovery was promoted at 28 days after middle cerebral artery occlusion. In cultured neurons, the Sema3A inhibitor decreased Rho family GTPase 1, increased R-Ras, which phosphorylates Akt and glycogen synthase kinase 3ß (GSK-3ß), selectively increased phosphorylated GSK-3ß in axons, and thereby enhanced phosphorylated high-molecular weight neurofilament-immunoreactive axons after oxygen-glucose deprivation. In cultured astrocytes, the Sema3A inhibitor suppressed activation of astrocytes induced by oxygen-glucose deprivation. Exosomes secreted from ischemic astrocytes treated with the Sema3A inhibitor further promoted axonal elongation and increased prostaglandin D2 synthase expression on microarray analysis. GSK-3ß+ and prostaglandin D2 synthase+ neurons were robustly increased after treatment with the Sema3A inhibitor in the peri-infarct area. Conclusions- Neuronal Rho family GTPase 1/R-Ras/Akt/GSK-3ß signaling, axonal GSK-3ß expression, and astrocyte-derived exosomes with prostaglandin D2 synthase expression contribute to axonal outgrowth and functional recovery after stroke.

Slow-Myofiber Commitment by Semaphorin 3A Secreted from Myogenic Stem Cells.

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell-membrane receptor (neuropilin2-plexinA3) → myogenin-myocyte enhancer factor 2D → slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2 -Sema3Afl °x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. Stem Cells 2017;35:1815-1834.

mTORC1 is a critical mediator of oncogenic Semaphorin3A signaling.

Aberration of signaling pathways by genetic mutations or alterations in the surrounding tissue environments can result in tumor development or metastasis. However, signaling molecules responsible for these processes have not been completely elucidated. Here, we used mouse Lewis lung carcinoma cells (LLC) to explore the mechanism by which the oncogenic activity of Semaphorin3A (Sema3A) signaling is regulated. Sema3A knockdown by shRNA did not affect apoptosis, but decreased cell proliferation in LLCs; both the mammalian target of rapamycin complex 1 (mTORC1) level and glycolytic activity were also decreased. In addition, Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation by oligomycin, but conferred resistance to decreased cell viability induced by glucose starvation. Furthermore, recombinant SEMA3A rescued the attenuation of cell proliferation and glycolytic activity in LLCs after Sema3A knockdown, whereas mTORC1 inhibition by rapamycin completely counteracted this effect. These results demonstrate that Sema3A signaling exerts its oncogenic effect by promoting an mTORC1-mediated metabolic shift from oxidative phosphorylation to aerobic glycolysis.

Semaphorin 3A deficiency improves hypoxia-induced myocardial injury via resisting inflammation and cardiomyocytes apoptosis.

Ischemia/hypoxia leads to heart injuries by inducing inflammation, cardiac fibrosis and cardiomyocyte apoptosis. Semaphorin 3A (Sema 3A) plays a regulatory role during all immune response stages, and has been demonstrated to be associated with multiple diseases. However, roles of Sema 3A during myocardial ischemia/hypoxia have not been studied in full. In this study, decline in Sema 3A was discovered in hypoxia-treated myocardial cells. When this decline was enhanced by silencing of Sema 3A gene, hypoxia-induced myocardial cell injury could be partially improved. Sema 3A deficiency can resist hypoxia-induced inflammatory factors (TNF-α, IL-1ß and IL-6) secretion, cell viability decline, cardiomyocyte apoptosis, ROS release, ATP generation decline as well as GSH/GSSG ratio decline in H9C2 cells. Besides, hypoxia-induced bcl-2 decrease and cleaved caspase-3 increase also can be partially reversed during Sema 3A deficiency. All these findings reflect that reduced Sema 3A is a protective strategy adopted by damaged myocardial cell. Our study first shows that Sema 3A deficiency can improve hypoxia-induced myocardial cell injury, which thus offers a new insight to treatment ischemic heart disease.

Sema3A maintains corneal avascularity during development by inhibiting Vegf induced angioblast migration.

Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1(Sema-) mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.

Semaphorin 3A inactivation suppresses ischemia-reperfusion-induced inflammation and acute kidney injury.

