Sema3A Expression Research Articles

The predictive value of semaphorins 3 expression in biopsies for biochemical recurrence of patients with low- and intermediate-risk prostate cancer

The class-3 semaphorins (Sema3A-F, Sema3s) are initially identified to play an important role in axonal guidance and cell migration. Our previous studies showed that Sema3s are also involved in the lymph node metastasis of prostate cancer, and are likely to modulate the behavior of prostate cancer with a pro-tumoral or an anti-tumoral effect, depending on their subtypes. However, no study has critically investigated the value of Sema3s expression in preoperative biopsy samples for the prediction of biochemical recurrence (BCR) after radical prostatectomy. In this study, we evaluated Sema3s expression by immunohistochemistry on 198 prostate biopsies with low- and intermediate-risk localized prostate cancer. The median follow-up was 42 months (range, 6-60) for all patients. Our results showed that Sema3A (OR: 0.19, P<0.001), Sema3B (OR: 0.38, P=0.003), Sema3E (OR: 0.39, P=0.007), and Sema3C (OR: 2.31, P=0.014) staining were independent predictors of BCR on multivariable analysis. Sema3A, 3B, 3C and 3E expression demonstrated potential values in predicting BCR upon survival analysis (P=0.001, P=0.003, P=0.029, P=0.037, respectively, Log-rank test). Our findings suggested that Sema3A, 3B, 3C, and 3E immunostaining in prostate biopsies, as supplements to clinicopathological parameters, could be used for predicting BCR in low- and intermediate-risk prostate cancer patients after radical prostatectomy. Specially, concurrent Sema3C-positive and Sema3A-negative, 3B-negative, 3E-negative staining is associated with an adverse prognosis. Further prospective studies in larger patient populations are needed to validate the current observations.

Biallelic SEMA3A defects cause a novel type of syndromic short stature

Chromosomal microarray testing is commonly used to identify disease causing de novo copy number variants in patients with developmental delay and multiple congenital anomalies. In such a patient we now observed an 150 kb deletion on chromosome 7q21.11 affecting the first exon of the axon guidance molecule gene SEMA3A (sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A). This deletion was inherited from the healthy father, but considering the function of SEMA3A and phenotypic similarity to the knock-out mice, we still assumed a pathogenic relevance and tested for a recessive second defect. Sequencing of SEMA3A in the patient indeed revealed the de novo in-frame mutation p.Phe316_Lys317delinsThrSerSerAsnGlu. Cloning of the mutated allele in combination with two informative SNPs confirmed compound heterozygosity in the patient. While the altered protein structure was predicted to be benign, aberrant splicing resulting in a premature stop codon was proven by RT-PCR to occur in about half of the transcripts from this allele. Expression profiling in human fetal and adult cDNA panels, confirmed a high expression of SEMA3A in all brain regions as well as in adult and fetal heart and fetal skeletal muscle. Normal intellectual development in the patient was surprising but may be explained by the remaining 20% of SEMA3A expression level demonstrated by quantitative RT-PCR. We therefore report a novel autosomal recessive syndrome characterized by postnatal short stature with relative macrocephaly, camptodactyly, septal heart defect and several minor anomalies caused by biallelic mutations in SEMA3A.

Modulation of semaphorin3A in perineuronal nets during structural plasticity in the adult cerebellum

