Sema3A Expression Research Articles

Expression of hypoxia-induced semaphorin 7A correlates with the severity of inflammation and osteoclastogenesis in experimentally induced periapical lesions

ObjectiveWhile hypoxia and inflammation are intimately linked, the effects of inflammatory hypoxia on the pathogenesis of periapical lesions remain largely unknown. The aim of this study was to examine hypoxia during the progression of experimentally induced rat periapical lesions, and to derive correlations between hypoxia-induced Semaphorin 7A (Sema7a) expression, severity of inflammation, and osteoclastogenesis in the lesions. DesignPeriapical lesions were developed after mandibular first molar pulp exposure in forty Sprague-Dawley rats. The animals were randomly divided into four groups and sacrificed at 0, 7, 14, and 28days after pulpal exposure. The bilateral mandibles containing the first molar were obtained and routinely prepared for histological, immunohistochemical, enzyme histochemical analyses and quantitative polymerase chain reaction detecting Sema7a mRNA expression. Data were analysed by one-way analysis of variance and the Pearson’s correlation and linear tendency test. ResultsPeriapical tissues become hypoxic during the development of experimentally induced periapical lesions, with steadily increasing numbers of HIF-1α-positive cells that positively correlate with the expression of Sema7a mRNA in the lesions. Furthermore, significant positive correlates were derived for the expression of Sema7a and the degree of inflammatory infiltration and osteoclast number, respectively. ConclusionsHypoxia-induced Sema7a participates in the pathogenesis of periapical lesions, providing a novel therapeutic target for the treatment of this inflammatory disease in the future.

Wedelolactone Enhances Osteoblastogenesis but Inhibits Osteoclastogenesis through Sema3A/NRP1/PlexinA1 Pathway.

Bone remodeling balance is maintained by tight coupling of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Thus, agents with the capacity to regulate osteoblastogenesis and osteoclastogenesis have been investigated for therapy of bone-related diseases such as osteoporosis. In this study, we found that wedelolactone, a compound isolated from Ecliptae herba, and a 9-day incubation fraction of conditioned media obtained from wedelolactone-treated bone marrow mesenchymal stem cell (BMSC) significantly inhibited tartrate-resistant acid phosphatase (TRAP) activity in RANKL-stimulated osteoclastic RAW264.7 cells. Addition of the semaphorin 3A (Sema3A) antibody to the conditioned media partially blocked the medium’s inhibitory effects on the RAW264.7 cells. In BMSC, mRNA expression of Sema3A increased in the presence of different wedelolactone concentrations. Blocking Sema3A activity with its antibody reversed wedelolactone-induced alkaline phosphatase activity in BMSC and concurrently enhanced wedelolactone-reduced TRAP activity in osteoclastic RAW264.7 cells. Moreover, in BMSC, wedelolactone enhanced binding of Sema3A with cell-surface receptors, including neuropilin (NRP)1 and plexinA1. Furthermore, nuclear accumulation of β-catenin, a transcription factor acting downstream of wedelolactone-induced Sema3A signaling, was blocked by the Sema3A antibody. In osteoclastic RAW264.7 cells, conditioned media and wedelolactone promoted the formation of plexin A1-NRP1, but conditioned media also caused the sequestration of the plexin A1-DNAX-activating protein 12 (DAP12) complex and suppressed the phosphorylation of phospholipase C (PLC)γ2. These data suggest that wedelolactone promoted osteoblastogenesis through production of Sema3A, thus inducing the formation of a Sema3A-plexinA1-Nrp1 complex and β-catenin activation. In osteoclastic RAW264.7 cells, wedelolactone inhibited osteoclastogenesis through sequestration of the plexinA1-DAP12 complex, induced the formation of plexinA1-Nrp1 complex, and suppressed PLCγ2 activation.

