Semaphorin Research Articles

Semaphorin 3A protects against alveolar bone loss during orthodontic tooth movement in mice with periodontitis.

This study investigated the effect of local semaphorin 3A (Sema3A) administration on alveolar bone loss during OTM in a mouse model of periodontitis. Orthodontic tooth movement (OTM) for patients with periodontal disease is known to increase the risk of exacerbating alveolar bone loss due to inflammation of the periodontal tissue. However, its mechanism of action and prevention remains unclear. Mice (male 7-8 weeks old, C57BL/6J, n= 12) were divided into six groups: untreated group (control), without OTM and recovered from induced periodontitis (RP), with OTM and administered PBS or Sema3A to the gingiva after induced periodontitis (VehPO, SemaPO), with OTM and administered PBS or Sema3A to the gingiva without periodontitis induction (VehNO, SemaNO). Samples were collected on 14 days, and bone loss, histological analysis, cytokine production level, and tooth movement were assessed. Cultured human periodontal ligament (hPDL) cells were stimulated with lipopolysaccharide (LPS) and compressive force (CF), and mRNA expression levels of Sema3A and its receptors were analyzed. The bone loss was significantly lower in the SemaPO group than in the VehPO group. The number of TRAP-positive cells in the SemaPO group was significantly lower than that in the VehPO group and was at the same level as that in the control group. The receptor activator of nuclear factor (NF)-kB-ligand/osteoprotegerin (RANKL/OPG) ratio and the levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, in the gingival tissues were significantly lower in the SemaPO group than in the VehPO group. Additionally, Sema3A mRNA expression in hPDL cells was significantly decreased by co-stimulation with LPS and CF compared with that in the control group. Finally, the distance moved (dist.) and the mesial tipping angle (θ) was significantly smaller in the SemaPO group than in the VehPO group and was not significantly different from that of VehNO. Pathological alveolar bone loss exacerbated by OTM in periodontitis might be prevented by local administration of Sema3A without inhibiting OTM.

054 IL-18 regulates nerve growth factor and semaphorin 3A in dermatomyositis-related pruritus

Pruritus is common in autoimmune diseases and a prominent symptom especially in patients with dermatomyositis (DM). In contrast, the severity of pruritus in cutaneous lupus erythematosus (CLE) is less severe. Importantly, pruritus is an important symptom to differentiate DM from CLE. It has been found that keratinocytes-derived interleukin (IL)-18 played a pathophysiologic role in DM and IL-18 potentially distinguishes DM from CLE. The present study aimed to investigate the association of IENFD in DM-related pruritus and the possible mechanism.

Efficacy of an Anti-Semaphorin 3A Neutralizing Antibody in a Male Experimental Retinal Vein Occlusion Mouse Model.

PurposeSemaphorin 3A (Sema3A) is a promising therapeutic target for macular edema in age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion (RVO). Anti-vascular endothelial growth factors (anti-VEGFs) are the current standard of care for many retinal diseases. This study investigated the Sema3A neutralizing antibody BI-X and/or anti-VEGF therapy (aflibercept) in an RVO mouse model. Treatment efficacy was examined and grouped by timing subsequent to the RVO mouse model induction: efficacy against the onset of intraretinal edema 1 day postinduction and protective effects at 7 days postinduction.MethodsWe examined the changes in expression of Sema3A in the retina of an RVO mouse model. In addition, changes in expression of tumor necrosis factor (TNF)-α and semaphorin-related proteins (neuropilin-1 and plexin A1) in the retina upon treatment were analyzed by Western blotting. The effects of BI-X and/or aflibercept were evaluated using measures of retinal edema, blood flow, and thinning of the inner nuclear layer.ResultsInduction of vein occlusion in the RVO mouse model significantly increased Sema3A expression in the retina, particularly in the inner nuclear layer. BI-X was effective as a monotherapy and in combination with anti-VEGF therapy, demonstrating a beneficial effect on intraretinal edema and retinal blood flow. Moreover, in the RVO mouse model, BI-X monotherapy normalized the changes in expression of TNF-α and semaphorin-related proteins.ConclusionsThese findings support targeting Sema3A to treat intraretinal edema and retinal ischemia.

