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Defective autophagy in vascular smooth muscle cells (VSMCs) in response to oxidative stress can lead to cellular apoptosis and plaque instability. Previous studies have revealed that the circadian clock system is involved in autophagic regulation and plaque progression. However, the mechanism by which circadian rhythmicity influences VSMC autophagy and plaque stability remains unclear. Our study described the circadian profiles in atheromatous plaques and verified the role of circadian misalignment in VSMC autophagy and apoptosis. We found that the mRNA expression levels of circadian locomotor output cycles protein kaput (CLOCK) and Beclin 1 were significantly decreased in unstable plaques compared with stable plaques. No significant differences were observed in other circadian rhythm genes. VSMCs treated with oxidized low-density lipoprotein (ox-LDL, 80 μg/ml) exhibited abnormal circadian rhythmicity and impaired autophagy, as evidenced by consistent decreases in CLOCK and Beclin 1 expression, suggesting a correlation between CLOCK and autophagy. CLOCK protein expression was inhibited by ox-LDL, accompanied by defective autophagy and an increased apoptosis rates (P

Introduction: Venous ulceration is a multifactorial disease, and whether hyperhomocysteinemia (HHcy) promotes deterioration from primary varicose veins to venous ulcers remains unproven. Methods: This study retrospectively analyzed clinical data from 717 patients to investigate the potential correlation between HHcy and primary varicose veins ulcer formation, including 611 patients without ulcers (control group) and 106 with ulcers (case group). Results: In this study, 46.2% (49/106) of patients in the case group and 17.5% (107/611, p < 0.001) in the control group suffered from HHcy. Multivariate logistic analysis revealed that HHcy was closely associated with the incidence of venous ulceration in patients with primary varicose veins ( p < 0.001). Propensity score matching created 101 matched pairs of patients with and without ulcers, and the analysis pointed to a potential link between HHcy and ulcer formation in the context of primary varicose veins ( p < 0.001). Additional experiments showed that HHcy could induce endothelial dysfunction and phenotypic switching of vascular smooth muscle cells. Conclusion: Both clinical and experimental findings implicated HHcy as a key factor in the development of venous ulceration. Further research is needed to appraise the effectiveness of HHcy-lowering therapy in the prevention of venous ulcers in patients with varicose veins.

Background Lower limb chronic venous disease (CVD), resulting from iliac vein compression syndrome (IVCS), manifests as a series of symptoms ranging from varicose veins to venous ulcerations. Stent implantation has been considered an effective treatment method; however, the management of CVD has rarely been reported. In the present study, we evaluated the treatment and outcomes of patients with CVD. Methods We performed a retrospective cohort study of patients with severe iliac vein stenosis with lower limb CVD. The patients were divided into two groups: group 1 had received stenting alone (n = 42), and group 2 had received stenting and high ligation/endovenous laser treatment (n = 29). We evaluated the clinical outcomes using the Venous Clinical Severity Score and visual analog scale, and assessed the quality of life (QoL) using the Chronic Venous Disease QoL questionnaire at a median follow-up point of 15 months (range, 6-25 months). Results In our cohort, the prevalence rate of nonthrombotic IVCS (NIVCS) was 11.7% (98 of 838 patients). The technical success rate was 100%, without severe complications. During the study period, three group 1 patients and two group 2 patients were lost to follow-up. The overall patency rate in the patients with NIVCS during a mean follow-up period of 15.0 months (range, 6-25 months) was 94.4%. For patients with a Clinical, Etiology, Anatomy, Pathophysiology (CEAP) clinical class of Conclusions The prevalence of NIVCS should not be overlooked. The proposed combination treatment is an effective therapeutic strategy for patients with NIVCS and advanced CVD (CEAP clinical class, ≥4) during short-term follow-up.

