MiR-15b-5p and PCSK9 inhibition reduces lipopolysaccharide-induced endothelial dysfunction by targeting SIRT4

Background Endothelial dysfunction and deregulated microRNAs (miRNAs) participate in the development of sepsis and are associated with septic organ failure and death. Here, we explored the role of miR-15b-5p on inflammatory pathways in lipopolysaccharide (LPS)-treated human endothelial cells, HUVEC and TeloHAEC. Methods The miR-15b-5p levels were evaluated in LPS-stimulated HUVEC and TeloHAEC cells by quantitative real-time PCR (qRT–PCR). Functional experiments using cell counting kit-8 (CCK-8), transfection with antagomir, and enzyme-linked immunosorbent assays (ELISA) were conducted, along with investigation of pyroptosis, apoptosis, autophagy, and mitochondrial reactive oxygen species (ROS) by cytofluorometric analysis and verified by fluorescence microscopy. Sirtuin 4 (SIRT4) levels were detected by ELISA and immunoblotting, while proprotein convertase subtilisin-kexin type 9 (PCSK9) expression was determined by flow cytometry (FACS) and immunofluorescence analyses. Dual-luciferase reporter evaluation was performed to confirm the miR-15b-5p–SIRT4 interaction. Results The results showed a correlation among miR-15b-5p, PCSK9, and SIRT4 levels in septic HUVEC and TeloHAEC. Inhibition of miR-15b-5p upregulated SIRT4 content, alleviated sepsis-related inflammatory pathways, attenuated mitochondrial stress, and prevented apoptosis, pyroptosis, and autophagic mechanisms. Finally, a PCSK9 inhibitor (i-PCSK9) was used to analyze the involvement of PCSK9 in septic endothelial injury. i-PCSK9 treatment increased SIRT4 protein levels, opposed the septic inflammatory cascade leading to pyroptosis and autophagy, and strengthened the protective role of miR-15b-5p inhibition. Increased luciferase signal validated the miR-15b-5p–SIRT4 binding. Conclusions Our in vitro findings suggested the miR-15b-5p–SIRT4 axis as a suitable target for LPS-induced inflammatory pathways occurring in sepsis, and provide additional knowledge on the beneficial effect of i-PCSK9 in preventing vascular damage by targeting SIRT4. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s11658-023-00482-5.