Semaphorin 3A: A potential target for prevention and treatment of nickel allergy

Metal allergy is one of the typical immune disorders encountered during the application of dental/medical materials and has a highly complex pathogenic mechanism. Semaphorin 3A (Sema3A), a member of the semaphorin family, is reported to be involved in various immune disorders. However, its role in metal allergy has not been clarified yet. Herein, we show that Sema3A expression was upregulated in nickel (Ni) allergy-induced mouse ear tissue and in NiCl2-stimulated mouse keratinocytes. Moreover, Sema3A regulated tumor necrosis factor-alpha production and mitogen-activated protein kinase activation in keratinocytes. The specific deletion of Sema3A in keratinocytes did not affect immune cell infiltration but reduced edema and ear swelling; it also impeded Th1 responses to cause a slight alleviation in Ni allergy in mice. Our results demonstrate that Sema3A promotes the development of metal allergy and should be explored as a potential target for the prevention and treatment of metal allergy.

Semaphorin 5A suppresses ferroptosis through activation of PI3K-AKT-mTOR signaling in rheumatoid arthritis

Abnormal activation of synovial fibroblasts (SFs) plays an important role in rheumatoid arthritis (RA), the mechanism of which remains unknown. The purpose of our study is to comprehensively and systematically explore the mechanism for Semaphorin 5A-mediated abnormal SF activation in RA. Here, we found that Semaphorin 5A levels were significantly higher in synovial fluid and synovial tissue from RA patients compared with osteoarthritis patients. We further found that the mRNA level and protein abundance of Plexin-A1 was elevated in RA SFs compared with OA SFs, while Plexin-B3 expression showed no significant difference. The increased Semaphorin 5A in RA synovial fluid was mainly derived from CD68+ synovial macrophages, and the elevation led to increased binding between Semaphorin 5A and its receptors, thereby promoting cytokine secretion, proliferation, and migration, and decreasing apoptosis. Moreover, the effect of Semaphorin 5A on enhancing activation (cytokine secretion, cell proliferation and migration) and reducing apoptosis of SFs was significantly abolished after knockdown of Plexin-A1 and Plexin-B3 by small interfering RNA. Transcriptome sequencing and protein array detection revealed that Semaphorin 5A activated the PI3K/AKT/mTOR signaling pathway and inhibited ferroptosis. Morphologically, transmission electron microscopy results showed that Semaphorin 5A could significantly eliminate the mitochondrial diminution, membrane density increased and crest ruptured of SFs induced by ferroptosis inducer RSL3. Mechanistically, Semaphorin 5A enhanced GPX4 expression and SREBP1/SCD-1 signaling by activating the PI3K/AKT/mTOR signaling pathway, thus suppressing ferroptosis of RA SFs. In conclusion, our study provided the first evidence that elevated Semaphorin 5A in RA synovial fluid promotes SF activation by suppressing ferroptosis through the PI3K/AKT/mTOR signaling pathway.

Semaphorin 3A in the Immune System: Twenty Years of Study.

Semaphorin 3A is a secreted glycoprotein, which was originally identified as axon guidance factor in the neuronal system, but it also possesses immunoregulatory properties. Here, the effect of semaphorin 3A on T-lymphocytes, myeloid dendritic cells and macrophages is systematically analyzed on the bases of all publications available in the literature for 20 years. Expression of semaphorin 3A receptors – neuropilin-1 and plexins A – in these cells is described in details. The data obtained on human and murine cells is described comparatively. A comprehensive overview of the interaction of semaphorin 3A with mononuclear phagocyte system is presented for the first time. Semaphorin 3A signaling mostly results in changes of the cytoskeletal machinery and cellular morphology that regulate pathways involved in migration, adhesion, and cell–cell cooperation of immune cells. Accumulating evidence indicates that this factor is crucially involved in various phases of immune responses, including initiation phase, antigen presentation, effector T cell function, inflammation phase, macrophage activation, and polarization. In recent years, interest in this field has increased significantly because semaphorin 3A is associated with many human diseases and therefore can be used as a target for their treatment. Its involvement in the immune responses is important to study, because semaphorin 3A and its receptors turn to be a promising new therapeutic tools to be applied in many autoimmune, allergic, and oncology diseases.

MiR-181b targets semaphorin 3A to mediate TGF-β-induced endothelial-mesenchymal transition related to atrial fibrillation.

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β–treated human AEECs, cardiac-specific TGF-β–transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β–treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β–transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.

