Hongwei Lu

Senescent endothelial dysfunction is attributed to the up-regulation of sphingosine-1-phosphate receptor-2 in aged rats

Sphingosine-1-phosphate (S1P) regulates an array of biological activities in various cell types. Many S1P-regulated functions are mediated by S1P receptor subtypes. Previous studies have shown that the S1P receptor-2 (S1P2 receptor) is involved in senescence of endothelial cells in vitro. However, the functional outcomes of S1P2 receptor-mediated signaling pathways in a physiological environment are currently unknown. The main aim of this study was to explore the underlying mechanisms of senescent-associated impairments in pulmonary microvascular endothelial cells (PMECs) isolated from young and aged rats in vivo. The expression of S1P receptor subtypes in PMECs was detected using RT-PCR and Western blot analyses. The chemotaxis of PMECs was measured using the transwell migration assay. The endothelial morphogenesis assay was performed in three-dimensional Matrigel. The wound-healing assay was performed using a scratch wound model on primary PMEC monolayers. We observed that the S1P2 receptor was markedly increased and S1P-regulated responses were significantly inhibited in PMECs isolated from aged rats. Furthermore, the senescent-associated impairments were revoked by the down-regulation of the S1P2 receptor in senescent PMECs. These results indicate that the impaired functions (such as chemotactic, wound healing, and morphogenetic responses) in senescent PMECs in vivo are mediated by the S1P2 receptor.

Role of sphingosine-1-phosphate receptor 1 and sphingosine-1-phosphate receptor 2 in hyperglycemia-induced endothelial cell dysfunction

The hyperglycemia-induced production of oxidative stress results in endothelial cell dysfunction. Previous studies have demonstrated that sphingosine-1-phosphate (S1P) regulates an array of biological activities in endothelial cells mediated by sphingosine-1-phosphate receptors (S1PRs). However, the role of S1PR-mediated signaling pathways in hyperglycemia-induced endothelial cell dysfunction is currently unknown. In the present study, we aimed to explore the role of S1PRs in endothelial cell dysfunction. For this purpose, hyperglycemia-induced oxidative stress was examined using human umbilical vein endothelial cells (HUVECs) cultured with either normal (5.6 mM) or high (25 mM) levels of glucose. The levels of reactive oxygen species (ROS) and nitric oxide (NO) were determined by flow cytometric (FCM) analysis and nitrate reductase, respectively. Endothelial morphogenesis assay was performed in three-dimensional Matrigel. The mRNA and protein expression levels of S1PRs in the HUVECs were determined by RT-qPCR and western blot analysis, respectively. In addition, ROS, NO and endothelial morphogenesis assays were conducted using the high glucose-treated endothelial cells transfected with adenoviral vector expressing exogenous S1PR1 gene (pAd-S1PR1) or with adenoviral vector expressing S1PR2-specific shRNA (pAd-shRNA-S1PR2). The expression levels of S1PR1 and S1PR2 in the endothelial cells treated with high levels of glucose decreased and increased, respectively. However, the effects of high levels of glucose on S1PR3 were minimal. In addition, high levels of glucose enhanced ROS generation and markedly reduced NO generation and morphogenetic responses. Nevertheless, all the aforementioned changes were completely reversed by transfection with pAd-S1PR1 or pAd-shRNA-S1PR2, which increased S1PR1 and decreased S1PR2 expression, respectively. It can thus be concluded that S1PR1 and S1PR2 play crucial roles in hyperglycemia-induced endothelial cell dysfunction.

The role of tumor necrosis factor-like weak inducer of apoptosis in atherosclerosis via its two different receptors (Review)

At present, it is commonly accepted that atherosclerosis is a chronic inflammatory disease characterized by disorder of the arterial wall. As one of the inflammatory cytokines of the tumor necrosis factor superfamily, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) participates in the formation and progression of atherosclerosis. TWEAK, when binding to its initial receptor, fibroblast growth factor inducible molecule 14 (Fn14), exerts adverse biological functions in atherosclerosis, including dysfunction of endothelial cells, phenotypic change of smooth muscle cells and inflammatory responses of monocytes/macrophages. However, accumulating data supports that, besides Fn14, TWEAK also binds to cluster of differentiation (CD)163, an anti-inflammatory cytokine and a scavenger receptor exclusively expressed by monocytes and macrophages. Furthermore, it has been demonstrated that CD163 is able to internalize TWEAK and likely elicits protective effects in atherosclerosis by terminating inflammation induced by TWEAK. In the present study, the role of TWEAK in atherosclerosis was reviewed, with a predominant focus on CD163 and Fn14 receptors.