Recent studies show that guidance molecules that are known to regulate cell migration during development may also play an important role in adult pathophysiologic states. One such molecule, semaphorin3A (sema3A), is highly expressed after acute kidney injury (AKI) in mice and humans, but its pathophysiological role is unknown. Genetic inactivation of sema3A protected mice from ischemia-reperfusion-induced AKI, improved tissue histology, reduced neutrophil infiltration, prevented epithelial cell apoptosis, and increased cytokine and chemokine excretion in urine. Pharmacological-based inhibition of sema3A receptor binding likewise protected against ischemia-reperfusion-induced AKI. In vitro, sema3A enhanced toll-like receptor 4-mediated inflammation in epithelial cells, macrophages, and dendritic cells. Moreover, administration of sema3A-treated, bone marrow-derived dendritic cells exacerbated kidney injury. Finally, sema3A augmented cisplatin-induced apoptosis in kidney epithelial cells in vitro via expression of DFFA-like effector a (cidea). Our data suggest that the guidance molecule sema3A exacerbates AKI via promoting inflammation and epithelial cell apoptosis.

Following experimental stroke, the recovering brain is vulnerable to lipoxygenase-dependent semaphorin signaling.

Recovery from stroke is limited, in part, by an inhibitory environment in the postischemic brain, but factors preventing successful remodeling are not well known. Using cultured cortical neurons from mice, brain endothelial cells, and a mouse model of ischemic stroke, we show that signaling from the axon guidance molecule Sema3A via eicosanoid second messengers can contribute to this inhibitory environment. Either 90 nM recombinant Sema3A, or the 12/15-lipoxygenase (12/15-LOX) metabolites 12-HETE and 12-HPETE at 300 nM, block axon extension in neurons compared to solvent controls, and decrease tube formation in endothelial cells. The Sema3A effect is reversed by inhibiting 12/15-LOX, and neurons derived from 12/15-LOX-knockout mice are insensitive to Sema3A. Following middle cerebral artery occlusion to induce stroke in mice, immunohistochemistry shows both Sema3A and 12/15-LOX are increased in the cortex up to 2 wk. To determine whether a Sema3A-dependent damage pathway is activated following ischemia, we injected recombinant Sema3A into the striatum. Sema3A alone did not cause injury in normal brains. But when injected into postischemic brains, Sema3A increased cortical damage by 79%, and again, this effect was reversed by 12/15-LOX inhibition. Our findings suggest that blocking the semaphorin pathway should be investigated as a therapeutic strategy to improve stroke recovery.

[The effect of semaphorin 3A in the process of apoptosis in oxygen induced retinopathy in rats].

