C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

BACKGROUND: This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS). METHODS AND RESULTS: EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. In vivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions. CONCLUSIONS: CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

Anatomical characteristics of aortic valve diseases: Implications for transcatheter aortic valve replacement.

Background: The etiology of aortic stenosis (AS) significantly impacts transcatheter heart valve (THV) implantation, with rheumatic etiology posing challenges. The concept of valve anchoring during transcatheter aortic valve replacement (TAVR) for patients with aortic regurgitation (AR) remains unclear. Objective: This study aims to investigate the clinical and CT anatomical characteristics of various aortic valve diseases. Methods: A retrospective analysis was conducted on consecutive patients who underwent CT for severe aortic diseases between April 2019 and February 2023. CT analysis was performed in eight anatomical landmarks: left ventricular outflow tract (LVOT), aortic annulus, sinus of Valsalva (SOV), sinotubular junction (STJ), ascending aorta (AAO), coronary height, aortic angle, and aortic valve calcification volume. Results: 121 patients with severe aortic valve disease were included, divided into AS (71 cases, 59%) and AR (50 cases, 41%) groups. In patients with AR, the absolute diameters of the annulus, LVOT, SOV, STJ, and AAO, as well as the heights of SOV and STJ and the cardiac angle, are larger than those in patients with AS (all P < 0.05). In normalized aortic root dimensions, the AR group had a higher SOV and STJ diameter-to-annulus ratio than the AS group (STJ-SOV-annulus: 1.51-1.44-1.00 vs 1.33-1.28-1.00). The bicuspid and rheumatic AS groups had smaller sinuses (STJ-SOV-annulus:1.27-1.35-1.00, 1.17-1.30-1.00, respectively), necessitating the downsizing of the THV. For 74% of AR patients, the sinotubular junction could not be used as a second anchoring zone, and anchoring relied primarily on the annulus. Conclusions: Patients with rheumatic etiology require smaller valves, and anchoring in AR patients depends on the valve annulus. These structural characteristics will influence TAVR selection.

Mechanism Analysis of Vascular Calcification Based on Fluid Dynamics.

Vascular calcification is the abnormal deposition of calcium phosphate complexes in blood vessels, which is regarded as the pathological basis of multiple cardiovascular diseases. The flowing blood exerts a frictional force called shear stress on the vascular wall. Blood vessels have different hydrodynamic properties due to discrepancies in geometric and mechanical properties. The disturbance of the blood flow in the bending area and the branch point of the arterial tree produces a shear stress lower than the physiological magnitude of the laminar shear stress, which can induce the occurrence of vascular calcification. Endothelial cells sense the fluid dynamics of blood and transmit electrical and chemical signals to the full-thickness of blood vessels. Through crosstalk with endothelial cells, smooth muscle cells trigger osteogenic transformation, involved in mediating vascular intima and media calcification. In addition, based on the detection of fluid dynamics parameters, emerging imaging technologies such as 4D Flow MRI and computational fluid dynamics have greatly improved the early diagnosis ability of cardiovascular diseases, showing extremely high clinical application prospects.

Screening of immune-related secretory proteins linking chronic kidney disease with calcific aortic valve disease based on comprehensive bioinformatics analysis and machine learning.

