Studies On The Role Of Axonal Guidance Molecule Sema4D In Cardiovascular And Obesity-related Diseases

Axonal guidance molecule Sema4D/CD100 is a member of the Semaphorin family that has been shown to be involved in axonal guidance, angiogenesis, bone resorption and the immune system. Sema4 D is a 150 kd transmembrane molecule highly expressed on platelets and immune cells. So far, studies on Sema4 D has been focused on its biological effects. Recent studies in our lab demonstrated that Sema4 D is involved in atherosclerosis, thrombosis formation and ischemia myocardial infarction. In this project we will study the potential role of Sema4 D in other cardiovascular diseases such as heart failure as well as obesity related diseases.Objectives:1 To study soluble Sema4 D levels in heart failure patients and its cellular origin.Heart failure is the common path of many serious heart diseases which have similar mortality to that of cancer. The treatment for heart failure is very limited right now and new diagnosis methods are also needed. Immune reactions play important parts in the process of heart failure and some inflammatory factors such as TNF could be use as biomarker for heart failure. Sema4 D is expressed on T cells and platelets and could be cleaved during cell activation, which will release a functional soluble fragment to blood. We hypothesize that Sema4 D participate in the pathogenesis of heart failure and could be used as a biomarker for heart failure. To verify this hypothesis, we used clinical specimen to(1) detect soluble Sema4 D levels in heart failure patients;(2) study the correlation between expression of Sema4 D and severity of heart failure;(3) and analyze the cellular origin of Sema4 D in heart failure patients.2 To study the role and mechanism of Sema4 D in obesity and hepatic steatosis.Obesity is and important risk factor for type 2 diabetes and many other cardiovascular diseases, while chronic inflammation in adipose tissue is a major link between obesity and obesity related diseases. We hypothesize that Sema4 D accelerates adipose inflammation based on its role in T cell activation; meantime, Sema4 D could also influence adipogenesis directly through its receptor Plexin B1, because Rho kinase, the downstream of Plexin B1 is closely involved in adipogenesis. To verify our hypothesis, We used genetic engineering mice and mouse model of high fat diet induced obesity and hepatic steatosis to(1) study the role of Sema4 D and its receptor Plexin B1 in diet induced obesity and hepatic steatosis;(2) and possible molecular mechanism.Methods:1 Establishment of an ELISA method and measurement of Soluble Sema4 D levels in healthy donors and heart failure patients.(1) Establishment of an ELISA method to detect soluble Sema4 D in human plasma.His-tagged soluble Sema4 D is purified with metal affinity and used as a standard in ELISA. The purified Sema4 D is used for the preparation of 10 monoclonal antibodies and two with different epitopes are used as coating antibody and detecting antibody respectively. The detecting antibody is conjugated with HRP. a proper standard curve was plotted.(2) Measurement of Soluble Sema4 D levels in healthy donors and heart failure patients.We collected peripheral blood from 126 healthy donors and soluble Sema4 D level is detected with the ELISA method we established. Analyze soluble Sema4 D levels in different genders and ages. Observe the effect of 17β estradiol on the expression of Sema4 D in Jurkat cells and platelets.We collected peripheral blood from 157 heart failure patients and characteristics of these patients such age, complications were analyzed. Soluble Sema4 D levels in heart failure patients were detected with ELISA, and the effects of age and sex were analyzed. Analyze the effect of DM and HP complications on Soluble Sema4 D levels in heart failure patients.2 Expression of Sema4 D on the surface of T cells, B cells and platelets(1) PBMC from HF and HD were purified for FACS. CD3 and CD19 antibodies were double stained with Sema4 D antibodies to detect expression of Sema4 D on the surface of T cells and B cells.(2) PBMC from HF and HD were purified for FACS. CD4 and CD8 antibodies were double stained with Sema4 D antibodies to detect expression of Sema4 D on the surface of CD4 and CD8 T positve cells.(3) Sema4 D on the surface of platelets were detected by double staining with Sema4 D and CD41 antibodies to detect expression of Sema4 D on the surface of platelets.3 Role of Sema4 D in HFD induced obesity and hepatic steatosis.(1) We collected peripheral plasma from healthy donor and type 2 diabetes patients and measureed soluble Sema4 D levels with ELISA. We also test the expression of Sema4 D and its receptors Plexin B1, Plexin B2 and CD72 with real time PCR in adipose tissue of mice fed chow diet and HFD.(2) WT and Sema4D-/- mice fed HFD for 8-10 weeks will be weighed every 5-7 days and the size and weight of adipose tissue will be measured. We will also test the blood lipids and glucose tolerance of WT and Sema4D-/- mouse fed on HFD.(3) Livers of WT and Sema4D-/- mice on HFD were weighed. OCT embedded liver were stained with oil red and paraffin embedded tissues were stained with HE to observe hepatic steatosis.(4) In order to test the immune cell infiltration, Genital fat is digested with collagenase I and centrifuged. Antibodies for detecting CD45+ leucocytes、CD4+T cells、CD8+T cells、F4/80+ macrophages、CD11+ M1 macrophages were added to the cells and detected by FACS.4 Role of Sema4 D in adipogenesis.Adipose derived mesenchymal stem cells(ADSC) were purified and verified by FACS. Then plasmids with or without the extracellular fragment of Sema4 D are transfected to ADSC and induced for adipogenesis. After 7 days, the cells are stained with oil red.5 Role of Plexin B1 in HFD induced obesity and hepatic steatosis.Adipose tissue from WT and Plexin B1-/- mouse fed HFD will be measured. Livers of WT and Plexin B1-/- on HFD will be stained with oil red to test hepatic steatosis.Results:1 Soluble Sema4 D levels increase in plasma of heart failure patients.