CHANGES OF SEMAPHORIN 3A EXPRESSION IN HEALING OF TIBIA FRACTURE AFTER TRAUMATIC BRAIN INJURY

To investigate the mechanism of Semaphorin 3A (Sema3A) in fracture healing after nerve injury by observing the expression of Sema3A in the tibia fracture healing after traumatic brain injury (TBI). A total of 192 Wistar female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into tibia fracture group (group A, n=48), TBI group (group B, n=48), TBI with tibia fracture group (group C, n=48), and control group (group D, n=48). The tibia fracture model was established at the right side of group A; TBI model was made in group B by the improved Feeney method; the TBI and tibia fracture model was made in group C; no treatment was given in group D. The tissue samples were respectively collected at 3, 5, 7, 14, 21, and 28 days after operation; HE staining, immunohistochemistry staining, and Western blot method were used for the location and quantitative detection of Sema3A in callus tissue. HE staining showed that no obvious changes were observed at each time point in groups B and D. At 3 and 5 days, there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups A and C. At 7 and 14 days, fibrous tissue grew from periosteum to fracture site in groups A and C; the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups A and C. The chondrocyte had bigger size, looser arrangement, and more osteoid in group C than group A. Group B had disorder periosteum, slight subperiosteal bone hyperplasia, and no obvious change of bone trabecula in group B when compared with group D. At 21 and 28 days, cartilage callus was gradually replaced by new bone trabecula in groups A and C. Group C had loose arrange, disorder structure, and low density of bone trabecula, big callus area and few chondrocyte and osteoid when compared with group A; group B was similar to Group D. Immunohistochemistry staining showed that Sema3A expression in chondrocytes in group C was higher than that in group A, particularly at 7, 14, and 21 day. Sema3A was significantly higher in osteoblasts of new bone trabecula in group A than group C, especially at 14 and 21 days (P<0.05). Western blot results showed that the Sema3A had the same expression trend during fracture healing in groups A and C. However, the expression of Sema3A protein was significantly higher in group C than group A (P<0.05) and in group B than group D (P<0.05) at 7, 14, 21, and 28 days. Abnormal expression of Sema3A may play a role in fracture healing after nerve injury by promoting the chondrocytes proliferation and reducing the distribution of sensory nerve fibers and osteoblast differentiation.

Co-stimulation with interleukin-4 and tumor necrosis factor-α increases epidermal innervation accompanied by suppression of semaphorin 3A

The density of epidermal nerve fibers is increased in patients with pruritic skin conditions such as atopic dermatitis (AD) and it has been speculated that epidermal hyperinnervation is responsible for itch sensitization. The extension of nerve fibers into the epidermis is probably regulated by the balance in nerve elongation factors and nerve repulsion factors, namely nerve growth factor (NGF) and semaphorin 3A (Sema3A), respectively, which are produced by keratinocytes. Although AD is considered as an allergic Th2-mediated disease, the involvement of Th2 cytokines in epidermal hyperinnervation has not been investigated. Here, we examined the effects of interleukin (IL)-4, a prototypic Th2 cytokine, and/or tumor necrosis factor (TNF)-α on the expression of NGF and Sema3A and the epidermal innervation in C57BL/6 mice skin. After two days of daily intradermal injections of IL-4 and/or TNF-α, analyses with real-time PCR and immunohistochemical staining revealed that expression of both of NGF and Sema3A in murine skin treated with IL-4 alone were decreased, while Sema3A but not NGF expression in murine skin treated with both IL-4 and TNF-α was decreased. As a result, the ratio of NGF to Sema3A expression was increased in murine skin treated with both TNF-α and IL-4, accompanied by an increase in epidermal innervation. Furthermore, the increase in the ratio of NGF to Sema3A and the increased epidermal innervation induced by treatment with IL-4 and TNF-α were not found in signal transducers and activator of transcription 6 (STAT6)-knockout mice. This is the first report showing significant involvement of Th2 cytokines in epidermal hyperinnervation and proposes a new mechanism by which Th2 cytokines cause itch in AD.

Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study.

Semaphorin 3A promotes activation of Pax7, Myf5, and MyoD through inhibition of emerin expression in activated satellite cells.

We previously showed that Semaphorin 3A (Sema3A) expression was induced when quiescent muscle satellite cells were stimulated by hepatocyte growth factor and became activated satellite cells (ASCs). However, how Sema3A regulates genes in the early phase of ASCs remains unclear. In this study, we investigated whether Sema3A signaling can regulate the early phase of ASCs, an important satellite cell stage for postnatal growth, repair, and maintenance of skeletal muscle. We showed that expression of the myogenic proliferation regulatory factors Pax7 and Myf5 was decreased in myoblasts transfected with Sema3A siRNA. These cells failed to activate expression MyoD, another myogenic proliferation regulatory factor, during differentiation. Interestingly, some of the Sema3A‐depleted cells did not express Pax7 and MyoD and had enlarged nuclei and very large cytoplasmic areas. We also observed that Pax7 and Myf5 expression was increased in Myc‐Sema3A overexpressing myoblasts. BrdU analysis indicated that Sema3A regulated proliferation of ASCs. These findings suggest that Sema3A signaling can modulate expression of Pax7, Myf5, and MyoD. Moreover, we found that expression of emerin, an inner nuclear membrane protein, was regulated by Sema3A signaling. Emerin was identified by positional cloning as the gene responsible for the X‐linked form of Emery–Dreifuss muscular dystrophy (X‐EDMD). In conclusion, our results support a role for Sema3A in maintaining ASCs through regulation, via emerin, of Pax7, Myf5, and MyoD expression.

