RapaLink-1 plays an antithrombotic role in antiphospholipid syndrome by improving autophagy both in vivo and vitro

https://doi.org/10.1016/j.bbrc.2020.02.084Get rights and content

Highlights

  • RapaLink-1 played an antithrombotic role in mice with antiphospholipid syndrome.

  • RapaLink-1 promoted autophagy in mice with antiphospholipid syndrome.

  • RapaLink-1 protected macrophages against lipids-induced injury via promoting autophagy.

Abstract

Background

RapaLink-1 is a third generation mammalian target of rapamycin (mTOR) inhibitor and displays superior inhibitory effect on mTOR complex 1 (mTORC1). mTOR pathway is known to block autophagy and inhibition of it can protect thrombosis-related diseases including atherosclerosis, antiphospholipid syndrome (APS) and stroke. The objective of this study was to investigate whether RapaLink-1 could exert anti-thrombotic effects on APS via improving autophagy.

Methods

BALB/c mice were injected with monoclonal anti-beta-2-GPI (β2GPI) antibodies to induce APS in vivo, and anti-β2GPI antibodies together with anticardiolipin (aCL) antibodies in mice serum were assessed. The aortas of mice were isolated, and oil red and haematoxylin and eosin (HE) staining were used for thrombus morphology. The levels of LC3B and CD68 were quantified. Human monocyte cell line THP-1 was stimulated with oxidized low-density lipoprotein (ox-LDL) and treated with RapaLink-1 in vitro. The cell viability, LDH activity, apoptosis rate and rate of fate-positive cells were detected. LC3 expression was quantified by immunofluorescence. Western blot was utilized to assess the protein expression of LC3-І, LC3-П, Beclin-1 and p62.

Results

The size of arterial thrombus plaque together with the level of anti-β2GPI antibodies and aCL was reduced by RapaLink-1. Immunostaining protocols confirmed that the application of RapaLink-1 inhibited plaque initiation and progression while decreased the extent of macrophage infiltration and enhanced the autophagy process. In vitro cultured THP-1 macrophages exposed to ox-LDL study showed that RapaLink-1 prevented cell apoptosis and enhanced autophagy of macrophages, indicated by the increasing expression of autophagy-related protein and morphological character under electron microscopy.

Conclusion

Our results revealed that Rapalink-1 has a potential to inhibit the formation of thrombus plaque in APS and these effects were dependent on facilitating cell autophagy both in vivo and in vitro.

Introduction

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis, affecting the venous or arterial vasculature, habitual abortion, and persistent positive anti-phospholipid antibodies (aPL), including lupus anticoagulants (LAC), anticardiolipin (aCL) antibodies, and anti-β-2-glycoprotein-I (aβ2GPI) antibodies [1]. APL-induced thrombosis can affect any body organ, thereby accelerating atherosclerosis, myocardial infarction and stroke in patients [2]. In a large prospective cohort study, 1000 patients with APS were followed for 10 years and results revealed that deep vein thrombosis was the most common clinical manifestation (39%), followed by thrombocytopenia (30%), livedo reticularis (24%), stroke (20%), pulmonary embolism (14%) and myocardial infarction (6%) [3]. APS is not only related to clinical adverse cardiovascular events, but also to subclinical cardiovascular risk factors responsible for endothelial cell dysfunction, such as increased carotid intima-media thickness, reduced blood flow-mediated dilatation, and increased carotid plaque frequency [4].

The leading cause of deaths from APS is thrombosis, thus thrombosis prevention has been the focus and difficulty in treatment of APS patients. At present, the treatment of APS patients mainly depends on long-term anticoagulation to prevent thrombosis and the combination of low-dose aspirin (LDA) and low-molecular-weight heparin (LMWH) to prevent obstetric manifestations. However, these treatments are not always effective due to the occasional occurrence of such side effects as bleeding and recurrent thrombotic events. Moreover, the use of long-term anticoagulation in some patients with APS is a common sense recommendation with no scientific basis [5]. Therefore, new treatment methods are urgently needed to specifically interfere in the pathological process of APS.