OBJECTIVE: To observe the change of semaphorin 3A (SEMA3A) expression in the retina of oxygen induced retinopathy (OIR) in rats and to investigate its influence on retinal degeneration in OIR. METHODS: Experimental study. Forty-eight newborn pups were randomly classified into four groups and only one eye was examined in each pup. Thirty-six pups was induced into OIR model through aspirating 50% and 10% oxygen every 24 hours alternatively while the control group (12 pups) was raised under normal atmosphere. OIR+SEMA3Aab group accepted intravitreous injection of rabbit anti-rat SEMA3A antibody (Abcam Co.LTD, 40 mg/L, 2 µl) while OIR+IgG group was injected the same amount of non-active rabbit IgG at postnatal day 7. And the OIR group had no injections. All the pups were executed at postnatal day 18. Histological changes of retinas were examined through HE stain while Immunoflurecence staining and TUNEL procedure were used to detect focal expression of SEMA3A and apoptosis respectively. Total tissue protein was extracted and the expression of SEMA3A and Caspase-3 p17subunit were examined by Western blot. The data including retinal thickness, cell counting of retinal ganglion cells layers (RGCL) , endothelia outside inner limiting membrane, retinal apoptosis index (AI), the relative expression of SEMA3A and Caspase3 p17 subunit were analyzed by One-way ANOVA and followed LSD-t test to compare group differences. RESULTS: There were significant differences of retinal thickness, cell counting of RGCL and endothelia outside inner limiting membrane among each groups respectively (F = 13.222, F = 22.537, F = 14.478;P < 0.01) . The retinal thickness was (117.07 ± 8.13) µm in normal control group, (70.93 ± 5.68) µm in OIR group, (91.28 ± 4.58) µm in OIR+SEMA3Aab group, and (67.27 ± 10.15) µm in OIR+IgG group; the cell counting of RGCL was 42.7 ± 3.6 in normal control group, 24.3 ± 3.1 in OIR group, 35.0 ± 6.2 in OIR+SEMA3Aab group, and 22.8 ± 4.3 in OIR+IgG group while the endothelia outside inner limiting membrane was 1.0 ± 0.3 in normal control group, 14.2 ± 3.2 in OIR group, 9.6 ± 1.1 in OIR+SEMA3Aab group, and 10.8 ± 1.6 in OIR+IgG group. The retinal thickness and cell counting of RGCL in OIR+SEMA3Aab group were significantly lower than those in the normal control group (P < 0.01) , but were apparently higher than the data of OIR group and OIR+IgG group respectively (P < 0.01) . There were no more endothelia on the vitreal side of the inner limiting membrane in OIR+SEMA3Aab group than OIR group or OIR+IgG group (P > 0.05) although these three groups had much more endothelia than it in the normal control group (P < 0.01). The retinal AI detected by TUNEL staining was 27.67 ± 2.51 in normal control, 58.33 ± 8.50 in OIR group, 37.33 ± 5.03 in OIR+SEMA3Aab group and 61.67 ± 6.65 in OIR+IgG group. There was significant difference of the retinal AI among the four groups (F = 19.250, P = 0.001) . Apoptotic cells were significantly reduced in OIR+SEMA3Aab group compared with OIR + IgG group or OIR group (P < 0.01). The difference of SEMA3A protein expression among the groups detected by Western blot was significant (F = 38.59, P = 0.000) . SEMA3A expression in OIR group was 0.97 ± 0.05, which was significantly upregulated compared with the normal control group (0.64 ± 0.03) ( P < 0.01) . And its expression was successfully neutralized in OIR + SEMA3Aab group (0.41 ± 0.02) with comparison with OIR+IgG group (1.03 ± 0.15) through Western blot (P < 0.01) . SEMA3A was detected apparently in the photoreceptors layer in the normal control while its fluorescence was stronger and much more scattered in the whole retina. However, anti-SEMA3Aab intravitreous injection successfully reduced its fluorescence compared with the IgG injection. The cleavage of Caspase-3 was not detected in the normal control while the relative expression of Caspase-3 p17 subunit was significantly different in the other three groups (F = 304.619, P < 0.01). OIR + SEMA3Aab group had much less Caspase-3 p17 subunit (0.12 ± 0.01) than OIR group (0.30 ± 0.02) or OIR +IgG group (0.27 ± 0.02) (P < 0.01) . CONCLUSIONS: The over regulation of SEMA3A probably enhances the aggravation of apoptosis in OIR rats. Inhibition of SEMA3A is helpful to protect neuroretina.

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord.

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

Syntaxin 1B suppresses macropinocytosis and semaphorin 3A-induced growth cone collapse.

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.

Semaphorin 3A contributes to sepsis­induced immunosuppression by impairing CD4+ T cell anergy.

Semaphorin 3A (Sema3A), a member of the Sema family of proteins, appears to serve an important role in sepsis and sepsis­induced immunosuppression and has been regarded as a crucial regulator involved in cellular immune response. However, the role of Sema3A in CD4+ T cell anergy during sepsis remains to be elucidated. In the present study, the cecal ligation and perforation model and lipopolysaccharide (LPS) were used to simulate sepsis and the role of Sema3A in sepsis­induced CD4+ T cell anergy was investigated in vivo and in vitro. In vivo, the serum concentration of Sema3A was enhanced and exacerbated sepsis­induced T cell immunosuppression and multiple organ dysfunction syndromes (MODS). Administration of (­)­epigallocatechin­3­gallate, an inhibitor of Sema3A, markedly improved sepsis­induced T cell immunosuppression and MODS. In vitro, both lymphoid and myeloid lineages secreted high concentration of Sema3A in LPS­induced sepsis, especially in the lymphoid lineage. Inhibition of Sema3A alleviated T cell anergy. The NF­κB signaling pathway was involved in Sema3A­mediated autocrine loop aggravating T cell immune dysfunction during LPS­induced sepsis. Inhibiting Sema3A exerted significant improvement of sepsis­induced immunosuppression and MODS, which was associated with improvement of CD4+ T cells anergy via regulation of the NF­κB signaling pathway.

