Changpo Lin

The rhythmic expression of clock genes attenuated in human plaque-derived vascular smooth muscle cells

BackgroundAcute myocardial infarction and stroke are more likely to occur in the early morning. Circadian pacemakers are considered to be involved in the process. Many peripheral tissues and cells also contain clock systems. In this study, we examined whether the primary cultured human plaque-derived vascular smooth muscle cells (VSMCs) process circadian rhythmicity; furthermore, we investigated the expression difference of clock genes between normal human carotid VSMCs and human plaque-derived VSMCs.MethodsFifty-six human carotid plaques provided the atherosclerotic tissue, and 21 samples yielded viable cultured primary VSMCs. The normal carotid VSMCs were cultured from donors’ normal carotids. The mRNA levels of the target genes were measured by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).ResultsAfter serum shock, both types of cells showed clear circadian expressions of Bmal1, Cry1, Cry2, Per1, Per2, Per3 and Rev-erbα mRNA; meanwhile the Clock mRNA show a rhythmic expression in plaque-derived SMCs but not in normal carotid VSMCs. The expression levels of these main clock genes were significantly attenuated in human plaque-derived VSMCs compared with normal human carotid VSMCs. The rhythm of Bmal1 mRNA in plaque-derived VSMCs was changed.ConclusionThe present results demonstrate that the human plaque-derived VSMCs possess different circadian rhythmicity from that of normal carotid VSMCs. The rhythm changes of clock genes in plaque-derived VSMCs may be involved in the process of atherosclerosis and finally promote the rupture of plaque.

hCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κB-Mediated Inflammatory Responses

Background. The human Circadian Locomotor Output Cycle protein Kaput (CLOCK) gene was originally discovered as a regulator of essential human daily rhythms. This seemingly innocuous gene was then found to be associated with a multitude of human malignancies, via several biochemical pathways. We aimed to further investigate the role of hCLOCK in the hypoxia-oxidative stress response system at the biochemical level. Methods. Expression levels of Rho GTPases were measured in normoxic and hypoxic states. The effect of hCLOCK on the hypoxic response was evaluated with the use of a retroviral shRNA vector system, a Rho inhibitor, and a ROS scavenger by analyzing expression levels of hCLOCK, Rho GTPases, and NF-κB pathway effectors. Finally, in vitro ROS production and tube formation in HUVECs were assessed. Results. Hypoxia induces ROS production via hCLOCK. hCLOCK activates the RhoA and NF-κB signaling pathways. Conversely, inhibition of hCLOCK deactivates these pathways. Furthermore, inhibition of RhoA or decreased levels of ROS attenuate these pathways, but inhibition of RhoA does not lead to decreased levels of ROS. Overall findings show that hypoxia increases the expression of hCLOCK, which leads to ROS production, which then activates the RhoA and NF-κB pathways. Conclusion. Our findings suggest that hypoxic states induce vascular oxidative damage and inflammation via hCLOCK-mediated production of ROS, with subsequent activation of the RhoA and NF-κB pathways.

CLOCK Promotes Endothelial Damage by Inducing Autophagy through Reactive Oxygen Species

A number of recent studies have implicated that autophagy was activated by reactive oxygen species (ROS). Our previous report indicated that CLOCK increased the accumulation of ROS under hypoxic conditions. In this study, we investigated the mechanisms by which CLOCK mediated endothelial damage, focusing on the involvement of oxidative damage and autophagy. Overexpression of CLOCK in human umbilical vein endothelial cells (HUVECs) showed inhibition of cell proliferation and higher autophagosome with an increased expression of Beclin1 and LC3-I/II under hypoxic conditions. In contrast, CLOCK silencing reversed these effects. Interestingly, pretreatment with 3-methyladenine (3-MA) resulted in the attenuation of CLOCK-induced cell autophagy and but did not influence the production of intracellular reactive oxygen species (ROS). Furthermore, Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt), a ROS scavenger, significantly attenuated CLOCK-induced cell autophagy. In addition, we found that overexpression of CLOCK had no significant effects on the production of ROS and expression of Beclin1 and LC3-I/II under normoxic conditions in HUVEC. In this present investigation, our results suggested a novel mechanism of action of CLOCK in HUVECs, opening up the possibility of targeting CLOCK for the treatment of vascular diseases.

