Thrombospondin-1 Signaling Through CD47 Induces Senescence In Endothelial Cells

Nowadays, longer life expectancy and falling birthrate are swelling the number of seniors. Aging population has become an increasingly striking social problem and put pressure on the country’s health and social systems. The outstanding health problem is the increasing prevalence rate of cardiovascular disease. Epidemic surveying has proved that aging is an important risk factor of cardiovascular disease for an impaired endothelial function exists in vascular aging. Aging vascular exhibits a decrease in compliance and self-healing, which ultimately resulting in diseases such as atherosclerosis and coronary heart disease(CHD). The root cause of these health problems is the senescence of vascular endothelial cells.Cellular senescence can occur in vivo or in vitro in response to various stressors,triggering alterations in the expression of a number of genes and leading to the suppression of cell proliferation and dysfunction. The main reasons for endothelial cell senescence are the replicative senescence and the peroxides in blood, which causing the formation of atherosclerosis plaque and thrombus. Therefore, delaying endothelial cell aging can be of great importance in reducing cardiovascular diseases.Thrombospondin-1(TSP1) is an extracellular matrix glycoprotein which was first found in platelets as an inactive precursor. In addition to release by platelets,TSP-1 can also be secreted by macrophages, monocytes, fibroblasts, vascular smooth muscle cells(SMC) and endothelial cells(EC) as extracellular matrix(ECM). TSP1 has multiple domains which can bind with various receptors such as CD36, CD47 and low-density lipoprotein receptor related protein 1(LRP1). Studies have shown that endogenous levels of TSP1 in vascular tissues and circulating in plasma serveimportant physiological and pathophysiological functions. TSP1, acting via CD47,inhibits the NO stimulation of s GC in both ECs and VSMCs, leading to the inhibition of vasodialation. TSP1 inhibits VEGF signaling resulting in a defect in angiogenesis.TSP1 can also upregulate TGF-? to promote the formation of atherosclerosis plaque.Studies have found the upregulation of TSP1 in aged tissues and this could be the reason in decreased blood perfusion in aged tissues.CD47, also named as Integrin-associated protein(IAP), is widely expressed on tissues and cells, with prominent expression on leukocytes, platelets, and erythrocytes.CD47 has multiple biological functions. In immune system, CD47 is important in regulating leukocyte adhesion, transmigration and immune cell apoptosis. CD47 inhibition of phagocytosis involves its binding to SIRPα on the phagocyte and delivers a “don’t eat me” signal. Although CD47 promotes survival function of cells by preventing phagocytic death, it also has a distinct cell-autonomous role in cell survival with a more negative function. Primary cells from CD47-/-mice, such as vascular endothelial cells and smooth muscle cells, proliferate more robustly than WT cells, but the mechanism is not well studied. A recent study reported that loss of CD47 enables self-renewal by regulating c-Myc expression in a TSP1 dependent way. In our study, we noticed that CD47-/-ECs are more proliferative and resistant to aging compared with WT ECs. Furthermore, CD47-deficient cells showed markedly improved cell cycle progression and this could explain the anti-aging phenomenon in CD47-deficient cells.First, we isolated ECs from the brain of 8-weeks-old WT or CD47-/- mice as our test subjects. CCK-8, Brd U and CFSE assay was performed to compare the proliferation ability, and tube formation assay was used to evaluate the ECs function in WT and CD47-/- ECs. Senescence-associated β-galactosidase(SA-β-gal) activity was performed using ECs after continuous passage culture and SA-β-gal positive cells(i.e., senescent cells) were identified as green-stained cells under standard light microscopy. We then detected the TSP1 level in WT and CD47-/- ECs to identify the ecpression changes in senescent cells. In further investigation, we compared them RNA expression levels of key cell cycle inhibitors(CKIs), p53, p21 and p16, which are known to be upregulated in senescent cells, cyclin-dependent kinase(CDK)4,CDK6, Cyclin D1, as well as E2F-mediated S-phase gene transcription, such as CDK1, CDK2, Cyclin A, Cyclin E1, Ki67, which are found to be downregulated in senescent cells. In the end, TSP1 or TSP1 antibody was added to WT and CD47-/- ECs separately to identify the function of TSP1-CD47 pathway in cellular senescence. The results were present below.1. ECs were prepared from the brain of 8-weeks-old WT or CD47-/- mice,cultured in vitro for 4 days, and the numbers of viable cells were assessed daily using the cell count kit-8. Although the numbers of viable cells were comparable between WT and CD47-/- EC cultures until day 2, the latter thereafter yielded significantly more viable cells. Moreover, CD47-/-ECs showed significantly increased Brd U incorporation and CFSE dilution compared to WT ECs. Together, these results demonstrated an increased potential of CD47-/- ECs to proliferate and expand compared to WT ECs.2. In vitro angiogenic potential of WT vs. CD47-/- ECs(at passage 2; P-2) was assessed