Construction of a Hierarchical Micro-/Submicro-/Nanostructured 3D-Printed Ti6Al4V Surface Feature to Promote Osteogenesis: Involvement of Sema7A through the ITGB1/FAK/ERK Signaling Pathway.

Constructing hierarchical hybrid structures is considered a facile method to improve the osseointegration of implants. Herein, a hierarchical micro-/submicro-/nanostructured surface feature of Ti6Al4V implants (3DAT group) was successfully constructed by combining the inherently formed three-dimensional (3D)-printed microscale topography, acid-etched sub-micropits, and anodized nanotubes. Compared with the classical SLA surface, the microscale topography and sub-micropits increased the three-dimensional space for the cell growth and mechanical stability of implants, while the modification of nanotubes dramatically improved the surface hydrophilicity, protein adsorption, and biomineralization. Most importantly, the 3DAT surface feature possessed excellent osteogenic performance in vitro and in vivo, with the involvement of semaphorin 7A (Sema7A) as revealed by RNA-seq through the ITGB1/FAK/ERK signaling pathway. The present study suggested that the hierarchically structured surface design strategy could accelerate the osseointegration rate of 3D-printed Ti6Al4V implants, promising personalized reconstruction of bone defects.

Sema3A Antibody BI-X Prevents Cell Permeability and Cytoskeletal Collapse in HRMECs and Increases Tip Cell Density in Mouse Oxygen-Induced Retinopathy.

PurposeSemaphorin 3A (Sema3A) is an axonal guidance molecule that inhibits angiogenesis by vasorepulsion and blocks revascularization in the ischemic retina. BI-X is an intravitreal anti-Sema3A agent under clinical investigation in patients with proliferative diabetic retinopathy (PDR) and diabetic macular ischemia (DMI).MethodsSurface plasmon resonance was used to determine binding affinity of BI-X to human and murine Sema3A. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to assess effects of BI-X on cell permeability and cytoskeletal collapse induced by Sema3A. In vivo, intravitreal BI-X or an anti-trinitrophenol control antibody was administered in both eyes in mice with oxygen-induced retinopathy (OIR). Retinal flat mounts were prepared, and avascular area and tip cell density were determined using confocal laser-scanning microscopy.ResultsDissociation constants for BI-X binding to human and murine Sema3A were 29 pM and 27 pM, respectively. In vitro, BI-X prevented HRMEC permeability and cytoskeletal collapse induced by Sema3A. In vivo, BI-X increased tip cell density by 33% (P < 0.001) and reduced avascular area by 12% (not significant). A significant negative correlation was evident between avascular area and tip cell density (r2 = 0.4205, P < 0.0001).ConclusionsBI-X binds to human Sema3A with picomolar affinity and prevents cell permeability and cytoskeletal collapse in HRMECs. BI-X also enhances revascularization in mice with OIR.Translational RelevanceBI-X is a potent inhibitor of human Sema3A that improves revascularization in a murine model of OIR; BI-X is currently being investigated in patients with laser-treated PDR and DMI.

Semaphorin-3 Promotes Specific Immunotherapy Effects on Experimental Food Allergy.

Sustaining higher frequency of mast cells in the allergic lesion site has been recognized. Factors causing high numbers of mast cells in the local tissues are not fully understood yet. RAS signaling plays a role in sustaining certain cell activities. This study is aimed at elucidating the role of RAS activation in the apoptosis resistance induction in mast cells and at employing semaphorin 3A to regulate RAS activities in sensitized mast cells and alleviating the allergic response in the intestine. A food allergy (FA) mouse model was developed. Mast cells were isolated from FA mouse intestinal tissues by flow cytometry. Mast cell apoptosis was assessed by staining with annexin V and propidium iodide. We found that aberrantly higher p21-activated kinase-1 (Pak1) expression in FA mast cells was associated with mast cell aggregation in the intestine. Sensitization increased Pak1 expression and apoptosis resistance in intestinal mast cells. RAS and Pak1 mutually potentiated each other in sensitized mast cells. Semaphorin 3A (sema3A) suppressed the Pak1 expression and RAS activation in mast cells. sema3A restored the apoptosis sensitivity in sensitized mast cells. Administration of sema3A potentiated allergen-specific immunotherapy in experimental FA. In conclusion, mast cells of FA mice showed higher Pak1 expression and high RAS activation status that contributed to apoptosis resistance in mast cells. Administration of sema3A restored the sensitivity to apoptosis inducers and promoted the therapeutic effects of specific immunotherapy on experimental FA.

