The Study Of CD248 Expression On The Microvessels Of Glioma And Its Role In The Pro-Angiogenesis Mediated By Pericytes

Tumor angiogenesis is important and critical for tumor growth, progression and metastasis. Therefore, anti-angiogenesis will be a suitable tumor therapies. Because malignant glioma is located in skull characterized by invasive growth, it is very difficult to be eradicated by conventional therapies. Since it resists to chemotherapy and radiotherapy, anti-angiogenesis will be an alternative choice or an adjunctive therapy. However, drugs such as Bevacizumab and Sunitinib, which target at endothelial cells do not always decrease tumor size. Thus, other angiogenesis-related cells such as pericytes may be another target for anti-angiogenesis therapies.The interaction of pericytes and endothelial cells can stabilize newly formed endothelial tubes and regulate blood flow and permeability, endothelial proliferation, differentiation, migration and survives. Pericytes can be differentiated into different mesenchymal cells, for example, during enlargement of vessels, pericytes can be differentiated into vSMC. In some cases, pericytes can be differentiated into fibroblast, adiocyte, and others.The markers of pericytes are different among different species in different organs and different development stages. At present, NG2, RGS5, PDGFRβ, desmin,α-SMA, 3G5ganglioside, Slug, XlacZ4 and aminopeptidase N are considered as the markers for pericytes. Recent studies have reported that CD248 (Endosialin/TEM1) is overexpressed in the pericytes of tumor vasculature, but is not expressed in the pericytes of normal tissue. CD248 is a single transmembrane glycoprotein, containing a c-type Lectin domain, a Sushi domain, three EGF like domain, a mucin-like region, a transmembrane segment and a short cytoplasmic tail. Four ligands are found for CD248: they are fibonectin, type I collagen, type IV collagen and Mac-2BP/90K.John MacFadyen et al. found that CD248 was expressed in the fibroblasts and the vasculature of lung, kidney, spleen, and liver, which was down regulated during development. In adult, CD248 is mainly expressed in uterus and glomerulus. CD248 is also expressed in endothelial precursor cells. CD248 expression is found in many tumors, such as sarcoma, glioma, breast cancer, skin carcinoma, corolectal carcinoma and neuroblastoma. CD248 expression in tumor vasculature indicates that CD248 is correlated with angiogenesis.In this study, fresh glioma frozen sections were used to perform double-staining with a rabbit anti-human CD248 antibody and mouse anti-human CD31 antibody or rabbit anti-human CD248 antibody and mouse anti-humanα-SMA antibody. My results indicate that CD248 is expressed on the pericytes of microvessels in glioma. In order to study the role of CD248 in angiogenesis, I isolated pericytes from glioma using immunomagnetic beads. Isolated pericytes were verified by immunohistochemistry, immunoflourescence and RT-PCR. Then purified pericytes were infected with CD248ShRNA lentivirus, and examined by immunoflourescence, RT-PCR and western blot, and their migration and proliferation capabilities were also examined. In order to examine the role of CD248 in the pericytes in vivo, U87 cells were mixed with the pericytes that were pre-infected with CD248ShRnA lentivirus or mock shRNA lentivirus and injected into nude mice. Overall results were:1. CD248 was specially expressed in the pericytes of glioma and its expression was correlated with glioma grade, but not gender or age. In order to verify CD248 was expressed in pericytes, 8 primary glioma frozen sections were double-stained with rabbit anti-human CD248 antibody and mouse anti-human CD31 antibody or rabbit anti-human CD248 antibody and mouse anti-humanα-SMA antibody. The data demonstrated that CD248 and CD31 were expressed on different cells, while CD248 andα-SMA were expressed on the same cells. CD248-positive cells were found around CD31-postive cells, and they were directly contacted with each. Thus, CD248-positive cells are pericytes, and CD248 can be a specific marker for detection of the tumor pericytes. One hundred and eighteen patients were included in the extensive examination of CD248 expression in the gliomas. Immunohistochemistry (IHC) showed that CD248 was negative in total 6 cases of gradeⅠsamples, but was detected in 15 cases in total 39 cases of gradeⅡ(38.46%), 26 cases in total 37 cases of gradeⅢ(70.27%), and 27 cases in total 36 cases of gradeⅣ(75%) samples. Statistical analysis demonstrated that there was no difference in gradeⅠand gradeⅡ(p > 0.05). Significant statistical difference was found when gradeⅢand gradeⅣwere compared to gradeⅠand gradeⅡ(p < 0.01), and there was no difference in gradeⅢand gradeⅣ(p > 0.05).2. I successfully isolated pericytes from gliomas. Before isolation, rabbit anti-human CD248 was incubated with suspension cells and the mixtures were incubated with immunomagnetic beads, the CD248-positive cells were isolated through magnetic field. IHC indicated that these cells were negative for both CD31 and GFAP, while positive for CD248, NG2 and PDGFR-β. Because both NG2 and PDGFR-βare well known markers for pericytes, these cells are pericytes. Flow cytometry analysis revealed that the purity of isolated pericytes was 91.0±5.6%. Examination of CD248 mRNA expression by RT-PCR demonstrated that the size of PCR product was about 230bp which matched the size as expected. Furthermore, the isolated pericytes could form tubular structures in ECM gel or 3D collagen scaffold.3. Downregulation of CD248 expression in the pericytes could inhibit their migration and proliferation. CD248 was decreased in the pericytes that were infected by CD248shRNA lentivirus as examined by immunofluoresence staining. RT-PCR showed that CD248 mRNA in the infected pericytes was deceased by 50% and western blot analysis also indicated that CD248 was significantly decreased after infection.4. We confirmed that CD248 participated in tumor angiogenesis. CD248shRNA infected pericytes or mock shRNA infected pericytes were mixed with U87 cells and subcutaneously injected into nude mice, respectively. After examination of tumor growth, we found that tumor volume of CD248shRNA infected pericytes-derived xenograft was significantly decreased as compared to mock-infected one (p = 0.022), and microvessels density was also decreased (p = 0.021), whereas there was no different in the microvessel diameter (p = 0.769), indicating that pericytes promoted angiogenesis via CD248. The possible underlying mechanism is that the ligands in the ECM (extral cellular martrix), including fibronectin, type I collagen, and typeⅣcollagen, contact with CD248 and enable pericytes to proliferate and secret MMP9 to induce CEM degeneration, resulting migration of pericytes and endothelial cells to form new microvessels.5. It has been known that CD248 stimulated by Mac-2BP/90K can upregulate the expression of TGFRβI, TGFRβII, PDGFRβ, and EDG-1, we speculate that the expression of CD248 in the pericytes is related to the pericyte recruitment. When CD248 is expressed in pericytes, tumor vessels can recruit pericytes to protect nascent endothelial tubes from death and increase angiogenesis.Conclusion: Overall, the expression of CD248 is correlated to glioma grade. CD248 can promote tumor angiogenesis. And downregulation of CD248 expression can inhibit pericyte migration and proliferation, so that the number of microvessels is decreased. Therefore, CD248 can be a novel anti-angiogensis target.