Analysis Of The Mechanism For CD47 Deficiency-Upregulated Tumor Angiogenesis

Angiogenesis plays an important role in tumor growth, metastasis and tumor pro-gression. The tumor microenvironment comprises numerous signaling molecules and pathways that influence the new blood vessel formation. Frequently, tumors overexpress proangiogenic factors, such as vascular endothelial growth factor, improvingangiogenetic activity.The inhibition of angiogenesis could be a potentapproach to cancertherapy.CD47 is a widely expressed 50 kD transmembrane receptor that hasextracellular N-terminal IgV domain, five transmembrane domains, a short C-terminal and belongs to the immunoglobulin superfamily. This receptor, also known as integrin-associated protein, mediates cell-to-cell communication by ligation to transmembrane signal-regulatory proteins SIRP-? and SIRP-? and interacts with integrins. CD47 is also implicated inextracellular matrix interactions via ligation with thrombospondins(TSPs). Binding of CD47 by recombinant C-terminal regions of TSP-1, TSP-1 peptides,or CD47 antibodies were also sufficient to inhibit NO-stimulatedphenotypicresponses and cGMP signaling in vascularcells. Furthermore, TSP-1 did not inhibit NO signaling inCD47-null vascular cells. Tumor cell proliferation is strongly sustained by continuous sprouting of newvessels. Recently, it has been reported that CD47 deficiency recalls stemness in endothelial cell through regulating c-myc and certain stem cell transcirption factors. In this study, we showed that CD47 deficiency increases tumor angiongenesis by promoting cell proliferation and decreases tumor necrosis. In further investigation, our data implicated that the loss of CD47 induces endothelial cell into an immortal-like state via down-regulating p53 and p21 in tumor vessel endothelial cell. Our results provide an opportunity for suppression of angiogenesis in tumor via regulation of CD47 expression and its downstream pathway.Recent studies indicate that, in addition to the canonical CD47-TSP-1 pathway, CD47 also affects endothelial cell function by regulating stemness of the endothelial cells, immortalizing adult endothelial cells and granting them the ability to differentiate.In our previous studies on the CD47-/- endothelial cells, we’ve obtained endothelial cells which can be passaged indefinitely with upregulated stemness markers, but the reproducibility was very bad. Further investigation on the function of CD47 indicated that loss of CD47 upregulated cell cycle-related factors in the endothelial cells, and delayed cell senescence. This result has also been verified in tumor models, in which tumors in CD47-/- mouse showed significantly higher angiogenesis and stronger resistance to oxidative stress.This study can be divided into two partsPart I: In vivo experimentsObjective: To clarify the role of CD47 in tumor angiogenesis, we constructed tumor model in mice to evaluate angiogenesis in wild-type and CD47-/- mice.Methods: RM1 tumor cells were revived and passaged, then injected subcutaneously at the flank. The mice were sacrificed on the 11 th day after injection and the tumors were isolated, which were sliced and stained with CD31 and CD144 antibody. Images were taken under a microscope and analyzed with Image pro-plus 6.0 software.Results: The tumors grew more vigorouslyin CD47-/- micewith significantly larger size compared to that in WT mice. Immunohistochemistry showed larger area of positive CD31 and CD144 staining in CD47-/- mice than in WT mice, suggesting more abundant blood vessels in CD47-/- mice than in WT mice.Conclusion: loss of CD47 promotes tumor angiogenesis, thereby promoting tumor growth.Part II: In vitro experimentsObjective: To analyzethe mechanism of CD47 deficiency promotes tumor angiogenesis in mice by molecular biological study.1. Isolation and purification in endothelial cellsMethods: endothelial cells were obtained from mouse tumor by magnetic bead sorting and APC sorting.Conclusion: Over 98% CD31+ cells were obtained.2. Effect of CD47 on senescence in endothelial cellsMethods: CD47-/- and WT endothelial cells were cultured for 2 d, 4 d and 6 d, followed by β-galactosidase staining to evaluate and compare senescence of the two.Conclusion: Theloss of CD47 has an anti-senescence effect.3. Effect of CD47 on tube formationin endothelial cellsMethods: The tube formation assay were performed for CD47-/- and WT endothelial cells, and morphology and length of the canaliculi were observed at 3h and 8h after induction under a light microscope.Conclusion: The loss of CD47 enhancestube formation.4. Effect of CD47 on cell cycle in endothelial cellsMethods: The endothelial cells were passaged to the 2nd, 3rd, 4th, and 5th generation(labeled P2, P3, P4, and P5), then cell cycle analysis was performed after PI staining to determine ratio of cells in G0/G1, S and G2 phases, and the results were compared between CD47-/- and WT endothelial cells.Conclusion: CD47 affects cell cycle progression in endothelial cells and CD47-/-cells showed more vigorous proliferation.5. The regulation of p53, p21 and c-myc by CD47Methods: The mRNA levels of p53, p21 and c-myc in CD47-/- and WT endothelial cells were analyzed by RT-PCR, and were compared between the two.Conclusion: CD47 affects expression of the cell cycle regulators. p53 and p21 weredownregulated in CD47-/- endothelial cells, promoting cell proliferation, while c-myc was upregulated, which enhances stemness and proliferation potential of the endothelial cells.