In the adult central nervous system (CNS) subsets of neurons are enwrapped by densely organized extracellular matrix structures, called perineuronal nets (PNNs). PNNs are formed at the end of critical periods and contribute to synapse stabilization. Enzymatic degradation of PNNs or genetic deletion of specific PNN components leads to the prolongation of the plasticity period. PNNs consist of extracellular matrix molecules, including chondroitin sulfate proteoglycans, hyaluronan, tenascins and link proteins. It has been recently shown that the chemorepulsive axon guidance protein semaphorin3A (Sema3A) is also a constituent of PNNs, binding with high affinity to the sugar chains of chondroitin sulfate proteoglycans. To elucidate whether the expression of Sema3A is modified in parallel with structural plasticity in the adult CNS, we examined Sema3A expression in the deep cerebellar nuclei of the adult mouse in a number of conditions associated with structural reorganization of the local connectivity. We found that Sema3A in PNNs is reduced during enhanced neuritic remodeling, in both physiological and injury-induced conditions. Moreover, we provide evidence that Sema3A is tightly associated with Purkinje axons and their terminals and its amount in the PNNs is related to Purkinje cell innervation of DCN neurons, but not to glutamatergic inputs. On the whole these data suggest that Sema3A may contribute to the growth-inhibitory properties of PNNs and Purkinje neurons may directly control their specific connection pattern through the release and capture of this guidance cue in the specialized ECM that surrounds their terminals.

Neurotropin inhibits the increase in intraepidermal nerve density in the acetone-treated dry-skin mouse model

Epidermal hyperinnervation is considered one cause of sensitization to itch, and is thought to regulated by keratinocyte-derived axonal guidance molecules, including nerve growth factor (NGF) and semaphorin (Sema)3A. Neurotropin (NTP) shows antipruritic effects in allergic disease and is also used for pain relief. Using cultured rat dorsal root ganglion neurones, we previously found that NTP inhibited NGF-induced neurite outgrowth. However, no such inhibitory effect has been shown in vivo. We therefore assessed the effects of intraperitoneal administration of NTP on nerve density and expression of NGF and Sema3A mRNAs in the epidermis of acetone-treated mice showing epidermal hyperinnervation. We found that NTP significantly reduced intraepidermal nerve growth in these acetone-treated mice. NTP significantly upregulated epidermal Sema3A mRNA, but had no effect on expression of epidermal NGF mRNA. These findings indicate that NTP may reduce intraepidermal nerve density by inducing expression of Sema3A in the epidermis.

The Guidance Molecule Semaphorin3A is Differentially Involved in the Arealization of the Mouse and Primate Neocortex

The visual cortex is organized into discrete domains characterized by their specific function, connectivity, chemoarchitecture, and cytoarchitecture. Gradients of transcription factors across the anteroposterior and mediolateral axes of the neocortex have previously been demonstrated to specify the main sensory regions. However, they do not account for the establishment of multiple areas in the primate visual cortex, which occupies approximately 50% of the neocortical surface. We demonstrate that the guidance molecule Semaphorin3A (Sema3A) is initially secreted in the cortical plate of the embryonic marmoset monkey and acts as an intrinsic cue to control the migration of subpopulations of neuronal progenitors and projection neurons expressing the receptor Neuropilin 1 (Npn1). During the first 2 postnatal weeks, Sema3A expression becomes primarily associated with ventral visual cortical areas, leading to the specific migration of Npn1+ neurons in the late maturing visual areas. In the mouse, Sema3A distribution is not arealized, but Npn1 expression becomes restricted to the posterior neocortex at embryonic day 16.5. The selective reduction in the striate cortex we observe in Sema3A-/- animals potentially results from the differential distribution of Npn1+ cells. Therefore, the Sema3A/Npn1 pathway participates to the parcellation of the visual neocortex in both the mouse and the marmoset, however, through different regulatory processes.

Sema3A regulates bone-mass accrual through sensory innervations

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a(-/-) mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a(-/-) mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1(-/-) and Sema3aosx(-/-) mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin(-/-) and Sema3anestin(-/-) mice) had low bone mass, similar to Sema3a(-/-) mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin(-/-) mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin(-/-) mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a(-/-) mice, such as olfactory development, were identified in Sema3asynasin(-/-) mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a(-/-) mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

Overexpression of sema3a in myocardial infarction border zone decreases vulnerability of ventricular tachycardia post‐myocardial infarction in rats