BACKGROUND: Chronic kidney disease (CKD) is one of the most significant cardiovascular risk factors, playing vital roles in various cardiovascular diseases such as calcific aortic valve disease (CAVD). We aim to explore the CKD-associated genes potentially involving CAVD pathogenesis, and to discover candidate biomarkers for the diagnosis of CKD with CAVD. METHODS: Three CAVD, one CKD-PBMC and one CKD-Kidney datasets of expression profiles were obtained from the GEO database. Firstly, to detect CAVD key genes and CKD-associated secretory proteins, differentially expressed analysis and WGCNA were carried out. Protein-protein interaction (PPI), functional enrichment and cMAP analyses were employed to reveal CKD-related pathogenic genes and underlying mechanisms in CKD-related CAVD as well as the potential drugs for CAVD treatment. Then, machine learning algorithms including LASSO regression and random forest were adopted for screening candidate biomarkers and constructing diagnostic nomogram for predicting CKD-related CAVD. Moreover, ROC curve, calibration curve and decision curve analyses were applied to evaluate the diagnostic performance of nomogram. Finally, the CIBERSORT algorithm was used to explore immune cell infiltration in CAVD. RESULTS: The integrated CAVD dataset identified 124 CAVD key genes by intersecting differential expression and WGCNA analyses. Totally 983 CKD-associated secretory proteins were screened by differential expression analysis of CKD-PBMC/Kidney datasets. PPI analysis identified two key modules containing 76 nodes, regarded as CKD-related pathogenic genes in CAVD, which were mostly enriched in inflammatory and immune regulation by enrichment analysis. The cMAP analysis exposed metyrapone as a more potential drug for CAVD treatment. 17 genes were overlapped between CAVD key genes and CKD-associated secretory proteins, and two hub genes were chosen as candidate biomarkers for developing nomogram with ideal diagnostic performance through machine learning. Furthermore, SLPI/MMP9 expression patterns were confirmed in our external cohort and the nomogram could serve as novel diagnosis models for distinguishing CAVD. Finally, immune cell infiltration results uncovered immune dysregulation in CAVD, and SLPI/MMP9 were significantly associated with invasive immune cells. CONCLUSIONS: We revealed the inflammatory-immune pathways underlying CKD-related CAVD, and developed SLPI/MMP9-based CAVD diagnostic nomogram, which offered novel insights into future serum-based diagnosis and therapeutic intervention of CKD with CAVD.

The myth of aortic valve annulus changes in aortic valve disease.

Background: The characteristics of aortic annulus changes in aortic regurgitation (AR) patients are poorly understood, and predictive factors among aortic valve disease are yet to be established. Objective: This study seeks to elucidate the pattern of annular size fluctuations across different cardiac phases in AR patients and to identify predictors for annular enlargement during either systole or diastole in aortic valve diseases. Methods: A retrospective analysis was conducted on 55 patients with severe aortic valve diseases, including 26 patients with aortic stenosis (AS) and 29 with AR, to discern the two groups' contrasting and analogous patterns of annular changes. The patient sample was expanded to 107 to investigate the factors influencing the size of the annulus during different cardiac phases. Based on our findings, patients were then divided into two groups: those with an annulus that is larger during systole (83 patients) and those where the annulus is larger during diastole (24 patients). Results: Typically, AR patients exhibit a dynamic annulus, with both perimeter and area being largest during mid-systole. These dimensions diminish progressively and then increase again in early diastole, a pattern consistent with observations in AS patients. Among 107 patients, 21% had diastolic enlargement. Systolic measurements would lead to prosthesis undersizing in 17% of these. Male gender and lower systolic annulus minimum relative to body surface area (AnMin index) were predictors of diastolic enlargement, with ROC curve areas of 0.70 and 0.87 for AR and AS, respectively. Conclusions: Systolic measurements are recommended for AR patients. Gender and the AnMin index are significant predictors, particularly potent in AS patients.

Fluid Shear Stress Ameliorates Prehypertension-Associated Decline in Endothelium-Reparative Potential of Early Endothelial Progenitor Cells.

This study investigated the effects of prehypertension and shear stress on the reendothelialization potential of human early EPCs and explored its potential mechanisms. Early EPCs from the prehypertensive patients showed reduced migration and adhesion in vitro and demonstrated a significantly impaired in vivo reendothelialization capacity. Shear stress pretreatment markedly promoted the in vivo reendothelialization capacity of EPCs. Although basal CXCR4 expression in early EPCs from prehypertensive donors was similar to that from healthy control, SDF-1-induced phosphorylation of CXCR4 was lower in prehypertensive EPCs. Shear stress up-regulated CXCR4 expression and increased CXCR4 phosphorylation, and restored the SDF-1/CXCR4-dependent JAK-2 phosphorylation in prehypertensive EPCs. CXCR4 knockdown or JAK-2 inhibitor treatment prevents against shear stress-induced increase in the migration, adhesion and reendothelialization capacity of the prehypertensive EPCs. Collectively, CXCR4 receptor profoundly modulates the reendothelialization potential of early EPCs. The abnormal CXCR4-mediated JAK-2 signaling may contribute to impaired functions of EPCs from patients with prehypertension.

