The Role Of Pattern Recognition Receptors In Intimal Hyperplasia And Vascular Smooth Muscle Cells Activation

Background and Objective: Vascular intimal hyperplasia is a response of the arterial wall upon a wide range of stimuli. It is also a prominent hallmark of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Activation of vascular smooth muscle cells (VSMC), which switch from quiescent state to proliferative phenotype, migrates into the intima to proliferate and produce extracellular matrix proteins, is a key event in intimal hyperplasia in response to inflammation or mechanical injury. More and more evidences show that infections and injurious stimuli play an important role in the activation of VSMC and intimal hyperplasia by triggering innate immune responses and promoting inflammation in the vessel wall. Yet, underlying molecular bases remain undefined.Pattern recognition receptors (PRRs) represent a diverse collection of germline-encoded receptors responsible for detecting danger signals by sensing pathogen-associated molecular patterns (PAMPs), and together with other immune components they are involved in the first line of defence. Two families of PRRs, toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) receptors have recently been identified and implicated in inflammatory diseases. Among them, TLRs are expressed on the surface of cells and responsible for detecting extracelluar PAMPs. To avoid recognition and escape from defense, bacteria have developed strategies for invading and crossing the epithelium. Upon arrival at the subepithelial space, bacteria encounter locally resident, as well as newly infiltrated, professional phagocytic cells. However, bacteria use a variety of strategies to avoid engulfment and degradation by the endolysosomal compartment by phagocytes. NOD receptors have the similar function of TLRs, which are cytoplasmic proteins, involved in intracellular bacterial detection, representing a means of cytosolic surveillance.Both TLRs and NOD receptors have a leucine rich repeat domain, which is involved in specific PAMPs recognition. Activation of TLRs and NOD receptors can initiate downstream cascade reaction of adapter molecules, which eventually lead the activation of nuclear factor-κB (NF-κB) and initiate the transcription of inflammatory cytokines and other host response elements, play a key role in the clearance of infecting pathogens. NF-κB signal transduction pathway play a central role in regulating VSMC proliferation, differentiation and apoptosis by inducing the expression of many kinds of proinflammatory cytokines. Quibus we suppose the signal pathway mediated by TLR or NOD receptors may play an important role in the VSMC activation and intimal hyperplasia.To explore the role of PRRs in the VSMC activation and intimal hyperplasia, and provide a novel insight for preventing atherosclerosis and restenosis, we explore the expression of TLR4/NF-κB and NOD1 signal pathway during the progression of intimal hyperplasia by a well established animal model of restenosis of rat balloon injury of the common carotid artery. We also observe the expression and function of NOD1 in the rat intimal smooth muscle cells (iSMC), and the NOD1 functions difference in iSMC and medial SMC (mSMC). We further investigate the expression of NOD1 and NOD2 in human VSMC, and the role of NOD1 and NOD2 mediated innate immune signaling pathway in the activation VSMC. We also explore the possible interaction of NOD2 signal pathways with TLR2 and 4 signal pathways in the induction of proinflammatory cytokines in VSMC.Methods and Results: Part one: We use immunohistochemistry, Western blotting and semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) to detect the expression of TLR4 and NF-κB on rat carotid arteries following angioplastic injury. We found the level of TLR4 both in mRNA and protein was increase following the neointimal formation. The expression of NF-κB was also up-regulated with intima hyperplasia.Part two: Using the methods of immunohistochemistry and semiquantitative RT-PCR, we found that expression of human homologue of NOD1 gene was highly up-regulated with the development of intima after angioplastic injury to rat carotid artery, and predominantly localized in