Inhibition of Connexin 43 reverses ox-LDL-mediated inhibition of autophagy in VSMC by inhibiting the PI3K/Akt/mTOR signaling pathway

Introduction

Atherosclerotic cardiovascular disease is the main contributor to the increase in global morbidity and mortality (Arnett et al., 2019). Atherosclerotic plaques are formed in the vessel wall due to the accumulation of lipids and other substances in the process of atherosclerosis (AS) (Poznyak et al., 2020). These lipid-rich cells in atherosclerotic plaques are called foam cells (FCs). Many past studies suggested that the majority of FCs originated from macrophages. However, recently it has been shown that most of the FCs in mouse and human atherosclerotic plaques originate from smooth muscle cell (SMC) (Wang et al., 2019a; Allahverdian et al., 2014). Accordingly, understanding the mechanism of SMC-derived FCs will contribute to the prevention and treatment of atherosclerotic diseases.

Autophagy is a physiological process in which eukaryotic cells maintain internal homeostasis (Qin, 2019). It is also involved in various physiological and pathological processes, including hypertension and the formation of coronary atherosclerotic plaques (Hughes, Beyer & Gutterman, 2020). In human atherosclerotic plaques, autophagy may be induced in VSMC, macrophages, and endothelial cells stimulated by lipids, metabolic stress, cytokines and reactive oxygen species (Grootaert et al., 2018). Over time, almost all types of cells have a certain degree of autophagy dysfunction, which negatively impacts plaque progression, but the underlying mechanisms are different (Grootaert et al., 2018). In addition, more studies have proven that regulation of autophagy may improve the occurrence and development of AS (Kumar et al., 2020; Shi et al., 2020). Rosuvastatin has been shown to enhance autophagic activity in macrophages by inhibiting the activation of the PI3K/Akt/mTOR signaling pathway and increasing autophagic flux, thus achieving the anti-AS effect (Zhang et al., 2021). The PI3K/Akt/mTOR signaling pathway is a major regulatory pathway that inhibits autophagy initiation. The PI3K/AKT/mTOR signaling pathway is a classical autophagic pathway. Meanwhile, it was also found that Danlou tablets (DLTs) can promote autophagy in macrophage by inhibiting this signaling pathway, thereby reducing FCs formation and improving AS (Liu et al., 2021). Despite lots of studies on the role of autophagy in anti-AS, those on the specific mechanism of autophagy is limited.

Gap junction channel (GJC), which is composed of connexin (Cx) as the basic unit, is a special channel to mediate direct communication at the intercellular (Beyer & Berthoud, 2018). Cx43 is the major Cx forming GJ in SMCs (Morel & Kwak, 2012). Gap26 is a specific Cx43 mimic peptide (Qing et al., 2021) that may inhibit Cx43 expression (Li et al., 2015, 2017) when used as a Cx43-GJ inhibitor. The increased expression of connexin 43 in intimal VSMC was observed in the early stages of human and mouse atherosclerotic lesions (Leybaert et al., 2017). This effect may be associated with various risk factors for AS, such as ox-LDL (Wang et al., 2019b) or angiotensin II (Lei et al., 2019), which may upregulate the expression of Cx43. Recently, studies have demonstrated that there is an important interaction between Cx and autophagy, that is Cx could be used not only as an autophagy substrate, but also an autophagy regulator (Iyyathurai et al., 2016). Besides that, it is reported that the regulation of Cx43 has impacts on the phosphorylation level of proteins in some signaling pathways, such as PI3K/Akt signaling pathway and related downstream pathways (Ji et al., 2019; Wang et al., 2020). This finding suggests that Cx43 and PI3K/Akt-related downstream pathways may be interrelated. Nevertheless, the connection between Cx43 in VSMC and autophagy remains elusive in atherosclerotic lesions. Therefore, it is worth exploring whether Cx43 takes part in regulating the PI3K/AKT/mTOR signaling pathway to have an impact on autophagy and understanding the role of Cx43 in autophagy may reveal its importance as a regulatory mechanism of disease development, making it a potential therapeutic target.

Materials and Methods

Results

Ox-LDL inhibited autophagy in VSMC via downregulation of autophagy-related protein

To examine the effect of ox-LDL on autophagy in VSMC, we monitored changes in the autophagy marker proteins p62, Beclin1, and LC3B, after they were treated with various concentrations of ox-LDL, and ox-LDL was used at different lengths of time. In our study, ox-LDL decreased Beclin-1 protein levels and the ratio of LC3II to LC3I at various time points in VSMC while the p62 protein levels were increased (Figs. 1A–1D). The p62 levels were increased with the elevation of ox-LDL concentrations, which was contrary to the results of Beclin 1 and LC3B (Figs. 1E–1H). These data indicated that ox-LDL inhibited autophagy in VSMC in a time-and concentration-dependent manner.

