PCSK9 Promotes Endothelial Dysfunction in Sepsis via TLR4/MyD88/NF-κB and NLRP3 Pathways

Longxiang Huang (  qqhlx20000@163.com ) The First A liated Hospital of Chongqing Medical University https://orcid.org/0000-0002-7606-1235 Yuanjing Li The First A liated Hospital of Chongqing Medical University Zhe Cheng Chongqing University Three Gorges Hospital Zi Lv Guangzhou Women and Children's Medical Center Suxin Luo The First A liated Hospital of Chongqing Medical University Yong Xia The Ohio State University


Introduction
Sepsis is a horrible disease, which usually leads to poor prognoses and even death for the patients [1].
Once sepsis enters the advanced stage, the in ammatory storm leads to multiple organ dysfunction and the disease becomes irreversible [2]. It is worth noting that hemodynamic disorder is one of the most crucial causes of disease progression [3]. In sepsis, the in ammatory factors damage blood vessels, which may present as disturbance of vascular tone regulation and a loss of effective blood volume due to excess uid spilling out of the vessels, and lead to blood pressure instability and shock [4].
Vascular endothelium is one of the most important components of blood vessels [5]. Although it is only distributed on the inner surface of blood vessels, it plays a very important role in regulating vascular function, such as the vasodilation function, vascular barrier function and anti-in ammatory effect [6].
Vascular endothelial dysfunction in sepsis could further aggravate the disease, and ultimately lead to individual death [7]. Recent studies have shown that protecting the endothelium is effective in treating the sepsis [8]. However, the mechanism of endothelial dysfunction in sepsis is not fully understood, and a further study is needed.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase and can regulate the LDL and cholesterol metabolism [9]. PCSK9 is associated with the formation of atherosclerotic plaques and inhibition of it has been showed to be bene cial to vascular function [10]. In addition, the negative role of PCSK9 in sepsis has also been gradually revealed by some studies [11,12].
Nevertheless, the relationship between PCSK9 and vascular endothelial function in sepsis remains unknown. This study explored the role of PCSK9 in septic endothelial dysfunction and further explored the possible mechanisms, providing new insights for the treatment of sepsis.

Cell viability assay
Cell viability was measured using the CCK-8) (Bimake, Shanghai, China) as described by the manufacturer. Brie y, cells were seeded in 96-well plates at a density of 1 × 104 cells/well and cultured for 1day. Next, different treatments were added in the medium of the HUVECs for 24h. The medium was removed and 100 µl of DMEM together with 10 µl of the CCK8 solution were added into each well. The plated were incubated at 37•C for 1-4 h. Then, OD450 was measured using a microplate spectrophotometer (Thermo Scienti c, USA).

Measurement of NO
The NO content in the medium was determined using a NO Detection kit (Beyotime, Shanghai, China) as described by the manufacturer. In brief, 50 µL/well standard or sample were added to a 96-well plate followed by Griess Reagent I and Griess Reagent II. The absorbance was measured at 540 nm with a a microplate spectrophotometer (Thermo Scienti c, USA).
Cecal Ligation and Puncture (CLP) CLP was performed to induce sepsis in mice and the procedure is described previously. Brie y, the mice were anesthetized with 2-3% iso urane in 100% oxygen, and an incision (1.0 cm) in the midline abdomen was performed. The cecum was exteriorized and 4-0 silk thread was used to ligate it at one half. 21-gauge needle was used to puncture the cecum and squeezed the cecum to assure patency of the puncture hole. The incision was sutured in layers after returning the cecum to the abdomen. For resuscitation, 5 mL/100 g prewarmed saline solution was injected to all the mice subcutaneously. Shamoperated mice received the same procedures except punctures. Animals were sacri ced by cervical dislocation, and the aortas were isolated to be used for subsequent experiments. Pep 2-8 (10 µg/kg) and KLA (1 µg/g) were administrated 2 h before surgery by intraperitoneal injection, with the dosage based on previous studies[13 , 14].

Western Blot Analysis
Proteins were extracted from the aorta and HUVECs with RIPA lysis buffer. Proteins were separated on 10~12% SDS-PAGE gels and transferred to polyvinylidene di uoride membranes. Skim milk in TBST buffer was used to block the membranes 1h at room temperature, and then the membranes were incubated with appropriate primary antibodies overnight at 4°C. The blots were then incubated with secondary antibodies conjugated with horseradish peroxidase and ultimately visualized using chemiluminescence.

Survival Study
After surgery, the animals in each group (n = 10) were observed for 48 h and No other experimental procedures were performed. Pep 2-8 (10 µg/kg) and KLA (1 µg/g) were injected intraperitoneally 2 h before surgery and re-administrated 24 h after CLP.