Background
Endothelial dysfunction, a critical hallmark of vascular homeostasis deregulation and cardiovascular disease pathogenesis [1,2], is related to sepsis, a severe condition leading to irreversible acute inflammatory response, multiple organ dysfunction, poor prognoses, susceptibility to secondary infections, and mortality [2][3][4].Although current evidence revealed that endothelium protection is functional in sepsis treatment [5], the detailed mechanisms of septic endothelial dysfunction are not fully understood.The endotoxin or lipopolysaccharide (LPS) is a potent proinflammatory element associated to endothelial cell (EC) injury and apoptosis [5,6].LPS elicits multiple endothelial responses, including the release of tissue factor, cytokines, and adhesion molecules, as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), interleukin-1β (IL-1β), IL-18, and monocyte chemoattractant protein-1 (MCP-1) [7].In EC, LPS increases mitochondrial reactive oxygen species (ROS) as well as Lectin-like ox-LDL receptor-1 (LOX-1) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression [8], belonging to the proprotein convertase family, regulating low-density lipoprotein (LDL) and cholesterol metabolism [9].PCSK9, the attractive molecular target for controlling and decreasing the risk of cardiovascular diseases [10], is involved in the evolution of atherosclerotic plaques, while the PCSK9 inhibitor (i-PCSK9), evolocumab, displays beneficial effects in vascular function [11].To date, the action of PSCK9 in sepsis has not been established [12].Indeed, PCSK9 has been reported as a valuable therapeutic target for sepsis [13].Furthermore, its loss in LPS-exposed EC exacerbates the proinflammatory response and increases LPS uptake, thus indicating that PCSK9 repression may counteract the therapeutic action of PCSK9 inhibition [14].
Evidence on the pathogenesis of endothelial dysfunction and sepsis revealed the prognostic and diagnostic potentials of microRNAs (miRNAs) [15][16][17][18][19], short noncoding single-stranded RNAs, with posttranscriptive gene silencing features arising by interaction with the 3′-untranslated region (3′-UTR) of target mRNAs [20,21].EC dysfunction and miRNAs represent key targets for the septic inflammatory process.Therefore, it is reasonable that miRNA regulation could represent a useful therapeutic tool for sepsis-induced EC damage.
Among the miR-15 family, miR-16 has been shown as vital player in sepsis [27][28][29].Deprivation of miR-16 notably reduces the mortality rates in different septic models [30,31], showing a positive correlation between miR-16 levels in serum and death in septic patients [32].It has been reported that miR-195 is able to promote apoptotic mechanism by targeting the SIRT1/eukaryotic translation initiation factor 2A (eIF2a) in intestinal epithelial cells infected with LPS, and increased miR-195-5p levels were also assessed in both human small airway epithelial cells exposed to LPS and in lung tissues from septic rats [33,34].MiR-15a-5p antagomiR denies the inflammatory pathway in both macrophages and septic mouse models via nuclear factor kappa B (NF-κB) signaling inhibition and targeting TNF-α induced protein 3 (NFAIP3)-interacting protein 2 (TNIP2) [35].MiR-15a and miR-16 expression are improved in the blood of septic newborn subjects and inhibit the LPS-promoted inflammation [36], and miR-15b drives the onset and evolution of sepsis in animal models [37].Research shows that miR-15b-5p expression predicts COVID-19 severity and it is involved in mammalian target of rapamycin (mTOR) signaling pathway in sepsis-induced acute kidney injury [37,38].However, the mechanistic role of miR-15b in septic EC has not yet been clearly explained.
Emerging evidence supports the role of sirtuins (SIRT1-7) in the progression and prognosis of sepsis acting on epigenetic profile [39][40][41][42], even if the involvement of SIRT4 in endothelial septic disorder is widely debated.Of note, SIRT4 emerges as a physiological player leading to hypo-inflammation and promoting sepsis recovery [43].SIRT4silenced EC showed an exacerbated inflammatory stress, while its upregulation reduces LPS-related inflammation [44].Therefore, SIRT4 might represent a promising candidate to counteract the in vitro endotoxin-mediated cytotoxicity.
Here, we provide the in vitro miR-15b-5p implication in the EC inflammatory response during sepsis and the possible relationship with SIRT4.In detail, we investigated the molecular mechanism(s) of EC dysfunction occurring under LPS exposure and the ability of miR-15b-5p inhibition (i-miR-15b) to oppose the LPS-derived pro-inflammatory and pro-autophagic phenomena by modulating SIRT4 and PCSK9 levels.In this context, an expanding body of research has revealed the strong interplay between PCSK9 and miRNA [45][46][47][48][49]. Therefore, given that an interesting relationship also exists between i-PCSK9 and endothelial function [11], we examined its ability in LPS-induced EC inflammation, thus contributing to the design of new approaches for sepsis treatment.

Viability and cytotoxicity detection
Cell viability and cytotoxicity assays were assessed as previously described [11,50], following the instructions of the manufacturer.The lactate dehydrogenase (LDH) release (%) in culture media was used to determine cytotoxicity.Data are from n = 3 independent experiments.

Nitric oxide (NO) levels
The amount of NO was assessed by Nitric Oxide assay kit, as previously described [50].The NO levels were estimated by interpolating the sample OD with the standard curve and reported as µM.
The 450 nm absorbance was detected with a reader (Bio-Rad, Hercules, CA, USA) and levels of cytokines, caspase-4, SIRT4, PCSK9, and LOX-1 in the samples were calculated using standard curve.