Importance of mitochondrial calcium uniporter in high glucose–induced endothelial cell dysfunction

Objective: Mitochondrial Ca2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment. Methods: Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2′,7′-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model. Results: High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively. Conclusion: Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.

Senescent endothelial dysfunctions were mediated by S1P2 receptor in cultured human umbilical vein endothelial cells

OBJECTIVE To investigate the variation of senescent endothelial function by regulating the sphingosine-1-phosphate receptor type 2 (S1P2) expression in cultured human umbilical vein endothelial cells (HUVECs). METHODS The S1P2 receptor expression was regulated by transfecting the cDNA or shRNA of S1P2 in cultured HUVECs. The expression levels of S1P2 receptor in HUVECs were detected by RT-PCR and Western blot. EC chemotaxis was measured by the transwell migration assay. The wound healing assay was performed by a scratch wound model on EC monolayer. Matrigel morphogenesis assay was employed to assess the in vitro angiogenic responses. RESULTS After up-regulating the S1P2 expression in young ECs, the S1P-stimulated formation of a tubular-like network in Matrigel was dramatically diminished in transfected ECs (P<0.05). Quantification of the wound healing assay showed that transfected ECs grew much slower than young ECs (P<0.05). The chemotactic capability was significantly decreased in transfected ECs (P<0.05). Furthermore, the senescent-associated impairments were revoked by the downregulation of S1P2 receptor in senescent HUVECs. CONCLUSION The impaired functions (chemotactic, wound-healing and morphogenetic responses) in senescent HUVECs in vitro are mediated by S1P2 receptor.

TWEAK/Fn14 promotes oxidative stress through AMPK/PGC‑1α/MnSOD signaling pathway in endothelial cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) contributes to dysfunction of endothelial cells via its receptor, Fn14. However, its role in the production of reactive oxygen species (ROS), particularly mitochondrial ROS (mtROS) and the subsequent decrease in nitric oxide (NO) in endothelial cells remains unclear. In this study, the effect of TWEAK/Fn14 on generation of ROS, mtROS and NO in endothelial cells and its potential mechanism was investigated. Human umbilical vein endothelial cells (HUVECs) were treated with TWEAK with Fn14 small interfering (si)RNA or negative control RNA. It was demonstrated that TWEAK induced the production of ROS and mtROS in HUVECs, which were detected by fluorescent microscope, and flow cytometry. In addition, TWEAK decreased the generation of NO as indicated using the Nitric Oxide Assay kit. Furthermore, TWEAK aggravated mtDNA damage as measured by quantitative polymerase chain reaction analysis. Inhibition of Fn14 by Fn14 siRNA decreased TWEAK‑induced ROS and mtROS production, as well as mtDNA damage, while it increased the production of NO in endothelial cells. In addition, TWEAK inhibited the expression of active AMP‑activated protein kinase (AMPK) and its downstream protein peroxisome proliferator‑activated receptor‑γ coactivator-1α (PGC‑1α) and manganese superoxide dismutase (MnSOD). Notably, Fn14 siRNA enhanced the expression of the aforementioned proteins. Taken together, TWEAK/Fn14 contributes to endothelial dysfunction through modulation of ROS and mtROS. In addition, the underlying mechanism is implicated in the AMPK/PGC‑1α/MnSOD signaling pathway.

S1PR2 antagonist protects endothelial cells against high glucose-induced mitochondrial apoptosis through the Akt/GSK-3β signaling pathway.

Vascular complications are the main cause of morbidity and mortality associated with type 2 diabetes mellitus. An early hallmark of the onset of vascular complications is endothelial dysfunction and apoptosis. We aimed to explore the role of sphingosine-1-phosphatereceptor 2 (S1PR2) in high glucose-induced endothelial cells apoptosis and to elaborate the underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were cultured in a high glucose with or without S1PR2 antagonist. The apoptosis of the cells was measured by flow cytometry and mitochondrial membrane permeability was detected by the fluorescent probe JC-1. The expression of the related protein was determined by western blot. Cell apoptosis and the loss of mitochondrial membrane permeability were induced under high glucose conditions in HUVECs. The expression of mitochondrial apoptosis related protein bax increased and bcl-2 decreased in high glucose-induced HUVECs. The level of cytochrome c released into the cytoplasm increased when cells were exposed to high glucose. In addition, the expression of p-AKT and p-GSK3β was reduced when HUVECs were treated with high glucose. However, these effects were reversed in HUVECs when cells treated with S1PR2 antagonist. In conclusion, S1PR2 antagonist protects endothelial cells against high glucose-induced mitochondrial apoptosis through the Akt/GSK-3β signaling pathway.