The Association Between Obstructive Sleep Apnea and Carotid Intima–Media Thickness: A Systematic Review and Meta-Analysis

Obstructive sleep apnea (OSA) has been suggested as a risk factor for carotid atherosclerosis. The present meta-analysis aimed to evaluate the association between OSA and carotid intima–media thickness (CIMT). Eighteen studies comparing CIMT of patients with OSA versus non-OSA patients were included. Quantitative data synthesis was used to pool weighted standardized difference in means (SMD) of CIMT in a random-effects model. Compared to healthy controls, patients with OSA had a significantly higher CIMT (SMD: 0.881; 95% confidence interval [CI]: 0.647-1.115; P < .001). Due to the great heterogeneity, a subgroup analysis was conducted based on the study design. The pooled SMD of CIMT between patients with OSA and healthy controls were 0.810 (95% CI: 0.676-0.943; P < .001) and 1.008 (95% CI: 0.506-1.510; P < .001) in matched and unmatched group, respectively. Moreover, the correlation of apnea–hypopnea index and CIMT was moderate (r = .389; 95% CI: 0.315-0.459; P < .001). After adjustment for several major confounders, OSA is an independent risk factor for CIMT. These findings remind clinicians to screen for cardiovascular diseases in patients with OSA.

Upregulation of the gene expression of CLOCK is correlated with hypoxia‑inducible factor 1α in advanced varicose lesions

According to previous literature, venous hypoxia and the hypoxia‑inducible factor (HIF) pathway may contribute to the pathogenesis of varicose veins (VVs). It is widely accepted that the circadian locomotor output cycles kaput (CLOCK) gene affects nucleotide excision repair, DNA damage checkpoints and apoptosis in mammalian organisms; however, the expression levels of CLOCK in varicose veins remain to be elucidated. The aim of the present study was to detect the expression of the circadian clock gene in initial and advanced varicose lesions and analyze the correlation between the CLOCK gene, HIF‑1α, and its target gene, vascular endothelial growth factor (VEGF), in VVs. Sections of the great saphenous veins (GSVs) were obtained from patients undergoing ligation and stripping for VVs (n=70) and a control group undergoing coronary artery bypass grafting with GSV harvest (n=11). All VV patients had incompetent GSVs, according to color flow duplex scanning. C‑class VVs were determined according to the clinical‑etiology‑anatomy‑pathophysiology classification for venous diseases following physical examination of the patients with VV. Reverse transcription‑quantitative polymerase chain reaction was used to determine the expression levels of the CLOCK gene, HIF‑1α and VEGF. Immunohistochemical analysis was also performed. The patients with VVs were divided into those with initial varicose lesions (C3 and C4) and advanced varicose lesions (C5 and C6). In total, 21 of the patients had C3 lesions, 23 had C4 lesions, 14 had C5 lesions and 12 had C6 chronic venous disease. The expression of the CLOCK gene was significantly higher in the VV lesions of the GSV, compared with the normal GSVs (P<0.0001). The same trend was found in the expression levels of HIF‑1α and its target gene, VEGF, in the VV lesions (P=0.003 and P<0.0001, respectively). Subgroup analysis revealed that the expression levels of the CLOCK gene, HIF‑1α and VEGF were significantly higher in the advanced stage varicose lesions, compared with the initial varicose lesions (P<0.0001, P=0.0014 and P<0.0001, respectively). However, no statistically significant difference was identified in the expression levels of the aforementioned genes in the C3 and C4 lesions. The results demonstrated that the expression gene levels of CLOCK, HIF‑1α and its target gene, VEGF, increased significantly in advanced stage varicose lesions. Therefore, upregulation of the CLOCK gene in the vessel walls of veins may be involved in the pathogenesis of VVs and the progression of venous disease.

Low molecular weight fucoidan attenuates experimental abdominal aortic aneurysm through interfering the leukocyte-endothelial cells interaction

Low molecular weight fucoidan (LMWF) is a sulfated polysaccharide extracted from Saccharina Japonica that presents high affinity for P-selectin and abolish selectin-dependent recruitment of leukocytes. We hypothesized that dietary intake of LMWF, as a competitive binding agent of P-selectin, could limit the inflammatory infiltration and aneurysmal growth in an Angiotensin II-induced abdominal aortic aneurysm (AAA) mouse model. The Gene Expression Omnibus database was used for gene expressions and gene set enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that focal adhesion was involved in the development of AAA. However, dietary intake of LMWF could limit the enlargement of AAA, decreasing maximal aortic diameter and preserving elastin lamellae. Although LMWF did not decrease the circulatory monocytes count and lower the expression of P-selectin in endothelium, it reduced macrophages infiltration in media and adventitia. Furthermore, matrix metalloproteinase expression was markedly downregulated, accompanied with reduced expression of inflammatory mediators, including interleukin 1β, tumor necrosis factor-α and monocyte chemotactic protein-1. The present study revealed a novel target for the treatment of AAA and the anti-inflammatory effects of LMWF.

