Interleukin-1/Toll-Like Receptor-Induced Nuclear Factor Kappa B Signaling Participates in Intima Hyperplasia after Carotid Artery Balloon Injury in Goto-Kakizaki Rats: A Potential Target Therapy Pathway

<div><p>The value of restenosis after percutaneous coronary intervention (PCI) is recognized worldwide, especially for diabetic patients. Interleukin-1/Toll-like receptor (IL-1/TLR) signaling is involved in innate and adaptive immune responses, but whether and how the IL-1/TLR-induced nuclear factor kappa B (NFκB) pathway plays key roles in intimal formation is unclear. The underlying mechanism of intima hyperplasia was investigated with a model of carotid balloon injury in Goto-Kakizaki (GK) and Wistar rats and with lipopolysaccharide-stimulated macrophages. Elastic-van Gieson staining showed the medial area peakedon Day 3 post-injury and decreased by Day 7 post-injury in both GK and Wistar rats. The N/M at Day 7 in GK rats was significantly higher than in Wistar rats (p<0.001). The percent of 5-ethynyl-2′-deoxyuridine (EdU) staining-positive cells on Day 3 post-injury was greater than seen on Day 7 post-injury in GK and Wistar rats. The percent of EdU-positive cells on Days 3 and 7 post-injury in Wistar rats was less than that found in GK rats (p<0.01; p<0.05). NFκBp65 immunostaining had increased by Day 7 post-injury. Agilent Whole Genome Oligo Microarray verified that the IL-1/TLR-induced NFκB pathway was activated by carotid balloon injury. TLR4, IL-1 receptor associated kinase, inhibitors α of NFκB, human antigen R, c-Myc (Proto-Oncogene Proteins), EGF-like module-containing mucin-like hormone receptor-like 1 and Interleukin-6 were up-regulated or down-regulated according to immunochemistry, quantitative real-time PCR, Western blotting and Enzyme linked immunosorbent assay. Overall, we conclude that the IL-1/TLR-induced NFκB pathway participates in the intimal hyperplasia after carotid injury in GK and Wistar rats and that GK rats respond more intensely to the inflammation than Wistar rats.</p></div

qRT-PCR of factors from common carotid arteries in Wistar and GK rats.

<p>mRNA levels of TLR4, IRAK4, HuR, IκBα, EMR1 and c-Myc in Wistar rats and GK rats(A, B, C, D, E, F). Data are represented as mean ± SEM. ^Δindicates versus uninjured p<0.05, ^ΔΔp<0.01, ^ΔΔΔp<0.001; *indicates versus Day 3 post-injury, p<0.05, **p<0.01, ***p<0.001; #indicates versus Day 7 post-injury, p<0.05, ^###p<0.001.</p

Levels of serum GLU, CHOL and TG in GK rats and Wistar rats.

<p>GLU: blood glucose, CHOL: total cholesterol, TG: triglycerides.</p

Immunohistochemistry for the expression of PCNA and NFκBp65 in common carotid arteries.

<p>PCNA-positive cells of uninjured in both rats (A–a, A–b). PCNA-positive cells of Day 3 post-injury (A–c, A–d) were more than those of Day 7 post-injury (A–e, A–f) in both rats and PCNA-positive cells in GK rats were more than those in Wistar rats (B). NFκBp65-positive cells (C) in GK rats were more than those in Wistar rats at Day 7 post-injury. Data are represented as mean ± SEM. ^ΔΔΔindicates versus uninjured, p<0.001; ***indicates versus Day 3 post-injury, p<0.001; ^##indicates versus Day 7 post-injury, p<0.01.</p

Agilent Whole Genome Oligo Microarray of common carotid arteries in Wistar and GK rats.

<p>Through three pairs of comparison (Wistar uninjured versus Wistar Day 7 post-injury (A), GK uninjured versus Day 7 post-injury (B), Wistar Day 7 post-injury versus GK Day 7 post-injury(C)), IL-1/TLR-induced NFκB activation signaling pathway was filtered out.</p

Representative images of EdU staining of common carotid arteries in Wistar and GK rats (400×).