5mC modification patterns provide novel direction for early acute myocardial infarction detection and personalized therapy.

Background: Most deaths from coronary artery disease (CAD) are due to acute myocardial infarction (AMI). There is an urgent need for early AMI detection, particularly in patients with stable CAD. 5-methylcytosine (5mC) regulatory genes have been demonstrated to involve in the progression and prognosis of cardiovascular diseases, while little research examined 5mC regulators in CAD to AMI progression. Method: Two datasets (GSE59867 and GSE62646) were downloaded from Gene Expression Omnibus (GEO) database, and 21 m5C regulators were extracted from previous literature. Dysregulated 5mC regulators were screened out by "limma." The least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) algorithm were employed to identify hub 5mC regulators in CAD to AMI progression, and 43 clinical samples (Quantitative real-time PCR) were performed for expression validation. Then a logistic model was built to construct 5mC regulator signatures, and a series of bioinformatics algorithms were performed for model validation. Besides, 5mC-associated molecular clusters were studied via unsupervised clustering analysis, and correlation analysis between immunocyte and 5mC regulators in each cluster was conducted. Results: Nine hub 5mC regulators were identified. A robust model was constructed, and its prominent classification accuracy was verified via ROC curve analysis (area under the curve [AUC] = 0.936 in the training cohort and AUC = 0.888 in the external validation cohort). Besides, the clinical effect of the model was validated by decision curve analysis. Then, 5mC modification clusters in AMI patients were identified, along with the immunocyte infiltration levels of each cluster. The correlation analysis found the strongest correlations were TET3-Mast cell in cluster-1 and TET3-MDSC in cluster-2. Conclusion: Nine hub 5mC regulators (DNMT3B, MBD3, UHRF1, UHRF2, NTHL1, SMUG1, ZBTB33, TET1, and TET3) formed a diagnostic model, and concomitant results unraveled the critical impact of 5mC regulators, providing interesting epigenetics findings in AMI population vs. stable CAD.

Assessment of atrial septal defect using 2D or real-time 3D transesophageal echocardiography and outcomes following transcatheter closure.

BACKGROUND: The assessment of interatrial septum (IAS) requires a standardized, systematic approach, including two-dimensional transthoracic echocardiography (2D TTE), 2D transesophageal echocardiography (2D TEE), and three-dimensional (3D) TEE. Although 2D TEE has been widely used for the preoperative assessment of atrial septal defect (ASD), its ability to provide reliable information is often limited due to the structural characteristics of IAS. The introduction of 3D TEE provides a unique "en face" view of IAS, which allows the visualization and accurate measurements of diameters, area, and rims of ASD. Hence, appropriate ASD imaging information is particularly important in successful transcatheter closure. METHODS: In this retrospective study, 2D TTE/TEE, and 3D TEE were performed before ASD closure, with 2D minimal and maximal diameters, areas, and residual rims being recorded. Adequate 3D TEE imaging data sets were collected and then analyzed. ASD related parameters were compared using different echocardiography. Patients who underwent ASD closure completed a clinical follow-up. RESULTS: The mean defect maximal diameter and aperture area by 3D TEE was significantly larger than that of the corresponding 2D TEE (P<0.05). There was no statistical difference in the minimal and maximal diameter or area by TEE for circular-shaped ASDs. For oval ASDs, mean minimal diameter on 2D TEE was larger than that on 3D TEE. The mean maximal diameter measured using 2D TEE was smaller than the 3D TEE measurement (16.0±7.1 vs. 19.8±8.6; P<0.05). For complex-shaped defects, there were statistical differences in minimal and maximal diameter between TEEs. Furthermore, 2D and 3D TEE had a longer superior vena cava (SVC) residual rim than did 2D TTE (P<0.05). The 3D TEE residual rims of the inferior vena cava (IVC) was significantly larger than the corresponding 2D TEE. There was a very strong correlation between the residual rim measurements using 3D and 2D TEE. However, the limits of agreement between 2D and real-time 3D TEE measurements were more apparent in the IVC rim group than in the other groups. CONCLUSIONS: Our study confirms the value of 3D TEE in assessing ASD shape and size reported by previous studies, and is also the first to accurately and systematically characterize ASD residual rim in complex ASDs.