Serum tumor necrosis factor α levels are associated with new ischemic brain lesions after carotid artery stenting

Objective New diffusion‐weighted imaging (DWI) lesions on magnetic resonance imaging (MRI) after carotid artery stenting (CAS) are associated with an increased risk of future cerebrovascular events. Therefore, we evaluated the association between the expression levels of serum inflammatory markers and new DWI lesions after CAS and the presence of intraplaque hemorrhage (IPH). We also explored the mechanisms underlying this association. Methods A total of 225 inpatients with severe carotid artery stenosis were consecutively enrolled in this cohort study. Serum inflammatory marker levels were detected in all patients by enzyme‐linked immunosorbent assay. In the final analysis, 128 patients who underwent CAS and received pretreatment and post‐treatment MRI scans were enrolled. DWI was performed to detect new ischemia brain lesions. T1‐weighted, T2‐weighted, and time‐of‐flight sequences were also conducted to identify IPH. Results Serum tumor necrosis factor &agr; (TNF‐&agr;) levels were significantly higher in symptomatic patients as well as in IPH+ patients identified by carotid MRI. New DWI lesions were identified in 50% of patients after CAS. Univariate analysis showed that DWI+ patients after CAS exhibited older mean age, higher mean TNF‐&agr; levels, and more IPH on preoperative MRI and were less likely to have right carotid stenosis than DWI− patients. Multivariate logistic regression analyses revealed that serum TNF‐&agr; concentrations were associated with new DWI lesions after CAS (odds ratio, 1.245; 95% confidence interval, 1.068‐1.451; P = .005). Finally, the specificity and sensitivity of serum TNF‐&agr; levels in predicting DWI+ patients after CAS were 0.828 and 0.453, respectively. Conclusions Higher serum TNF‐&agr; levels are associated with a higher likelihood of new DWI lesions after CAS and the presence of IPH. Therefore, TNF‐&agr; is a potentially valuable predictor of acute ischemic cerebral lesions after CAS and the presence of IPH.

Extensive screening for occult malignancy in unprovoked venous thromboembolism: A meta-analysis

BACKGROUND The present meta-analysis aimed to evaluate the efficacy and sensitivity of an extensive screening strategy for occult malignant diseases in patients with unprovoked venous thromboembolism (VTE). METHODS We conducted a systematic search of PubMed, Cochrane, EMBASE, and relevant article references. Meta-analysis was used to pool weighted relative risks (RR) for the rate of missed diagnosis, all-cause mortality, and cancer-related mortality. Heterogeneity test was performed using the inconsistency index. Furthermore, pooled analysis of the sensitivity and the proportion of false-positive findings of PET/CT were conducted. RESULTS A total of 5 controlled studies were included with 1,115 and 1,159 unprovoked VTE patients receiving limited or extensive screening strategy, respectively. The risk of missed diagnosis (RR, 0.51; 95% CI, 0.20-1.28; P=0.15) was not significantly different between the limited and extensive screening group. Moreover, there was no statistically significant difference in all-cause mortality (RR, 0.86; 95% CI, 0.58-1.27; P=0.44) and cancer-related mortality (RR, 0.86; 95% CI, 0.46-1.62; P=0.65) between the two groups. The pooled sensitivity and proportion of false-positive findings of PET/CT as a screening tool for occult malignancy in patients with unprovoked VTE was 95% (95% CI, 38%-100%) and 33% (95% CI, 20%-47%), respectively. CONCLUSIONS Extensive screening strategy did not show a clinically significant benefit over limited screening strategy. Considering the high cost and the additional physical and emotional harm, current evidence did not support extensive screening for each patient in the setting.

Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells

BackgroundThe clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions.MethodsSeven health donors’ normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits.ResultThe circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs.ConclusionThe results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.