The expression of the chemorepellent Sema3a is inversely related to sympathetic innervation. We investigated whether overexpression of Sema3a in the myocardial infarction (MI) border zone could attenuate sympathetic hyper-innervation and decrease the vulnerability to malignant ventricular tachyarrhythmia (VT) in rats. Survived MI rats were randomized to phosphate buffered saline (PBS, n = 12); mock lentivirus (MLV, n = 13) and lentivirus-mediated overexpression of Sema3a (SLV, n = 13) groups. Sham-operated rats served as control group (CON, n = 20). Cardiac function and electrophysiological study (PES) were performed at 1 week later. Blood and tissue samples were collected for histological analysis, epinephrine (EPI), growth-associated factor 43 (GAP43) and tyrosine hydroxylase (TH) measurements. QTc intervals were significantly shorter in SLV group than in PBS and MLV groups (168.6 ± 7.8 vs. 178.1 ± 9.5 and 180.9 ± 8.2 ms, all P < 0.01). Inducibility of VT by PES was significantly lower in the SLV group [30.8% (4/13)] than in PBS [66.7% (8/12)] and MLV [61.5% (8/13)] groups (P < 0.05). mRNA and protein expressions of Sema3a were significantly higher and the protein expression of GAP43 and TH was significantly lower at 7 days after transduction in SLV group compared with PBS, MLV and CON groups. Myocardial EPI in the border zone was also significantly lower in SLV group than in PBS and MLV group (8.73 ± 1.30 vs. 11.94 ± 1.71 and 12.24 ± 1.54 μg/g protein, P < 0.001). Overexpression of Sema3a in MI border zone could reduce the inducibility of ventricular arrhythmias by reducing sympathetic hyper-reinnervation after infarction.

Insufficient OPC migration into demyelinated lesions is a cause of poor remyelination in MS and mouse models

Failure of remyelination of multiple sclerosis (MS) lesions contributes to neurodegeneration that correlates with chronic disability in patients. Currently, there are no available treatments to reduce neurodegeneration, but one therapeutic approach to fill this unmet need is to promote remyelination. As many demyelinated MS lesions contain plentiful oligodendrocyte precursor cells (OPCs), but no mature myelinating oligodendrocytes, research has previously concentrated on promoting OPC maturation. However, some MS lesions contain few OPCs, and therefore, remyelination failure may also be secondary to OPC recruitment failure. Here, in a series of MS samples, we determined how many lesions contained few OPCs, and correlated this to pathological subtype and expression of the chemotactic molecules Semaphorin (Sema) 3A and 3F. 37 % of MS lesions contained low numbers of OPCs, and these were mostly chronic active lesions, in which cells expressed Sema3A (chemorepellent). To test the hypothesis that differential Sema3 expression in demyelinated lesions alters OPC recruitment and the efficiency of subsequent remyelination, we used a focal myelinotoxic mouse model of demyelination. Adding recombinant (r)Sema3A (chemorepellent) to demyelinated lesions reduced OPC recruitment and remyelination, whereas the addition of rSema3F (chemoattractant), or use of transgenic mice with reduced Sema3A expression increased OPC recruitment and remyelination. We conclude that some MS lesions fail to remyelinate secondary to reduced OPC recruitment, and that chemotactic molecules are involved in the mechanism, providing a new group of drug targets to improve remyelination, with a specific target in the Sema3A receptor neuropilin-1.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1112-y) contains supplementary material, which is available to authorized users.

