Impact of CD137-CD137L signaling mediated exocytosis of autophagosome within vascular smooth muscle cells on the formation of atherosclerotic calcification

Abstract

Objective: To explore whether CD137-CD137L signaling mediated exocytosis of autophagosome within vascular smooth muscle cells (VSMCs) could influence the formation of atherosclerotic calcification. Methods: Fifteen 8-week-old male ApoE(-/-)(C57BL/6J-KO) mice fed with high fat diet for 5 weeks were randomly divided into three groups by using stochastic indicator method as follows: control group, n=5; agonist-CD137 group: agonist-CD137 antibody 200 μg/2 weeks for 4 weeks, ip, n=5; anti-CD137 group: 200 μg anti-CD137 antibody+ 200 μg agonist-CD137 antibody/2 weeks for 4 weeks, ip, n=5. Von Kossa staining was applied to observe the calcification of the thoracic aortic atherosclerotic plaque in each group. Immunohistochemistry was used to detect the expression of LC3 and Beclin1 which were the autophage markers of early-to-mid stage; Western blot was adopted to quantify protein level of microtubule-associated proteins 1 light chain 3B(LC3B) and mammalian ortholog of the yeast autophagy-related gene 6 (Beclin1). Transmission electron microscope (TME) was used to observe the formation of autophagosome in plaque. C57BL/6J mouse VSMCs were cultured by using tissue piece inoculation method. Groups of in vitro studies were the same as in vivo study: control group, agonist-CD137 group, anti-CD137 group, the agonist-CD137 groups was treated with agonist-CD137 antibody (10 μg/ml) and anti-CD137 group was treated with anti-CD137 antibody (10 μg/ml) for 30 minutes, followed by agonist-CD137 antibody (10 μg/ml). Von Kossa staining and osteogenesis phenotypic alkaline phosphatase (ALP) activity detection were adopted to observe calcification in VSMCs. Autophagosomes were separated from the supernatant of the agonist-CD137 group with density gradient centrifugation method. VSMCs were divided into two groups: positive group (containing complete medium with above autophagosomes to a final concentration 15 μg/ml) and negative group (only complete medium) after being pretreated with mixed inflammatory cytokines (IL-1β、IFN-γ and TNF-α, final concentration was 25 ng/ml respectively) for 24 hours and calcium deposition and osteogenesis phenotypic marker bone morphogenetic protein 2(BMP2) were then detected. Results: (1) Compared with the control group, activation of the CD137-CD137L signal significantly increased the formation of calcification area in thoracic aortic atherosclerotic plaque of ApoE(-/-) mice((1.82±0.15)×10(4) μm(2) vs. (0.34±0.08)×10(4) μm(2,) P<0.01), this effect was significantly attenuated by inhibiting this signal ((0.83±0.30)×10(4) μm(2) vs. (1.82±0.15)×10(4) μm(2,) P<0.05); positive autophagy makers LC3B and Beclin1 were detected in both agonist-CD137 group and anti-CD137 groups and the expression of LC3B and Beclin1 was substantially higher in anti-CD137 group. Western blot analysis indicated that the expression of LC3B and Beclin1 in agonist-CD137 group was significantly upregulated compared with the control group (0.17±0.01 vs. 0.03±0.08, P<0.05, and 0.12±0.02 vs. 0.06±0.02, P<0.05), which could be significantly downregulated in anti-CD137 group (0.28±0.09 vs. 0.17±0.01, P<0.05 and 0.17±0.02 vs. 0.12±0.02, P<0.05). TME showed that the number (QTY /HP) of autophagosome of agonist-CD137 group and anti-CD137 group in plaque were both increased (14.67±2.52 vs. 3.67±1.53, P<0.01, and 15.33±2.08 vs. 3.67±1.53, P<0.01), while in the agonist-CD137 group, the number of extracellular autophagosome within thoracic aortic atherosclerotic plaque of ApoE(-/-) mice increased more substantially (5.33±1.53 vs. 1.33±0.58, P<0.01). (2) In vitro study showed that activating CD137-CD137L signal could promote calcium deposition in extracellular matrix and the activity of osteogenesis phenotypic ALP((6.73±0.02) μmol/mg protein vs. (1.07±0.03) μmol/mg protein, P<0.05), and ((563.20±0.72) U/mg protein vs. (117.50±0.64) U/mg protein, P<0.05), while these effects were significantly blunted in anti-CD137 group ((1.94±0.05) μmol/mg protein vs. (6.73±0.02) μmol/mg protein, P<0.05, and (236.10±0.14) U/mg protein vs. (563.20±0.72) U/mg protein, P<0.05). TME showed that the number of intracellular autophagosome in agonist-CD137 group and anti-CD137 group was both significantly higher than in control group ((21.65±1.34) μg/ml vs. (8.32±1.58) μg/ml, P<0.01, and (15.42±1.65) μg/ml vs. (8.32±1.58) μg/ml, P<0.05). After the density gradient centrifugation, exocytotic autophagosome in the medium of agonist-CD137 group was markedly higher than in control group ((14.67±1.53) μg/ml vs. (2.33±1.15) μg/ml, P<0.01). (3) Compared with the control group, autophagosomes isolated from culture supernatant (final concentration: 15 μg/ml) could significantly stimulate calcium deposition((2.30±0.10) μmol/mg protein vs. (0.15±0.40) μmol/mg protein, P<0.05) and enhance the expression of bone morphogenetic protein 2 (2.10±0.04 vs. 0.30±0.01, P<0.05). Conclusion: CD137-CD137L signaling could mediate exocytosis of autophagosome within VSMCs, thus influence the formation of atherosclerotic calcification.