Atmospheric particulate matter impedes autophagic flux by impairing lysosomal milieu and integrity in human umbilical vein endothelial cells (HUVECs)

https://doi.org/10.1016/j.scitotenv.2020.143290Get rights and content

Highlights

  • After 24 h, autophagic flux was blocked in HUVECs following exposure to PM SRM1648a.

  • PM SRM1648a causes defective autophagy in HUVECs.

  • Lysosomes are damaged in HUVECs in response to PM SRM1648a.

  • Particles-induced lysosomal impairment is responsible for dysfunction of autophagic flux.

Abstract

Autophagy is a dynamic process for waste disposal and cell equilibrium. Previous studies have demonstrated that atmospheric particulate matter (APM) induces autophagy and enhances LC3II expression in human vascular endothelial cells. However, the underlying mechanism of autophagosome accumulation in human vascular endothelial cells under the exposure to APM has not been understood. In principle, the upregulation of LC3II or autophagosomes accumulation is presumably caused by the enhancement of autophagic ability, or alternatively, by the abnormal autophagic degradation. Therefore, in the current study, autophagic ability and autophagic flux are systemically studied to decipher the exact cause of autophagosomes accumulation in human umbilical vein endothelial cells (HUVECs) in response to a standard urban particulate matter, PM SRM1648a. As a result, it was observed that after 24 h of exposure, PM SRM1648a significantly increases LC3II expression with apparent autophagosomes accumulation in HUVECs. Compared with the control group, there is a time-dependent increase in p62, a protein of autophagic substrate that can be preliminarily used to evaluate the autophagic degradation, in the PM SRM1648a-exposed HUVECs, which suggested that normal function of autophagic degradation was probably impaired. Additionally, mRFP-GFP-LC3 assay and LAMP-2/LC3B co-localization suggested that autolysosomes (fusion between autophagosomes and lysosomes) were partially inhibited in PM SRM1648a-treated HUVECs. Furthermore, LC3II turn-over assay hinted that after 24 h, LC3II upregulation is attributed to the blockage of autophagic flux instead of the enhancement of autophagic induction. Mechanistically, the blockade of autophagic flux can be explained by the detrimental effects of PM SRM1648a on lysosomal function, including lysosomal destabilization, lysosomal alkalization and hydrolase inactivation, which are involved in the blockade of fusion between autophagosomes and lysosomes, further disrupting autophagic degradation and waste disposal. These observations provide evidence that PM SRM1648a destroys the equilibrium of lysosomal stability and thus results in the dysfunction of autophagic flux, eventually contributing to endothelial cell damage.

Introduction

Atmospheric particulate matter (APM) is a ubiquitous component of air pollution, mainly produced by anthropogenic activities, such as industrial emissions, automotive exhaust and construction operations (Yang et al., 2018). According to the published information, human beings can be adversely influenced by urban particulate matter through inhalation, dermal exposure, ingestion and absorption (Wang et al., 2017c). APM is clearly an emerging public concern in different nations, producing generic risk of health, including observable cardiovascular and pulmonary consequences as reported in these papers (Atkinson et al., 2014; Honda et al., 2018; Huang et al., 2018; Hwang et al., 2017).

Both environmental and laboratory investigations support a conclusion that short-term exposure to APM would result in acute inflammatory responses and oxidation-antioxidation system imbalance (Ferguson et al., 2013; Wang et al., 2018). Moreover, long-term exposure could adversely impact multiple systems, leading to chronic damage, or disease onset or the aggregation of systemic dysfunction (Honda et al., 2018; Stockfelt et al., 2017). The cardiovascular system is vulnerable to APM directly and indirectly (Nelin et al., 2012). Specifically, the soluble components of particles could gain easy access to the body circulation by penetrating the air-blood barrier. Both local and systemic inflammatory cytokines have been observed to cause lung and circulatory damage. It has been demonstrated that these pollutants disturb the equilibrium of the cardiovascular system by damaging vascular endothelial cells and cause the occurrence of cardiovascular-related events and disease, such as hypertension and atherosclerosis (Honda et al., 2018; Huang et al., 2018; Yin et al., 2017).