Naringin ameliorates bone loss induced by sciatic neurectomy and increases Semaphorin 3A expression in denervated bone.

Naringin maintains bone mass in various osteoporosis models, while its effect on bone in disuse osteoporosis has not been reported. The present study explores whether naringin can prevent disuse osteoporosis induced by unilateral sciatic neurectomy (USN) and whether the Semaphorin 3A-induced Wnt/β-catenin signalling pathway is involved in the osteoprotection of naringin. Naringin dose-dependently prevented the deterioration of bone mineral density (BMD), trabecular structure and biomechanical strength in femur due to USN. Naringin increased bone formation but inhibited resorption, as indicated by bone-turnover markers in blood and urine and the histological staining of Osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) in femur. Semaphorin 3A (Sema3A) and active β-catenin protein decreased after USN and could be restored by naringin to the levels of the sham-operated rats. In addition, naringin in vitro promoted the differentiation of osteoblasts and inhibited osteoclastic differentiation. Our studies suggest that the down-regulation of Sema3A and the subsequent inactivation of Wnt/β-catenin signalling may be some of the mechanisms involved in USN-induced osteoporosis. Naringin could increase the expression of Sema3A and the activation of Wnt/β-catenin signalling to prevent disuse osteoporosis induced by denervation. Thus, naringin functions in bone maintenance and could be a promising therapeutic alternative in preventing disuse osteoporosis.

The Axon Guidance Protein Semaphorin 3A Is Increased in the Motor Cortex of Patients With Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disorder that leads to progressive paralysis of skeletal muscles and death by respiratory failure. There is increasing evidence that ALS is at least in part an axonopathy and that mechanisms regulating axonal degeneration and regeneration might be pathogenetically relevant. Semaphorin 3A (Sema3A) is an axon guidance protein; it acts as an axon repellent and prevents axonal regeneration. Increased Sema3A expression has been described in a mouse model of ALS in which it may contribute to motor neuron degeneration. This study aimed to investigate Sema3A mRNA and protein expression in human CNS tissues. We assessed Sema3A expression using quantitative real-time PCR, in situ hybridization, and immunohistochemistry in motor cortex and spinal cord tissue of 8 ALS patients and 6 controls. We found a consistent increase of Sema3A expression in the motor cortex of ALS patients by all 3 methods. In situ hybridization further confirmed that Sema3A expression was present in motor neurons. These findings indicate that upregulation of Sema3A may contribute to axonal degeneration and failure of regeneration in ALS patients. The inhibition of Sema3A therefore might be a promising future therapeutic option for patients with this disease.

The Chemorepulsive Protein Semaphorin 3A and Perineuronal Net-Mediated Plasticity.

During postnatal development, closure of critical periods coincides with the appearance of extracellular matrix structures, called perineuronal nets (PNN), around various neuronal populations throughout the brain. The absence or presence of PNN strongly correlates with neuronal plasticity. It is not clear how PNN regulate plasticity. The repulsive axon guidance proteins Semaphorin (Sema) 3A and Sema3B are also prominently expressed in the postnatal and adult brain. In the neocortex, Sema3A accumulates in the PNN that form around parvalbumin positive inhibitory interneurons during the closure of critical periods. Sema3A interacts with high-affinity with chondroitin sulfate E, a component of PNN. The localization of Sema3A in PNN and its inhibitory effects on developing neurites are intriguing features and may clarify how PNN mediate structural neural plasticity. In the cerebellum, enhanced neuronal plasticity as a result of an enriched environment correlates with reduced Sema3A expression in PNN. Here, we first review the distribution of Sema3A and Sema3B expression in the rat brain and the biochemical interaction of Sema3A with PNN. Subsequently, we review what is known so far about functional correlates of changes in Sema3A expression in PNN. Finally, we propose a model of how Semaphorins in the PNN may influence local connectivity.

Potential Role of Semaphorin 3A and Its Receptors in Regulating Aberrant Sympathetic Innervation in Peritoneal and Deep Infiltrating Endometriosis.

