Albumin leak across human pulmonary microvascular vs. umbilical vein endothelial cells under septic conditions.
Human pulmonary microvascular endothelial cell (HPMVEC) injury is central to the pathophysiology of human lung injury. However, septic HPMVEC barrier dysfunction and the contribution of neutrophils have not been directly addressed in vitro. Instead, human EC responses are often extrapolated from studies of human umbilical vein EC (HUVEC). We hypothesized that HUVEC was not a good model for investigating HPMVEC barrier function under septic conditions. HPMVEC was isolated from lung tissue resected from lung cancer patients using magnetic bead-bound anti-PECAM-1 antibody. In confluent monolayers in 3-mum cell-culture inserts, we assessed trans-EC Evans-Blue (EB)-conjugated albumin leak under basal, unstimulated conditions and following stimulation with either lipopolysaccharide or a mixture of equal concentrations of TNF-alpha, IL-1beta and IFN-gamma (cytomix). Basal EB-albumin leak was significantly lower across HPMVEC than HUVEC (0.64 +/- 0.06% vs. 1.13 +/- 0.10%, respectively, P < 0.001). Lipopolysaccharide and cytomix increased leak across both HPMVEC and HUVEC in a dose-dependent manner, with a similar increase relative to basal leak in both cell types. The presence of neutrophils markedly and dose-dependently enhanced cytomix-induced EB-albumin leak across HPMVEC (P < 0.01), but had no effect on EB-albumin leak across HUVEC. Both cytomix and lipopolysaccharide-induced albumin leak was not associated with a loss of cell viability. In conclusion, HPMVEC barrier dysfunction under septic conditions is dramatically enhanced by neutrophil presence, and HUVEC is not a suitable model for studying HPMVEC septic barrier responses. The direct study of HPMVEC septic responses will lead to a better understanding of human lung injury.
Shelton
Jennifer L
JL
Division of Respirology, Department of Medicine, Centre for Critical Illness Research, Lawson Health Research Institute, London Health Sciences Center, University of Western Ontario, South Street Campus, London, Ontario, Canada.
Wang
Lefeng
L
Cepinskas
Gediminas
G
Sandig
Martin
M
Inculet
Richard
R
McCormack
David G
DG
Mehta
Sanjay
S
D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000098:blood supply* / Q000166:cytology / Q000378:metabolism*; D008833:Microcirculation / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism*
D008070:Lipopolysaccharides; D012709:Serum Albumin; D004492:Edetic Acid