Ox-LDL increased Cx43 protein levels in VSMC

Next, we investigated the effect of ox-LDL on Cx43 in VSMC. The results appeared that ox-LDL treatment (80 μg/ml, 24 h) obviously elevated the protein (Figs. 2A and 2B) and mRNA (Fig. 2C) levels of Cx43 in VSMC. Moreover, the semi-quantitative results of immunofluorescence appeared that the relative fluorescence intensity of Cx43 were evidently enhanced after ox-LDL treatment, and Cx43 was mainly distributed in the cytoplasm and cell membrane (Figs. 2D and 2E). Thus, the above results prompted us that ox-LDL-inhibited autophagy in VSMC was accompanied by the upregulation of Cx43 protein levels.

Involvement of Cx43 in VSMC autophagy and FCs formation mediated by Ox-LDL

To identify whether Cx43 was involved in ox-LDL-inhibited autophagy in VSMC, we assessed the role of Gap26, an Cx43-specific inhibitor, on autophagy. The results demonstrated that Gap26 treatment increased Beclin 1 protein levels and the induction of LC3II in VSMC incubated with ox-LDL, which was the opposite of p62 protein levels (Figs. 3A–3D). And the immunofluorescence results were consistent with those of the Western blot (Figs. 3E–3J). At the ultrastructural level, autophagosomes presents a double-membraned structure, which contains undigested cytoplasmic contents. The TEM studies indicated that Gap26 treatment increased the number of autophagosomes compared with that in the ox-LDL group (Fig. 3K). These results revealed that inhibiting Cx43 could improve the autophagy level of VSMC. To clarify whether alternation of autophagy levels affected transformation of VSMC into foam cells, we evaluated the changes in lipid droplets (Fig. 3L). The findings suggested that inhibition of Cx43 prevented the ox-LDL-induced lipid droplet accumulation in VSMC, thereby suppressing FCs formation.

Inhibition of Cx43 up-regulated the autophagy levels of VSMC after ox-LDL treatment via inhibiting the PI3K/AKT/mTOR signaling pathway

We pretreated VSMC with LY294002 and Gap26 to explore the potential mechanism by which Cx43 is involved in ox-LDL-inhibited autophagy in VSMC. As compared with ox-LDL-treated VSMC, LY294002 markedly inhibited p62 protein levels, increased Beclin-1 protein levels, and promoted the conversion of LC3-I to LC3-II (Figs. 4A–4D), indicating that PI3K/AKT/mTOR signaling pathway might be involved in ox-LDL-mediated autophagy of VSMC. Next, ox-LDL up-regulated the protein levels of p-PI3K, p-AKT, and p-mTOR, which was partially reversed by Gap26 (Figs. 4E–4H), suggesting that Cx43 inhibition prevented the activation of this pathway. To further analyse the relationship between Cx43 and this signaling pathway, LY294002 treatment was used to examine changes in Cx43 protein levels in ox-LDL-treated VSMC. Cx43 protein levels was decreased as VSMC was exposed to LY294002 (Figs. 4I and 4J).

Discussion

VSMC are an important source of FCs in atherosclerotic lesions and have received increased attention in recent years. Many studies have reported that FCs formation may be inhibited by regulating autophagy in atherosclerotic lesions (Kumar et al., 2020; Shi et al., 2020; Hui et al., 2021). We demonstrated that ox-LDL-inhibited autophagy induction in VSMC via the downregulation of autophagy-related protein. Cx43 protein levels were also found to increase after ox-LDL treatment. The inhibition of Cx43 could activate autophagy and reduced ox-LDL-mediated lipid droplet accumulation by inhibiting PI3K/Akt/mTOR signaling pathway.

There are many risk factors for the formation of AS, and one of which is ox-LDL (Khatana et al., 2020). It is closely associated with the formation of FCs. Consequently, we used the atherogenic factor ox-LDL (Zhao et al., 2020) to induce the formation of VSMC-derived FCs. There is increasing in vitro evidence that autophagy is present in atherosclerotic plaques (Martinet & De Meyer, 2009). However, autophagy has dual effects on AS (Martinet & De Meyer, 2008, 2009) and may be either protective or harmful. Therefore, we examined the effect of ox-LDL on VSMC autophagy and found that it regulated autophagy in a time-dependent manner (Li et al., 2014). Additionally, the effects of different doses of ox-LDL on autophagy in SMC were also different (Ding et al., 2013). In the early stage of AS, a low concentration of ox-LDL increased the protective autophagy in SMCs, while a high concentration of ox-LDL weakened the protective effect of autophagy and enhanced autophagy-induced cell death (Hassanpour et al., 2019). ox-LDL downregulated the accumulation of LC3-II and Beclin1 protein levels in macrophage transformation into the foam cell (Cao et al., 2019). Nevertheless, ox-LDL was found to increase the LC3II/LC3I ratio and Beclin1 protein levels in vascular endothelial cells in a cell injury model of vascular endothelial cells induced by ox-LDL (Li et al., 2018). This finding implied that there were differences in the influence of ox-LDL on autophagy in different cell models.