Previous studies have demonstrated the involvement of nerve repellent factors in regulation of the imbalanced innervation of endometriosis. This prospective study aims to explore the role of Sema 3A in regulating aberrant sympathetic innervation in peritoneal and deep infiltrating endometriosis. Ectopic endometriotic lesion were collected from patients with peritoneal endometriosis (n = 24) and deep infiltrating endometriosis of uterosacral ligament (n = 20) undergoing surgery for endometriosis. Eutopic endometrial samples were collected from patients with endometriosis (n = 22) or without endometriosis (n = 26). Healthy peritoneum (n = 13) from the lateral pelvic wall and healthy uterosacral ligament (n = 13) were obtained from patients who had no surgical and histological proof of endometriosis during hysterectomy for uterine fibroids. Firstly, we studied the immunostaining of Sema 3A, Plexin A1 and NRP-1 in all the tissues described above. Then we studied the nerve fiber density (NFD) of endometriosis-associated (sympathetic) nerve and para-endometriotic (sympathetic) nerve by double immunofluorescence staining. Finally we analyzed the relationship between expression of Sema 3A in stromal cells of endometriotic lesion and the aberrant innervation of endometriosis. Semi-quantitative immunostaining demonstrated that (1) Higher immunostaining of Sema 3A were found in the eutopic endometrial glandular epithelial cells from patients with endometriosis (p = 0.041) than those without endometriosis; (2) Sema 3A immunostaining was higher in glandular epithelial cells of peritoneal endometriosis (P<0.001) and deep infiltrating endometriotic lesions of uterosacral ligament (P = 0.028)compared with glandular epithelial cells of the endometrium from women with endometriosis, while its expression in ectopic stormal cells in both groups were significantly lower than that from eutopic endometrium of women without endometirosis (P<0.001, P<0.001, respectively). NFDs of Anti-TH (+) endometriosis-associated sympathetic nerve of peritoneal endometriosis (p<0.001) and deep endometriosis of uterosacral ligament (p<0.001) were significantly lower than NFDs of para-endometriotic sympathetic nerve. Our results suggest that Sema 3A may contribute to the regulation of aberrant sympathetic innervation in peritoneal and deep infiltrating endometriosis.

Differential expression of sema3A and sema7A in a murine model of multiple sclerosis: Implications for a therapeutic design

We characterised the expression of semaphorin (sema)3A, sema7A and their receptors in the immune and the central nervous system (CNS) at different stages of experimental autoimmune encephalomyelitis (EAE). We also studied their expression in neonatal and adult oligodendrocyte progenitor cell (OPC) and in mature oligodendrocyte cultures. Our results show that sema3A is increased in the CNS and decreased in the immune system upon EAE induction. However, sema7A expression is increased in both the CNS and the immune system during EAE. We also detected sema3A, sema7A and their receptors in neonatal and adult OPCs and in mature oligodendrocytes.These data suggest that sema3A and sema7A are involved in the pathogenesis of EAE, in the modulation of the immune response and in the neurodegeneration that take place in the CNS. Sema7A may represent an intriguing potential therapeutic target for the treatment of both the neurodegenerative and immune-mediated disease processes in MS.

Expression of semaphorin 3A, semaphorin 7A and their receptors in multiple sclerosis lesions

Background: Studies in multiple sclerosis (MS) and in experimental models point to a critical role of semaphorin (sema)3A and sema7A in MS pathogenesis. Objective: The objective of this paper is to characterise the expression of sema3A, sema7A, and their receptors in MS lesions. Methods: We included 44 demyelinating lesions from MS patients, 12 lesions with acute cerebral infarct, 11 lesions with progressive multifocal leucoencephalopathy and 10 non-neurological control patients. MS lesions were classified according to inflammatory activity and all samples were immunostained for sema3A, sema7A, neuropilin 1 (Np-1), α1-integrin, and β1-integrin. Results: In MS-damaged white matter sema3A and Np-1 were both detected in microglia/macrophages, whereas reactive astrocytes expressed only sema3A. Otherwise, sema7A, α1-integrin and β1-integrin were observed in reactive astrocytes, and microglia/macrophages only expressed β1-integrin. The expression of sema3A, sema7A and their receptors is more relevant in MS than in other demyelinating diseases. Sema3A and sema7A expression correlated with the inflammatory activity of the MS lesions, suggesting their involvement in the immunological process that takes place in MS. Conclusions: The expression pattern of sema3A, sema7A and their receptors in MS lesions suggests that both molecules contribute to create a negative environment for tissue regeneration, influencing the ability to regenerate the damaged tissue.

