A potential role of protein extractions from Metagonimus yokogawai in amelionating inflammation in patients with ankylosing spondylitis.

Helminth infections and their components has been recognized to have a positive impact on the immune system. This study aimed to investigate the potential of Metagonimus yokogawai-derived proteins (MYp) to provide protection against ankylosing spondylitis (AS) through modulation of immune responses. The cytotoxicity of MYp at various doses was first assessed using MTS and flow cytometry. Peripheral blood mononuclear cells (PBMCs) were collected from AS patients, and the production of inflammatory cytokines was analyzed through flow cytometry. In the experiments with SKG mice, MYp or vehicle was administered and inflammation was evaluated through immunohistochemistry and enzyme-linked immunosorbent assay. The results showed that MYp did not decrease cell viability of PBMCs even after 48 h. Additionally, the frequencies of IFN-γ and IL-17A producing cells were significantly reduced after MYp treatment in the PBMC cultures. Furthermore, MYp treatment significantly suppressed arthritis and enthesitis in the SKG mouse model. The results suggest the first evidence that MYp can effectively alleviate clinical symptoms and restore cytokine balance in patients with AS.

Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis.

BACKGROUND: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. RESULTS: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. CONCLUSION: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

CCL20 inhibition for treating inflammation in ankylosing spondylitis.

OBJECTIVES: Th17 cells are known to play a significant role in AS. C-C motif chemokine ligand 20 (CCL20) binds to C-C chemokine receptor 6 (CCR6) on Th17 cells, promoting their migration to inflammation sites. The aim of this research is to examine the effectiveness of CCL20 inhibition in treating inflammation in AS. METHODS: Mononuclear cells from peripheral blood (PBMC) and SF (SFMC) were collected from healthy individuals and AS. Flow cytometry was used to analyse cells producing inflammatory cytokines. CCL20 levels were determined using ELISA. The impact of CCL20 on Th17 cell migration was verified using a Trans-well migration assay. The in vivo efficacy of CCL20 inhibition was evaluated using an SKG mouse model. RESULTS: The presence of Th17 cells and CCL20 expressing cells was higher in SFMCs from AS patients compared with their PBMCs. The CCL20 level in AS SF was significantly higher than in OA patients. The percentage of Th17 cells in PBMCs from AS patients increased when exposed to CCL20, whereas the percentage of Th17 cells in SFMCs from AS patients decreased when treated with CCL20 inhibitor. The migration of Th17 cells was found to be influenced by CCL20, and this effect was counteracted by the CCL20 inhibitor. In the SKG mouse model, the use of CCL20 inhibitor significantly reduced joint inflammation. CONCLUSION: This research validates the critical role of CCL20 in AS and suggests that targeting CCL20 inhibition could serve as a novel therapeutic approach for AS treatment.

Breast Tumor Cell-Stimulated Bone Marrow-Derived Mesenchymal Stem Cells Promote the Sprouting Capacity of Endothelial Cells by Promoting VEGF Expression, Mediated in Part through HIF-1α Increase.

Breast tumor cells recruit bone marrow-derived mesenchymal stem cells (BM-MSCs) and alter their cellular characteristics to establish a tumor microenvironment. BM-MSCs enhance tumor angiogenesis through various mechanisms. We investigated the mechanisms by which BM-MSCs promote angiogenesis in response to breast tumor. Conditioned media from MDA-MB-231 (MDA CM) and MCF7 (MCF7 CM) breast tumor cells were used to mimic breast tumor conditions. An in vitro spheroid sprouting assay using human umbilical vein endothelial cells (HUVECs) was conducted to assess the angiogenesis-stimulating potential of BM-MSCs in response to breast tumors. The ROS inhibitor N-acetylcysteine (NAC) and JAK inhibitor ruxolitinib attenuated increased HIF-1α in BM-MSCs in response to MDA CM and MCF7 CM. HIF-1α knockdown or HIF-1ß only partially downregulated VEGF expression and, therefore, the sprouting capacity of HUVECs in response to conditioned media from BM-MSCs treated with MDA CM or MCF7 CM. Inactivation of the VEGF receptor using sorafenib completely inhibited the HUVECs' sprouting. Our results suggest that increased HIF-1α expression under normoxia in BM-MSCs in response to breast tumor cells is mediated by ROS and JAK/Stat3, and that both HIF-1α-dependent and -independent mechanisms increase VEGF expression in BM-MSCs to promote the angiogenic sprouting capacity of endothelial cells in a VEGF-dependent manner.