intimal lesion. Studies using isolated iSMC showed that stimulation of NOD1 by its ligand diaminopimalic acid (DAP) led to marked induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in time-depend and dose-depend manner. Stimulation of NOD1 also led to a transient up-regulation of tumor necrosis factor-α?TNF-α??in iSMC. Rat iSMCs and mSMC were stimulated with DAP and/or IFN- ?for 24 hours. Expression of NOD1 and receptor-interacting protein 2(RIP2) and iNOS were assessed by Western blot analysis. In spite of comparable levels of NOD1 protein in iSMCs and mSMC under basal condition, activation of RIP2 and expression of iNOS were elicited merely in iSMC subsequent to the stimulation. In addition, a synergistic effect of DAP and IFN- ?was shown in the induction of NO production by iSMC whereas such effect could not be found in mSMC.Part three: (1) Human coronary artery SMCs were in vitro stimulated with NOD1 receptor agonist PGN(10μg/ml)for 0, 3, 6 and 24 hours. The mRNA expression of NOD1 and fibroblast growth factor-2(FGF-2) were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of interleukin-8(IL-8) and TNF-αwas determined by ELISA. We found human SMC constitutively expressed a low level of NOD1 under resting condition. Upon PGN stimulation, the expression of NOD1 mRNA was up-regulated in SMC, from 0.164±0.005 to 0.231±0.027 (p< 0.05). Stimulation of NOD1 also led to a transient up-regulation of FGF-2, which reaches the peak at 6 hour(22.72±0.58), then decrease quickly(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。ELISA showed the concentration in the culture supernatants of IL-8 and TNF-αincreased from 0.12±0.01ng/ml to 2.31±0.11 ng/ml and from 4.22±0.50 pg/ml to 143.11±12.58 pg/ml, respectively (p< 0.01).(2) Human coronary artery SMCs were in vitro stimulated with NOD2 agonist Muramyl dipeptide (MDP), TLR2 agonist Pam3CSK4(PAM3)and TLR4 agonist lipopolysaccharides (LPS) alone or MDP in synergy with either PAM3 or LPS. The mRNA expression of NOD2 and FGF-2 were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of IL-8 and TNF-αwas determined by ELISA. VSMC proliferation viability was analyzed by the MTT assay. MDP can up-regulate the expression of NOD2 mRNA as a time-dependent manner (0h: 0.028±0. 001; 3h: 0.045±0.002; 6h: 0.053±0.002; 24h: 0.162±0.013) in VSMC. It can also up-regulate the expression of FGF-2 mRNA (9.32±0.37 vs control 7.44±0.25; p<0.05) in VSMC, induce the production of IL-8 and TNF-α(2.41±0.22 ng/ml vs control 1.02±0.13 ng/ml; 51.9±4.73 pg/ml vs control 29.4±3.73 pg/ml; respectively, p < 0.05) and increase the proliferation viability of VSMC (0.87±0.05 vs control 0.72±0.02; p<0.05). Additional, MDP can synergy with LPS and PAM3 to increase the proliferation viability of VSMC and induce the production of IL-8 and TNF-α.Conclusion: 1.In the model of vascular restenosis by balloon injury, the levels of TLR4 mRNA and protein of carotid arteries were significantly increased and companied with activation of NF-κB. It implied that TLR4/ NF-κB pathway may play an important role in the developing of restenosis. 2. NOD1 is also expressed in rat tissue and exhibits highly homology with the human NOD1. Rat NOD1 has an increased expression during intimal hyperplasia in a rat carotid artery injury model and preferentially expressed by iSMCs. Activation of NOD1 can robustly induce the expression of iNOS and TNF-α, which regulated by NF-κB signal pathway. It implied that NOD1 is an intimal specific innate immune response mediator. The functional difference of NOD1 in iSMC and mSMC maybe due to the difference of their downstream mediator. The mSMC lacks RIP2 protein, a key component of NOD1 signal pathway.3. The activation of NOD1 mediated innate immune signaling pathway can induce the production of proinflammatory cytokines such as IL-8 and TNF-αin VSMC. It can also induce the expression of FGF-2. This may represent a novel innate immune activation pathway in vascular inflammation and intimal formation.4. The activation of NOD2 mediated innate immune signaling pathway can increase the proliferation viability of VSMC and induce the production of proinflammatory cytokines in VSMC. It had a synergistic effect with those of TLR2 and TLR4 mediated signaling pathways in this process.