Decreased Semaphorin3A expression correlates with disease activity and histological features of rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is an autoimmune disease of which the pathogenetic mechanisms are not fully understood. Semaphorin3A (Sema3A) has an immune regulatory role. Neuropilin1 (NRP1), the primary receptor for Sema3A, is also a receptor for vascular endothelial growth factor 165 (VEGF165). It has been shown that Sema3A competitively antagonizes VEGF165 signaling. This study investigated whether Sema3A is expressed in synovial tissues, and is associated with disease activity and the histological features of synovial tissues from RA patients.MethodsHuman synovial tissues samples were obtained from RA and osteoarthritis (OA) patients. Disease activity of RA patients was calculated using the 28-joint Disease Activity Score based on C-reactive protein (DAS28-CRP). The histological features of RA synovial tissues were evaluated using Rooney’s inflammation scoring system. The localization of Sema3A, VEGF165 and NRP1 positive cells was immunohistochemically determined in synovial tissues. Expression levels of Sema3A, VEGF-A and NRP1 mRNA were determined using quantitative real-time polymerase chain reaction (qPCR).ResultsIn OA specimens, Sema3A, VEGF165 and NRP1 proteins were expressed in the synovial lining and inflammatory cells beneath the lining. Immunohistochemistry revealed the protein expression of Sema3A in synovial lining cells was decreased in RA tissues compared with OA samples. qPCR analysis demonstrated a significant reduction of Sema3A mRNA levels in RA synovial tissue samples than in OA and a significant correlation of the ratio of Sema3A/VEGF-A mRNA expression levels with DAS28-CRP (R = −0.449, p = 0.013). Sema3A mRNA levels also correlated with Rooney’s inflammation score, especially in perivascular infiltrates of lymphocytes (R = −0.506, p = 0.004), focal aggregates of lymphocytes (R = −0.501, p = 0.005) and diffuse infiltrates of lymphocytes (R = −0.536, p = 0.002).ConclusionsReduction of Sema3A expression in RA synovial tissues may contribute to pathogenesis of RA.

Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic colon segment and functional intestinal obstruction. The RET proto-oncogene is the major gene associated to HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In addition, many other genes have been described to be associated with this pathology, including the semaphorins class III genes SEMA3A (7p12.1) and SEMA3D (7q21.11) through SNP array analyses and by next-generation sequencing technologies. Semaphorins are guidance cues for developing neurons implicated in the axonal projections and in the determination of the migratory pathway for neural-crest derived neural precursors during enteric nervous system development. In addition, it has been described that increased SEMA3A expression may be a risk factor for HSCR through the upregulation of the gene in the aganglionic smooth muscle layer of the colon in HSCR patients. Here we present the results of a comprehensive analysis of SEMA3A and SEMA3D in a series of 200 Spanish HSCR patients by the mutational screening of its coding sequence, which has led to find a number of potentially deleterious variants. RET mutations have been also detected in some of those patients carrying SEMAs variants. We have evaluated the A131T-SEMA3A, S598G-SEMA3A and E198K-SEMA3D mutations using colon tissue sections of these patients by immunohistochemistry. All mutants presented increased protein expression in smooth muscle layer of ganglionic segments. Moreover, A131T-SEMA3A also maintained higher protein levels in the aganglionic muscle layers. These findings strongly suggest that these mutants have a pathogenic effect on the disease. Furthermore, because of their coexistence with RET mutations, our data substantiate the additive genetic model proposed for this rare disorder and further support the association of SEMAs genes with HSCR.

Effect of endostar combined with cisplatin on expression of VEGF and Sema3A of Lewis lung cancer rats

ObjectiveTo evaluate the therapeutic effect of endostar (ED) combined with cisplatin(DDP) on model of C57BL/6 rats, and to further investigate the inhibiting mechanism of endostar from tumor angiogenesis. MethodsLewis lung cancer cells were inoculated in C57BL/6 mouse, then the mouse were randomized into control group (group A), ED (group B), DDP (group C) and ED/DDP (group D). They were treated according to the plan. And the expressions of VEGF and Sema3A were evaluated by immunhistochemisty. ResultsThe weight of tumor increased in group A and B. It was decreased in group C and D. The tumor volume was increased in all the 4 groups. The VEGF expression of group D was obviously lower than the other group 3, but the Sema3A expressed of group D was significantly strengthener than the other group 3. The VEGF expression of group B and group D were obviously low especially in the 4th-8th days. Pearson correlated analysis showed that the expression VEGF and Sema3A were negatively correlated (r=-0.72, P<0.05). ConclusionsED combined with DDP could control the tumor growth effectively, and avoid weight loss. ED could reduce VEGF expression, and enhance Sema3A expression. Tumor vessel presents transient normalization. It is easy for DDP perfusion, and to kill tumor cells.

