Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype

Supplemental Digital Content is available in the text. Objectives: Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro. Design: Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression. Setting: Research laboratory. Subjects: Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (Cd44+/+/Prg4+/+), Cd44–/– (Cd44tm1HbgPrg4+/+), Prg4GT/GT (Cd44+/+ Prg4tm2Mawa/J), and double knockout (Cd44tm1Hbg Prg4tm2Mawa/J) lung microvascular endothelial cells. Interventions: Cells were treated with 100 or 250 ng/mL lipopolysaccharide-Escherichia coli K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ–Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma. Measurements and Main Results: Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60–73% in human lung microvascular endothelial cells. In Cd44–/– genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that Cd44 is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls. Interpretation and Conclusions: Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.