A Potential Predictive Role of the Scalp Microbiome Profiling in Patients with Alopecia Areata: Staphylococcus caprae, Corynebacterium, and Cutibacterium Species.

Little is known about the scalp bacterial composition of alopecia areata (AA) patients. The aim of this study was to investigate the differences in the scalp microbiome of AA patients according to their prognosis, in addition to healthy controls. A total of 33 AA patients and 12 healthy controls (HC) were included in this study. The microbiomes were characterized by sequencing 16S rRNA genes on the Illumina MiSeq platform. The scalp microbiome was more diverse in AA patients compared to HC, but not significantly different according to the severity of AA. Nevertheless, the higher proportion of Corynebacterium species and the lower proportion of Staphylococcus caprae among the Staphylococcus species were noticed in severe AA patients compared to HC or mild AA. The higher ratio of Cutibacterium species to S. caprae was noticed in severe AA. We highlight the potential predictive role of scalp microbiome profiling to a worse prognosis of patients with alopecia areata.

Bone Marrow-Derived Vasculogenic Mesenchymal Stem Cells Enhance In Vitro Angiogenic Sprouting of Human Umbilical Vein Endothelial Cells.

Vasculogenic properties of bone marrow-derived mesenchymal stem cells (MSCs) have been reported, but it is still unclear whether the vasculogenic properties are restricted to some populations of MSCs or whether the entire population of MSCs has these properties. We cultured two different populations of MSCs in different culture media and their vasculogenic properties were evaluated using In vitro spheroid sprouting assay. Neither population of MSCs expressed markers of endothelial progenitor cells (EPCs), but they were different in the profiling of angiogenic factor expression as well as vasculogenic properties. One population of MSCs expressed basic fibroblast growth factor (bFGF) and another expressed hepatocyte growth factor (HGF). MSCs expressing HGF exhibited In vitro angiogenic sprouting capacity in response to bFGF derived from other MSCs as well as to their autocrine HGF. The vasculogenic mesenchymal stem cells (vMSCs) derived from the bone marrow also enhanced In vitro angiogenic sprouting capacity of human umbilical vein endothelial cells (HUVECs) in an HGF-dependent manner. These results suggest that MSCs exhibit different vasculogenic properties, and vMSCs that are different from EPCs may contribute to neovascularization and could be a promising cellular therapy for cardiovascular diseases.

Investigation of Oxidation Methods for Waste Soy Sauce Treatment.

To obtain a suitable oxidation method for removing the color and lowering the chemical oxygen demand (COD) of waste soy sauce, Fenton (Fe2+), Fenton-like (Fe3+), and ozone (O3) oxidation methods are used as the target reactions. In experimental conditions for Fenton oxidation, the dose of Fe2+ and Fe3+ was varied between 100 mg/L and 300 mg/L. The dose of hydrogen peroxide for the reaction was injected from 100-1000 mg/L. For ozone oxidation, the pH was increased from 3 to 14 and the O3-containing gas was supplied continuously for 30 min through a gas diffuser at the bottom of the reactor at different applied O3 doses (10-90 mg/L). We subjected it to a simple 1:20 dilution with deionized water to identify the comparison result in detail. O3 oxidation shows the highest efficiencies of color removal (81.1%) and COD lowering (64.9%) among the three oxidation methods. This is mainly due to the fact that it has a relatively large amount of hydroxyl radical, resulting in the degradation of organics. Thus, O3 oxidation could be a promising method for removing the color and lowering the COD of waste soy sauce. The critical parameters (pH and applied O3 dose) were varied systematically to optimize O3 oxidation. It was found that the optimum pH and applied O3 dose are 11.0 mg/L and 50.0 mg/L, respectively (color removal = 34.2%, COD removal = 27.4%).

Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3.