(1) Establishment of an ELISA method to detect soluble Sema4 D in human plasma.His-tagged soluble Sema4 D was purified with metal affinity and used as a standard in ELISA. The purified Sema4 D was used for the preparation of 10 monoclonal antibodies and two with different epitopes were used as coating antibody and detecting antibody respectively. The detecting antibody is conjugated with HRP. a proper standard curve(R2=0.996) were ploted.(2) Soluble Sema4 D levels in heart failure patients, especially those complicated with diabetes increase significantly.We collected peripheral blood from 126 healthy donors and soluble Sema4 D levels were detected with ELISA method we established. Plasma Sema4 D levels are higher in men than that in women(5.15 ± 3.30 ng/m L, n=63, vs. 4.19 ± 2.39 ng/m L, n=63, P<0.05), While there is no difference of Sema4 D in different age groups. 17β estradiol inhibits the expression of Sema4 D on the surface of Jurkat cells and collagen induced Sema4 D shedding in Platelets.In HF patients, plasma Sema4 D levels were significantly higher than that in healthy controls(8.94 ± 5.89 ng/m L, n=157 vs. 4.67 ± 2.99 ng/m L, n=126, P<0.0001).There is no difference of Sema4 D between male and female HF patients(P=0.19). Plasma Sema4 D levels are significantly higher in heart failure patients with diabetes mellitus(DM)(10.45 ± 5.76 ng/m L, n=40, P<0.05). HP has no effect on Soluble Sema4 D levels in heart failure patients.2 Expression of Sema4 D on the surface of T cells, B cells and platelets.(1) There is a higher percentage of Sema4 Dhigh CD3+T cells(P<0.01), including CD4+T cells(P<0.001) and CD8+ T cells(P<0.05) in samples from HF patients that that in healthy donors.(2) There is no difference of Sema4 D expression in B cells and platelets between HF patients and healthy donors.3 Sema4 D knock out accelerates diet induced obesity and hepatic steatosis.(1) Sema4 D increases in the plasma of type 2 diabetes patients and adipose tissue of mice fed HFD.ELISA results show that soluble Sema4 D level is significantly higher in type 2 diabetes patients than that in healthy controls(P<0.01). Expression of Sema4 D in adipose tissue of mouse significa ntly increases after HFD(P<0.05), While its receptors Plexin B1(P<0.05) and Plexin B2(P<0.05) decrease significantly. HFD has no effect on the expression of CD72(P=0.64).(2) Sema4 D knock out accelerates diet induced obesityBoth male and female Sema4D-/- mice weight more than WT mice on HFD, and the size and weight of genital fat(P<0.01) and perirenal fat(P<0.01) increase significantly. Knock out of Sema4 D also decreases glucose tolerance significantly after HFD( P<0.05). TG, TC and LDL-C increase significantly in Sema4D-/- mice while HDL-C does not change significantly.(3) Sema4 D knock out accelerates diet induced hepatic steatosis.Livers(P<0.0001) and spleens(P<0.01) from Sema4D-/- mice weigh more than that from WT mice on HFD. HE staining and oil red staining showed that Sema4D-/-mice develop much more serious hepatic steatosis.(4) Sema4 D knock out increases immune cell infiltration in adipose tissue.In order to test the immune cell infiltration, Genital fat is digested with collagenase I and centrifuged. FACS is used for detecting the number and percentage of immune cells. We found that the number of CD45+ leucocytes( P=0.0009)、CD4+T cells(P=0.02), CD8+T cells(P=0.002) and F4/80+ macrophages(P=0.02) increase significantly in adipose tissue of Sema4D-/- mice. The percentage of CD45+ leucocytes in all cells and the percentage of CD4+T cells(P=0.29), CD8+T cells(P=0.09) of CD45+ leucocytes does not change, while the percentage of F4/80+ macrophage(P=0.0005) and CD11+M1 macrophage of CD45+ leucocytes increase significantly.4 Sema4 D inhibit in vitro adipogenesisAdipose derived mesenchymal stem cells(ADSC) were purified and verified by FACS. CD45 is not detected while most cells are CD90 and CD105 positive. To test the role of Sema4 D on adipogenesis, plasmids with or without the extracellular fragment of Sema4 D were transfected to ADSCs and induction medium added. After 7 days, the cells were stained with oil red, and significantly much less lipid is detected in cells transfected with Sema4 D.5 Plexin B1-/- knock out accelerates diet induced obesity and hepatic steatosis.RT-PCR results show that Plexin B1 is expressed in adipose tissue and liver, with a higher expression in liver. We also found a significantly decreased expression of Plexin B1 in liver of mice fed HFD compared to mice fed chow diet.WT and Plexin B1-/- mice were fed on HFD and adipose tissue from WT and Plexin B1-/- mouse show that Plexin B1-/- knock out accelerates diet induced obesity significantly. Oil red staining shows that hepatic steatosis is more severe in Plexin B1-/-mice. Expression of PPAR?1 and PPAR?2 in livers of Plexin B1-/- mice increases significantly.Conclusions:1. Sema4 D level is higher in HF patients than that in healthy donors, with the highest levels being in HF patients with diabetes mellitus(DM).2. There was a higher percentage of Sema4 Dhigh CD3+, including CD4+ T and CD8+ T-cells in samples from HF patients, but no changes in Sema4 D expression levels in B cells and platelets.3. Sema4 D knock out accelerates HFD induced obesity and steatosis.4. Role of Sema4 D in HFD is mediated by its receptor Plexin B1.5. Sema4 D inhibits adipogenesis in vitro.