Hypoxia-induced increase in NO production and eNOS expression by murine pulmonary microvascular endothelial cells

Morphology, function and gene-expression profile of endothelial cells (ECs) are specific for particular organs, vascular beds or microenvironments. ECs lining different segments along the pulmonary arterial bed vary in the expression of surface molecules. Histochemical labeling of living murine lung sections with Bandeiraea simplicifolia lectin revealed a preferential staining of ECs coating the capillary network of alveolar septa. These ECs were isolated employing magnetic beads coated with Bandeiraea simplicifolia lectin, expanded and characterized. After propagation up to passage 20 the ECs remained their capacity to bind Bandeiraea simplicifolia lectin and were able to take up DiI-Ac-LDL, indicating their microvascular origin. Endothelial nitric oxide synthase (eNOS) is widely expressed within endothelial cells (ECs) lining the vasculature and plays a pivotal role in regulation of the vascular tone. NO released by ECs diffuses into the underlying smooth muscle cells causing them to relax. Additionally, NO is related to non-contractile functions such as regulation of gene expression. Immunohistochemical analysis of the isolated microvascular ECs demonstrated a predominantly nuclear localization of eNOS. Exposure to hypoxia (90min) elevated NO production as detected by enhanced DAF-2T fluorescence which also dominated in the nucleus. Increased expression of eNOS mRNA was found after 6h of hypoxia, and enhanced protein levels after 15h of hypoxia. Thus, NO released from pulmonary microvascular ECs appears to be involved in short-term and subacute adaptation of the alveolar region to hypoxia.

Furthermore, the isolated endothelial cells possess the equipment for remodelling of the surrounding extracellular matrix: in the culture supernatants matrix metalloproteinase-2 (MMP-2) activity (gelatine zymography) as well as tissue inhibitors of metalloproteinases 2 and 3 (TIMP-2, -3; Western blot) were detectable.