Satellite cells produce neural chemorepellent semaphorin 3A upon muscle injury

Regenerative mechanisms that regulate intramuscular motor innervation. including configuration of the neuromuscular connections are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of satellite cells as a key source of a secreted neural chemorepellent semaphorin 3A (Sema3A) expression. In order to verify this concept, there is still a critical need to provide direct evidence to show the up-regulation of Sema3A protein in satellite cells in vivo upon muscle injury. The present study employed a Sema3A/MyoD double-immunohistochemical staining for cryo-sections prepared from cardiotoxin injected gastrocnemius muscle of adult mouse lower hind-limb. Results clearly demonstrated that Sema3A expression was up-regulated in myogenic differentiation-positive satellite cells at 4-12 days post-injury period, the time that corresponds to the cell differentiation phase characterized by increasing myogenin messenger RNA expression. This direct proof encourages a possible implication of satellite cells in the spatiotemporal regulation of extracellular Sema3A concentrations, which potentially ensures coordinating a delay in neurite sprouting and re-attachment of motoneuron terminals onto damaged muscle fibers early in muscle regeneration in synchrony with recovery of muscle-fiber integrity.

Semaphorin 7a+ Regulatory T Cells Are Associated with Progressive Idiopathic Pulmonary Fibrosis and Are Implicated in Transforming Growth Factor-β1–induced Pulmonary Fibrosis

Lymphocytes are increasingly associated with idiopathic pulmonary fibrosis (IPF). Semaphorin 7a (Sema 7a) participates in lymphocyte activation. To define the relationship between Sema 7a and lymphocytes in IPF. We characterized the significance of Sema 7a+ lymphocytes in humans with IPF and in a mouse model of lung fibrosis caused by lung-targeted, transgenic overexpression of TGF-β1. We determined the site of Sema 7a expression in human and murine lungs and circulation and used adoptive transfer approaches to define the relevance of lymphocytes coexpressing Sema7a and the markers CD19, CD4, or CD4+CD25+FoxP3+ in TGF-β1-induced murine lung fibrosis. Subjects with IPF show expression of Sema 7a on lung CD4+ cells and circulating CD4+ or CD19+ cells. Sema 7a expression is increased on CD4+ cells and CD4+CD25+FoxP3+ regulatory T cells, but not CD19+ cells, in subjects with progressive IPF. Sema 7a is expressed on lymphocytes expressing CD4 but not CD19 in the lungs and spleen of TGF-β1-transgenic mice. Sema 7a expressing bone marrow-derived cells induce lung fibrosis and alter the production of T-cell mediators, including IFN-γ, IL-4, IL-17A, and IL-10. These effects require CD4 but not CD19. In comparison to Sema 7a-CD4+CD25+FoxP3+ cells, Sema7a+CD4+CD25+FoxP3+ cells exhibit reduced expression of regulatory genes such as IL-10, and adoptive transfer of these cells induces fibrosis and remodeling in the TGF-β1-exposed murine lung. Sema 7a+CD4+CD25+FoxP3+ regulatory T cells are associated with disease progression in subjects with IPF and induce fibrosis in the TGF-β1-exposed murine lung.