3D transesophageal echocardiography assists in evaluating the morphology, function, and presence of thrombi of left atrial appendage in patients with atrial fibrillation.

BACKGROUND: Left atrial appendage (LAA) is significantly more likely to form thrombi in patients with atrial fibrillation (AFib). Two-dimensional transesophageal echocardiography (2D TEE) is considered the gold standard for assessing and studying LAA morphology and anatomy. However, 2D TEE can only visualize one plane at any given time. Real-time three-dimensional echocardiography (RT-3D TEE) imaging can preserve spatial and temporal resolution, which is a safe, accurate, and reproducible imaging modality. There are few reports of the usage of RT-3D TEE to study LAA in AFib patients. In our research, RT-3D TEE helps to provide detailed LAA information and identifying the presence or absence of thrombi from pectinate muscles in paroxysmal and long-standing AFib patients. METHODS: LAA morphology was analyzed in detail by 2D TEE and RT-3D TEE in 320 patients with paroxysmal or long-standing AFib. The LAA flow pattern, as maximal LAA emptying flow velocity (LAAeV), was retrieved from 2D and 3D TEE imaging. LAA morphological parameters, spontaneous echo contrast (SEC), and thrombi were also detected by 2D and 3D TEE in all patients. In addition, LAA lobes and types were classified according to the morphology by 3D TEE, and the relationship between LAA types and the incidence of thrombi was evaluated. RESULTS: Long-standing AFib had greater enlargement of LAAs (orifice diameters and area), significantly more severe SEC, and a higher thrombi clot incidence rate by 3D-TEE compared with paroxysmal AFib patients (P<0.05). In addition, cauliflower morphology in long-standing AFib patients was associated with a higher LAA thrombus (OR 2.1, 95% CI: 1.1-8.5, P=0.031) and increased prevalence of SEC. Moreover, the uncertainty of thrombi detection was significantly decreased by 3D TEE compared with 2D TEE (P<0.001), and the certainty of thrombi detection by 3D TEE also decreased slightly (P=0.06). CONCLUSIONS: RT-3D TEE is a safe and real-time option for the evaluation of LAA morphology and function. Long-standing AFib has greater LAA and SEC, as well as a higher incidence of thrombi than the paroxysmal group. Cauliflower LAA type was associated with a higher prevalence of SEC and thrombi.

LncRNA-mRNA co-expression analysis discovered the diagnostic and prognostic biomarkers and potential therapeutic agents for myocardial infarction.

Currently, the role of lncRNA in myocardial infarction (MI) is poorly understood. 17 co-expression modules were determined, specifically, the greenyellow, saddlebrown, grey60, royalblue, lightgreen, white, and pink modules were specifically expressed in the acute phase of MI, and brown, darkred, and royalblue, while greenyellow modules were specifically expressed in MI compared with CAD. 12 time-dependent of lncRNA/mRNA clusters with consistent expression trends were also identified. MI-associated modules were mainly enriched to immune, cell cycle, and metabolic pathways. We further obtained a network of 1816 lncRNA-mRNAs with higher expression correlations among these lncRNAs by analyzing the topological properties of the network. Herein, lncRNA RP11-847H18.2 and KLHL28, SPRTN, and EPM2AIP1 were determined as gene markers specifically expressed in MI, and they demonstrated a high predictive performance for MI diagnosis and prognosis. Three drugs, namely, Calcium citrate, Calcium Phosphate, and Calcium phosphate dihydrate, were identified as potential precursors of MI. Finally, gene and lncRNA diagnostic models were developed based on these genes and lncRNAs, with their AUCs averaged above 0.89 in both training and validation datasets. The findings of this study improve the diagnosis and prognosis of MI and personalized treatment of MI.