Statistical Analysis
All data are expressed as the mean ± SD. All statistical analyses were performed using GraphPad Prism 8.0 software. Statistical evaluation was performed by using one-way analysis of variance followed by the Bonferroni post hoc test. Survival curves for all groups were performed using Kaplan-Meier survival curves and analyzed using log-rank tests. Differences were considered statistically signi cant at P < 0.05.

Results
LPS decreased eNOS and VE-Cadherin expression and increased PCSK9 expression in HUVECs in a dosedependent manner.
The expression of eNOS and VE-Cadherin re ects endothelial function. We treated HUVECs with different concentrations of LPS for 24 h, and the expression of eNOS, VE-Cadherin and PCSK9 was detected by Western blot. As shown in Fig. 1A-D, LPS decrease the eNOS and VE-Cadherin expression and increased PCSK9 expression in HUVECs in a dose-dependent manner (P<0.05, n=4). NO, a vasodilators, is produced under eNOS catalysis, and the content of it in the culture medium of HUVECs was determined. As shown in Fig. 1E, NO content decreased with the increase of LPS concentration (P<0.05, n=4). CCK8 experiment demonstrated that the cell viability of HUVEC was decreased by LPS in a dose-dependent manner (Fig. 1F,   P<0.05, n=4). Since 10 µg/ml LPS treatment leads to the most obvious changes, this concentration was selected and used in subsequent experiments.
Inhibition of PCSK9 reversed LPS-induced declines in eNOS and VE-Cadherin expression, NO content and cell viability.
To investigate the role of PCSK9 in septic endothelial dysfunction, we used EVC to speci cally inhibit PCSK9 in HUVECs. The results showed that LPS induced signi cant declines in eNOS ( Fig. 2A and B) and VE-Cadherin ( Fig. 2A and C) expression, NO content (Fig. 2E) and cell viability (Fig. 2F), and up-regulation of PCSK9 expression (P<0.05, n=4). However, these effects could be reversed by EVC treatment in a dosedependent manner (P<0.05, n=4). Since the 200µM EVC treatment leads to the most obvious changes, this concentration was selected and used in subsequent experiments.
Inhibition of PCSK9 reversed LPS-induced activation of TLR4/MyD88/NF-κB and NLRP3 pathways and increased production of in ammatory cytokines.
The therapeutic effects of inhibiting PCSK9 in septic endothelial dysfunction were counteracted by agonists of TLR4.
To determine the role of TLR4/MyD88/NF-κB and NLRP3 pathways in PCSK9-induced septic endothelial dysfunction, we used KLA to speci cally activate TLR4. The Western blot results showed that inhibition of PCSK9 with EVC, compared with LPS group, could increase the expression of eNOS ( Fig. 4A and B) and VE-Cadherin ( Fig. 4A and C) and decrease the expression of TLR4 (Fig. 4A and E), MyD88 (Fig. 4A and F), p-p65 ( Fig. 4A and G), NLRP3 ( Fig. 4A and H), ASC ( Fig. 4A and I) and Caspase1 p20 ( Fig. 4A and J) (P<0.05, n=4). However, these effects was diminished by KLA treatment (P<0.05, n=4). In addition, compared with LPS group, inhibition of PCSK9 with EVC decreased mRNA expression of TNF-α (Fig. 3H), IL-1 (Fig. 3I) and IL-18 (Fig. 3J) and increased of NO content and cell viability(P<0.05, n=4), while, these effects was reversed by KLA treatment (P<0.05, n=4). Although EVC could induce a decrease of PCSK9  To determine the relationship between PCSK9 and septic endothelial dysfunction in vivo, CLP was performed on mice to induce sepsis and PCSK9 inhibitor Pep 2-8 were used. The results showed that CLP decreased the expression of eNOS and VE-Cadherin and increased the expression of TLR4 (Fig. 4A and E), MyD88 (Fig. 4A and F), p-p65 (Fig. 4A and G), NLRP3 (Fig. 4A and H), ASC ( Fig. 4A and I) and Caspase1 p20 (Fig. 4A and J) and the mRNA expression of TNF-α (Fig. 3H), IL-1 (Fig. 3I)

