Metabolic regulation and dysregulation of endothelial small conductance calcium activated potassium channels

The vascular endothelium is an important regulator of vascular reactivity and preserves the balance between vasoconstrictor and vasodilator tone during normal physiologic conditions. Example endothelial-derived vasoconstrictors include endothelin-1 and thromboxane A2; example vasodilators include nitric oxide and prostacyclin. A growing body of evidence points to the existence of a non-nitric oxide, non-prostacyclin endothelium-derived vasodilatory factor of currently unclear identity, often referred to as endothelium-derived hyperpolarizing factor (EDHF). Recent research testifies to the significance of EDHF in endothelium-dependent vascular smooth muscle relaxation. Special emphasis has been placed on the role of small conductance calcium-activated potassium channels (SK) in facilitating the endothelial and vascular responses to EDHF across the microcirculation, including coronary, mesenteric, and pulmonary vascular beds. Meanwhile, decreased activity of endothelial SK channel activity has been implicated in the pathology of a variety of disease states that alter the balance between vasodilator and vasoconstrictor tone. Hence the primary goal of this review is to characterize the physiology of endothelial SK channels in the microvasculature under normal and pathological conditions. Themes of regulation and dysregulation of SK channel activity through the action of protein kinases, reactive oxygen species, and byproducts of intermediary metabolism provide unifying principles to tie together vascular pathology in altered metabolic states ranging from hypertension to diabetes, to ischemia-reperfusion. A comprehensive understanding of SK channel pathophysiology may provide a foundation for development of new therapeutics targeting SK channels, particularly SK channel potentiators, that may have widespread application for many chronic disease states.     

COX-2 is involved in vascular oxidative stress and endothelial dysfunction of renal interlobar arteries from obese Zucker rats

Obesity is related to vascular dysfunction through inflammation and oxidative stress and it has been identified as a risk factor for chronic renal disease. In the present study, we assessed the specific relationships among reactive oxygen species (ROS), cyclooxygenase 2 (COX-2), and endothelial dysfunction in renal interlobar arteries from a genetic model of obesity/insulin resistance, the obese Zucker rats (OZR). Relaxations to acetylcholine (ACh) were significantly reduced in renal arteries from OZR compared to their counterpart, the lean Zucker rat (LZR), suggesting endothelial dysfunction. Blockade of COX with indomethacin and with the selective blocker of COX-2 restored the relaxations to ACh in obese rats. Selective blockade of the TXA₂/PGH₂ (TP) receptor enhanced ACh relaxations only in OZR, while inhibition of the prostacyclin (PGI₂) receptor (IP) enhanced basal tone and inhibited ACh vasodilator responses only in LZR. Basal production of superoxide was increased in arteries of OZR and involved NADPH and xanthine oxidase activation and NOS uncoupling. Under conditions of NOS blockade, ACh induced vasoconstriction and increased ROS generation that were augmented in arteries from OZR and blunted by COX-2 inhibition and by the ROS scavenger tempol. Hydrogen peroxide (H₂O₂) evoked both endothelium- and vascular smooth muscle (VSM)-dependent contractions, as well as ROS generation that was reduced by COX-2 inhibition. In addition, COX-2 expression was enhanced in both VSM and endothelium of renal arteries from OZR. These results suggest that increased COX-2-dependent vasoconstriction contributes to renal endothelial dysfunction through enhanced (ROS) generation in obesity. COX-2 activity is in turn upregulated by ROS.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

Specific Activation of Insulin-like Growth Factor-1 Receptor by Ginsenoside Rg5 Promotes Angiogenesis and Vasorelaxation

Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE^−/− mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and G_i-mediated phospholipase C/Ca²⁺/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC₅₀ of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.     

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities

Purpose

Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs).

Methods

Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25 mmol/l for 24 h. l-Glucose was used at a concentration of 25 mmol/l as a control.

Results

Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB⁺FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment.

Conclusion

These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.     

Tubulogenesis during blood vessel formation

The ability to form and maintain a functional system of contiguous hollow tubes is a critical feature of vascular endothelial cells (ECs). Lumen formation, or tubulogenesis, occurs in blood vessels during both vasculogenesis and angiogenesis in the embryo. Formation of vascular lumens takes place prior to the establishment of blood flow and to vascular remodeling which results in a characteristic hierarchical vessel organization. While epithelial lumen formation has received intense attention in past decades, more recent work has only just begun to elucidate the mechanisms controlling the initiation and morphogenesis of endothelial lumens. Studies using in vitro and in vivo models, including zebrafish and mammals, are beginning to paint an emerging picture of how blood vessels establish their characteristic morphology and become patent. In this article, we review and discuss the molecular and cellular mechanisms driving the formation of vascular tubes, primarily in vivo, and we compare and contrast proposed models for blood vessel lumen formation.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

胰蛋白酶诱导人脐静脉内皮细胞分泌白细胞介素-8

目的:研究胰蛋白酶对IL-8释放的影响。方法:分离、培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)、倒置显微镜观察形态变化,流式细胞术检测内皮细胞标志和蛋白酶活化受体-2(proteinase-activated receptor-2,PAR-2)表达,ELISA检测HUVECs培养上清中IL-8水平。结果:HUVECs表达内皮细胞标志和PAR-2。刺激16 h,1 g/ml胰蛋白酶和100M PAR-2激活肽组HUVECs单层均匀性降低。胰蛋白酶能够显著刺激HUVECs释放IL-8,PAR-2激活肽也诱导IL-8水平升高。蛋白酶抑制剂和PAR-2抑制肽均能够显著抑制胰蛋白酶诱导的IL-8释放。PAR-2激活肽和胰蛋白酶诱导升高的IL-8水平之间成正相关性。结论:胰蛋白酶很可能通过PAR-2激活促进血管内皮细胞释放IL-8。