Aims: Diabetic foot results in frequent amputation and quality-of-life reduction in diabetes population. These lesions are featured by a prolonged and exaggerated inflammation with a significant impairment in local bacterial invasion. Negative pressure wound therapy (NPWT) attenuates hyperinflammation in the healing of diabetic foot wounds, but the potential mechanism of NPWT down-regulated inflammatory reaction still remains elusive. This study aims to explore the inflammatory signaling involved in the effect of NPWT on diabetic ulcer. Methods: Thirty patients with diabetic foot ulceration were divided into NPWT group (treated with NPWT, n = 10), NPWT + FK565 group (treated with NPWT combined with FK565 which is NOD1 receptor ligand, n = 10) and control group (n = 10). After two weeks treatment, samples were harvested and analyzed by histochemistry for infiltration of inflammatory cells, immunofluorescence stain for NOD1, western blotting for NOD1, RIP2 (Receptor interacting protein 2), IL-1β, TAK1 (Transforming growth factor-β-activated kinase1), p65 and real time-PCR for expression of NOD1 and RIP2. Results: NPWT could notably accelerate the diabetic wound healing through alleviating inflammatory reaction. The immunofluorescence analysis results revealed that NOD1 was mainly expressed in the cytoplasm and noticeably decreased after the NPWT treatment. And NPWT obviously decreased both the mRNA and protein level of NOD1 and RIP2. Moreover, The protein expression of IL-1β, TAK1 and p65 in the NPWT-group were significant decreased. Conclusion: NPWT effectively promotes wound healing by suppressing the wound inflammation in diabetic foot, which is mediated at least in part by suppression of NOD1 receptor.

A chronic change in hemodynamic forces might activate the pathophysiological process of maladaptive venous remodeling. Biomechanical stretching stimulates venous smooth muscle cells (SMCs) in the media, and biomechanical loads exceeding physiological levels affect the intrinsic circadian rhythm and cellular phenotype. This study aimed to investigate the changes in the expression patterns of circadian clock genes under biomechanical stretching and their role in the regulation of the SMC phenotype.Circadian genes were detected in venous specimens and venous SMCs from patients with varicose veins (VVs) and patients with autologous vein grafts (normal veins). Molecular mechanism studies of SMC phenotypic switching under biomechanical stretching were performed in human umbilical venous SMCs (HUVSMCs).CLOCK upregulation was observed in VVs. The circadian rhythm was disrupted in venous SMCs derived from VVs. In addition, CLOCK expression and cell proliferation and migration were increased in HUVSMCs exposed to biomechanical stretch. CLOCK overexpression activated NF-κB signaling and phenotypic transformation in HUVSMCs, whereas CLOCK depletion had inhibitory effects on these pathways. Further experiments revealed that the CLOCK protein regulates phenotypic and functional transformation via the RHOA/ROCK1 pathway.Our results demonstrate that CLOCK is a crucial regulator of the SMC phenotype under mechanical stretch. The CLOCK/RHOA/ROCK1 pathway is important in phenotypic adaptation, and targeting RHOA/ROCK1 could potentially reverse stretch-induced phenotypic switching.

BACKGROUND This study aimed to investigate the clinical results of endovenous thermal ablation combined with stab phlebectomy for unilateral varicose veins based on diabetic management. METHODS The study reviewed 501 patients who underwent endovenous thermal ablation combined with stab phlebectomy for unilateral varicose veins, including 337 nondiabetics (control group) and 164 diabetics. Diabetics with hemoglobin A1c ≥7% were classified as the poor glycemic control group, and hemoglobin A1c

Carotid plaque rupture with thrombosis is responsible for the majority of acute cerebrovascular events. The study aimed to identify key genes associated with plaque destabilization. In our study, expression profiles regarding carotid plaque stability (GSE41571 and GSE120521) were obtained from the Gene Expression Omnibus (GEO) database and used to analyze differentially expressed genes (DEGs), key biological processes (BP) and intersecting pathways. And the expression of the DEGs was validated in clinical and cell samples. A total of 113 DEGs were identified and gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. Among the BP annotations, changes in “extracellular matrix (ECM) were closely related to plaque stability. “Focal adhesion”, ECM -receptor interaction”, “vascular smooth muscle contraction” and “regulation of actin cytoskeleton” were key pathways associated with plaque stability. Genes with top 15 scores (CytoHubba) were selected as hub genes, including ACTA2, MYLK, CALD1, MYH10, ACE, HMOX1, CYBA, NOX4, LUM, RAC2, COL4A5, SDC1, UCP2, SORBS1, and FCER1G. Pathway enrichment analysis showed that selected hub genes were also significantly enriched in “vascular smooth muscle contraction” and “focal adhesion”. Further detection in tissue and cell samples suggested MYLK, NOX4, COL4A5, SDC1 and CYBA might have potential clinical significance.

