Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Pharmacology and Toxicology

Supervisor

Dr Sanjay Mehta

Abstract

Abstract

Background. Neutrophils and nitric oxide (NO) derived from inducible NO synthase (iNOS) contributes importantly to the pathophysiology of acute lung injury (ALI) and pulmonary microvascular endothelial cell (PMVEC) injury. However, the mechanism of neutrophil and neutrophil iNOS dependent PMVEC injury has not been addressed. In our studies, we assessed PMVEC activation under septic conditions, and defined the role of PMVEC vs. bone-marrow polymorphonuclear leukocytes (PMN) iNOS in this septic PMVEC activation.

Methods and Results. We isolated PMVEC from iNOS+/+ and iNOS-/- mice lungs magnetically by microbeads attached to anti-PECAM antibodies, sorted by flow cytometry (FACS) by DiI-acetylated low density lipoprotein (Dil-Ac-LDL) uptake by cells. Bone-marrow PMNs were isolated from femurs and tibia of iNOS+/+ and iNOS-/- mice followed by percoll gradient isolation. Cultured PMVEC monolayers showed that basal E-selectin expression was significantly lower in iNOS-/- vs iNOS+/+ PMVEC (MFI 16±2 vs 59±7, p<0.05). After lipopolysaccharide (LPS) stimulation, E-selectin expression in iNOS+/+ PMVEC increased at 1 hr (MFI 146±15, p<0.01), peaked at 2 hrs (MFI 284±38, p<0.01), and then gradually declined over 12 hrs. iNOS-/- PMVEC had a similar response to iNOS+/+ PMVEC in the timing and magnitude of E-selectin upregulation, with a similar peak expression (MFI 257±29, p=NS vs iNOS+/+). PMVEC responded to cytomix similarly to LPS, with PMVEC E-selectin upregulation in a time dependent and dose dependent manner which was similar in iNOS+/+ and iNOS-/- PMVEC. As compared to E-selectin, basal plasminogen activation inhibitor (PAI)-1 expression was similar in iNOS+/+ and iNOS-/- PMVEC. LPS-induced upregulation of PAI-1 in iNOS+/+ PMVEC peaked at 4 hrs (MFI 3.4±0.1 vs 1.7±0.1 for control medium, p<0.01), and was sustained until 12 hrs and this pattern of LPS-stimulated PAI-1 expression was similar in iNOS-/- PMVEC. In contrast, cytomix induced PAI-1 in iNOS+/+ PMVEC peaked at 2 hrs and was sustained until 8 hrs, and was similar in iNOS-/- PMVEC. The presence of PMN with PMVEC synergistically enhanced LPS induced PMVEC E-selectin expression in a dose dependent manner. However, this LPS-induced PMN-dependent PMVEC E-selectin induction was independent of PMN iNOS. The presence of either iNOS+/+ or iNOS-/- PMN with PMVEC did not upregulate PMVEC E-selectin post cytomix stimulation. Moreover, iNOS+/+ or iNOS-/- PMN did not affect PMVEC PAI-1 protein expression or mRNA expression after either LPS or cytomix treatment.

Conclusions. PMVEC are activated under different septic conditions (both LPS and cytomix) as reflected by increased E-selectin and PAI-1 expression. However, PMVEC iNOS did not affect LPS or cytomix induced PMVEC activation. PMN enhanced septic PMVEC E-selectin expression following LPS treatment, and this endothelial activation was independent of PMN iNOS. These data suggest that septic PMVEC activation in isolation or in the presence of PMN, is independent of both PMVEC and PMN iNOS.