B75-31 Investigating The Mechanistic Role Of Proprotein Convertase Subtilisin-kexin Type 9 In Acute Respiratory Distress Syndrome Through Pulmonary Endothelial And Epithelial Cell Models

Rationale

Proprotein Convertase Subtilisin-Kexin type 9 (PCSK9) is a key regulator of lipid metabolism. Recently, a pro-inflammatory role for PCSK9 has been hypothesised, through reduced bacterial phospholipid clearance. Elevated circulating PCSK9 levels in patients with Acute Respiratory Distress Syndrome (ARDS) has been reported in a single cohort, leading to the hypothesis that PCSK9 plays a role in ARDS pathogenesis. This work aimed to explore the expression and pro-inflammatory impact of PCSK9 in alveolar epithelial and endothelial cells as clinically relevant models of ARDS.

Methods

Human primary pulmonary microvascular endothelial (HPMEC) and small airway epithelial (SAEC) cells, as an alveolar epithelial model, were incubated with 10% ARDS patient plasma/bronchoalveolar lavage (BAL) fluid respectively for 6-24hrs. PCSK9 mRNA and protein expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). HPMECs/SAECs were also incubated for 24hrs with 2.5µg/mL PCSK9, followed by quantification of cytokine expression via ELISA and assessment of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation through western blot for phosphorylated-p65. Epithelial sodium channel (ENaC) mRNA was measured via RT-PCR in PCSK9-treated SAECs (2.5µg/mL, 24hrs), and surface expression analysed through western blot on membrane fractions. Air-liquid interface (ALI)-polarised SAECs were also treated with PCSK9 (2.5µg/mL, 24hrs) and assessed for transepithelial electrical resistance (TEER) and ENaC surface expression through confocal microscopy.

Results

PCSK9 mRNA and protein are expressed by HPMECs/SAECs and significantly upregulated upon ARDS plasma (Figure 1A and B, n = 5, p < 0.0001) and BAL fluid stimulation (Figure 1C and D, n = 6, p < 0.0001) respectively. Incubation with PCSK9 significantly increased interleukin-6 (median 153.3 vs 688.8pg/mL) and interleukin-8 (median 695.5 vs 1977pg/mL) secretion compared to controls in SAECs (both n = 6, p < 0.0022), but not HPMECs, and induced p65 phosphorylation. PCSK9 treatment also significantly downregulated ENaC mRNA expression in SAECs (Figure 1E, n = 6, p < 0.0001) and reduced protein levels in membrane fractions (Figure 1F, n = 3). In ALI-polarised SAECs, PCSK9 incubation decreased TEER values significantly (median 700 vs 438Ω*cm2, n = 3, p < 0.0022) and reduced ENaC expression at the apical surface (n = 3).

Conclusions

Distal lung epithelial and endothelial cells significantly increase expression of PCSK9 when stimulated with ARDS BAL fluid/plasma respectively, to mimic the ARDS microenvironment. PCSK9 activates NF-κB in, and increases inflammatory cytokine secretion by SAECs, but not HPMECs. Interestingly, it also reduces ENaC mRNA and protein expression, decreases TEER values and ENaC surface expression in ALI differentiated cells, potentially increasing permeability and reducing alveolar fluid clearance in ARDS.

This abstract is funded by: Department for the Economy Northern Ireland Ph.D. studentship awarded to EC, and an Association of Physicians of GB and Ireland Young Investigator’s award to JS