Interleukin-1 Receptor-Associated Kinase 1/4 as a Novel Target for Inhibiting Neointimal Formation After Carotid Balloon Injury.

AIM: Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 play essential roles in the induction of inflammatory gene products. We aimed to investigate the effect of the inhibition of IRAK1 and IRAK4 kinase activities on neointimal formation in rats with carotid artery balloon injuries using the IRAK1/4 inhibitor N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, a cell-permeable benzimidazole compound. METHODS: Wistar rats and vascular smooth muscle cells (VSMCs) isolated from the thoracic aortas were used. Toll-like receptor 4 (TLR4)-mediated nuclear factor kappa B (NFκB) signaling pathway was revealed by microarrays analysis. In addition, the differential expression of the TLR4 pathway genes, including TLR4, IRAK1, IκBα, and interleukin-1ß (IL-1ß), was confirmed by quantitative real-time polymerase chain reaction. Immunohistochemical staining, elastic-van Gieson and Masson staining, 5-ethynyl-2´-deoxyuridine staining, enzyme-linked immunosorbent assay, transwell migration assay and western blotting were also contributed for relevant detection. RESULTS: The expression of TLR4 protein gradually increased at days 1, 3, 7, and 21 after balloon injury compared with the uninjured group. The dual inhibition of IRAK1 and IRAK4 attenuated neointimal formation and fibrotic remodeling after injury in vivo and suppressed VSMC proliferation and migration in vitro. The production of mediators such as tumor necrosis factor-α and IL-1ß in injured arteries were also reduced by the inhibition of IRAK1 and IRAK4. The expression of NFκB p65- and F4/80-positive cells in inhibitor rats were fewer than those in control rats at day 7, while IRAK1 expression was markedly higher at day 3 in inhibitor rats. Furthermore, western blotting analysis revealed that the IRAK1/4 inhibitor suppressed the IRAK1 and IRAK4 kinase activities and the activation of the TLR4-mediated NFκB pathway in vivo and in vitro. CONCLUSIONS: This study suggested that IRAK1/4 could serve as a potential therapeutic target to suppress neointimal formation in carotid arteries after balloon injury through the TLR4/NFκB signaling pathway.

Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2'-deoxyuridine.

BACKGROUND: Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU) incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU) antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima. METHODS: The carotid artery balloon injury model was established in Goto-Kakizaki (GK) and Wistar rats. A Cell-LightTM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA) and p-Akt (Ser473), as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells. RESULTS: At day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473) and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats. CONCLUSION: By intraperitoneal injections of EdU at a dose of 100 mg/kg three times, EdU incorporation can detect carotid arterial DNA synthesis caused by neointimal formation in GK rats and Wistar rats at day 7 after balloon injury by the EdU click reaction quickly and effectively. Moreover, more obvious DNA synthesis in the vascular neointima could be observed in GK rats than in Wistar rats.

Inhibition on the production of collagen type I, III of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid.

AIM: To investigate the inhibition effects on the production of collagen type I, III secreted by activated rat hepatic stellate cells (rHSCs) by antisense tissue inhibitors of metalloproteinase 1 (TIMP-1) recombinant plasmid through elevating interstitial collagenase activity. METHODS: rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN) separately. Cells were selected after growing in DMEM containing 400 microg/ml G418 for 2-3 weeks. Expression of exogenous gene was assessed by Northern blot, and expression of TIMP-1 in rHSCs was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate. Ultimately, we quantified the type I, III collagen by Western blot. RESULTS: The exogenous antisense TIMP-1 recombinant plasmid could be expressed in rHSCs well, which could block the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNA level (P<0.05); the ratio of TIMP-1/beta-actin was 0.31, 0.98 and 1.32 separately at protein level (P<0.05); It might elevate active and latent interstitial collagenase activity, the collagenase activity was 0.3049, 0.1411 and 0.1196 respectively. (P<0.05), which led to promotion the degradation of type I, III collagen, the ratio of collagen I/beta-actin was 0.63, 1.78 and 1.92 separately (P<0.05); and the ratio of collagen III/beta-actin was 0.59, 1.81 and 1.98 separately (P<0.05). CONCLUSION: These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on the production of type I, III collagens secreted by activated rHSCs in vitro. It could be a novel method to reverse hepatic fibrosis in the future.