We also determined that ox-LDL increased Cx43 expression on VSMC. However, few studies have shown a relationship between Cx43 and autophagy in VSMC and the roles of these factors. It has been demonstrated that Cxs could regulate autophagy (Iyyathurai et al., 2016). Cx43 located on the plasma membrane may directly interact with autophagy-related gene 16 (Atg16), which is the initial step in the formation of autophagosomes. Therefore, Cxs may act as negative regulators of basal autophagy by binding to the components of the initiation complex. Other Cx subtypes (Cx26 or Cx32) may also have negative effects on autophagic flux, suggesting that some members of the Cx family may regulate autophagy. Pharmaceuticals including tamoxifen and lindane have been shown to increase autophagy in Cx43-expressing cells but not in Cx43-knockout cells (Iyyathurai et al., 2016), which suggests that Cx43 may take part in the regulation of autophagy. Other studies have found the mRNA level of LC3B in Cx43-knockout group was significantly increased compared with untreated porcine early embryos (Shin et al., 2020). However, there have been few reports on the interaction between Cx43 and autophagy in AS (Iyyathurai et al., 2016). Our results showed that the autophagic flux in VSMC was impaired after ox-LDL incubation, and this effect was partly reversed by the specific Cx43 inhibitor, Gap26.

Furthermore, studies have demonstrated that autophagy takes part in lipid metabolism. Autophagy may be effective in the decomposition of lipid droplets to provide free fatty acids for cells (Martinez-Lopez & Singh, 2015). The role of autophagy in lipid metabolism is associated with triglycerides breakdown and cholesterol stored in lipid droplets by selective lysosome-dependent macrophages (Liu & Czaja, 2013). Many important metabolic disorders, such as fatty liver, obesity, and AS, may be associated with impaired lipid autophagy (Robichaud et al., 2021; Khawar, Gao & Li, 2019). Our results show that ox-LDL-induced FC formation may be inhibited by activating autophagy (Ko et al., 2020) and that autophagy levels increased and intracellular lipid droplets decreased significantly after the inhibition of Cx43 in VSMC. This effect may be associated with the activation of autophagy and the increase of autophagosomes, which can engulf more lipid droplets and bind to lysosomes to degrade lipids.

The PI3K/Akt/mTOR signaling pathway has been demonstrated that it plays an important role in regulating autophagy in AS (Jiang et al., 2017) and our study has shown that ox-LDL could activate this pathway (Zhuo et al., 2019). Propofol reduces cell adhesion by inhibiting Cx43 and the downstream PI3K/Akt/NF-κB signaling pathway. Cx43 was overexpressed (or knocked down) on monocytes, resulting in a decrease of the related protein p-AKT with the downregulation of Cx43, and vice versa (Ji et al., 2019). Other studies have shown that resveratrol inhibits Akt and its downstream pathway by upregulating Cx43 in colorectal cancer cell lines (Wang et al., 2020). This finding suggests that there is a relationship between Cx43 and the PI3K/AKT pathway. Therefore, we used the PI3K-specific inhibitor LY294002 (Fang et al., 2020) and Gap26 to confirm whether there is a connection between Cx43 and the PI3K/AKT/mTOR signaling pathway. We found that the promoting effect of Cx43 on autophagy may be produced by inhibiting the activation of this pathway, thus preventing FCs formation. Furthermore, we found that LY294002 decreased Cx43 protein levels. This effect may be associated with the fact that when autophagy is induced (such as during hunger), internalized Cx43 will be degraded by autophagy (Catarino et al., 2020). Autophagy has been identified as an important pathway in the degradation of different types of Cxs, and autophagy induced by different factors will directly affect the expression level of Cxs (Iyyathurai et al., 2016; D’hondt et al., 2016). We hypothesized that VSMC autophagy was activated by LY294002, which resulted in the degradation of internalized Cx43, thereby reducing Cx43 expression (Bi et al., 2017). These findings suggest that Cxs can be used as autophagy substrates and as autophagy regulators.

Based on the above results, this study still has some limitations. Although some studies have reported that in podocytes (MPC5 cell line), silencing of Cx43 expression improved autophagy flow experimentally impaired by exposure of the cells to high glucose levels (Ji et al., 2019), the role of Cx43 in ox-LDL-mediated autophagy of primary VSMC still needs to be verified by more experimental techniques, such as knockdown Cx43 by transfection technology. In addition, we can also explore the role of gap junction intercellular communication (GJIC) function in VSMC autophagy. It has been found that GJIC negatively regulated autophagy and inhibited autophagy flux (Zou et al., 2020). In summary, ox-LDL prevented VSMC autophagy. The inhibition of Cx43 significantly reversed this effect. The underlying mechanism may be mediated by inhibiting the PI3K/Akt/mTOR signaling pathway. These findings may become an important regulatory mechanism for disease development, making it a potential target for the treatment of AS.

Supplemental Information