Cell death detection
The different mechanisms of cell death as pyroptosis, lysosome accumulation, autophagy, and apoptosis were evaluated by fluorescent image acquisition and flow cytometry analyses, as previously described [11,50,51].

Confocal laser scanning microscopy
Immunofluorescence detection of PCSK9 was assessed in EC as previously reported [52].Images were obtained with a LSM 700 confocal microscope while the analysis was estimated with ImageJ 1.52n software.

Statistical analysis
Statistical analyses were carried by GraphPad Prism software (La Jolla, CA, USA), version 9.1.2,and the results are shown as the mean ± standard deviation (SD) of three independent experiments.To determine statistically significant differences, Student's t-tests were used between two groups, and one-way ANOVAs for more than two groups.Differences with p < 0.05 were evaluated as statistically significant.

LPS effects on PCSK9 levels
Given the crucial role of PCSK9 in EC dysfunction and vascular diseases, the effects of LPS on PCSK9 expression have been investigated.Treatment with endotoxin led to PCSK9 accumulation in EC lines, as assessed by different experimental approaches (Fig. 2 and Additional file 1: Fig. S2).In detail, treated EC showed upregulated PCSK9 content in culture media (p < 0.01), mRNA (p < 0.05), and protein expression (p < 0.01) (Fig. 2 and Additional file 1: Fig. S2).The immunoblotting data were confirmed by intracellular PCSK9 detection through FACS evaluation and immunofluorescent analysis (p < 0.001) (Fig. 2D-F and Additional file 1: Fig. S2).

LPS triggered cell death
The ascertained LPS-mediated inflammatory cascade prompted us to investigate the occurrence of programmed cell death mechanisms, as apoptosis and pyroptosis, and their association with the autophagic phenomenon.Exposure to LPS led to the induction of pyroptosis and inflammasome formation, as confirmed by increased NLRP3 protein levels (p < 0.01) (Fig. 3A-C and Additional file 1: Fig. S3).Treatment with endotoxin also induced lysosome accrual and autophagic flux, accompanied by extended pro-autophagic LC3B II/I ratio in TeloHAEC cells (p < 0.001) (Fig. 3D-H and Additional file 1: Fig. S3).The activation of these mechanisms was accompanied by LPS-promoted mitochondrial ROS production (p < 0.01) and apoptotic cell death (p < 0.001) (Fig. 3I-K and Additional file 1: Fig. S3).

