PAI-1Protects Against Cardiac Fibrosis And Proteinuria Through Vitronectin

Vitronectin-binding PAI-1protects against cardiac fibrosis in uninephrectomy/high salt/AngⅡ miceBackgroundThe profibrotic effects of plasminogen activator inhibitor-1(PAI-1) are postulated reflect decreased extracellular matrix degradation due to decreased plasmin activity. However, recent findings suggest that PAI-1can promote or abate fibrotic processes occuring in different organs. In addition to the protease inhibitroy effect, PAI-1also binds Vitronectin(Vn), a matrix protein that interacts with cell-surface integrins. The study was designed to determine the importance of plasmin vs. vitronectin pathways in angiotensin Ⅱ (AngⅡ)-induced cardiac fibrosis.MethodsUninephrectomized (UNx) mice were fed a high salt diet and infused with Ang II for8weeks. Different human stable PAI-1proteins, including AK (retaining protease inhibitory effects of native PAI-1but not binding to Vn), or1RR (competitive blocker of Vn), or CPAI (control PAI-1, retaining all known functions of native PAI-1), or PBS was injected daily.ResultsIncreased systolic blood pressure and heart weight were found in all mice with Ang II infusion. In the heart, AK injected mice showed significantly more extensive myocardial and interstitial fibrosis compared with the other AngⅡ-infused mice. Ang II increased the level of endogenous total mouse PAI-1in heart tissue, this was further elevated by AK. AngⅡ signifcantly incresed the number of fibroblasts in the heart, this was reverted by1RR. Active plasmin and heart vitronectin expression were not changed by Ang II and were not different among the groups treated with PAI-1proteins.ConclusionPAI-1promotes extracellular matrix accumulation through the protease inhibitory pathway, while protecting against cardiac fibrosis through its binding to vitronectin. PartⅡVitronectin-binding PAI-1protects against cardiac fibrosis through interaction with fibroblastsBackgroundPlasminogen activator inhibitor-1(PAI-1) is actively involved in fibrotic processes of multiple organs. Our previous in vivo studies indicated that the protease inhibitory PAI-1protein increased profibrotic responses in the mouse heart exposed to chronic angiotensin II (All) infusion and uninephrectomy, the vitronectin (Vn)-binding PAI-1variant rather decreased expression of cardiac fibroblast markers. The aim of the present study was to elucidate how Vn-binding and protease inhibitory PAI-1differentially impact cardiac fibroblast functions.MethodsFibroblasts from normal adult human ventricle (NHCF-V) were stimulated with AngⅡ for48hours. One of the human stable PAI-1proteins including, AK (retaining protease inhibitory effects, but not binding to Vn),1RR (binding Vn normally, but no inhibitory activity toward any proteinase), CPAI (retaining all known functions of native PAI-1) was added simultaneously.ResultsProtease inhibitory AK and CPAI proteins increased supernatant fibronectin while decreasing activities of plasminogen activator, plasmin. Vitronectin-binding1RR and CPAI proteins significantly reduced the fibroblast supernatant vitronectin, fibroblast integrin β3, and fibroblast adhesion to vitronectin compared with the non vitronectin-binding AK. Further, vitronectin-binding1RR and CPAI proteins decreased anti-apoptotic and proliferative activities. Compared with AK and CPAI groups,1RR decreased the migration of fibroblasts.ConclusionOur in vitro data showed protective effects of vitronectin-binding PAI-1against Ang Ⅱ-induced cardiac fibrosis. By binding with vitronectin, PAI-1may block integrin αvβ3action, induce apoptosis, and suppress proliferation, adhesion and migration of cardiac fibroblasts. Part ⅢVitronectin-binding PAI-1protects against proteinuria through interaction with podocytesBackgroundGlomerular PAI-1expression increases in chronic kidney disease, which accompanies more matrix and macrophage infiltration. It has been found that PAI-1plays different roles in glomerulonephropathy through plasmin suppression vs. vitronectin-binding pathways. Vitronectin-binding PAI-1may have direct cell affection. The aim of this study is to investigate the mechanism of PAI-1on proteinuria and podocyte by using mutant human PAI-1variants.MethodsUninephrectomized mice fed a high salt diet and infused with angiotensin II were followed for8weeks. All mice were injected with different stable PAI-1variants by i.p, and divided into five groups:AK group, injected with AK which retains protease inhibitory effects of native PAI-1but does not binds vitronectin.1RR group, injected with1RR which binds vitronectin but does not inhibits plasminogen activator. CPAI group, injected with CPAI which retains all functions of native PAI-1. AngⅡ group, which was injected with PBS. Control group, uninephrectomized mice were fed a high salt diet. Also primary cultured podocyte were injured by puromycin, added by different stable PAI-1variants or PBS, and followed for48hours.ResultsIncreased systolic blood pressure and proteinuria were found in all mice with Ang II infusion.1RR and CAPI decreased proteinuria without BP modification. There were no differences in endogenous PAI-1expression among groups. Only the vitronectin-binding PAI-1variants, CPAI and1RR, resulted in human PAI-1deposition along glomerular basement membranes, while stasis in microcirculation was found in AK group. In parallel, CPAI and1RR maintained synaptopodin and decreased desmin on podocytes, while AK and PBS did not. In vitro study showed that anti-apoptosis, maintained cytoskeleton and podocyte differentiation marker, and decreased migration in1RR and CAPI treated podocytes.ConclusionIn the Unx/Salt/AngⅡ induced renal injury model, PAI-1attenuated podocyte injuy, decreased proteinuria through its Vn-binding pathway.