Inhibition of Semaphorin3A Promotes Ocular Dominance Plasticity in the Adult Rat Visual Cortex.

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity.

Pharmacokinetics, Biodistribution, and Toxicity Evaluation of Anti-SEMA3A (F11) in In Vivo Models.

BACKGROUND/AIM: The aim of our study was to investigate the pharmacokinetics (PK), tissue distribution and toxicity of F11 antibody to semaphorin 3A in mouse models and explore its anti-angiogenic and tumor-inhibitory effect. MATERIALS AND METHODS: Patient-derived xenograft (PDX) models were established via subcutaneous implantation of glioblastoma multiforme (GBM) cells and treated with F11. RESULTS: F11 significantly attenuated tumor growth and angiogenesis in the GBM PDX model. Within the range of administered doses, the PK of F11 in serum demonstrated a linear fashion, consistent with general PK profiles of soluble antigen-targeting antibodies. Additionally, the clearance level was detected at between 4.63 and 7.12 ml/d/kg, while the biological half-life was measured at 6.9 and 9.4 days. Tissue distribution of F11 in kidney, liver and heart was consistent with previously reported antibody patterns. However, the presence of F11 in the brain was an interesting finding. CONCLUSION: Collectively, our results revealed angiogenic and tumor-inhibitory effect of F11 antibody and its potential therapeutic use within a clinical framework based on PK, biodistribution and toxicity evaluation in mouse models.

Sema3E/PlexinD1 inhibition is a therapeutic strategy for improving cerebral perfusion and restoring functional loss after stroke in aged rats.

Brain tissue survival and functional recovery after ischemic stroke greatly depend on cerebral vessel perfusion and functional collateral circulation in the ischemic area. Semaphorin 3E (Sema3E), one of the class 3 secreted semaphorins, has been demonstrated to be a critical regulator in embryonic and postnatal vascular formation via binding to its receptor PlexinD1. However, whether Sema3E/PlexinD1 signaling is involved in poststroke neovascularization remains unknown. To determine the contribution of Sema3E/PlexinD1 signaling to poststroke recovery, aged rats (18 months) were subjected to a transient middle cerebral artery occlusion. We found that depletion of Sema3E/PlexinD1 signaling with lentivirus-mediated PlexinD1-specific-shRNA improves tissue survival and functional outcome. Sema3E/PlexinD1 inhibition not only increases cortical perfusion but also ameliorates blood-brain barrier damage, as determined by positron emission tomography and magnetic resonance imaging. Mechanistically, we demonstrated that Sema3E suppresses endothelial cell proliferation and angiogenic capacity. More importantly, Sema3E/PlexinD1 signaling inhibits recruitment of pericytes by decreasing production of platelet derived growth factor-BB in endothelial cells. Overall, our study revealed that inhibition of Sema3E/PlexinD1 signaling in the ischemic penumbra, which increases both endothelial angiogenic capacity and recruitment of pericytes, contributed to functional neovascularization and blood-brain barrier integrity in the aged rats. Our findings imply that Sema3E/PlexinD1 signaling is a novel therapeutic target for improving brain tissue survival and functional recovery after ischemic stroke.

Inhibition of semaphorin-3a suppresses lipopolysaccharide-induced acute kidney injury.