Discussion
The above results represent the first evidence of the role of miR-15b-5p in the endothelial impairment caused by sepsis.We found that during sepsis, the elevated miR-15b-5p levels were related to vascular endothelial cell damage, robust inflammation, activation of the NLRP3 pathway, increased PCSK9 levels, and downregulation of SIRT4.Our data suggest that miR-15b-5p, by targeting SIRT4, could represent a potential target in septic EC and advanced comprehension on the pleiotropic beneficial effects of i-PCSK9, able to ameliorate the endothelial inflammatory response during LPS treatment.
Sepsis is a complex pathophysiology with features of excessive inflammatory response leading to endothelial impairment [53].Accordingly, our data show that the LPS displayed cytotoxicity in EC by increasing LDH content and NO levels, and promoting the massive release of inflammatory modulators as cytokines VCAM1, ICAM1, and MCP-1.The resulting systemic inflammation increases miR-15b-5p levels, as well as the expression of PCSK9, while a notable downregulation of SIRT4 protein levels was determined.In the sepsis-induced inflammatory mechanisms, evidence describes that miRNA deregulation might be related to clinical symptoms and disease severity.
Clinical studies have reported that patients with sepsis had increased sera levels of PCSK9, indicating PCSK9 as a biomarker of septic disorder [67], while other studies showed that PCSK9 content is positively associated with liver and kidney damage in septic mouse models [68].Our study showed that PCSK9 is upregulated in EC during in vitro sepsis condition, while its pharmacological inhibition reduces vascular inflammation and improves endothelial function.Recent studies have described the ability of PCSK9 inhibitors, potent lipid-lowering agents, in decreasing the risk of sepsis [69][70][71].Our previous data have shown that PCSK9 caused inflammatory stress and endothelial dysfunction, and that the pleiotropic protective effects of i-PCSK9 in EC might be mediated, at least in part, by NAD-dependent deacetylase SIRT3 [11].Here, we substantiate previous data by showing that i-PCSK9 opposed the LPS-related inflammatory cascade, promoted SIRT4 upregulation, counteracted pyroptosis and autophagic mechanisms, and enhanced the positive action of miR-15b-5p repression on septic EC.Reports on the role of SIRT4 during sepsis are scarce.SIRT4 overexpression inhibits the mitochondrial metabolism, inflammation, and degranulation in LPS-stimulated mast cells, while the endotoxin triggers mitochondrial impairment via SIRT4 inhibition in LC-540 Leydig cells [72,73].Of note, both PCSK9 and miR-15b-5p repression exerted modulatory action on SIRT4 protein levels, counteracting the LPS-induced depletion, suggesting mitochondrial SIRT4 as a promising target in the septic phenotype.The functional effects mediated by miR-15b-5p can be attributed, at least partly, to its capacity to modulate SIRT4 expression.However, the role of SIRT4 in damaged endothelium and its involvement in septic disease require further investigations.The limitations of our study are due to the experiments based on in vitro cell line model.The use of animal models should be used to further understand the pathogenesis of sepsis.Among these models, cecal ligation and puncture in rodents may contribute to the definition of mechanistic roles of miR-15b in the regulation of EC pathophysiological responses associated with sepsis, as well as to clarify the expression modulation of sepsis-related miRNAs in EC [29,74].Following this experimental strategy, Wu et al. identified a panel of sepsisinduced miRNAs and their transcription regulators by array and immunoprecipitation analyses performed on blood samples from septic mice that had undergone cecal ligation and puncture [74].The use of genomic and sequencing approaches could enhance comprehension on the intricate networks regulating the gene expression mediated by miR-15b in septic EC.Because miR-15b sharing the same cluster could emphasize its functional cooperation on LPS-mediated endothelial dysfunction, further investigations are needed to understand the contributing role of miR-15/16 family members in the development and progression of human septic conditions.In this scenario, it has to be taken into account the fundamental regulatory role of other noncoding RNAs, as long noncoding RNAs (lncRNAs), able to negatively modulate miRNA functions.Indeed, additional studies are undoubtedly necessary to expand the cell-based results with a mechanistic approach and translate this notion to human or animal diseases, which may improve knowledge to better understand and treat septic conditions.

Conclusions
The present study provides in vitro evidence on PCSK9 and miR-15b-5p repression as potent antiinflammatory, antipyroptotic, and antiautophagic mechanisms by modulating the mitochondrial SIRT4 protein levels.In summary, miR-15b-5p inhibition opposed the NLRP3 pathways related to inflammatory stress and impaired endothelium during sepsis.Furthermore, suppression of PCSK9 may be a promising tool in septic therapy when miR-15b-5p is silenced.Overall, these results demonstrate that i-PCSK9 protected endothelium in sepsis via SIRT4, indicating this epigenetic modulator as a possible innovative target against endothelial dysfunction induced by sepsis.

Fig. 3
Fig. 3 LPS outcomes on programmed cell mechanisms.A Representative images and B FACS detection of pyroptosis.C Intracellular NLRP3 levels assessed by FACS analysis.Representative images and cytofluorimetric evaluation of D, E lysosomes, F, G autophagy, and H immunoblotting of LC3B II/I.I Representative images and J FACS-detected mitochondrial ROS levels.K Representative annexin V-FITC and PI-staining detected by FACS analysis.Mean ± SD, n = 3. M, molecular weight markers; lane 1, Ctr; lane 2, LPS.Scale bars = 100 μm.‡ p < 0.01 versus Ctr, ¶ p < 0.001 versus Ctr.Statistical analysis of data was performed using Student's t-tests