Autophagy downregulates thrombin-induced VSMCs proliferation through lysosomal pathway

Abstract

of ceroid which is an insoluble protein complex bound with oxidized lipids found in almost all atherosclerotic lesions [2]. Although there is evidence that induction of autophagy through beclin1 activation can inhibit cell proliferation in several cancer cell lines, while as yet, knowledge about the impact of autophagy in thrombin-induced VSMCs proliferation is still limited. Hence, we conducted the current research to uncover the link between autophagy and proliferation of VSMCs. The experimental VSMCs were obtained from the abdominal aorta of healthy SD rat and cultured. The passage 4 VSMCs were used in the experiments and were divided into the following groups: (1) control group, (2) thrombin group (1 U/ml), (3) thrombin (1 U/ ml)+trehalose group, (4) thrombin (1 U/ml)+3-methyladenine (3-MA) (10 ng/ml) group, (5) thrombin (1 U/ml)+3-methyladenine (3-MA)+pepstatin A group (10 ng/ml). Immunofluorescence and western blot analysis were used to detect the proliferation related protein PCNA expression after treatment of trehalose, which is a novel mTOR-independent autophagy enhancer, and autophagy inhibitor 3-methyladenine (3-MA). Thereafter, we further discussed the potential mechanism herein; pepstatin A was used to inhibit the lysosomal pathway to inspect its involvement in the process. Our results showed that autophagy up-regulation can suppress thrombin-induced proliferation of VSMCs, in contrast, down-regulation of autophagy played a positive role in thrombin-stimulated VSMCs proliferation, and furthermore, this function can be inhibited by the treatment of pepstatin A.Tofurtherunderstandthepotentialmechanismof theinhibitoryroleof autophagy in thrombin-induced VSMCs proliferation, the expression of PCNA was detected after inhibition of lysosomal signaling pathway with pepstatin A.Combination use of pepstatin Awith trehalosecan neutralize the down-regulated role of autophagy on thrombin-induced VSMCs proliferation, which suggested the involvement of autophagy–lysosome pathway in this mechanism. With the awareness of the essential significance of VSMCs proliferation in cardiovascular diseases, antiproliferative strategies have aroused tremendous attention. In the present stage, antiproliferative strategies mainly include long-term and shortterm treatments with therapeutic demerits both, for example, the shortterm cell-inhibitory agents may not be so effective for such a diffuse disease like atherosclerosis, while long-term agents may weaken the fibrous cap of lesions and followed by increased susceptibility to rupture [3,4]. Autophagy was initially described as a positive role for cell survival, defective autophagy is involved in various diseases such as infections, cardiovascular diseases and cancers, etc. [5], and it can be triggered by some factors such as oxidized lipids, inflammationstress states in terms of atherosclerosis [6]. In the current study, we have observed the expression of proliferating cell nuclear antigen (PCNA) and microtubule-associated protein light chain 3 (MAP1-LC3) under treatment of autophagy inducer trehalose and autophagy inhibitor 3methyladenine (3-MA) (Fig.1). We found that the thrombin-induced proliferation of VSMCs decreased under up-regulation of autophagy, while in the sharp contrast, down-regulation of autophagy can increase thrombin-stimulated VSMCs proliferation, which can be inhibited by the treatment of pepstatin A, suggesting the involvement of autophagy–lysosome signal pathway in this process. In this investigation, we chose trehalose as autophagy inducer rather than the classic one rapamycin, for the former one is an mTORindependent agent while the later increases cell proliferation through PI3K/mTOR signal pathway [7] .T hesefindings suggest that autophagy acts as an adverse effect in thrombin-induced VSMCs proliferation through autophagy–lysosome pathway. VSMCs antiproliferative strategies based upon the regulation of autophagy may emerge as a novel approach in atherosclerosis treatment. Several studies demonstratedthatectopicexpressionofautophagy-relatedgenebeclin1