Comparative analysis of semaphorin 3A in soleus and EDL muscle satellite cells in vitro toward understanding its role in modulating myogenin expression

Resident myogenic stem cells, satellite cells, up-regulate a secreted multi-functional modulator, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle-crush injury and treatment with hepatocyte growth factor (HGF) or basic fibroblast growth factor (FGF2). Here, we add evidence that the Sema3A expression and secretion induced by the growth factors is significantly higher in primary cultures from adult rat soleus than from the fast-twitch extensor digitorum longus (EDL) muscle. The higher Sema3A response, revealed by quantitative PCR and Western blotting of cell lysates and conditioned media, may account for the higher myogenin expression of soleus muscle satellite cells early in differentiation since addition of recombinant Sema3A stimulates myogenin expression in cultures. These experiments also showed that mRNA expression of plexin A2, which together with neuropilins, constitutes Sema3A composite-receptors, was higher in satellite cells from soleus than EDL with no difference in plexin A1 and A3 and neuropilin-1 and 2 levels. These comparative studies, therefore, highlight a possible Sema3A-plexin A2-myogenin signaling axis that may ensure promoting early differentiation by soleus muscle satellite cells.

Time‐coordinated prevalence of extracellular HGF, FGF2 and TGF‐β3 in crush‐injured skeletal muscle

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3A upon in vivo muscle-crush injury. The study also revealed that Sema3A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)-β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-β3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescence-Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2-8 days post-crush, whereas active TGF-β3 increases after 12 days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-β3 that drive regulation of Sema3A expression during regenerative intramuscular moto-neuritogenesis.

Semaphorin 3A – a marker for disease activity and a potential putative disease-modifying treatment in systemic lupus erythematosus

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen-induced arthritis. Sema3A was also found to be involved in other immune-mediated diseases, e.g. psoriasis and allergic rhinitis. In this review we concentrated on the involvement of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and on the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients. We demonstrated the expression of sema3A in renal biopsies from lupus glomerulonephritis patients. This expression was found to be inversely correlated with proteinuria and kidney function tests. Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (disease control) and lower yet than in normal individuals. Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage and the presence of anti-cardiolipin antibodies. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, we demonstrated that when sema3A was co-cultured with CpG-ODN-stimulated memory B cells of SLE patients, their TLR-9 expression was significantly reduced by almost 50% (p = 0.001). These findings, along with the observation of sema3A being reduced in SLE patients in correlation with disease severity and autoimmunity, and memory B cells being beneficially responsive to sema3A, suggest this regulatory molecule may be considered as a potential therapy for SLE. Such focused therapies will help in achieving the maintenance of self-tolerance and alter pro-inflammatory status in lupus.

Semaphorin 7a Links Nerve Regeneration and Inflammation in the Cornea

We determined Semaphorin 7a (Sema7a) localization and abundance in naive corneas and in corneas after nerve-transecting lamellar flap surgery, and determined the effect of Sema7a supplementation on corneal nerve regeneration and inflammation. Immunolocalization and Western blot analyses were performed to evaluate the abundance of Sema7a in naive corneas and corneas undergoing nerve regeneration after lamellar corneal surgery in thy1-YFP+ neurofluorescent mice. We used compartmental cultures of dissociated trigeminal ganglion cells to determine the effect of Sema7a exposure on neurite outgrowth in vitro. Finally, a Sema7a pellet was implanted under the corneal flap after lamellar transection surgery to determine the neuronal and inflammatory effects of Sema7a supplementation in vivo. Sema7a was expressed in the corneal epithelium and stromal keratocytes, but was more abundant in the epithelium (74.3%) compared to the stroma (25.7%, P = 0.02). Sema7a expression was increased significantly in the cornea after lamellar corneal surgery and was localized to stromal cells near the regenerating nerve fronds. Exposure of trigeminal neurites to Sema7a (20 nM) in the side compartment increased neurite length significantly. The implanted Sema7a pellet increased significantly YFP+ inflammatory cell influx into the cornea as well as increased corneal nerve length. Sema7a is expressed constitutively in the cornea, and potently stimulates nerve regeneration and inflammatory cell influx. Therefore, this immune semaphorin links nerve regeneration and inflammatory processes in the cornea.