<p>EdU staining (A–a, b, c, d), Hoechst33342 staining (A–e, f, g, h), images of merging EdU staining and Hoechst33342 staining (A–j, k, m, n). Percentage of EdU-positive cells of Day 3 post-injury were more than those of Day 7 post-injury in both rats and percentage of EdU-positive cells in GK rats were more than those in Wistar rats (B). Data are represented as mean ± SEM. **indicates versus Day 3 post-injury, p<0.01, ***p<0.001; ^#indicates versus Day 7 post-injury, p<0.05.</p

Disodium Benzodipyrrole Sulfonate as Neutral Hole-Transporting Materials for Perovskite Solar Cells

Hole-transporting material (HTM) is an indispensable constituent in organic electronic devices, generally comprising a donor/dopant combination. We report that a disodium salt of substituted benzo­[1,2-<i>b</i>:4,5-<i>b</i>′]­dipyrrole bearing two racemic alkanediylsulfonate anion side chains (BDPSOs) serves as a neutral, nonhygroscopic, dopant-free HTM for lead perovskite (MAPbI₃) solar cells. These organic/inorganic hybrid molecules are useful for tunable orbital level and controllable solubility. A fluorinated BDPSO has an energy level matched with MAPbI₃, affording an inverted-structure solar cell that performs with 17.2% efficiency with minimal hysteresis. The solar cell devices fabricated using BDPSOs showed remarkable storage and operational stability

IL-1/TLR-induced NFκB signaling pathway.

<p>After the stimulation, IL-1R/TLR recruits adaptor molecule myeloid differentiation factor 88(MyD88) to their TIR domain, which further recruits and activates IRAK4. Then IRAK4, TRAF6 and IRAKs combine into a complex. After the coalition of Pellino2 and TAK1, the new complex is divided into at least two parts: complex including TAK1, activating NFκB through IκBα phosphorylation and degradation, and complex including IRAK4 which phosphorylates p38 and binds to the ARE-binding proteins like HuR and c-Myc. Two complexes both promote the release of cytokines and chemokines like IL-6 to further promote inflammation response. During the process EMR1 keeps growing.</p

LPS-induced activation of factors in BMM.

<p>Representative western blotting bands of TLR4, IRAK4, IκBα, p-p38, HuR in BMM from Wistar rats and GK rats (<b>A</b>). Bar diagrams depicting the relative protein levels after normalization to β-actin (<b>B, </b><b>C, D, E, F</b>). Changes of IL-6 with the LPS stimulation by ELISA in both rats (<b>G</b>). Data are represented as mean ± SEM. ^Δindicates versus 0 h, p<0.05, ^ΔΔΔp<0.001; <b>*</b>indicates versus 2 h, p<0.05, **p<0.01, ***p<0.001; ^# indicates versus 4 h, p<0.05,^##p<0.01, ^###p<0.001; ^$indicates 12 h, p<0.05, ^$$p<0.01,^$$$ p<0.001; ^☆indicates 24 h, p<0.05, ^☆☆☆p<0.001.</p

EVG staining of common carotid arteries in Wistar and GK rats (100×).

<p>EVG staining of uninjured (A-a, A-b), Day 3 post-injury (A-c, A-d) and Day 7 post-injury (A–e, A–f) in Wistar and GK rats. Neointimal emerged at Day 7 post-injury (B), the media peaked at Day 3 post-injury (C) and N/M ratio in GK rats was higher than that in Wistar rats (D) at Day 7 post-injury. White arrows pointed to the media. Data are represented as mean ± SEM. ^ΔΔΔindicates versus uninjured, p<0.001; *indicates versus Day 3 post-injury, p<0.05, **p<0.01; ^#indicates versus Day 7 post-injury, p<0.05, ^###p<0.001.</p