Höfundar:
Sarah McGarrity, Kevin Leahy, Alexía Rós Viktorsdóttir, Adrián López García de Lomana, Arnar Ingi Vilhjálmsson, Óttar Rolfsson, William Oldham
Endothelial cells (ECs) are covered by a proteoglycan rich layer, the endothelial glycocalyx (EGL). The EGL mediates cell interactions, EC nitric oxide (NO) production by nitric oxide synthase 3 (NOS3) is dependent on NOS3 being present near the inside of the cell membrane and may connect with the transmembrane portion of the EGL, NO is a key vaso-relaxant. Inflammation and adrenal hyperactivity adversely affect vascular health and cause shedding of the endothelial glycocalyx. Increased levels of EGL shed into plasma predict poor outcome in sepsis as does altered NOS3 activity.
By stimulating microvascular ECs with TNFalpha and adrenaline then measuring the release of heparan sulfate by ELISA we have shown the effect of these stimuli in vitro. We used colorimetric plate-based enzyme assays, RNAseq data and western blotting to assess the expression and activity of these proteins in parallel samples. We knocked down NOS3 using siRNA. Finally, we have enzymatically removed heparan sulfate from the EGL and then measured enzyme expression and activity.
Microvascular ECs stimulated with TNFalpha showed an initial reduction in NOS activity and then a final increase in NOS activity. TNFalpha and adrenaline stimulation caused an increase in both NOS3 expression at the mRNA level.
Knock-down of NOS3 altered the ICAM response to TNFalpha. Removing EGL from cells altered the activity of NOS.
NOS3 activity in response to inflammatory stimulation is modulated be the make-up of the EGL, which is also altered by TNFalpha stimulation. In the future this may have implications for the treatment of inflammation.
