Interleukin-1 Receptor-Associated Kinase 1/4 as a Novel Target for Inhibiting Neointimal Formation After Carotid Balloon Injury
Cardiovascular System & Cardiology; in-vivo; smooth-muscle-cells; signaling pathways; hyperplasia; NF kappa B; arterial injury; bacterial infections; Carotid artery balloon injury; intimal; irak-4 protein; IRAK1/4; muscle cell; Neointimal formation; NF kappa B; TLR4; toll-like-receptor; toll-like-receptor-4 expression; Vascular smooth
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 (NF kappa B) signaling pathway was revealed by microarrays analysis. In addition, the differential expression of the TLR4 pathway genes, including TLR4, IRAK1, I kappa B alpha, and interleukin-1 beta (IL-1 beta), 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-alpha and IL-1 beta in injured arteries were also reduced by the inhibition of IRAK1 and IRAK4. The expression of NF kappa B 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 NF kappa B 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/NF kappa B signaling pathway.
Bai S R; Li D Y; Zhou Z M; Cao J L; Xu T D; Zhang X T; Wang Y; Guo J S; Zhang Y B
Journal of Atherosclerosis and Thrombosis
2015
2015
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.5551/jat.29421" target="_blank" rel="noreferrer noopener">10.5551/jat.29421</a>
Detecting Dna Synthesis Of Neointimal Formation After Catheter Balloon Injury In Gk And In Wistar Rats: Using 5-ethynyl-2 '-deoxyuridine
alkynes; arterial injury; balloon injury; Cardiovascular System & Cardiology; carotid-artery; Catheter; cell-cycle progression; click chemistry; diabetes-mellitus; diabetes-mellitus; DNA synthesis; EdU; eluting stents; Endocrinology & Metabolism; in-vitro; mammalian target; Neointimal formation; PCNA; proliferation; terminal
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-Light (TM) 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.
Guo J S; Li D Y; Bai S R; Xu T D; Zhou Z M; Zhang Y B
Cardiovascular Diabetology
2012
2012-12
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1186/1475-2840-11-150" target="_blank" rel="noreferrer noopener">10.1186/1475-2840-11-150</a>
Affinity Manipulation Of Surface-conjugated Rgd Peptide To Modulate Binding Of Liposomes To Activated Platelets
cyclic RGD; delivery systems; drug-eluting stents; Engineering; insight; integrin alpha(iib)beta(3); liposomes; Materials Science; nanoparticles; peptide modification; platelet-targeting; responses; restenosis; therapy
Platelet adhesion, activation and fibrinogen-mediated aggregation are primary events in vascular thrombosis and occlusion. An injectable delivery system that can carry thrombolytics selectively to the sites of active platelet aggregation has immense potential in minimally invasive targeted therapy of vascular occlusion. To this end we are studying liposomes surface-modified by fibrinogen-mimetic RGD motifs that can selectively target and bind integrin GPIIb-IIIa on activated platelets. Here we report liposome surface-modification with a conformationally constrained high affinity cyclic RGD motif to modulate the GPIIb-IIIa-binding capability of the liposomes. Such affinity enhancement is important for practical in vivo applications to compete with native fibrinogen towards binding GPIIb-IIIa. The platelet-binding of RGD-modified liposomes was studied by fluorescence and scanning electron microscopy, and flow cytometry, in vitro. Binding of RGD-modified liposomes was also tested in vivo in a rat carotid injury model and analyzed ex vivo by fluorescence microscopy. The results from all experiments show that cyclic RGD-liposomes bind activated platelets significantly higher compared to linear RGD-liposomes. Hence, the results establish the feasibility of modulating the platelet-targeting and binding ability of vascularly targeted liposomes by manipulating the affinity of surface-modifying ligands. (c) 2008 Elsevier Ltd. All rights reserved.
Huang G F; Zhou Z M; Srinivasan R; Penn M S; Kottke-Marchant K; Marchant R E; Gupta A S
Biomaterials
2008
2008-04
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1016/j.biomaterials.2007.12.015" target="_blank" rel="noreferrer noopener">10.1016/j.biomaterials.2007.12.015</a>