Semaphorin 3A Is Effective in Reducing Both Inflammation and Angiogenesis in a Mouse Model of Bronchial Asthma

Semaphorin 3A (sema3A) belongs to the sub-family of the immune semaphorins that function as regulators of immune-mediated inflammation. Sema3A is a membrane associated molecule on T regulatory cells and on B regulatory cells. Being transiently ligated to the cell surface of these cells it is suggested to be a useful marker for evaluating their functional status. In earlier studies, we found that reduced sema3A concentration in the serum of asthma patients as well as reduced expression by Treg cells correlates with asthma disease severity. Stimulation of Treg cells with recombinant sema3A induced a significant increase in FoxP3 and IL-10 expression. To find out if sema3A can be of benefit to asthma patients, we evaluated the effect of sema3A injection in a mouse model of asthma. BALB\c-mice were sensitized using ovalbumin (OVA) + adjuvant for 15 days followed by OVA aerosol inhalation over five consecutive days. Four hours following air ways sensitization on each of the above days- 15 of these mice were injected intraperitoneally with 50 μg per mouse of recombinant human sema3A-FR and the remaining 15 mice were injected with a similarly purified vehicle. Five days later the mice were sacrificed, broncheo-alveolar lavage (BAL) was collected and formalin-fixed lung biopsies taken and analyzed. In sema3A treated mice, only 20% of the bronchioles and arterioles were infiltrated by inflammatory cells as compared to 90% in the control group (p = 0.0079). In addition, eosinophil infiltration was also significantly increased in the control group as compared with the sema3A treated mice. In sema3A treated mice we noticed only a small number of mononuclear and neutrophil cells in the BAL while in the control mice, the BAL was enriched with mononuclear and neutrophil cells. Finally, in the control mice, angiogenesis was significantly increased in comparison with sema3A treated mice as evidenced by the reduced concentration of microvessels in the lungs of sema3A treated mice. To conclude, we find that in this asthma model, sema3A functions as a potent suppressor of asthma related inflammation that has the potential to be further developed as a new therapeutic for the treatment of asthma

Semaphorins in Angiogenesis and Tumor Progression

The semaphorins were initially described as axon guidance factors, but have recently been implicated in a variety of physiological and developmental functions, including regulation of immune response, angiogenesis, and migration of neural crest cells. The semaphorin family contains more than 30 genes divided into seven subfamilies, all of which are characterized by the presence of a sema domain. The semaphorins transduce their signals by binding to one of the nine receptors belonging to the plexin family, or, in the case of the class 3 semaphorins, by binding to one of the two neuropilin receptors. Additional receptors, which form complexes with these primary semaphorin receptors, are also frequently involved in semaphorin signaling. Recent evidence suggests that some semaphorins can act as antiangiogenic and/or antitumorigenic agents whereas other semaphorins promote tumor progression and/or angiogenesis. Furthermore, loss of endogenous inhibitory semaphorin expression or function on one hand, and overexpression of protumorigenic semaphorins on the other hand, is associated with the progression of some tumor types

Plexin-A2 enables the proliferation and the development of tumors from glioblastoma derived cells

Abstract The semaphorin guidance factors receptor plexin-A2 transduces sema6A and sema6B signals and may mediate, along with plexin-A4, the anti-angiogenic effects of sema6A. When associated with neuropilins plexin-A2 also transduces the anti-angiogenic signals of sema3B. Here we show that inhibition of plexin-A2 expression in glioblastoma derived cells that express wild type p53 such as U87MG and A172 cells, or in primary human endothelial cells, strongly inhibits cell proliferation. Inhibition of plexin-A2 expression in U87MG cells also results in strong inhibition of their tumor forming ability. Knock-out of the plexin-A2 gene in U87MG cells using CRISPR/Cas9 inhibits cell proliferation which is rescued following plexin-A2 re-expression, or expression of a truncated plexin-A2 lacking its extracellular domain. Inhibition of plexin-A2 expression results in cell cycle arrest at the G2/M stage, and is accompanied by changes in cytoskeletal organization, cell flattening, and enhanced expression of senescence associated β-galactosidase. It is also associated with reduced AKT phosphorylation and enhanced phosphorylation of p38MAPK. We find that the pro-proliferative effects of plexin-A2 are mediated by FARP2 and FYN and by the GTPase activating (GAP) domain located in the intracellular domain of plexin-A2. Point mutations in these locations inhibit the rescue of cell proliferation upon re-expression of the mutated intracellular domain in the knock-out cells. In contrast re-expression of a plexin-A2 cDNA containing a point mutation in the semaphorin binding domain failed to inhibit the rescue. Our results suggest that plexin-A2 may represent a novel target for the development of anti-tumorigenic therapeutics

Semaphorin-3D and semaphorin-3E inhibit the development of tumors from glioblastoma cells implanted in the cortex of the brain.

Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients

Semaphorin3A: A Potential Therapeutic Tool for Lupus Nephritis

BackgroundThe immune regulatory properties of semaphorin3A (sema3A) (both innate and adaptive) are well established in many in vitro studies. The injection of sema3A into a mice model of rheumatoid arthritis was proven to be highly beneficial, both in attenuating clinical symptoms and in decreasing inflammatory mechanisms.ObjectivesThis study was designed in order to assess the possible therapeutic benefits of sema3A following its injection into female NZB/W mice.MethodsForty-eight NZB/W mice were recruited for this study. Thirty mice were treated as a “prevention group” and 18 were used as a “treatment group.” Eight-week-old mice were acclimated and then divided into the two abovementioned groups.ResultsThe injection of sema3A into young mice (at week 12) before the onset of disease (the prevention group) delayed the appearance of proteinuria. Here, the median time to severe proteinuria was 110 days, 95% CI: 88–131. However, in mice in which the empty vector was injected, the median time to severe proteinuria was 63 days, 95% CI: 0–139. sema3A treatment, significantly reduced renal damage, namely, it prevented the deposition of immune complexes in the glomeruli. When sema3A was injected at the onset of proteinuria (the treatment group), aiming to treat rather than to prevent disease in these mice, survival was increased and the deterioration of proteinuria was delayed.ConclusionSemaphorin3A is highly beneficial in reducing lupus nephritis in NZB/W mice. It delays the appearance and deterioration of proteinuria, and increases the survival rates in these mice. The regulatory mechanisms of sema3A involve both innate and adaptive immune responses. Further studies will establish the idea of applying sema3A in the treatment of lupus nephritis

Favorable outcome of empagliflozin treatment in two pediatric glycogen storage disease type 1b patients.