Although aβ2GPI antibodies are the main pathogenic antibodies in APS, the mechanisms by which aPL induces thrombosis and pregnancy loss are not fully understood. With the deepening of research on APS, it has been evidenced that the activation of mammalian target of rapamycin (mTOR) pathway, which regulates cell growth, proliferation, and autophagy, plays an important role in endothelial proliferation and intimal hyperplasia in aPL positive patients, leading to proliferation of peripheral vascular smooth muscle cells (VSMC) and endothelial cells. Moreover, a recent study showed that immunoglobulin G (IgG) antibodies from APS patients activated mTOR complex (mTORC) via the phosphatidylinositol 3-kinase (PI3K)-AKT pathway in vitro. APS patients who received sirolimus (mTOR inhibitor) had no recurrence of vascular disease and reduced vascular proliferation, compared with patients who did not receive sirolimus [6]. Since then, increasing studies further confirmed the activation of AKT/mTOR pathway in APS at the molecular level, the central role of it in the process of vascular injury induced by APS vascular endothelial activation and the efficacy of treatment with sirolimus in patients with APS [7,8].

Much of the research regarding the potential role of mTOR pathway in APS derived from the use of sirolimus. mTOR exists in two distinct complexes, mTORC1 and mTORC2. Sirolimus specifically targets the mTORC1 with IC50 value in the high picomolar range, and has been approved by FDA to be used for the prevention of organ rejection after transplantation owing to its immunosuppressive effect [9]. However, the shortcomings of sirolimus limit its clinical use, and second and third generations of mTORC1 inhibitors have been developed. RapaLink-1 is a recently developed third generation mTOR inhibitor, which was generated by covalently linked rapamycin and an mTOR kinase inhibitor, thus resulting in a relative longer durability of mTORC1 [10]. Studies have confirmed the superior efficacy of RapaLink-1in inhibition of the mTORC1 pathway and treating glioblastoma [11]. In addition, it is well-established that mTOR is involved in blocking autophagy and AKT/mTOR signaling-mediated inhibition of autophagy has been implied in preventing angiogenesis and aggravating fibrosis [12,13]. However, whether mTOR/autophagy pathway was connected with thrombosis in APS remains to be elucidated.

Taken together, the objective of this study is to elucidate the effect of novel mTOR inhibitor RapaLink-1 on the pathogenesis of APS thrombotic events, and verify whether these effects was dependent on mTOR/autophagy pathway.

Section snippets

Animals

The Specific pathogen Free (SPF) male BALB/c mice at 10–12 weeks of age, were purchased from Harlan Israel and kept in ventilated cages. Mice were divided into four groups: control, APS, APS+0.5 mg/kg RapaLink-1 and APS+1 mg/kg RapaLink-1. RapaLink-1 was supplemented in the diet of mice daily, and control or APS group was fed with normal diet. All experiments were approved and executed according to the protocols for animal experimentation.

Induction of APS in mice

Experimental APS was induced in BALB/c male mice by

RapaLink-1 blunted the progression of thrombosis in APS mice

First of all, to investigate the effects of RapaLink-1 on aortic thrombosis in vivo, we injected aβ2GPI antibodies into mice to generate APS mice model and utilized FeCl3 to induce thrombosis in carotid artery of mice. Analyses of the total aorta en face surface stained with Oil Red O revealed larger lesions in APS mice, and the quantitative data showed decreased area of plaque in the presence of 0.5 mg/kg or 1 mg/kg RapaLink-1, compared with APS mice that were fed with normal diet (Fig. 1A).

Discussion

This study is the first to shed light on the protective effect of a novel generation mTOR inhibitor Rapalink-1 on thrombosis. Our results demonstrated that in FeCl3-treated APS mice, mice that were fed with RapaLink-1 displayed smaller thrombus area and alleviative macrophage infiltration compared with mice did not receive RapaLink-1. At the same time, the autophagy was also enhanced by RapaLink-1 both in vivo and in vitro.

In fact, the evidence about the modulatory role of mTORC pathway in APS

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authorship

ZC and FM: the conception and design of the study; FM and YJ: acquisition of data; FA and HW: analysis and interpretation of data; QY and ZC drafting the article or revising it critically for important intellectual content; ZC final approval of the version to be submitted.

Declaration of competing interest

None declared.

References (19)

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