Exosome-delivered syndecan-1 rescues acute lung injury via a FAK/p190RhoGAP/RhoA/ROCK/NF-κB signaling axis and glycocalyx enhancement

https://doi.org/10.1016/j.yexcr.2019.111596Get rights and content

Highlights

  • Syndecan-1, a novel therapeutic agent for acute lung injury, is proposed.

  • Exosomes (SDC1-high-Exos) serve as a new carrier for delivering syndecan-1.

  • SDC1-high-Exos ameliorate lung inflammation, edema and preserve glycocalyx.

  • The mechanism relies on FAK/p190RhoGAP/RhoA/ROCK/NF-κB pathway.

Abstract

Acute lung injury (ALI) is characterized by protein-rich pulmonary edema, critical hypoxemia, and influx of pro-inflammatory cytokines and cells. There are currently no effective pharmacon therapies in clinical practice. Syndecan-1 in endothelial cells has potential to protect barrier function of endothelium and suppress inflammation response. Thus, the present study was to identify whether exosomes with encapsulation of syndecan-1 could achieve ideal therapeutic effects in ALI. Exosomes were isolated from the conditional medium of lentivirus-transfected mouse pulmonary microvascular endothelial cells (MPMVECs) and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting. ALI mouse models were induced via intratracheal administration of lipopolysaccharide (LPS) and treated with exosomes. Lung edema, inflammation, and glycocalyx thickness were examined. The possible mechanism was verified by immunoblotting in MPMVECs. The purified exosomes included SDC1-high-Exos and SDC1-low-Exos which loaded with up-regulated syndecan-1 and down-regulated syndecan-1 respectively. Compared with SDC1-low-Exos, administration of SDC1-high-Exos could ameliorate lung edema and inflammation, attenuate number of cells and protein levels in bronchoalveolar lavage fluid (BALF), and preserve glycocalyx. Furthermore, SDC1-high-Exos also mitigated the expression of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 following LPS challenge. In MPMVECs, SDC1-high-Exos decreased stress fiber formation and ameliorated monolayer hyper-permeability after LPS stimulation. Western blotting analysis demonstrated that FAK/p190RhoGAP/RhoA/ROCK/NF-κB signaling pathway may be involved in LPS-induced ALI. In conclusion, SDC1-high-Exos play a pivotal role in ameliorating LPS-stimulated ALI models and may be served as a potential therapeutic agent for clinical application in the future.

Introduction

Sepsis is a common and serious systemic inflammatory response, which is responsible for multiple organ failure and high mortality [[1], [2], [3]]. Millions of lives are endangered worldwide every year, with one fifth of the deaths occurring and the incidence increasing year by year [4,5]. The inflammatory consequences of sepsis are particularly involved in pulmonary circulation, which lead to acute lung injury. More than 40% of sepsis patients suffered from ALI, which was characterized by breakdown of pulmonary microvascular endothelial barrier and acute inflammatory response [6]. The development of ALI significantly exacerbated the prognosis of patients with septic shock, increasing the intensive care unit mortality rate from 11% to 38% [7].

Endothelial cells (ECs) line the entire vascular system, and lung ECs account for about 50% of all pneumonocytes and receive the entire cardiac output. Therefore pulmonary ECs are always exposed to circulating pathogens and bacterial endotoxins such as LPS, which are crucial to the etiopathogenesis of ALI [8]. Robust endothelial barrier functions are the basis of pulmonary vascular permeability [9], while breakdown of endothelial barriers are responsible for the lung edema, inflammatory cells influx, coagulation factor activation, and pro-inflammatory cytokine secretion [10]. LPS, major external component of gram-negative bacteria, induces considerable endothelial dysfunctions, including derangement of cytoskeletal structure, increased endothelial permeability, and release of inflammatory cytokines [11].

We have previously reported that the MyD88 and GEF-H1-RhoA pathways are responsible for NF-κB activation and IL-8 expression in ECs [12]. In addition, we have also confirmed that GSK-3 beta exacerbated pulmonary vascular permeability and escalated acute lung injury via GEF-H1/ROCK signaling axis [13]. However, the mechanisms involved in LPS-induced pulmonary microvascular permeability have not been well understood.

