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PAYWALLED: CD9 induces cellular senescence and aggravates atherosclerotic plaque formation

ageing cardiovascular diseases

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#1 Engadin

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Posted 01 May 2020 - 07:06 PM


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P A Y W A L L E D   S O U R C E :   nature_Cell Death and Differentiation

 

 

 

 

 

Abstract
 
CD9, a 24 kDa tetraspanin membrane protein, is known to regulate cell adhesion and migration, cancer progression and metastasis, immune and allergic responses, and viral infection. CD9 is upregulated in senescent endothelial cells, neointima hyperplasia, and atherosclerotic plaques. However, its role in cellular senescence and atherosclerosis remains undefined. We investigated the potential mechanism for CD9-mediated cellular senescence and its role in atherosclerotic plaque formation. CD9 knockdown in senescent human umbilical vein endothelial cells significantly rescued senescence phenotypes, while CD9 upregulation in young cells accelerated senescence. CD9 regulated cellular senescence through a phosphatidylinositide 3 kinase-AKT-mTOR-p53 signal pathway.
 
CD9 expression increased in arterial tissues from humans and rats with age, and in atherosclerotic plaques in humans and mice. Anti-mouse CD9 antibody noticeably prevented the formation of atherosclerotic lesions in ApoE−/− mice and Ldlr−/− mice. Furthermore, CD9 ablation in ApoE−/− mice decreased atherosclerotic lesions in aorta and aortic sinus. These results suggest that CD9 plays critical roles in endothelial cell senescence and consequently the pathogenesis of atherosclerosis, implying that CD9 is a novel target for prevention and treatment of vascular aging and atherosclerosis.
 
 
 
Fig. 1.jpg
 
Figure 1. Knockdown of CD9 in senescent HUVECs rescues cellular senescence. Senescent HUVECs (PD >50) were transfected with CD9 or negative control siRNA, and then incubated for 6 days at 37°C. A. Actin staining (red) with rhodamin-conjugated phalloidin. Scale bar: 5 m. B. Ki67 immunostaining (red) and the percentages of Ki67 positive cells. The nuclei (blue) were stained with DAPI. Scale bar: 20 m. C. Flow cytometry of BrdU incorporation. Representative data are shown and values are the means ± SD of three independent experiments. Y, young cells; siCon, negative control siRNA; siCD9, CD9 siRNA; NT, not treated; *p < 0.05.
 
 
 
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Figure 2. Ectopic expression of CD9 in young HUVECs (PD<20) accelerates cellular senescence. Young cells were transduced with recombinant human CD9 or negative control adenovirus and incubated for 6 days at 37°C. A. CD9 immunostaining (red) in young cells transduced with control or recombinant CD9 adenovirus. Scale bar: 20 m. B. Actin staining (red) in young cells transduced with control or recombinant CD9 adenovirus. Scale bar: 20 m. C. Ki67 immunostaining (red) and the percentages of Ki67 positive cells in young cells transduced with control or recombinant CD9 adenovirus. Scale bar: 20 m. D. Flow cytometry of BrdU incorporation. E. H2AX foci (green) and the percentages of H2AX positive cells in the nuclei of control or CD9 adenovirus cells. Scale bar: 20 m. F. Western blotting of PARP-1/2 and caspase-3 in control or CD9 adenovirus cells. The nuclei (blue) were stained with DAPI. Scale bar: 20 m. Representative data are shown and values are the means ± SD of three independent experiments. Ad/Con, negative control recombinant adenovirus; Ad/CD9, recombinant human CD9 adenovirus; NT, not treated; MOI, multiplicity of infection; **p < 0.01.
 
 
 
Fig. 3.jpg
 
Figure 3. Effects of CD9 upregulation in cells transfected with siRNA or pretreated with rapamycin or LY294002. A. Relative levels of CD9 and p16 mRNAs in p53 or p16 siRNA cells. B. SA--gal staining in p53 or p16 siRNA cells. Scale bar: 30 m. C. Relative levels of CD9 and ATM mRNAs in ATM siRNA cells. D. SA--gal staining in ATM siRNA cells. Scale bar: 30 m. E. SA--gal staining in rapamycin or LY294002-pretreated cells. Scale bar: 30 m. F. SA--gal staining in PIK3CA or PIK3CB siRNA cells. Scale bar:  30 m. G. Proposed signal transduction pathway in CD9-mediated cellular senescence. Representative data are shown and values are the means ± SD of three independent experiments. Y, young cells; S, senescent cells; siCon, negative control siRNA; sip53, p53 siRNA; sip16, p16 siRNA; siATM, ATM siRNA; DMSO, dimethyl sulfoxide; LY, LY294002; Rap, rapamycin; siPIK3CA, PIK3CA siRNA; siPIK3CB, PIK3CB siRNA; Ad/Con, negative control recombinant adenovirus; Ad/CD9, recombinant human CD9 adenovirus; NT, not treated.
 