Semaphorin 3A regulates alveolar bone remodeling on orthodontic tooth movement

Semaphorin 3A (Sema3A) promotes osteoblast differentiation and inhibits osteoclast differentiation. In the present study, we observed the regulation of alveolar bone remodeling by Sema3A during orthodontic tooth movement (OTM). Four inflammatory cytokines (IL-1β, IL-6, TNFα, and INF-γ) involved in OTM were applied to osteoblasts in vitro, and Sema3A expression was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In vivo, springs were attached to the maxillary first molars of C56BL/6J mice (OTM model) and the localization of Sema3A was confirmed by immunofluorescent. Recombinant Sema3A (rSema3A) was locally injected into the OTM model. Inflammatory cytokine localization in the OTM model was confirmed by immunohistochemistry. In vivo, more Sema3A was observed on the tension side in the OTM group. Injection of rSema3A into the OTM model increased mineralization on the tension side and decreased the number of osteoclasts on the compression side. In vitro, IL-1β significantly increased Sema3A mRNA levels. Immunohistochemistry for IL-1β in vivo showed more concentrated staining in the periodontal ligament on the tension side than on the compression side. In summary, our findings revealed the distribution of Sema3A in the periodontal ligament and demonstrated that rSema3A administration promotes bone formation and inhibits bone resorption during OTM.

Smooth Muscle‐Specific Deletion of Neuropilin‐1 Increases Vascular Contractility and Blood Pressure

IntroductionNeuropilin‐1 (NRP1) is a transmembrane glycoprotein that acts as a co‐receptor for the Class 3 Semaphorin (SEMA) ligand SEMA3A and mediates the inhibition of RhoA signaling. NRP1 is expressed in vascular smooth muscle cells (VSMCs), however, its role in regulating VSMC contractility and blood pressure in vivo is unknown.HypothesisWe hypothesize that SEMA3A‐NRP1 signaling is essential in mitigating RhoA activity in VSMCs and that loss of NRP1 increases vascular tone and blood pressure in vivo.MethodsTo study the role of NRP1 in SMCs, mice with inducible, smooth muscle cell‐specific deletion of NRP1 (SM22a‐CreERT2; Nrp1flox/flox) were generated. Following recombination using 4‐hydroxy tamoxifen (SM‐NRP1 KO) in male and female adult mice (8‐12 weeks), systolic blood pressure (SBP) was measured using a tail cuff and compared to age‐ and sex‐matched mice that did not receive tamoxifen (control). Vascular reactivity to various contractile agonists was measured using ex vivo tension myography.ResultsSBP was significantly increased in SM‐NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Aortas of SM‐NRP1 KO mice displayed significantly enhanced contractile response to phenylephrine, KCl, and the thromboxane agonist U44619, and reduced relaxation to the endothelium‐independent vasodilator sodium nitroprusside. In vitro, treatment of murine primary aortic VSMC with SEMA3A decreased angiotensin II‐induced Rho‐GTP activation. Gene expression of Sema3a in the aorta was downregulated in two models of hypertension, angiotensin II‐dependent and genetic model of hypertension, as identified by qPCR and transcriptomic analysis respectively, further supporting the role of SEMA3A‐NRP1 signaling in basal blood pressure regulation. Additionally, control and SM‐NRP1 KO mice (starting at 2 weeks post‐recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM‐NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, gene expression of atrial natriuretic peptide was increased (p=0.06) in hearts of angiotensin II‐infused SM‐NRP1 KO mice. Preliminary analyses indicated an increase in aortic medial thickness at baseline and an increase in aortic adventitial collagen following angiotensin II infusion in SM‐NRP1 KO mice compared to controls.ConclusionTogether, our data point to the role of NRP1 as a novel regulator of basal vascular tone and blood pressure, and the loss of NRP1 leads to the onset of hypertension and exacerbates pathological vascular remodeling and cardiac dysfunction.

