Longhua Fan

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.

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.

Comparison of beraprost and ticlopidine in Chinese patients with chronic peripheral arterial occlusion: a multicenter, single-blind, randomized, controlled study

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<0.05). ABI was significantly increased with both beraprost and ticlopidine at 6 weeks versus baseline (P<0.001 and P<0.01, respectively), suggesting that this pharmacologic action may have led to their beneficial effect on various symptoms. The tolerability analysis included 123 patients (65 and 58 patients in the beraprost and ticlopidine groups, respectively). The numbers of patients who (1) experienced adverse events (AEs), (2) experienced adverse drug reactions, and (3) withdrew due to AEs were significantly smaller in the beraprost group than in the ticlopidine group (P<0.001, P<0.05, and P<0.05, respectively). CONCLUSIONS In this study population of patients with CPAO, beraprost ameliorated cool sensation in the limbs, intermittent claudication, limb pain, and ischemic/leg ulcers. Beraprost was more efficacious in relieving CPAO symptoms and was better tolerated than ticlopidine. Beraprost may be useful for the treatment of patients with CPAO, but more studies are needed.

A Positive Feedback Loop of Profilin-1 and RhoA/ROCK1 Promotes Endothelial Dysfunction and Oxidative Stress

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.

Significance of the distribution of platelet-derived growth factor B chain in arteriosclerosis plaque in type- 2 diabetic patients with arteriosclerosis obliterans of lower extremity

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. Reversetranscription 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.