Vascular endothelial cells are the potential location of contact once APM reaches the blood. Blood circulation is the most effective way for the transmission of particles to other sites beyond the pulmonary area. Under this circumstance, it is inevitable to encounter the interaction between particles or their components with endothelial cells. Additionally, dysfunction of endothelial cells is an independent risk factor for cardiovascular diseases, which could be induced by particles exposure. Although a large number of previous studies have discussed the adverse influences of APM on the endothelial cells to some extent, such as endothelial nitric oxide synthase (eNOS) uncoupling, inflammatory reaction and apoptosis (Bourdrel et al., 2017; Chen et al., 2018; Fiordelisi et al., 2017; Rao et al., 2018; Wang and Tang, 2019a, Wang and Tang, 2019b), autophagy and autophagy-related mechanisms under APM exposure are still in infant stage. APM has been observed to increase the expression of LC3II and result in autophagosomes accumulation in an extensive field of immortal cell lines and primarily cultured cells (Deng et al., 2013; Liu et al., 2015), including vascular endothelial cells (Ding et al., 2017; Zhou et al., 2018; Wang and Tang, 2020). Looking at the subcellular system, although progressively increasing number of studies concentrated on mitochondria and endoplasmic reticulum, few studies have focused on lysosomes (Laing et al., 2010; Qi et al., 2019; Wang et al., 2020a; Wang and Tang, 2020; Wang et al., 2020b; Zhou et al., 2017). Lysosomes are a kind of organelle responsible for waste degradation, thus protecting cellular homeostasis, which are closely linked to autophagy, particularly in the late stage of autolysosomes production and cargo degradation (Yu et al., 2018). Therefore, in the current study, apart from autophagy itself, lysosomes are studied to discuss their role in PM SRM1648a-induced autophagosome accumulation in human vascular endothelial cells.

Several studies have studied autophagosomes/autophagic vacuoles by TEM observations, or inducible signaling pathways for autophagy stimulation under APM exposure. These have involved AMPK and PI3K/AKT/mTOR signal transduction, TFEB nucleus translocation and ULK1/Beclin 1 phosphorylation (Mihaylova and Shaw, 2011; Settembre et al., 2011; Munson and Ganley, 2015). However, the later stage of autophagy (autophagic flux) has been scarcely discussed. Therefore, apart from the LC3II expression, we have considered autophagic machinery, especially the autophagic degradation. This has been undertaken using a classical human vascular endothelial cell model, human umbilical vein endothelial cells (HUVECs).

Our objective was intended to detect autophagic flux, and to determine what functional changes could be observed in lysosomes in HUVECs after exposure to PM SRM1648a. Furthermore, the role of lysosomal microenvironment and stability in the autophagic process was discussed. Accordingly, the results indicate that PM SRM1648a triggers lysosomal impairment, including lysosomal destabilization, lysosomal alkalization and hydrolase inactivation, which are crucial risk factors for the blockade of autophagic flux and thus exerts detrimental effects on vascular endothelial cell survival.

Section snippets

Reagents and materials

Atmospheric particulate matter (PM SRM1648a) was obtained from the National Institute of Standards and Technology (NIST, USA). The Certificate, and Material Safety Data Sheet and other important information of this material could be referenced to the webpage of https://www-s.nist.gov/srmors/view_detail.cfm?srm=1648A.

All components, the characteristics of hydrodynamic particle size, potential and endotoxin detection, with the treatment and exposure methods were recorded in previous paper (Wang

PM SRM1648a induces LC3II upregulation in vascular endothelial cells, and autophagy itself confers protection against apoptosis

HUVECs were exposed to different concentrations of PM SRM1648a, 0, 5, 10 and 20 μg/cm2 for 24 h. These correspond to the concentrations of 0, 16, 32 and 64 μg/mL when 3 mL of solutions was added in each of the 6-well cell culture plates. Fig. 1A & B provides a demonstration that autophagic marker proteins of LC3II and p62 were significantly upregulated from the minimum dosage level used, while Beclin 1 kept stable within the dose spectrum, suggesting that after 24 h of exposure, PM SRM1648a can

Discussion

As the first line, endothelial cells form a barrier layer throughout the entire blood vessel wall, which plays a vital role in maintaining the normal function of blood vessels (H. Li et al., 2014; Zhou et al., 2012). When APM enters the blood circulation, interactions between endothelial cells and particles are bound to occur. Endothelial cells may be attacked by particles and their components directly, and by inflammatory cytokines from the respiratory system and circulating blood indirectly(

Conclusion

The current study has indicated that lysosomes are potential targets of PM SRM1648a in HUVECs. Lysosomal damage may eventually lead to defective autophagy. After 24 h of treatment, PM SRM1648a no longer enhances autophagic activity, but blocks autophagic flux in HUVECs. Lysosomal impairment appears to be the underlying mechanism for PM SRM1648a inducible dysfunction of autophagic flux with a subsequent failure in waste degradation. It can be concluded that PM SRM1648a contributes to lysosomal

CRediT authorship contribution statement

Yan Wang: Conceptualization, Investigation, Data curation, Data analysis & interpretation, Writing - original draft, review & editing. Na Liu, Xiaoquan Huang, Wangcheng Hu & Ying Ma: Methodology, Investigation & Data analysis. Ying Liang & Wenjing Xie: Data analysis. Meng Tang: Supervision, Project administration, Resources & Funding acquisition.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by National Natural Science Foundation of China (grant numbers 21876026, 31671034, and 81473003), and Natural Science Foundation of Jiangsu Province (grant number BK20180371).

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