Intermittent concurrent use of clopidogrel and proton pump inhibitors did not increase risk of adverse clinical outcomes in Chinese patients with coronary artery disease.

BACKGROUND: Proton pump inhibitors (PPIs) are frequently prescribed to patients with coronary heart disease (CHD) under antiplatelet therapy to prevent gastrointestinal (GI) bleeding. However, its clinical impact is still under debate, especially in Asian population. This study was undertaken to explore the effects of concurrent use of clopidogrel and PPIs on the clinical outcomes in Chinese patients with CHD in secondary prevention. METHODS: A single-center retrospective study was conducted in 638 patients with CHD on consecutive clopidogrel therapy for at least 1 year. After 18-month follow-up, adverse clinical events were collected. Cox regression was used to calculate hazard ratios (HR) and 95% confidence interval (CI) for the effect of PPI use on the outcomes. A total of 638 patients were recruited from 2014 to 2015 in this study, among whom 201 were sustained PPI users, 188 were intermittent PPI users and the remaining 249 were non-PPI users. RESULTS: Compared with sustained PPI users, intermittent use of PPIs was associated with a lower risk of stroke, major adverse cardiac events (MACE) and net adverse clinical event (NACE) (stroke: adjusted HR: 0.109, 95% CI 0.014-0.878, p = 0.037; MACE: adjusted HR: 0.293, 95% CI 0.119-0.722; p = 0.008; NACE: adjusted HR: 0.357, 95% CI 0.162-0.786, p = 0.011). Subgroup analysis further revealed the benefit of intermittent PPI use was significant in male CHD patients over 60 years old, with hypertension or chronic kidney disease, and undergoing percutaneous coronary intervention during hospitalization. CONCLUSION: The current findings suggest that the intermittent concurrent use of PPIs and clopidogrel is not associated with an increased risk of 18-month adverse clinical outcomes, and intermittent use of PPIs is associated with a lower rate of MACE and NACE.

Comparison of intravascular ultrasound-guided with angiography-guided double kissing crush stenting for patients with complex coronary bifurcation lesions: Rationale and design of a prospective, randomized, and multicenter DKCRUSH VIII trial.

BACKGROUND: Double kissing (DK) crush approach for patients with coronary bifurcation lesions, particularly localized at distal left main or lesions with increased complexity, is associated with significant reduction in clinical events when compared with provisional stenting. Recently, randomized clinical trial has demonstrated the net clinical benefits by intravascular ultrasound (IVUS)-guided implantation of drug-eluting stent in all-comers. However, the improvement in clinical outcome after DK crush treatment guided by IVUS over angiography guidance for patients with complex bifurcation lesions have never been studied in a randomized fashion. TRIAL DESIGN: DKCRUSH VIII study is a prospective, multicenter, randomized controlled trial designed to assess superiority of IVUS-guided vs angiography-guided DK crush stenting in patients with complex bifurcation lesions according to DEFINITION criteria. A total of 556 patients with complex bifurcation lesions will be randomly (1:1 of ratio) assigned to IVUS-guided or angiography-guided DK crush stenting group. The primary end point is the rate of 12-month target vessel failure, including cardiac death, target vessel myocardial infarction, or clinically driven target vessel revascularization. The secondary end points consist of the individual component of primary end point, all-cause death, myocardial infarction, and in-stent restenosis. The safety end point is the incidence of definite or probable stent thrombosis. An angiographic follow-up will be performed for all patients at 13 months and clinical follow-up will be continued annually until 3 years after the index procedure. CONCLUSIONS: DKCRUSH VIII trial is the first study designed to evaluate the differences in efficacy and safety between IVUS-guided and angiography-guided DK crush stenting in patients with complex true bifurcation lesions. This study will also provide IVUS-derived criteria to define optimal DK crush stenting for bifurcation lesions at higher complexity.

Importance of ß2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells.