Discussion
This study mainly explored the role of PCSK9 in septic endothelial dysfunction. It was found that, in sepsis, PCSK9 damaged vascular endothelial cells and decreased endothelium-dependent vasodilation function and barrier function, in which the TLR4/MyD88/NF-κB and NLRP3 pathways were involved. Our study suggests that the role of PCSK9 and the impairment of vascular endothelial function should be considered in the treatment of patients with sepsis.
Sepsis is a terrible systemic disease and causes damage to almost all organs of the body [15]. Pathogens and in ammatory cytokines spread throughout the body along the bloodstream, and severely damage the function of various organs [16]. In its advanced stages, multiple organ dysfunction usually occurs and individual death ensues [17]. Modern medicine has made great progress in the treatment of sepsis. Nevertheless, it still kills a large number of patients each year [18]. Exploring its mechanism does favor to further understand this disease and nd more effective treatment.
In sepsis, vascular dysfunction leads to hemodynamic instability and decreases circulating blood volume, and severe hypotension and even shock occur [19]. The blood stream is unable to bring the oxygen, nutrients and therapeutic drugs to the organs, and the patient's chances of survival are slim [20]. In addition, the blood vessels are ubiquitous in the body, and they are not only the channels for transporting blood, but also important components of each organ [21]. Thus, protecting vascular function in sepsis is of great signi cance.
Endothelium is attached to the inner surface of blood vessels and plays an important role in regulation of vascular function [22]. It regulates tension of blood vessel, prevents too much uid from leaking from blood into tissues, and reduces in ammation [23]. Endothelial nitric oxide synthase (eNOS) and Vascular endothelial cadherin (VE-Cadherin) directly affect endothelial cell function. eNOS is an important enzyme catalyzing nitric oxide synthesis and plays an important role in vasodilation regulation [24]. VE-Cadherin promotes the endothelial cell-to-cell adhesion and maintains endothelial integrity [25]. In our study, LPS and CLP decreased the expression of eNOS and VE-Cadherin, suggesting that endothelial function impairment occurred in sepsis.
PCSK9 is involved in lipid metabolism [26]. Clinical studies have proved that the level of PCSK9 in serum of patients with sepsis increased, and PCSK9 is regarded as a biomarker of sepsis [27]. Another studies have shown that the expression level of PCSK9 is positively correlated with the pathological damage of liver and kidney in septic mice [12,28]. In the current study, we found for the rst time that PCSK9 increased in LPS-treated HUVECs and the aortas of CLP-induced septic mice. Subsequently, we found that inhibition of PCSK9 reversed the sepsis-induced decline in eNOS and VE-Cadherin expression in vitro and in vivo. This con rms our hypothesis that the elevation of PCSK9 in sepsis induces endothelial dysfunction.
Toll-like receptor 4 (TLR4) is a vital component of innate immunity system [29]. A number of exogenous and endogenous ligands bind the TLR4 and activate TLR4/MyD88/NF-κB signaling pathway [30]. p65 is an important subunit of NF-κB. Phosphorylation of p65 promotes NF-κB translocation to the nucleus and increases transcription of in ammatory cytokines to induce in ammation [31]. Our previous study has manifested that this pathway involves in septic endothelial dysfunction [32]. Recent studies showed that PCSK9 could activate TLR4 to induce in ammatory responses in atherosclerosis [33]. NLRP3 in ammasomes is a complex composed of NLRP3,ASC and Caspase1. which play a critical role in vascular endothelial function impairment in sepsis [34]. Activation of NF-KB increases the transcription of each component of NLRP3 in ammasomes [35]. Next, stimulated by some substances, NLRP3 was activated and caspase1 is recruited and cleaved into caspase1 p20 which increase the transcription of IL-1and IL-18 [36]. Therefore, we hypothesized that the TLR4/MyD88/NF-κB and NLRP3 pathways is associated with the PCSK9-induced vascular endothelial cells in sepsis. In this study, we found that activation of the TLR4/MyD88/NF-κB and NLRP3 pathways and up-regulated mRNA expression levels of in ammatory cytokines can be reversed by PCSK9 inhibition. However, speci c activation of TLR4 by KLA could abolish the protective effect of PCSK9 inhibition, although PCKS9 expression is still inhibited. Our data suggest that PCSK9 activated the TLR4/MyD88/NF-κB and NLRP3 pathways to induces an in ammatory response that leads to vascular endothelial injury.
Vascular reactivity test and survival study of septic mice can more directly re ect the vascular function and status of the organism. Results from these con rmed our conclusion that inhibiting PCSK9 to prevent the activation of TLR4 in sepsis could improve the vascular function and survival rates of septic mice.
In summary, our study suggests that PCSK9 activates TLR4/MyD88/NF-κB and NLRP3 pathways to trigger in ammatory response and damage vascular endothelial function in sepsis. Inhibition of PCSK9 may have a great potential in clinical treatment. Consent for Publication The manuscript is approved by all authors for publication.

Declarations
Availability of Data and Materials The data and materials used in this study are available from the corresponding author on reasonable request.
Competing Interests The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
Funding This work was supported by the National Natural Science Foundation of China (82070238).