Semaphorin-3a (Sema3A), a soluble axon guidance cue, appears to play an important role in the development of acute kidney injury (AKI) and has been regarded as an early diagnostic marker to evaluate the progression of AKI. However, the role of Sema3A in sepsis-associated AKI remains unknown. In this study, lipopolysaccharide (LPS) was used to simulate sepsis-associated AKI and the role of Sema3A in LPS-induced AKI was investigated in vivo and in vitro. In our in vivo study, Sema3A was found in tubular epithelial cells (TECs), which presented a higher level after LPS treatment. Meanwhile, the results of our in vitro experiment showed that Sema3A was also elevated in NRK-52E cells treated by LPS. Notably, inhibition of Sema3A by (-)-epigallocatechin-3-gallate (EGCG) could significantly reduce kidney inflammation and apoptosis in mice. Likewise, EGCG intervention also ameliorated the inflammation and apoptosis of cells in vitro. Furthermore, our research also found that the Rac1/NF-κB p65 and JNK pathways were possibly involved in the Sema3A-mediated inflammation and apoptosis of TECs, respectively. Our findings suggest that Sema3A play a pathogenic role by promoting inflammation and apoptosis of TECs in LPS-induced AKI. It might serve as a useful treatment target in ameliorating sepsis-associated AKI. KEY MESSAGES: Sema3A is upregulated in LPS-induced AKI. Inhibition of Sema3A attenuates inflammation and apoptosis of TECs in LPS-induced AKI. Sema3A enhances the LPS-induced inflammation of TECs through the Rac1/NF-κB p65 pathway. Sema3A exacerbates the LPS-induced apoptosis of TECs through the JNK pathway.

Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth.

PURPOSE: Glioblastoma (GBM) is classified as one of the most aggressive and lethal brain tumor. Great strides have been made in understanding the genomic and molecular underpinnings of GBM, which translated into development of new therapeutic approaches to combat such deadly disease. However, there are only few therapeutic agents that can effectively inhibit GBM invasion in a clinical framework. In an effort to address such challenges, we have generated anti-SEMA3A monoclonal antibody as a potential therapeutic antibody against GBM progression. MATERIALS AND METHODS: We employed public glioma datasets, Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas, to analyze SEMA3AmRNA expression in human GBM specimens. We also evaluated for protein expression level of SEMA3A via tissue microarray (TMA) analysis. Cell migration and proliferation kinetics were assessed in various GBM patient-derived cells (PDCs) and U87-MG cell-line for SEMA3A antibody efficacy. GBM patient-derived xenograft (PDX) models were generated to evaluate tumor inhibitory effect of anti-SEMA3A antibody in vivo. RESULTS: By combining bioinformatics and TMA analysis, we discovered that SEMA3A is highly expressed in human GBM specimens compared to non-neoplastic tissues. We developed three different anti-SEMA3A antibodies, in fully human IgG form, through screening phage-displayed synthetic antibody library using a classical panning method. Neutralization of SEMA3A significantly reduced migration and proliferation capabilities of PDCs and U87-MG cell line in vitro. In PDX models, treatment with anti-SEMA3A antibody exhibited notable tumor inhibitory effect through down-regulation of cellular proliferative kinetics and tumor-associated macrophages recruitment. CONCLUSION: In present study, we demonstrated tumor inhibitory effect of SEMA3A antibody in GBM progression and present its potential relevance as a therapeutic agent in a clinical framework.

The Semaphorin 3A inhibitor SM-345431 preserves corneal nerve and epithelial integrity in a murine dry eye model.

Dry eye disease (DED) is a common disorder causing discomfort and ocular fatigue. Corneal nerves are compromised in DED, which may further cause loss of corneal sensation and decreased tear secretion. Semaphorin 3A (Sema3A) is expressed by the corneal epithelium under stress, and is known as an inhibitor of axonal regeneration. Using a murine dry eye model, we found that topical SM-345431, a selective Sema3A inhibitor, preserved corneal sensitivity (2.3 ± 0.3 mm versus 1.4 ± 0.1 mm in vehicle control, p = 0.004) and tear volume (1.1 ± 0.1 mm versus 0.3 ± 0.1 mm in vehicle control, p < 0.001). Fluorescein staining area of the cornea due to damage to barrier function was also reduced (4.1 ± 0.9% in SM-345431 group versus 12.9 ± 2.2% in vehicle control, p < 0.001). The incidence of corneal epithelial erosions was significantly suppressed by SM-345431 (none in SM-345431 group versus six (21%) in vehicle control, p = 0.01). Furthermore, sub-epithelial corneal nerve density and intraepithelial expression of transient receptor potential vanilloid receptor 1 (TRPV1) were significantly preserved with SM-345431. Our results suggest that inhibition of Sema3A may be an effective therapy for DED.