Enzymatic conversion of natural glycosides to their corresponding hydroxylated products using cytochromes P450 has significant advantages over synthetic chemistry and even enzyme-catalyzed glycosylation of chemicals. At present, the basic strategy for making glycosides of stilbenoid compounds is to use the glycosylation activity of enzymes, such as glycosyltransferases. Here, an efficient synthesis of a valuable (E)-astringin, a piceatannol glucoside, was developed using CYP102A1 via the highly regioselective C-3' hydroxylation of polydatin, a resveratrol glucoside. (E)-astringin is a high added value compound found in plants and wine. Benzylic hydroxylation of polydatin provides an attractive route to (E)-astringin, a catechol product. Thus far, chemical and enzymatic methods of producing (E)-astringin have not been developed. In the present study, a set of CYP102A1 mutants from Bacillus megaterium was found to catalyze regioselective hydroxylation of polydatin at the C-3' position to generate an (E)-astringin, a piceatannol glucoside.

Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry.

Multiple crystal structures of CYP2B4 have demonstrated the binding of the detergent 5-cyclohexyl-1-pentyl-ß-D-maltoside (CYMAL-5) in a peripheral pocket located adjacent to the active site. To explore the consequences of detergent binding, X-ray crystal structures of the peripheral pocket mutant CYP2B4 F202W were solved in the presence of hexaethylene glycol monooctyl ether (C8E6) and CYMAL-5. The structure in the presence of CYMAL-5 illustrated a closed conformation indistinguishable from the previously solved wild-type. In contrast, the F202W structure in the presence of C8E6 revealed a detergent molecule that coordinated the heme-iron and extended to the protein surface through the substrate access channel 2f. Despite the overall structural similarity of these detergent complexes, remarkable differences were observed in the A, A', and H helices, the F-G cassette, the C-D and ß4 loop region. Hydrogen-deuterium exchange mass spectrometry (DXMS) was employed to probe these differences and to test the effect of detergents in solution. The presence of either detergent increased the H/D exchange rate across the plastic regions, and the results obtained by DXMS in solution were consistent in general with the relevant structural snapshots. The study provides insight into effect of detergent binding and the interpretation of associated conformational dynamics of CYP2B4.

Effect of onion peel extract on endothelial function and endothelial progenitor cells in overweight and obese individuals.

OBJECTIVES: Acute or chronic intake of polyphenol-rich foods has been reported to improve endothelial function. Quercetin, found abundantly in onion, is a potent antioxidant flavonoid. The aim of this study was to investigate whether consumption of onion peel extract (OPE) improves endothelial function in healthy overweight and obese individuals. METHODS: This was a randomized double-blind, placebo-controlled study. Seventy-two healthy overweight and obese participants were randomly assigned to receive a red, soft capsule of OPE (100 mg quercetin/d, 50 mg quercetin twice daily; n = 36 participants) or an identical placebo capsule (n = 36) for 12 wk. Endothelial function, defined by flow-mediated dilation (FMD), circulating endothelial progenitor cells (EPCs) by flow cytometry, and laboratory test were determined at baseline and after treatment. RESULTS: Baseline characteristics and laboratory findings did not significantly differ between the two groups. Compared with baseline values, the OPE group showed significantly improved FMD at 12 wk (from 12.5 ± 5.2 to 15.2 ± 6.1; P = 0.002), whereas the placebo group showed no difference. Nitroglycerin-mediated dilation did not change in either group. EPC counts (44.2 ± 25.6 versus 52.3 ± 18.6; P = 0.005) and the percentage of EPCs were significantly increased in the OPE group. When FMD was divided into quartiles, rate of patients with endothelial dysfunction defined as lowest quartile (cutoff value, 8.6%) of FMD improved from 26% to 9% by OPE. CONCLUSION: Medium-term administration of OPE an improvement in FMD and circulating EPCs.

Functional importance of a peripheral pocket in mammalian cytochrome P450 2B enzymes.

The functional importance of a peripheral pocket found in previously published X-ray crystal structures of CYP2B4 and CYP2B6 was probed using a biophysical approach. Introduction of tryptophan within the pocket of CYP2B4 at F202 or I241 leads to marked impairment of 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) or 7-benzyloxyresorufin O-dealkylation efficiency; a similar substitution at F195, near the surface access to the pocket, does not affect these activities. The analogous CYP2B6 F202W mutant is inactive in the 7-EFC O-dealkylation assay. The stoichiometry of 7-EFC deethylation suggested that the decreased activity of F202W and I241W in CYP2B4 and lack of activity of F202W in CYP2B6 coincided with a sharp increase in the flux of reducing equivalents through the oxidase shunt to produce excess water. The results indicate that the chemical identity of residues within this peripheral pocket, but not at the mouth of the pocket, is important in substrate turnover and redox coupling, likely through effects on active site topology.