Amino acid F223 in neuroimmune semaphorin 4A is essential for stabilization of human regulatory T cells

Abstract Class IV semaphorin family members, Sema4A and Sema4D, displayed opposite effects on human Treg cells in vitro. In PBMC cultures, Sema4A potentiated the CD4+CD25+Foxp3+ cell numbers and increased CD25/Foxp3 expression while Sema4D inhibited them. Sema4A downregulated lung inflammation in allergen-induced experimental asthma whereas Sema4D was necessary for optimal allergic response. Allergen treatment decreased lung local and lymphoid tissue Treg cell numbers in Sema4A−/− mice and increased them in Sema4D−/− mice as compared to similarly treated WT counterparts. Sema4A and Sema4D competitively bind to Plexin B1 in microplate-based binding studies. Using Bayesian Partitioning with Pattern Selection (BPPS) and Structurally Interacting Pattern Residues’ Inferred Significance (SIPRIS), we identified Sema4A residues likely responsible for its functional specificity. We mutated the Sema4A residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. We evaluated their stimulatory activity toward human Treg cells and assayed them for binding to Plexin B1. The F223A mutant was unable to stimulate Treg stability in the in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to WT and M198A proteins. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing novel immunotherapeutics for asthma. This work was supported by NIH grant R01AI 122631 and the Merit Review Award Number I01 BX001850 from the US Department of Veterans Affairs Biomedical R&amp;D Service (both awarded to A.D.K.) and by NIH-NIGMS grant R01GM125878 to A.F.N.

Developmental Hypothyroidism Influences the Development of the Entorhinal-Dentate Gyrus Pathway of Rat Offspring.

BackgroundDevelopmental hypothyroidism impairs learning and memory in offspring, which depend on extensive neuronal circuits in the entorhinal cortex, together with the hippocampus and neocortex. The entorhinal-dentate gyrus pathway is the main entrance of memory circuits. We investigated whether developmental hypothyroidism impaired the morphological development of the entorhinal-dentate gyrus pathway.MethodsWe examined the structure and function of the entorhinal-dentate gyrus pathway in response to developmental hypothyroidism induced using 2-mercapto-1-methylimidazole.Results1,1´-Dioctadecyl-3,3,3´,3´-tetramethylindocarbocyanine perchlorate tract tracing indicated that entorhinal axons showed delayed growth in reaching the outer molecular layer of the dentate gyrus at postnatal days 2 and 4 in hypothyroid conditions. The proportion of fibers in the outer molecular layer was significantly smaller in the hypothyroid group than in the euthyroid group at postnatal day 4. At postnatal day 10, the pathway showed a layer-specific distribution in the outer molecular layer, similar to the euthyroid group. However, the projected area of entorhinal axons was smaller in the hypothyroid group than in the euthyroid group. An electrophysiological examination showed that hypothyroidism impaired the long-term potentiation of the perforant and the cornu ammonis 3–cornu ammonis 1 pathways. Many repulsive axon guidance molecules were involved in the formation of the entorhinaldentate gyrus pathway. The hypothyroid group had higher levels of erythropoietin-producing hepatocyte ligand A3 and semaphorin 3A than the euthyroid group.ConclusionWe demonstrated that developmental hypothyroidism might influence the development of the entorhinal-dentate gyrus pathway, contributing to impaired long-term potentiation. These findings improve our understanding of neural mechanisms for memory function.

457 Semaphorin7a expression in breast cancer promotes susceptibility to immune checkpoint blockade

OBJECTIVES/GOALS: The goal of this study is to decipher the mechanisms by which breast cancers evade the immune system to facilitate metastasis through the lymphatic vasculature. We believe this is caused, in part, by semaphorin 7A (SEMA7A) as its expression is associated with increased lymphatic vessels, tumor-associated macrophages, and metastasis in breast cancer. METHODS/STUDY POPULATION: We have observed that breast cancer cells, lymphatics, and macrophages exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to determine if SEMA7A expression makes tumors more susceptible to immune checkpoint blockade. E0771 and 66cl4 mouse mammary carcinoma cells were injected orthotopically into the #4 mammary fat pads. Once tumors reached 50-100 mm^3 mice were injected intraperitoneally with 250ug of anti-PD-1, anti-PD-L1, or IgG control every third day. Tumors were measured with calipers every other day and harvested for flow cytometry to determine the effect of immune checkpoint blockade on the TME in SEMA7A+ tumors. RESULTS/ANTICIPATED RESULTS: We reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls – all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a pro-tumor microenvironment. DISCUSSION/SIGNIFICANCE: SEMA7A expression establishes a pro-tumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ breast cancers have high rates of metastasis, more specific treatments for these patient populations should be explored.

