Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype
Authors: Richendrfer Holly A. , Levy Mitchell M. , Elsaid Khaled A. , Schmidt Tannin A. , Zhang Ling , Cabezas Ralph , Jay Gregory D. 
Published: June 2020

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
–/– (Cd44
tm1Hbg
Prg4
+/+), Prg4
GT/GT (Cd44
+/+
Prg4
tm2Mawa/J), and double knockout (Cd44
tm1Hbg
Prg4
tm2Mawa/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.