Effects of electroacupuncture on endothelial function and circulating endothelial progenitor cells in patients with cerebral infarction.

This study evaluated the effects of electroacupuncture (EA) on endothelial function and endothelial progenitor cells (EPC) in patients with cerebral infarction. In a randomized, placebo-controlled, crossover study, 20 patients with cerebral infarction were randomized into two treatment groups: EA or placebo. Before and after each intervention, pulse amplitude tonometry (PAT) was used to assess endothelial function and peripheral blood was analyzed for the number of EPCs. Circulating EPCs were quantified by flow cytometry as CD45(low) CD34(+) KDR2(+) cells. Plasma vascular endothelial growth factor (VEGF) and interleukin (IL)-10 levels were measured. Seven days later, crossover was performed on each group, with each group receiving the other treatment using the same protocol. The PAT hyperemia ratio ranged from 1.57 ± 0.41 to 2.04 ± 0.51 after EA, representing a significant improvement (P = 0.002); however, there was no improvement in the placebo group (P = 0.48). Circulating EPCs, as measured by flow cytometry, increased to 110.6 ± 74.3/100 µL in the EA group (P = 0.001) but did not change in the placebo group (45.9 ± 35.3/100 µL, P = 0.08). The increases in the number of EPCs and the PAT ratio after treatment were correlated (r = 0.78, P < 0.001). Plasma VEGF levels increased with EA compared to baseline (261.2 ± 34.0 vs 334.9 ± 80.5 pg/mL, P = 0.003). The number of circulating EPCs was positively correlated with plasma levels of VEGF (r = 0.50, P = 0.02). In conclusion, EA induced improvement of EPC levels and the PAT ratio in patients with cerebral infarction.

Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison.

X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.

Impact of lysophosphatidylcholine on survival and function of UEA-1(+)acLDL (+) endothelial progenitor cells in patients with coronary artery disease.

Lysophosphatidylcholine (LPC) generated from oxidized low-density lipoprotein by lipoprotein-associated phospholipase A2 plays a key role in plaque inflammation and vulnerability. Endothelial progenitor cells (EPCs) can repair injured endothelium and exert anti-inflammatory effects of vulnerable plaque. We study the impact and mechanisms of LPC on UEA-1 and acLDL binding EPCs (UEA-1(+)acLDL(+) EPCs). UEA-1(+)acLDL(+) EPCs from coronary artery disease (CAD) patients were cultured and exposed to LPC at different concentrations and different timepoints. We determined the significant concentration (40 µM). UEA-1(+)acLDL(+) EPCs were preincubated for 30 min with pravastatin (20 µM) with LY249002, a specific inhibitor of the Akt signaling pathway, and exposed for 24 h to LPC 40 µM. The survival, migration, adhesion, and proliferation of UEA-1(+)acLDL(+) EPCs were assessed. To examine the mechanisms of LPC toxicity and pravastatin effects, phosphorylated Akt and endothelial nitric oxide synthase (eNOS) levels and the ratio of Bcl-2/Bax protein expression were assessed. LPC induced apoptosis and impaired migration and adhesion of UEA-1(+)acLDL(+) EPCs significantly. The detrimental effects of LPC were attenuated by pravastatin. However, when UEA-1(+)acLDL(+) EPCs were pretreated with pravastatin and LY249002, a specific inhibitor of the Akt signaling pathway, simultaneously, the beneficial effects of pravastatin were abolished. Furthermore, LPC suppressed Akt and eNOS phosphorylation and increased Bcl-2/Bax expression. The effects of LPC on Akt/eNOS and Bcl-2/Bax activity were reversed by pravastatin. In conclusion, LPC inhibited UEA-1(+)acLDL(+) EPCs survival and impaired its functions, and these were attributable to inhibition of the Akt/eNOS and Bcl-2/Bax pathway. Pravastatin reversed the detrimental action of LPC. These findings suggest that LPC inhibition can be a possible strategy for CAD through EPC revitalization.

Additional Effects of Back-Shu Electroacupuncture and Moxibustion in Cardioprotection of Rat Ischemia-Reperfusion Injury.