Glycogen storage disease type 1b (GSD1b) is an ultra-rare autosomal recessive disorder, caused by mutations in gene. Affected patients present with episodes of fasting hypoglycemia and lactic acidosis, hepatomegaly, growth retardation, hyperlipidemia and renal impairment. In addition, patients present neutropenia, neutrophil dysfunction and oral, and skin infections as well as a significant predisposition to develop inflammatory bowel disease (IBD). Low neutrophil counts and function is related to the toxic accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P). Recently, several reports have shown that off-label treatment with empagliflozin (EMPA), an inhibitor of the renal glucose transporter SGLT2, decreased blood 1,5-anhydroglucitol (1,5-AG), and neutrophil 1,5-AG6P, thus resulting in a new therapeutic option for neutropenia and neutrophil dysfunction in patients. Off-label treatment with EMPA was established in two GSD1b patients after signed informed consent. The patients were followed clinically. We monitored neutrophil counts and function, 1,5-AG levels in plasma and its renal clearance before and during EMPA treatment. A 17 year-old girl who had long standing oral ulcers and developed IBD, requiring systemic steroid and regular granulocyte colony-stimulating factor (GCSF) therapy and an 8 year-old boy who had steady non healing oral lesions were treated with empagliflozin during 18-24 months. Treatment led to increase of neutrophil counts and function with substantial clinical improvement. This included remission of IBD in the first patient which allowed to discontinue both GCSF and steroid therapy and resolution of oral lesions in both patients. The concentration of 1,5-AG in blood was greatly decreased within two weeks of treatment and remained stable thereafter. Repurposing of empagliflozin to treat neutropenia in two GSD1b patients was safe and resulted in the urinary excretion of 1,5-AG, the normalization of neutrophil function, and a remarkable improvement of neutropenia-related clinical traits. We showed for the first time that empagliflozin increases concomitantly the renal clearance of both 1,5-anhydroglucitol and glucose in GSD1b patients

The effect of class-3 semaphorins on the development of subcutaneous tumors derived from u87 mg glioblastoma cells:

<p>U87MG cells infected with control lentivirus (control) or infected with lentiviruses directing expression of the designated semaphorins (Sema3X) were implanted subcutaneously in balb\c nu/nu mice as described. (A, C) The average volume of the developing tumors was measured as described. (B, D, E) The average weight of the tumors was determined at the end of the experiment as described.</p

The effect of class-3 semaphorins on the development of tumors from U87MG glioblastoma cells implanted in the brain cortex:

<p>(<b>A</b>) U87MG cells infected with control lentivirus or infected with lentiviruses directing expression of six different semaphorins (Sema3x) were implanted in the brain cortex of CD1 nu/nu mice (8 mice/group) as described. Tumor growth was followed in <i>vivo</i> using bioluminescence imaging following administration of D-luciferin as described. Shown are representative pictures of pseudo colored overlay images of the bioluminescence signal intensity superimposed on mice. The pictures were taken 22 days after the implantation of the tumor cells. (<b>B</b>) Quantification of luminescence in mice in which control or semaphorin expressing tumor cells were implanted as described above. Luminescence was measured 22 days after the implantation of the cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042912#s4" target="_blank">materials and methods</a>. Each bar represents the average luminescence from 8 different mice. (<b>C</b>) Brains from mice injected with U87MG cells infected with empty control lentiviruses (Control), or lentiviruses directing expression of sema3D or sema3E were sliced transversely and the slices were photographed. A large brain tumor is seen in the control (yellow arrows) whereas no tumors can be detected in brains in which sema3D or sema3E expressing U87MG cells were implanted. (<b>D</b>) Tumor sections from the brain cortex of mice implanted with U87MG cells infected with empty control lentiviruses (Control), or lentiviruses directing expression of sema3G (sema3G) were sectioned perpendicularly at the injection site, fixed in formalin, and embedded in paraffin. Paraffin sections (4 µm) were stained with anti-vWF using a fluorescent microscope as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042912#s4" target="_blank">materials and methods</a>. Microscopic fields were photographed and the vessel area per microscopic field was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042912#s4" target="_blank">materials and methods</a>.</p

The effect of the expression of class-3 semaphorins on the microvascular density of subcutaneous tumors derived from U87MG glioblastoma cells:

<p>Tumors excised from mice injected subcutaneously with U87MG cells infected with empty control lentiviruses (control), or lentiviruses directing expression of the designated class-3 semaphorin were embedded in OCT and frozen as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042912#s4" target="_blank">materials and methods</a>. Frozen sections (20 µm) were stained with anti CD-31 antibody (red) and nuclei were stained with DAPI (blue), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042912#s4" target="_blank">materials and methods</a>. (<b>A</b>) Representative pictures (20×) of microscopic fields taken from tumor areas in which the density of blood vessels was highest. Pictures were taken using a fluorescent microscope. (<b>B</b>) The area of CD-31 stained blood vessels in fields of equal area was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042912#s4" target="_blank">materials and methods</a>.</p