Identifying Function Determining Residues in Neuroimmune Semaphorin 4A.

Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants.

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.

Local treatment with Sema3a could promote the osseointegration of hydroxyapatite coated titanium rod in diabetic rats.

Recently, semaphorin 3A (Sema3A) has been identified as a critical gene for osteogenic differentiation of mesenchymal stem cells and increases osteoblastic bone formation. However, in current research studies, there is a lack of focus on whether Sema3a can affect the osseointegration of titanium rods in diabetes and through what biological mechanisms. Therefore, the present work was aimed to evaluate the effect of local administration with Sema3A on hydroxyapatite coated titanium rod osseointegration in diabetic rat model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with Sema3A and high glucose and induced to osteogenesis, and the cell viability, osteogenic activity was observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase staining, Alizarin Red Staining, and Western Blot. In vitro experiments, CCK-8, ALP, and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by Sema3A, as well as a higher levels of protein expressions including Runt-Related Transcription Factor 2, silent mating type information regulation 2 homolog-1(SIRT1), catalase (CAT), superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2). In vivo experiment, a better stability and osseointegration of the titanium rod were observed after treatment with Sema3A, as well as a higher SOD1, SOD2, CAT, and SIRT1 gene expression. The present study indicates that local treatment with Sema3A was associated with increased osseointegration of titanium rod by reducing the oxidative stress of osteoblasts and enhancing the function of osteoblasts in a diabetic rat.

Semaphorin 6C Suppresses Proliferation of Pancreatic Cancer Cells via Inhibition of the AKT/GSK3/β-Catenin/Cyclin D1 Pathway.

Semaphorins (SEMAs) are axon guidance factors that participate in axonal connections and nerve system development. However, the functional roles of SEMAs in tumorigenesis are still largely uncovered. By using in silico data analysis, we found that SEMA6C was downregulated in pancreatic cancer, and its reduction was correlated with worse survival rates. RNA sequencing revealed that cell cycle-related genes, especially cyclin D1, were significantly altered after blockage of SEMA6C by neutralizing antibodies or ectopic expressions of SEMA6C. Mechanistic investigation demonstrated that SEMA6C acts as a tumor suppressor in pancreatic cancer by inhibiting the AKT/GSK3 signaling axis, resulting in a decrease in cyclin D1 expression and cellular proliferation. The enhancement of cyclin D1 expression and cyclin-dependent kinase activation in SEMA6C-low cancer created a druggable target of CDK4/6 inhibitors. We also elucidated the mechanism underlying SEMA6C downregulation in pancreatic cancer and demonstrated a novel regulatory role of miR-124-3p in suppressing SEMA6C. This study provides new insights of SEMA6C-mediated anti-cancer action and suggests the treatment of SEMA6C-downregulated cancer by CDK4/6 inhibitors.

Basolateral Amygdala Mediates Central Mechanosensory Feedback of Musculoskeletal System.

Musculoskeletal diseases, such as osteoporosis and sarcopenia, are tremendous and growing public health concerns. Considering the intimate functional relationship between muscle and bone throughout development, growth, and aging, muscle provides the primary source of skeletal loading through contraction force. However, significant gaps exist in our knowledge regarding the role of muscle in bone homeostasis and little is known regarding the mechanism through which the central nervous system responds and regulates unloading-induced bone loss. Here, we showed that the basolateral amygdala (BLA) and medial part of the central nucleus (CeM) are anatomically connected with the musculoskeletal system. Unloading-induced bone loss is accompanied by a decrease in serum semaphorin 3A (Sema3A) levels as well as sensory denervation. In vivo fiber photometry recordings indicated that the mechanical signal is integrated by the BLA and CeM within 24 h and subsequently regulates bone remodeling. Moreover, chemogenetic activation of BLACaMKII neurons mitigates severe bone loss caused by mechanical unloading via increased serum levels of Sema3A and sensory innervation. These results indicate that the BLA integrates the mechanosensory signals rapidly and mediates the systemic hormonal secretion of Sema3A to maintain bone homeostasis.