Many preclinical studies show that electroacupuncture (EA) on PC6 and ST36 can reduce infarct size after ischemia-reperfusion (IR) injury. Yet studies to enhance the treatment effect size are limited. The purpose of this study was to explore whether EA has additional myocardial protective effects on an ischemia-reperfusion (IR) injury rat model when back-shu EA and moxibustion are added. SD rats were divided into several groups and treated with either EA only, EA + back-shu EA (B), or EA + B + moxibustion (M) for 5 consecutive days. Transthoracic echocardiography and molecular and immunohistochemical evaluations were performed. It was found that although myocardial infarct areas were significantly lower and cardiac function was also significantly preserved in the three treatment groups compared to the placebo group, there were no additional differences between the three treatment groups. In addition, HSP20 and HSP27 were expressed significantly more in the treatment groups. The results suggest that adding several treatments does not necessarily increase protection. Our study corroborates previous findings that more treatment, such as prolonging EA duration or increasing EA intensity, does not always lead to better results. Other methods of increasing treatment effect size should be explored.

Effects of sildenafil on nanostructural and nanomechanical changes in mitochondria in an ischaemia-reperfusion rat model.

Sildenafil exerts cardioprotective effects by activating the opening of mitochondrial ATP-sensitive potassium channels to attenuate ischaemia-reperfusion (IR) injury. In the present study, we used atomic force microscopy (AFM) to investigate changes in mitochondrial morphology and properties to assess sildenafil-mediated cardioprotection in a rat myocardial infarction model. To investigate the cardioprotective effects of sildenafil, we used an in vivo Sprague-Dawley rat model of IR. Rats were randomly divided into three groups: (i) sham-operated rats (control; n = 5); (ii) IR-injured rats treated with vehicle (normal saline; IR; n = 10); and (iii) IR-injured rats treated with 0.75 mg/kg, i.p., sildenafil (IR + Sil; n = 10). Morphological and mechanical changes to mitochondria were analysed by AFM. Infarct areas were significantly reduced in sildenafil-treated rats (7.8 ± 3.9% vs 20.4 ± 7.0% in the sildenafil-treated and untreated IR groups, respectively; relative reduction 62%; P < 0.001). Analysis of mitochondria by AFM showed that IR injury significantly increased the areas of isolated mitochondria compared with control (24 150 ± 18 289 vs 1495 ± 1139 nm(2) , respectively; P < 0.001), indicative of mitochondrial swelling. Pretreatment with sildenafil before IR injury reduced the mitochondrial areas (7428 ± 3682 nm(2) ; P < 0.001; relative reduction 69.2% compared with the IR group) and ameliorated the adhesion force of mitochondrial surfaces. Together, these results suggest that sildenafil has cardioprotective effects against IR injury in a rat model by improving the morphological and mechanical characteristics of mitochondria.

Biochemical and clinical correlation of intraplaque neovascularization using contrast-enhanced ultrasound of the carotid artery.

OBJECTIVE: Several biomarkers reflecting inflammatory or proteolytic activity have been known to represent plaque vulnerability. Moreover, a recent study confirmed that contrast-enhanced ultrasound (CEUS) can visualize intraplaque neovascularization (IPN) and demonstrate plaque vulnerability. In this study, we tried to demonstrate that IPN detected by CEUS was correlated with several well-known biomarkers and clinical outcome in patients with coronary artery disease (CAD). METHODS: Patients with stable CAD were screened by conventional carotid ultrasound and patients with carotid plaque thickness more than 2 mm were performed by CEUS for the presence of IPN. Plasma levels of biomarkers and clinical outcomes were evaluated. RESULTS: Among consecutive 89 patients fulfilled the inclusion criteria, 30 patients without IPN (group 1) and 59 patients with IPN (group 2) were analyzed. There were no significant difference in baseline characteristics except for mean age (62.9±10.1 yrs versus 68.4±9.6 yrs, p=0.015). On multivariate analysis, only MMP-9 (p=0.021, 95% CI 1.002-1.027) showed a significant association with IPN. But patients with IPN showed only trend for a history of cardiovascular disease (CVD) (44% versus 30%, p=0.19) and one-year cardiovascular events (CVE) (6.8% versus 3.3%, p=0.50) compared to group 1. Maximum plaque thickness (p=0.04, 95% CI 1.230-6.322) showed a significant correlation with the clinical outcome including CVD or CVE. CONCLUSION: MMP-9 correlated with IPN on CEUS. For clinical implication, however, large prospective studies are needed.