by EC tube formation assay. CD47-/- ECs showed a significant increase in endothelial tube length and branch point numbers compared to WT ECs. We also compared the in vivo angiogenic potential of WT and CD47-/- ECs using Matrigel plug assay, in which EC-containing Matrigels were subcutaneously implanted into WT or CD47-/-mice, and the plugs were removed two weeks later for analysis.Matrigel plugs with CD47-/- ECs, harvested from both WT and CD47-/- mice, showed significantly increased microvessel density compared to Matrigel plugs with WT ECs, demonstrating that CD47 deficiency can promote angiogenesis in vivo. The observed neovascularization was attributed to the injected ECs, as microvessel formation was not detected in Matrigel plugs without ECs in either WT or CD47-/-mice. Interestingly, both CD47-/-and WT ECs showed increased neovascularization potential, as shown by higher microvessel density, when injected into CD47-/- mice compared to WT mice, indicating that the environment of CD47-/- mice may enhance angiogenesis. Together, these results indicate that CD47 deficiency in both ECs and the microenvironment may promote angiogenesis.3. Both WT and CD47-/-ECs showed a clear increase in SA-?-gal+ cells overtime, however, the increase was markedly less in the latter group and the percentages of SA-?-gal+ cells in CD47-/- ECs at P-4 and P-6 were significantly lower compared to WT ECs at the same passages. One of the most important features of senescence is cell cycle arrest, which is an indispensable marker for identifying the senescence of cells in vivo and in vitro. Cell cycle distribution was analyzed by measuring cell DNA content. The proportion of cells in G1 phase increased with passage, as reflected by the decreased ratio of S phases in both WT and CD47-/-ECs. However, the percentages of cells in S phase were significant greater in CD47-/- ECs compared to those in WT ECs.We further analyzed m RNA expression levels of the key cell cycle regulators.We first compared the m RNA expression levels of key cell cycle inhibitors(CKIs),p53, p21 and p16, which are known to be upregulated in senescent cells. Consistent with the cell cycle analysis results, the expression of cell cycle inhibitors p53, p21,and p16 was significantly reduced in CD47-/-WT ECs compared to WT ECs during the course of replicative senescence. Accordingly, CD47-/- ECs expressed a significantly higher levels of cell cycle promoters including cyclin-dependent kinase(CDK)4, CDK6, Cyclin D1. Cyclin D1 and CDK4/6 co-regulate cell cycle progression and past the restriction point by phosphorylating and thus inhibiting RB,which allows E2F-mediated S-phase gene transcription, such as CDK1, CDK2,Cyclin A, Cyclin E1, Ki67. We noticed the upregulation in E2F-mediated S-phase gene including CDK1, CDK2, Cyclin A1 and Ki67. Together, these results demonstrated a critical role for CD47 in the regulation of EC cell cycle progression and senescence.4. Because CD47 is an important receptor for TSP1-induced inhibition of angiogenesis, we measured the levels of TSP1 production in EC cultures. A significant increase in TSP1 production overtime was seen in WT, but not CD47-/-ECs. To further determine the role of TSP1-CD47 signaling in EC function, we compared the effect of exogenous TSP1 on the angiogenic potential of WT vs.CD47-/-ECs at P-2, in which neither of these ECs produced significant amount of TSP1. Addition of TSP1 significantly inhibited the angiogenic potential of WT, butnot CD47-/-, P-2 ECs. Addition of TSP1 also led to a significant increase in the ratio of SA-?-gal+ senescent cells in the cultures of WT, but not CD47-/- ECs(at P-2).Addition of neutralizing anti-TSP1 antibodies had no effect on WT P-2 EC senescence, likely due to the low level of TSP1, but mediated significant protection against senescence in WT P-4 ECs. However, anti-TSP1 had no detectable effect on either P-2 or P-4 CD47-/-ECs. Taken together, these results indicate that TSP1 signaling through CD47 not only inhibits angiogenesis, but also induces EC senescence.TSP1 also inhibits cell cycle progression in ECs. Addition of TSP1 to WT EC cultures led to a significant upregulation of cell cycle inhibitors p53, p21 and p16,and downregulation of cell cycle promoters CDK4, CDK6, and Cyclin D1.Interestingly, in CD47-/-EC cultures TSP1 did not significantly affect the expression of the most cell cycle regulators measured, there was a reduction in Ki67 expression.Although the observed effects of TSP1 on ECs was predominantly mediated by CD47, this result suggests that some of the effects might be CD47-independent.Above all, we draw a conclusion that CD47-TSP1 pathway promoted endothelial cell senescence possibly via regulating the key cell cycle inhibitors and cyclin-dependent kinase to manage the cell cycle progression. The expression of cell cycle inhibitors p53, p21, and p16 was significantly reduced in CD47-/- WT ECs compared to WT ECs during the course of replicative senescence. Accordingly,CD47-/-ECs expressed a significantly higher levels of cell cycle promoters including cyclin-dependent kinase(CDK)4, CDK6, Cyclin D1. Together, these results demonstrated a critical role for CD47 in the regulation of EC cell cycle progression and senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.