BACKGROUND: Physiological fluid shear stress has been shown to have a beneficial impact on vascular homeostasis. Endothelial progenitor cells (EPCs) make a significant contribution to maintaining endothelial integrity. Therefore, we hypothesised that shear stress-induced endothelium protection plays a role in hydrogen sulphide (H2S) production and up-regulation of cystathionine γ-lyase (CSE) expression in EPCs. METHODS: Human EPC-derived CSE activity was detected by colorimetric assay, and H2S production was evaluated by membrane adsorption method. Cell proliferation, migration, and adhesion were assessed by MTT, Transwell, and endothelial cell-mediated adhesion assays, respectively. Real-time polymerase chain reaction (RT-PCR) was carried out to analyse gene expression. Protein expression was analysed by western blot. RESULTS: Human EPCs were treated with shear stress levels of 5-25 dyn/cm2 for up to 3 h, and 25 dyn/cm2 for up to 24 h. H2S production and CSE mRNA expression in the EPCs were increased by shear stress in a dose-dependent manner in vitro. Likewise, time-dependent shear stress also significantly enhanced CSE protein expression. Compared to static condition, shear stress improved EPCs proliferation, migration and adhesion capacity. Knockdown of CSE expression by small interfering RNA substantially eliminated the shear stress-induced above functions of human EPCs in vitro. CONCLUSIONS: This study gives new insight into the regulatory effect of physiological shear stress on the CSE/H2S system in human EPCs. Our findings may contribute to the development of vascular protective research, although the relevant evidence is admittedly indirect.

Oxidized low-density lipoprotein inhibits the degradation of cyclophilin A via the lysosome in vascular smooth muscle cells.

BACKGROUND: Cyclophilin A (CyPA) plays an important role in the progression of atherosclerosis. Additionally, it has been reported that lysosomal function is markedly impaired in atherosclerosis induced by oxidized low-density lipoprotein (ox-LDL). As the CyPA degradation pathway remains to be elucidated, we aimed to uncover the role of lysosomes and ox-LDL in the degradation of CyPA. METHODS: We exploited RNA interference (RNAi) in combination with either the lysosomal inhibitor chloroquine (CQ) or the proteasomal inhibitor MG-132 to examine CyPA turnover. We also investigated the role of ox-LDL in lysosomal function and the CyPA degradation pathway and determined whether CyPA interacts with the selective autophagy adaptor p62. RESULTS: CQ markedly reversed the CyPA downregulation induced by RNAi and increased intracellular levels of LC3 and p62. MG-132 significantly suppressed polyubiquitinated protein degradation but did not inhibit RNAi-induced CyPA downregulation. Additionally, neither CQ nor MG-132 influenced the gene-silencing efficiency of CyPA siRNA. Moreover, ox-LDL induced cytosolic accumulation of p62 was inconsistent with increased expression of LC3-II. Meanwhile, ox-LDL inhibited RNAi-induced downregulation of CyPA. Immunofluorescence indicated colocalization of endogenous CyPA with ubiquitin and with p62 in response to CQ treatment, and co-immunoprecipitation analysis confirmed interaction between CyPA and p62. CONCLUSION: CyPA is degraded by a lysosome-dependent pathway that may involve p62-mediated selective autophagy. Furthermore, ox-LDL modulates the degradation of CyPA via its inhibitory role in lysosomes, contributing to increased expression of CyPA in atherosclerotic plaques.

ß2AR-dependent signaling contributes to in-vivo reendothelialization capacity of endothelial progenitor cells by shear stress.