Semaphorin7A Promotion of Tumoral Growth and Metastasis in Human Oral Cancer by Regulation of G1 Cell Cycle and Matrix Metalloproteases: Possible Contribution to Tumoral Angiogenesis.

BackgroundSemaphorins (SEMAs) consist of a large family of secreted and membrane-anchored proteins that are important in neuronal pathfinding and axon guidance in selected areas of the developing nervous system. Of them, SEMA7A has been reported to have a chemotactic activity in neurogenesis and to be an immunomodulator; however, little is known about the relevance of SEMA7A in the behaviors of oral squamous cell carcinoma (OSCC).MethodsWe evaluated SEMA7A expression in OSCC-derived cell lines and primary OSCC samples using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and semiquantitative immunohistochemistry (sq-IHC). In addition, SEMA7A knockdown cells (shSEMA7A cells) were used for functional experiments, including cellular proliferation, invasiveness, and migration assays. We also analyzed the clinical correlation between SEMA7A status and clinical behaviors in patients with OSCC.ResultsSEMA7A mRNA and protein were up-regulated significantly (P<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. The shSEMA7A cells showed decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors (p21Cip1 and p27Kip1) and down-regulation of cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6); and decreased invasiveness and migration activities by reduced secretion of matrix metalloproteases (MMPs) (MMP-2, proMMP-2, pro-MMP-9), and expression of membrane type 1- MMP (MT1-MMP). We also found inactivation of the extracellular regulated kinase 1/2 and AKT pathways, an upstream molecule of cell-cycle arrest at the G1 phase, and reduced secretion of MMPs in shSEMA7A cells. sq-IHC showed that SEMA7A expression in the primary OSCCs was significantly (P = 0.001) greater than that in normal counterparts and was correlated with primary tumoral size (P = 0.0254) and regional lymph node metastasis (P = 0.0002).ConclusionOur data provide evidence for an essential role of SEMA7A in tumoral growth and metastasis in OSCC and indicated that SEMA7A may play a potential diagnostic/therapeutic target for use in patients with OSCC.

Decreased expression of neuropilin-1 as a novel key factor contributing to peripheral microvasculopathy and defective angiogenesis in systemic sclerosis

ObjectivesIn systemic sclerosis (SSc), vascular involvement is characterised by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) system disturbances. Neuropilin-1 (NRP1), a receptor for both class-3 semaphorins (Sema3s) and VEGF-A, is...

Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts.

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed an unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan-2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan-2, 4 was abundant (two magnitudes higher than syndecan-1, 3) in early-differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2-induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non–knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan-2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2-syndecan-2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery.

Semaphorins and neuropilins expression in salivary gland tumors.

Salivary gland tumors (SGT) account for 3-10% of all head and neck neoplasms, and little is known about their angiogenic properties. Despite semaphorins and neuropilins have been demonstrated to be prognostic determinants in many human cancers, they remain to be investigated in SGT. Therefore, the objective of this study was to analyze the clinical significance of the expression of class 3 semaphorins A (Sema3A) and B (Sema3B) and neuropilins-1 (Np-1) and neuropilins-2 (Np-2), in SGT. Two hundred and forty-eight SGT were organized in tissue microarray paraffin blocks and expression of CD34, Sema3A, Sema3B, Np-1, and Np-2 was determined through immunohistochemistry. The immunoreactions were quantified using digital algorithms and the results correlated with clinicopathological parameters. Malignant tumors had an increased vascular density than their benign counterparts and their increased vascular area significantly correlated with recurrences (P<0.05). Patients older than 40years and the presence of recurrences determined an inferior survival rate (P=0.0057 and P=0.0303, respectively). In normal salivary glands, Np-1 and Np-2 expression was restricted to ductal cells, whereas Sema3A and Sema3B were positive in the serous acinar compartment. Tumors were positive for all markers and the co-expression of Np-1/Np-2 significantly correlated with the presence of paresthesia and advanced stages of the tumors (P=0.01 and P=0.04, respectively). Sema3A, Sema3B, Np-1, and Np-2 may be involved in the pathogenesis of SGT, but their expression did not present a statistically significant prognostic potential in this study.

