Abstract
Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH2 and SLIGKV-NH2, respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.
Highlights
IntroductionDigest and promote extracellular killing of invading microorganisms
Human neutrophils engulf, digest and promote extracellular killing of invading microorganisms
Elastase-and PR3induced von Willebrand Factor (vWF) release was abolished in the presence of the serine protease inhibitor, alpha-1 anti-trypsin (Fig. S2) indicating that serine protease induced vWF is solely dependent on proteolytic activity
Summary
Digest and promote extracellular killing of invading microorganisms This function is aided by the release of the serine proteases, PR3 and elastase, and by the formation of serine protease-containing neutrophil extracellular traps (NETS) [1]. Infusion of neutrophil serine proteases, such as elastase, through renal arteries leads to localization of the enzyme on the glomerular capillaries and transient proteinuria [8] Both PR3 and elastase have been implicated in the glomerular endothelial cell (GEC) activation/injury that occurs during vasculitic glomerulonephritis. In-vitro treatment of endothelial cells with serine proteases (1–5 mg/ml) has been shown to induce a behavioral shift towards to a more pro-adhesive and proinflammatory phenotype within endothelial cells and HUVEC [10] Taken together, these findings suggest a direct link between serine protease release and renal disease, regulated at the endothelial level
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