BACKGROUND: Endothelial progenitor cells (EPCs) play a crucial role in the endothelial repair after arterial injury. Shear stress has a beneficial effect on modulating EPC functions. The molecular mechanism underlying the influence of EPCs on the endothelial integrity and shear stress effects on EPC regulation remained unclear. Herein, we investigated the influence of ß2 adrenergic receptor (ß2AR)-dependent signaling on in-vitro shear stress-mediated function and in-vivo reendothelialization capacity of human EPCs. METHOD: The human EPCs from healthy population were exposed to in-vitro 5, 10, and 20 dyn/cm shear stress for 15 h, and 10 dyn/cm for 5, 10, and 15 h, respectively. The in-vitro proliferation was assessed by CCK8 and BrDU tests. The migration and adhesion were evaluated by Transwell system and human umbilical vein endothelial cells (HUVECs) incorporation assays. Meanwhile, the angiogenic cytokine stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) concentration of supernatant were tested by ELISA. Phosphorylated ß2AR, Akt, and eNOS were detected by western blot. In an in-vivo study, mice carotid injury models were produced through denuding the endothelium with a curved flexible wire, and thereafter CM-Dil-labeled EPCs were injected intravenously. After 3 days, cells recruited to the injury sites were detected by fluorescent microscopy, and the in-vivo reendothelialization capacity was assessed by Evans blue dye. RESULTS: Shear stress improved in-vitro functions and in-vivo reendothelialization capacity of human EPCs. In parallel, shear stress up-regulated the phosphorylation of ß2AR, Akt, and eNOS, and promoted vascular endothelial growth factor (VEGF) secretion of human EPCs. With ICI118,551 (a ß2AR inhibitor) treatment, shear stress-induced Akt and eNOS phosphorylation as well as VEGF secretion were suppressed. After ß2AR/PI3K/Akt/eNOS pathway of EPCs was blocked, the effects of shear stress on in-vitro functions and in-vivo reendothelialization capacity of EPCs were inhibited. CONCLUSION: The present study provided the novel data that shear stress-induced ß2AR/Akt/eNOS pathway enhanced reendothelialization capacity of EPCs. Shear stress-induced ß2AR-dependent pathway may be a novel and important therapeutic target for endothelial repair.

Plasma D-dimer as a predictor of intraluminal thrombus burden and progression of abdominal aortic aneurysm.

AIM: Intraluminal thrombus (ILT) is presented in most abdominal aortic aneurysms (AAAs) and is suggested to promote AAA expansion. D-dimer, a breakdown product in the thrombus remodeling, may have prognostic value for AAA. This study investigated the interrelation between plasma D-dimer level, ILT volume, AAA size and progression. MAIN METHODS: This was a retrospective observational study that involved 181 patients with infra-renal AAA. They were divided into small and large AAA groups according to AAA diameter. 24 of them had repeated abdominal computed tomography angiography (CTA) scan and were divided into slow-growing and fast-growing AAA groups according to the median value of AAA growth rate. Baseline and follow-up plasma D-dimer level, maximum diameter of AAA, total infra-renal aortic volume and ILT volume were analyzed. KEY FINDINGS: Plasma D-dimer level was positively correlated with ILT volume (R = 0.382, P < 0.001) and maximum diameter of AAA (R = 0.442, P < 0.001). Increasing value of plasma D-dimer was positively associated with the accelerated growth rate of AAA (R = 0.720, P < 0.01). ILT volume showed positive correlation with maximum diameter (R = 0.859, P < 0.001) and growth rate of AAA (R = 0.490, P < 0.05). After adjusting the baseline ILT volume, the positive correlations remained to be statistically significant between plasma D-dimer level and AAA size (R = 0.200, P < 0.05), as well as increasing value of plasma D-dimer and growth rate of AAA (R = 0.642, P < 0.05). SIGNIFICANCE: Plasma D-dimer level reflected ILT burden in AAAs. Plasma D-dimer level and ILT volume were positively correlated with AAA size. Increasing value of plasma D-dimer and baseline ILT volume could be predictors of AAA progression.

CC chemokine receptor 2 functions in osteoblastic transformation of valvular interstitial cells.