Vascular endothelial dysfunction is considered critical development in the progression of cardiovascular events and is associated with vascular damage and oxidative stress. Previous studies have shown that profilin-1 could be induced by advanced glycation end products (AGEs) and contributes to vascular hyperpermeability; however, the mechanisms are not fully understood. In this study, we sought to assess whether reactive oxygen species (ROS) were involved in profilin-1-mediated RhoA/ROCK1 signaling. Treatment with AGEs significantly induced the expression of profilin-1 and ROS production in human umbilical vein endothelial cells (HUVECs), whereas knockdown of profilin-1 diminished AGE-induced RhoA and ROCK1 activation and ROS production. Moreover, ectopic overexpression of profilin-1 also increased RhoA and ROCK1 activation and ROS production under low AGE concentration. Furthermore, blockage of RhoA/ROCK1 with the inhibitors CT04 and Y27632 significantly attenuated the profilin-1-mediated cell damage and ROS production. Additionally, ROS inhibition resulted in a significant decrease in profilin-1-mediated RhoA/ROCK1 expression, suggesting that the regulation of RhoA/ROCK1 signaling was partly independent of ROS. Taken together, these results suggested that the RhoA/ROCK1 pathway activated by excessive ROS is responsible for profilin-1-mediated endothelial damage.

The expression and secretion of infla-mmation‑associated cytokines are induced by hypoxia. Circadian locomotor output cycles protein kaput (CLOCK) has previously been shown to activate the nuclear factor‑κB (NF‑κB) pathway, which is a key transcription factor during hypoxia. The present study evaluated the role of the NF‑κB pathway in the CLOCK‑induced inflammatory response. Under hypoxic conditions, the expression levels of NF‑κB and proinflammatory cytokines, including interleukin (IL)‑1, IL‑1β, IL‑6, intercellular adhesion molecule 1, cyclooxygenase 2 and tumor necrosis factor alpha, were significantly increased compared with under control conditions. Conversely, human umbilical vein endothelial cells (HUVECs) that were transfected with small hairpin RNA against human CLOCK exhibited reversed effects. Furthermore, inhibition of NF‑κB with pyrrolidine dithiocarbamate (PDTC) reduced the expression of proinflammatory cytokines in HUVECs treated under hypoxic conditions. In addition, the CLOCK‑induced inflammatory response was abolished with PDTC treatment. These findings suggest that the mechanism by which CLOCK induces inflammation mainly involves activation of the NF‑κB signaling pathway.

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.

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

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.

Autogenous arteriovenous fistula (AVF) is the first choice for hemodialysis access in renal failure with uremia. However, AVF cannot be performed in some patients due to small and narrow veins in the forearm. In this study, a Fogarty catheter was used to establish autogenous radiocephalic hemodialysis access in patients with small caliber cephalic veins, and the patency rate and complications of this method were observed. Sixty-seven patients with uremia were divided into a treatment group (40 cases, caliber of cephalic veins

This study aims to explore the expression of platelet-derived growth factor (PDGF) in arteriosclerosis plaque on type 2 diabetes mellitus (DM) with arteriosclerosis obliterans (ASO) of the lower extremity. A total of 24 samples from 12 patients with lower-extremity ASO who underwent surgical treatment were collected in this study. Among these samples were 12 iliofemoral arterial plaques and 12 popliteal or distal arterial plaques. Immunohistochemistry staining was performed on the plaque intima to determine the expression level of platelet-derived growth factor receptors α and β. Reverse-transcription polymerase chain reaction was carried out to detect the mRNA level of the PDGF A and B chains. PDGFR-α and PDGFR-β were excessively expressed in lower-extremity arteriosclerosis plaques. The mean PDGF B chain mRNA level in the popliteal arterial plaques of ASO patients with type-2 DM was increased when compared with that in the iliofemoral arterial plaques. The PDGF B chain mRNA level was higher in the popliteal arterial plaques in type-2 DM with ASO than in iliofemoral artery plaques. This finding confirmed that the PDGF B chain has a key function in arteriosclerosis development. Key words: Arteriosclerosis obliterans, platelet-derived growth factor, type-2 diabetes.