Abstract 346: Functional role for axonal guidance molecule Sema7A in breast cancer metastasis: Epithelial to mesenchymal transition and tumor metastasis

Abstract Reactivation of embryogenesis related processes such as neurogenesis is a powerful mechanism used by tumor cells to enhance their survival and propagation. Recent studies have shown that many types of solid tumors express neurogenic molecules independent of the tumor's geography. Semaphorins are a compelling example of these pleiotropic neuronal molecules. Semaphorins were originally characterized for their role in axonal guidance and several of them have been found to play a role in cancer and other inflammatory diseases. Of the Semaphorins, Semaphorin7A (Sema7A) has not yet been studied in the context of cancer. It has however been associated with fibrosis, inflammation and immune modulation. Most semaphorins are repellent cues and inhibit cellular migration, but Sema7A is the only vertebrate semaphorin that serves as an axon chemoattractant. Sema7A also promotes dendricity, adhesion and motility during neuronal development, monocyte differentiation and osteoclast development. All these cellular properties have been attributed to Sema7A's ability to activate p38 mitogen-activated protein kinase pathway (MAP-kinase) through α1β1 integrins. We have recently discovered that Sema7A is over-expressed in the tumor tissue of breast cancer patients compared to the adjacent normal tissue. Within the tumor tissue, the mesenchymal cancer stem cells had the highest expression of Sema7A. We have also found that breast cancer cell lines of a mesenchymal phenotype express high levels of Sema7A and that transforming growth factor beta-1 can induce Sema7A expression in breast epithelial cells. Further, we show that mice bearing Sema7A gene silenced mammary tumors had decreased tumor growth and metastasis with enhanced survival compared to the control tumors. Our studies aim to unravel the functional role of Sema7A in breast cancer progression. We hypothesize that Sema7A expression in tumor cells may lead to increased tumor cell dendricity, migration, and adhesion through the activation of the MAP-kinase pathway and mediating epithelial to mesenchymal transition (EMT). Similar to its role in neurogenesis, in the tumor microenvironment Sema7A could contribute to tumor cell migration and tissue remodeling. Therefore, Sema7A could prove to be a novel therapeutic target to limit tumor growth and metastasis not only for breast cancer but for other solid tumors as well. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 346. doi:1538-7445.AM2012-346

Semaphorin 3A overcomes cancer hypoxia and metastatic dissemination induced by antiangiogenic treatment in mice

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

Tongluo Jiunao Injection, a Chinese herbal fomula protects the focal ischemic brain injury and decreases the expression of Semaphorin 3A and its receptor Neuropilin 1

Tongluo Jiunao (TLJN) injection has been shown to exhibit protective effects following ischemia injury in vivo and in vitro. We hypothesized that TLJN injection alleviates the injury of neuron and brain blood barrier by inhibiting the expression of Semaphorin 3A (Sema 3A) and its receptor Neuropilin 1 (Nrp‐1) following ischemia stroke. A permanent middle cerebral artery occlusion (PMCAO) surgery was induced in the study on SD rats. Brain edema, infarct volume and spatialtemporal expression of Sema 3A and Nrp‐1 were detected. Triphenyltetrazolium chloride staining results revealed that TLJN injection reduced the infarct volume from 1d to 7d (p&lt;0.01) and edema of brain at 1d (p&lt;0.01) post injury compared to model group. Double‐labeled immunofluorescence showed that Sema 3A and Nrp‐1 positive cells increased in cortex and hippocampus in ipsilateral hemisphere at all the time points after the onset of PMCAO. Enzyme‐linked immunosorbent assay results showed that TLJN decreased Sema 3A. Reverse‐transcription polymerase chain reaction and western blot results showed that TLJN injection decreased the expression of Sema 3A and Nrp‐1 in the ischemic brain tissues of rats. These results suggested that TLJN injection reduced the damage of brain by decreasing the infarction volumn and edema of brain which is related to its inhibitory of Sema 3A and Nrp‐1.