AIMS: Calcific aortic valve disease (CAVD) emerges as a challenging clinical issue, which is associated with high cardiovascular mortality. It has been demonstrated that osteoblastic transformation of AVICs is a key mechanism of CAVD and C-C motif chemokine receptors (CCRs) may favor this process. Thus, we aimed to investigate whether CCRs were involved in osteoblastic transformation of AVICs during the development CAVD. MAIN METHODS: We first analyzed microarray data (GSE51472 and GSE12644) to identify differentially expressed genes between CAVD aortic valve tissue and normal samples, followed by verification of immunohistochemistry, qPCR and western blotting. Primary aortic valvular interstitial cells (AVICs) were incubated with specific inhibitors and/or siRNA of CCR2 and CCL2 under pro-calcifying medium. The levels of CCL2 in the medium were measured by ELISA. In addition, we used recombinant CCL2 to activate CCR2 in calcifying AVICs. Alizarin red S staining and calcium deposition were used to evaluate the degree of calcification. Western blotting was used to determine osteoblastic transformation of AVIC and total Akt and phosphorylated Akt expression. KEY FINDING: CCR2 levels were remarkably up-regulated in calcified aortic valve and calcifying AVICs. Silencing CCR2 inhibited the osteoblastic transformation and calcification of AVICs. In addition, recombinant CCL2 activated CCR2 and accelerated AVICs calcification through PI3K/Akt pathway. SIGNIFICANCE: We characterize abnormal activation of CCL2/CCR2 axis as a promoter of AVICs osteoblastic transformation and calcification. Our findings implicate the CCL2/CCR2-PI3K/Akt pathway as a potential target for treatment of CAVD.

Pharmacodynamic changes of platelet reactivity status in patients with chronic kidney disease after coronary artery stenting.

OBJECTIVES: This study was to evaluate platelet reactivity over time among patients with chronic kidney disease (CKD) receiving standard dose of clopidogrel after percutaneous coronary intervention (PCI). The effect of CYP2C19 loss-of-function genotypes on platelet reactivity was also determined. METHODS: Patients with CKD (n = 138) on maintenance dose of clopidogrel after PCI were enrolled. Platelet reactivity was assessed by measuring P2Y12 reaction units (PRU) with VerifyNow P2Y12 assay, and platelet reactivity index (PRI) with flow cytometric using vasodilator-stimulated phosphoprotein (VASP) at baseline and 2 weeks later, respectively. The genotypes of CYP2C19 were also measured concurrently. RESULTS: The proportion of patients with high platelet reactivity (HPR) ranged from 23.2% to 59.4%, and almost 1 in 5 patients had a dual conversion between HPR and non-HPR status. Patients carrying CYP2C19 loss-of-function genotypes showed a higher platelet reactivity than non-carriers, but with an undetermined HPR status between the first and second visits. The individual switch of HPR to non-HPR status existed in both loss-of-function genotype carriers and non-carriers. CONCLUSIONS: HPR conversions occur in a significant proportion of CKD patients with maintenance doses of clopidogrel treatment post-PCI, and this conversion was not confined to CYP2C19 loss-of-function genotype carriers. Risk stratification for treatment adjustment in personalized antiplatelet therapy should be investigated in future research.

Hydrogen sulfide improves vascular repair by promoting endothelial nitric oxide synthase-dependent mobilization of endothelial progenitor cells.

BACKGROUND: Endothelial progenitor cells (EPCs) play a crucial role in endothelial repair after arterial injury. Hydrogen sulfide (H2S) is a novel gasotransmitter that regulates vascular homeostasis. METHOD: We investigated whether exogenous H2S could facilitate EPCs in repairing arterial injury. RESULTS: Sodium hydrosulfide (NaHS), a precursor of H2S, promoted re-endothelialization and inhibited neointima formation in a rodent carotid artery injury model. Flow cytometric analysis revealed that NaHS treatment significantly increased the yield of EPCs after vascular injury. Furthermore, NaHS enhanced the capacity of EPCs to the luminal surface of injured arteries in wild-type mice, which had received a bone marrow transplantation from tie2-GFP donor mice. However, this enhancing effect was greatly attenuated in endothelial nitric oxide synthase knockout mice (eNOS). In-vitro incubation of human EPCs with NaHS not only increased the yield of EPCs, but also enhanced their adhesion and colony formation capacities. Treatment with an eNOS inhibitor (L-NAME) blocked the effects of NaHS on EPCs functions. CONCLUSION: H2S enhances eNOS-dependent mobilization of bone marrow-derived EPCs and facilitates re-endothelialization following vascular injury.