The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs)

Background and AimsImmune semaphorins are a large family of proteins involved in the pathogenesis of inflammatory diseases through the regulation of immune homeostasis and tissue inflammation. We aim to assess the possible involvement of semaphorin3A (sema3A) and 4A (sema4A) in peripheral immune responses and bowel tissue inflammation of patients suffering from Crohn’s disease (CD) and ulcerative colitis (UC).Patients and MethodsTwenty-seven CD patients and 10 UC patients were studied and compared to 10 patients followed for acute diverticulitis (disease control) and 12 healthy individuals. All were evaluated for sema3A expression on T regulatory cells (Tregs), serum levels of sema3A and sema4A, and tissue expression of sema3A and sema4A in bowel biopsies.ResultsThe percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals.ConclusionsAltered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.

Abstract 173: Upregulation of Sema7A Induced by Disturbed Flow Contributes to the Development of Atherosclerosis

Background: Atherosclerosis preferentially develops in areas of arteries with arches and branches, where the flow is disturbed (D-flow). D-flow upregulates proatherogenic genes and promotes the dysfunctional endothelial cell phenotype. Semaphorin 7A (Sema7A), a membrane-associated glycosylphosphatidylinositol (GPI)-linked cell-surface glycoprotein, was recently found to be expressed in the endothelial cells and played important roles in several pathophysiological processes. We hypothesize that Sema7A is involved in the proatherogenic process induced by D-flow. Methods and Results: We performed partial carotid ligation (PCL) on C57BL/6 mice by ligation of the external carotid artery (ECA), internal carotid artery (ICA), and ophthalmic artery (OA) of the left carotid artery (LCA), leaving the superficial temporal artery (STA) untied. The ultrasound image was used to monitor blood flow. Successful partial ligation was obtained as blood flow velocity was reduced by 70% (n=3) and D-flow was generated in the LCA while the blood flow in RCA remained normal. After 48h ligation, mice were sacrificed and total RNA was isolated from carotid arteries for the analysis of Sema7A gene expression by real-time PCR. Results showed that Sema7A expression in the LCA was 2.6-fold higher (P=0.0237, n=3) in D-flow condition compared to RCA. To ask whether Sema7A is involved in D-flow-induced atherosclerosis, we generated Sema7A-/-ApoE-/- mice and Sema7A+/+ApoE-/- mice, and performed PCL at 2 weeks on high fat diet. The results showed that atherosclerotic lesion size in the LCA under disturbed condition was decreased by 50.74% (P=0.0072) in ApoE-/-Sema7A-/- mice (0.2411±0.05265%, n=6) compared to Sema7A+/+ApoE-/- control mice (0.4894 ± 0.05186%, n=6). Conclusion: Our data showed that D-flow promotes the upregulation of Sema7A gene expression in endothelial cells. Deletion of Sema7A attenuates atherosclerotic plaque formation induced by D-flow.

Semaphorin 3A: an immunoregulator in systemic sclerosis

Semaphorin 3A (sema3A) plays a regulatory role in immune responses, mainly affecting the activation of regulatory T cells. It has been found to correlate with disease activity in rheumatoid arthritis and systemic lupus erythematosus (SLE). To investigate the expression of sema3A in patients with systemic sclerosis (SSc) compared to healthy controls and SLE disease controls and to correlate it with clinical characteristics, 27 SSc patients, 42 SLE patients and 28 healthy controls were enrolled. Serum level of sema3A was measured by ELISA, and expression of sema3A on regulatory T cells was evaluated by FACS analysis. SSc patients were evaluated for demographics, clinical manifestations, routine laboratory results, nailfold videocapillaroscopy, pulmonary function tests, echocardiograms, modified Rodnan skin score, and disease activity and severity scores. Serum levels of semaphorin 3A were lower in SSc compared to healthy controls 14.38±5.7 versus 27.14±8.4ng/ml, p<0.0001 and similar to SLE 15.7±4.3ng/ml. The expression of semaphorin 3A on regulatory T cells was also lower in SSc compared to healthy controls 61.7±15.7 versus 88.7±3. 7% (p<0.0001). Semaphorin 3A serum level inversely correlated with the duration of disease: r=-0.4, p=0.036 and with low C4 level r=0.66, p=0.026. SCL-70 antibody positivity was associated with a lower semaphorin 3A level (difference in mean of 3.44, p=0.06). Sema3A expression is low in SSc serum and more specifically on regulatory T cells. This may help explain the reduced activation of regulatory T cells in SSc.

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by insilico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.