Background: Chronic peripheral arterial occlusion (CPAO) is a progressive disease that is associated with a variety of symptoms, the 4 most common being a sensation of coolness in the limbs, intermittent claudication (in which pain occurs on walking), limb pain (which occurs spontaneously at rest), and ischemic leg ulcers. Beraprost sodium is an oral prostaglandin I2 analogue that may ameliorate these symptoms. Objective: The aim of this study was to compare the efficacy and tolerability of beraprost sodium and ticlopidine hydrochloride in the treatment of patients with CPAO in China. Methods: In this multicenter, single-blind, controlled study, patients with CPAO were randomly assigned to receive beraprost 120-μg tablet TID or ticlopidine 500-mg tablet BID, both administered orally. The clinical efficacy of the drugs was assessed using the 4 main symptoms of CPAO. Ankle-brachial index (ABI) also was measured as a clinical pharmacologic procedure. Adverse events were assessed throughout the study. Results: A total of 124 patients (96 men, 28 women; mean [SD] age, 65 [12] years) were enrolled in 3 hospitals. Data from 119 patients (93 men, 26 women; mean [SD] age, 65 [12] years) were included in the efficacy analysis (64 and 55 patients in the beraprost and ticlopidine groups, respectively). Although all 4 symptoms of CPAO were ameliorated after 3 and 6 weeks of treatment with both drugs, only the cool sensation was significantly improved with beraprost compared with ticlopidine at 6 weeks (P

OBJECTIVE To evaluate the clinical effect of endovenous laser treatment (EVLT) for patients with varicose veins. METHODS Our series included 117 patients who underwent EVLT combined with high ligation and stripping since the introduction of the technique in our institution. All EVLT procedures were performed with local skin cooling to prevent skin burns, as well as stripping after exsanguinations to prevent thrombotic phlebitis. RESULTS A total of 146 limbs in 117 patients were ablated by EVLT. Bilateral EVLT was performed in 29 patients, with the remaining 88 patients undergoing unilateral EVLT. The mean age of the patients was 57 years ± 12 years (range: 21 years to 80 years), and 56 were male and 61 were female. Follow-up for all patients lasted three to six months. The most common complication was induration and swelling, which was observed in 64 patients, followed by paraesthesia in 27, and skin burns in 12. CONCLUSION The treatment with endovenous laser treatment for patients with varicose veins is safe and effective.

OBJECTIVE: Piwi-interacting RNAs (piRNAs) represent a molecular feature shared by all nonaging biological systems, including the germline and somatic cancer stem cells, which display an indefinite renewal capacity and lifespan-stable genomic integrity and are potentially immortal. Here, we tested the hypothesis that piRNA is a critical genetic determinant of aging in humans. METHODS: Expression of transcriptome-wide piRNAs (n=24k) was profiled in the human prefrontal cortex of 12 subjects (84.9±9.5, range 68–100, years of age) using microarray technology. We examined the correlation between these piRNAs’ expression levels and age, adjusting for covariates including disease status. RESULTS: A total of 9,453 piRNAs were detected in brain. Including seven intergenic and three intronic piRNAs, ten piRNAs were significantly associated with age after correction for multiple testing (|r|=0.9; 1.9×10(−5)≤p≤9.9×10(−5)). CONCLUSION: We conclude that piRNAs might play a potential role in determining the years of survival of humans. The underlying mechanisms might involve the suppression of transposable elements (TEs) and expression regulation of aging-associated genes.