In recent years, many studies have demonstrated that LPS-induced ECs dysfunction was involved in cell surface glycoprotein glycocalyx damage, the main component of which is syndecan-1 glycoprotein. Syndecan-1, on the surface of ECs, plays a pivotal role in protecting endothelial barrier function [14]. Lined between blood stream and ECs, glycocalyx significantly affects the vascular permeability [15,16]. The loss of the glycocalyx in rat myocardial microvascular induces myocardial edema [17]. In addition, syndecan-1 overexpression in SDC-1 knock-out mice could attenuate inflammatory response after myocardial infarction [18]. Fresh frozen plasma resuscitation mitigates the pulmonary vascular hyperpermeability and inflammation in hemorrhagic shock due to increased syndecan-1 expression [19]. Syndecan-1 can also affect the expression of ZO-1 and occludin via the STAT3 signaling pathway, and maintain the integrity of the monolayers of intestinal epithelial cells [20]. Therefore, syndecan-1 plays an important role in protecting ALI and inhibiting inflammation. However, LPS stimulation can significantly attenuate the expression of syndecan-1 compared with control mice [21]. In addition, shedding of syndecan-1 induced by sepsis is extremely common in critically ill patients [22]. Hence, how to preserve and improve syndecan-1 levels in ECs is crucial to treating ALI currently.

Exosomes, extracellular vesicles, derive from internal endocytic compartments and multi-vesicular bodies which are associated with intercellular communication [23]. Recently exosomes have proven to be a promising medicine delivery carrier evidenced by excellent biocompatibility, efficient transportation, and hypo-immunogenicity [24]. Some studies have used exosomes as novel carriers to deliver specific siRNA or miRNA to targeting organ through intravenous administration [25,26]. However, the role of protein delivery in exosomes has been remained unclear.

In this study, we hypothesized that intravenous administration of SDC1-high-Exos would ameliorate ALI via delivery of syndecan-1. With LPS-induced ALI models and monolayer transfection assays, we identified that SDC1-high-Exos ameliorated the severity of ALI, whereas SDC1-low-Exos did not. Our results may provide a novel therapeutic strategy for ALI via systemic administration of SDC1-high-Exos.

Section snippets

Animals

Eight-week-old C57BL/6J male mice were used in this experiments and mice were bred in a pathogen-free animal facility in Shanghai Ruijin hospital. Mouse studies were authorized by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.

Cell culture

MPMVECs were purchased from Cell Biologics and cultured in complete mouse endothelial cell medium (Cell Biologics, Cat. M1168, Chicago, USA) in a humidified 37 °C, 5% CO2 incubator. The primary MPMVECs were used at 3–6 passages and medium

Characterization of exosomes

Nanoparticle tracking analyzer (NTA) demonstrated that the particle size was mainly ranging from 30 to 120 nm and the number of particles was mainly distributed around 100 nm (Fig. 1C). Tetraspanin proteins (CD9, CD63, CD81) were positive in all samples by Western blotting (Fig. 1B) [44]. In addition, exosome morphology was identified by transmission electron microscopy (TEM) and exhibited a cup-shaped appearance (Fig. 1A). MPMVECs were incubated with SDC1-high-Exos or SDC1-low-Exos which were

Discussion

Although vast majority of therapeutic proteins are able to treat a variety of illnesses, the majority of the available protein-based drugs have been confined to extracellular mechanisms of action. Protein transportation or lipid nanoparticle-based protein delivery methods have been suggested for direct delivery of protein into target cells and tissues. However, considerable obstacles limit the successful application of these methods in vivo, including low purification efficiency, inability to

Conclusions

In summary, we demonstrated that SDC1-high-Exos intravenous administration significantly ameliorated ALI symptoms by reducing pro-inflammatory cytokine production, lung edema, and enhancing the thickness of glycocalyx. In addition, SDC1-high-Exos also attenuated LPS-induced stress fiber formation and hyperpermeability of MPMVECs monolayers in vitro. Furthermore, we identified that the underlying mechanism lies in FAK/p190RhoGAP/RhoA/ROCK/NF-κb singling pathway and enhancement of the glycocalyx.

Acknowledgments

We thank the Institute of Chinese Academy of Sciences for help with the packaging plasmids (psPAX2 and pMD2.G). This work was supported by the National Natural Science Foundation of China (no. 81772077, 81772078, and 81801910).

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