Crp Promotes Atherosclerosis By Increasing LDL Transcytosis Across Endothelial Cells

Objective:Atherosclerosis (AS) is the pathological basis of various cardio-and cerebrovascular diseases. However, the pathogenesis of AS is far from fully understood. Currently, more and more evidences have demonstrated that the subendothelial retention of apoB100containing lipoproteins (e.g. Low density lipoprotein, LDL etc) is the initial step of atherogenesis, and is usually termed "response to retention hypothesis". The only pathway for the LDL particles to traffic across the intact endothelial barrier is through a transporting process termed transcytosis. Whether or not C-reactive protein (CRP) can directly affect the transcytosis of LDL is not clear. We therefore aim to determine the effect of CRP on the transcytosis of LDL across endothelial cells (ECs) and the underlying mechanisms, and further explore the effect of CRP on the formation of atherosclerotic lesion.Methods:The effects of CRP and inhibitors on the transcytosis of LDL across ECs were investigated by the in-vitro transcytosis model we have established; Lipid rafts (LRs) fractions were isolated by discontinuous sucrose density gradient ultracentrifugation and further analyzed the expression of proteins involved in LDL endocytosis (Caveolin-1, Cavin-1, and Dynamin2) and exocytosis (NSF, a-SNAP, VAMP3/Cellubrevin and Syntaxin4) after incubating with CRP or inhibitors. After incubated with CRP or inhibitors, the levels of intracellular ROS were determined by measuring fluorescence of2’,7’-dichlorofluorescein (DCF-DA); PKC (Protein kinase C, PKC) and Src kinase activity were measured by ELISA kit; meanwhile, LDL uptake by cultured ECs and LDL retention in isolated vessel walls were visualized under confocal microscope. The aortic roots were stained with Oil Red O and plaque size was quantified in ApoE-/-mice; CD154were examined by immunohistochemical analyses.Results:With the established in-vitro transcytosis model, we found that CRP significantly increased the transcytosis of LDL. An NADPH oxidase inhibitor, diphenylene iodonium (DPI), and the disulfide-reducing agent, dithiothreitol (DTT) could partly or completely block the CRP-stimulated increase of LDL transcytosis. Similarly, the effect of CRP was inhibited not only by the inhibitors of transcytosis, N-ethylmaleimide (NEM) and Methyl-β-cyclodextrin (MβCD), but also by protein kinase C (PKC) inhibitor, bisindolylmaleimide I (BIM I), and Src kinase inhibitor, PP2. Meantime, these inhibitors blocked the trafficking of the molecules responsible for transcytosis (Caveolin-1、Cavin-1、Dynamin-2、NSF、α-SNAP、VAMP3/Cellubrevin、Syntaxin4). Moreover, exogenous H2O2largely increased LDL transcytosis, which was very similar to the effect of CRP. In addition, CRP significantly upregulated intracellular ROS levels and the activities of PKC and Src kinase in ECs. Confocal imaging analysis revealed that CRP stimulated a significant increase of LDL uptake in ECs and the subendothelial retention of LDL in vessel walls. In contrast, pretreatment with MβCD, NEM, DPI, DTT, BIM I or PP2, significantly diminished CRP-stimulated LDL uptake and retention. In ApoE-/-mice, CRP significantly promoted multiple atherosclerotic lesion formation characterized by the predominance of lipid deposition in the subendothelial space, which were substantially blocked by inhibitors of transcytosis (MβCD、NEM), kinase (BIM I、PP2) and ROS (DTT). Compared to the groups treated with the inhibitors (MβCD、NEM、 DTT、BIM I、PP2), aortic plaques in CRP-treated ApoE-/-mice showed strong CD154staining, which is used as a marker of arterial inflammation and correlates with early Conculsions:In conclusion, the present study demonstrates that CRP is able to increase the LDL transcytosis across ECs, promotes LDL retention in human vascular walls and therefore accelerates the progression of AS. Mechanistically, it might be associated with a ROS→PKC/Src→Caveolae or SNARE signaling pathway in ECs. Objective:Inflammasome, which is a large multiprotein complex in the cytosol and regulates IL-1β production, plays an important role in AS. NLRP3(Nucleotide-binding domain and leucine-rich repeat protein3) inflammasome is the most widely studied inflammasome. What is more, recent studies have identified NLRP3as a predictor of CRP levels. This section aims to investigate the effect of CRP on the activation of the NLRP3inflammasome in ECs and the underlying mechanisms, and further explore the effect of the NLRP3inflammasome on CRP-induced LDL transcytosis across ECs.Methods:Transfecting the small interfering RNA target human NLRP3gene (NLRP3siRNA) into the ECs or pre-treated with Caspase-1inhibitor, and then incubated with CRP, the effect of CRP on NLRP3inflammasome was measured by Western blot. In addition, the effect of NLRP3siRNA and Caspase-1inhibitor on CRP-induced LDL transcytosis was assayed by the in-vitro transcytosis model. By using an ELISA-based transcriptional factor-DNA binding activity assay, the CRP-induced NF-κB activity was analyzed. What is more, the effect of different inhibitors on CRP-stimulated the expression of NLRP3, Pro-caspase-1, Caspase-1, Pro-IL-1β and IL-1β in ECs was also detected by Western blot.Results:The expression of proteins involved in NLRP3inflmmasome signaling passway (NLRP3、Caspase-1、IL-1β) was up-regulated after incubating with CRP, while NLRP3siRNA or Caspase-1inhibitor (Z-VAD-FMK) could significantly decrease CRP-induced activation of NLRP3. Meanwhile, NLRP3siRNA or Z-VAD-FMK could largely block the CRP-induced LDL transcytosis. By using an ELISA-based transcriptional factor-DNA binding activity assay, we found that CRP could significantly activate NF-κB. What is more, FcyR-blocking antibodies (CD32A、CD64Ab)、NF-κB inhibitor (Bay)、ROS inhibitors (DPI、DTT)、Caspase-1inhibitor、P2X7R inhibitor (oATP) and broad spectrum inhibitor of cysteine proteases (E-64d) could attenuate the CRP-induced increased expression of those proteins (NLRP3、Caspase-1、IL-1β).Conculsions:In conclusion, CRP can activate NLRP3inflammasome in ECs. Meantime, NLRP3inflammasome plays an important role in CRP-induced LDL transcytosis across ECs. Contributing to the binding of CRP to CD32and CD64, the upregulation of ROS levels, the activation of NF-κB, the effect of K+efflux as well as the activation of P2X7R, the release of cathepsins, CRP can significantly activate NLRP3inflammasome in ECs.