Respiratory complications are major causes of patient morbidity and mortality. Amongst these, Acute Lung Injury (ALI) and the Acute Respiratory Distress Syndrome (ARDS) remain challenging clinical entities with only supportive therapeutic options. Our previous work with Endothelium-derived microparticles (EMPs) has shown their ability to induce oxidative injury, inflammation, and increase pulmonary capillary permeability with granulocyte cotreatment.
High mobility group box 1 (HMGB-1) is a major histone protein with cytokine-like effects capable of inducing further inflammation. Previous studies have shown that HMGB-1 release leads to increased pulmonary endothelial permeability. The early onset of these changes closely match those seen in EMP-induced ALI. In this study we hypothesize that a functional inhibitor of extracellular HMGB-1, Glycyrrhizic acid, will protect against EMP-induced pulmonary endothelium permeability changes.
60,000 human pulmonary artery endothelial cells (HPAEC, Lonza, Switzerland) were seeded in an 8 chambered slide coated with 1% gelatin and cultured to confluence in 4% FBS medium. EMPs were generated from plasminogen activator inhibitor-1 (PAI-1; 10ng/ml) stimulated human umbilical vein endothelial cells (HUVECs). HL-60 cells (Human Promyelocytic Leukemia) were transformed into mature granuloctyes by incubating them with 1.2% dimethyl sulfoxide (DMSO) for 5-7 days.
Resistance measurements were made in the presence and absence of EMPs (500,000/ml) and HL-60 cells (200,000/mL). Cells were either treated with HMGB-1 (20ug/ml), or Glycyrrhizic acid (1mM) or both in the presence or abscess of EMPs and HL-60s. 2.5U/ml Thrombin (Sigma) was used as positive control. Individual well impedance was measured using Applied Biophysics ECIS Z Theta (electric cell substrate impedance sensing) equipment. Statistical analysis was performed using a two way ANOVA.
Repeated resistance measurements show increased HPAEC permeability in cells co-treated with EMPs and HL-60 cells or with HMGB-1 respectively at 3-4 hours with subsequent plateau (p<.0001). Cells treated with the combination of HMGB-1 and Glycyrrhizic acid show a partial reversal of the permeability changes (p<.001). However, this same reversal was not seen in the EMP/HL-60 treated HPAECs.
Previous studies have shown the important role of HMGB-1 in the inflammatory cascade and its prominent role in human ALI/ARDS. While HMGB-1 induced permeability changes occur in three to four hours after endothelial cell treatment with subsequent plateau and match the pattern of EMP/granulocyte injury, attempts to abrogate this effect using glycyrrhizic acid were unsuccessful. This lack of effect could be due to inadequacy if glycyrrhizic acid to block other B box binding domains (eg RAGE) or HMGB1-independent mechanism. Glycyrrhizic acid, a commercially available direct HMGB-1 inhibitor, only partially protects against HMGB1-induced permeability changes at high doses (1mM). Identifying a more complete inhibitor or receptor blocker is an important next step. EMP-induced granulocyte activation and the role of additional cytokine activation are ongoing areas investigation.