 
 
Fig. 4.jpg
 
Figure 4. Upregulation of CD9 in the atherosclerotic lesions and senescent cells. A. CD9 immunostaining (brown) in atherosclerotic lesions of Ldlr-/- mice. Scale bar: 200 m. B. The levels of CD9 protein and mRNA in young and senescent HUVECs measured by Western blotting and real-time PCR. C. The level of CD9 mRNA in adriamycin-treated cells examined by RT-PCR. (D-E) Rhodamine-conjugated CD9 antibody or isotype IgG and FITC-conjugated CRKRLDRNC peptide were injected intraventricularly into ApoE-/- male mice fed with a high fat diet and CD9 expression were analyzed in the atherosclerotic lesions and the normal regions of aortas. D. CD9 expression and colocalization of CD9 with IL4R in cryosection tissues of atherosclerotic lesions and normal regions of aortas of ApoE-/- mice. Arrows indicate the endothelial layer in atherosclerotic lesions. Scale bar: 30 m. E. CD9 expression and colocalization of CD9 with IL4R in the surface of atherosclerotic lesions and normal regions of aoric en face in ApoE-/- mice. Scale bar: 20 m. Ldlr-/-, low-density lipoprotein receptor deficient mouse; AP, atherosclerotic plaque; L, lumen; ApoE-/-, apolipoprotein E deficient mouse. Representative data are shown and values are the means ± SE (n=3). IL4R, interleukin 4 receptor; Y, young cells; S, senescent cells; Adr, adriamycin. **p<0.01.
 
 
 
Fig. 5.jpg
 
Figure 5. Effects of a CD9 blocking antibody and CD9 ablation on the formation of atherosclerotic lesions in ApoE-/- mice. (A-G) MS-1 cells and RAW264.7 cells were pretreated with 10 g/ml of a rat monoclonal antibody against mouse CD9 (rCD9), or 10 g/ml of rat IgG (rIgG) for 2 h and then treated with 10 g/ml LDL or oxLDL for 4 days. oxLDL-induced cellular senescence was measured. ApoE-/- mice fed a high fat diet were treated with 100 g of rCD9 or rIgG every 3.5 days for 15 weeks. Body weight and food intake were measured. A. SA--gal staining in MS-1 cells and RAW264.7 cells treated with oxLDL. Scale bar: 20 m. B. Oil-red O staining (red) for lipid accumulation in MS-1 cells and RAW264.7 cells treated with oxLDL. Scale bar: 20 m. C. Changes in body weights of ApoE-/- mice treated with CD9 antibody. D. Food intake in ApoE-/- mice treated with CD9 antibody. E. The levels of total cholesterol (T-chol), triglycerides (TG), HDL-cholesterol (HDL-chol) and LDL-cholesterol (LDL-chol) in serum from ApoE-/- mice treated with rCD9 or rIgG (each, n=5). F. The percentages of SAG positive area in fatty streaks, and atherosclerotic plaque and SAG positive area in aortic arches (each, n=3). G. Relative levels of pAKT, p53, p21, p16, CD9, and pS6K proteins in aortas with atherosclerotic lesions (each, n=4). (H) Ldlr-/- mice were fed with a high fat diet for 4 weeks. After withdrawal of a high fat diet, the mice were treated with 100 µg of rCD9 or rIgG every 3.5 days for 6 weeks, and sacrificed. H. Oil-red O staining (red), SAG staining (blue), and the percentages of SAG positive area in fatty streaks, and atherosclerotic plaque and SAG positive area in aortic arches of Ldlr-/- mice (each, n=3). Scale bar: 1 mm. (I-L) CD9+/+ApoE-/-, CD9+/-ApoE-/-, and CD9-/-ApoE-/- mice were fed with a high fat diet for 9-14 weeks and then sacrificed. I. Body weight changes in CD9+/+ApoE-/-, CD9+/-ApoE-/-, and CD9-/-ApoE-/- mice. Inset, Western blot analysis of CD9 in aortas of CD9+/+ApoE-/-, CD9+/-ApoE-/-, and CD9-/-ApoE-/- mice. J. Oil-red O staining (red) in aorta en face of female mice. Animal numbers are indicated in the parenthesis. Scale bar: 5 mm. K. Oil-red O staining (red) of atherosclerotic lesion in aortic sinuses of female mice. Scale bar: 1 mm. L. The levels of T-chol, TG, HDL-chol and LDL-chol in serum. M. CD9+/+ApoE-/-, CD9+/-ApoE-/-, and CD9-/-ApoE-/- mice were fed with a normal chow diet for 54 weeks and then sacrificed. The formation of atherosclerotic lesions in aortic en face was analyzed by Oil-red O staining. Scale bar: 5 mm. N. The quantification of colocalization of p16 and p53 with PECAM, a marker of endothelial cells in atherosclerotic lesions (each, n=3). O. The quantification of colocalization of p16 and p53 with F4/80, a marker of macrophages in atherosclerotic lesions (each, n=3). Representative data are shown and values are the means ± SE. NT, not treated; rCD9, a rat monoclonal antibody against mouse CD9; rIgG, rat IgG; HDL, high-density lipoprotein; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein. *p < 0.05 and **p < 0.01. 
 
 
 
 
 
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Edited by Engadin, 01 May 2020 - 07:09 PM.






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