Related Experiment Video
Updated: Dec 14, 2025
Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis
Published on: December 9, 2022
Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced
Holly A Richendrfer1,2, Mitchell M Levy3, Khaled A Elsaid4
1Department of Emergency Medicine, Warren Alpert School of Medicine, Brown University, Providence, RI.
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.
Insights
Recombinant human proteoglycan-4 effectively reduced interleukin-6 (IL-6) in endothelial cells during sepsis-like conditions. This suggests proteoglycan-4 may be a promising adjunctive therapy for sepsis patients.
Area of Science:
- Biomedical research
- Immunology
- Vascular biology
Background:
- Sepsis is a critical condition with high mortality, characterized by systemic inflammatory response syndrome.
- Targeting toll-like receptors (TLRs) 2 and 4, and CD44 receptors on vascular endothelial cells is a strategy to mitigate sepsis-induced inflammation.
- Proteoglycan-4 (PRG4) interacts with CD44 and TLR4, potentially blocking the NOD-like receptor pyrin domain-containing-3 (NLRP3) inflammasome pathway.
Purpose of the Study:
- To investigate the effect of recombinant human proteoglycan-4 (rhPRG4) on interleukin-6 (IL-6) expression in endothelial cells under sepsis-like conditions.
- To test the hypothesis that rhPRG4 treatment reduces IL-6 expression in endothelial cells induced into a sepsis phenotype in vitro.
Main Methods:
- Utilized human umbilical vascular endothelial cells (HUVECs) and human lung microvascular endothelial cells (HLMECs), as well as wild-type and knockout mouse lung microvascular endothelial cells (MLMECs) with varying CD44 and PRG4 genotypes.
- Induced a sepsis phenotype using lipopolysaccharide (LPS) from Escherichia coli K12 or plasma from sepsis patients.
- Measured IL-6 protein and gene expression via enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), respectively, following rhPRG4 treatment.
Main Results:
- LPS treatment significantly increased IL-6 protein and gene expression in HUVECs, HLMECs, and MLMECs compared to controls.
- rhPRG4 treatment significantly reduced IL-6 levels in both human and mouse endothelial cells.
- rhPRG4 reversed LPS-induced IL-6 expression in 80% of HUVEC sepsis samples and 60-73% of HLMEC sepsis samples; CD44 was not essential for rhPRG4's anti-inflammatory effect in the TLR4 agonist model.
Conclusions:
- Recombinant human proteoglycan-4 demonstrates potential as an adjunctive therapy for sepsis.
- Further in vivo studies are warranted to validate the efficacy of rhPRG4 in sepsis treatment.
