Reduction of high glucose and phorbol-myristate-acetate-induced endothelial cell permeability by protein kinase C inhibitors LY379196 and hypocrellin A

Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. Although recent studies have established a link between protein kinase C (PKC) pathway and hyperglycaemic-induced vascular permeability, it is unclear which PKC isoforms involve increased endothelial cell permeability. In the present study, we investigated whether high glucose induced endothelial hyperpermeability via distinct PKC isoforms in human umbilical vein endothelial cells (HUVECs) and whether increased endothelial permeability could be substantially reversed by PKC inhibitors LY379196 and hypocrellin A (HA). High glucose (20 mM) and phorbol-myristate-acetate (PMA)-induced endothelial hyperpermeability was almost abolished by 150 nM HA and partially reduced by 30 nM PKC beta inhibitor (LY379196). LY379196 and HA inhibited the membrane fraction of PKC activity in a dose-dependent manner. Western blot analysis revealed high-glucose-induced overexpression of PKC alpha and PKC beta2 in the membrane fraction of HUVECs. LY379196 (30 and 150 nM) selectively inhibited PKC beta2 with no significant effect on PKC alpha expression. HA (150 nM) significantly reduced PKC alpha expression with no inhibitory effect on PKC beta2. At higher concentrations (300 nM), both LY379196 and HA were no longer selective for PKC beta or alpha, respectively. This study showed that both PKC alpha and beta2 contributed to endothelial hyperpermeability. Since reduction of endothelial hyperpermeability was greater with inhibition of PKC alpha rather than PKC beta2, we conclude that PKC alpha may be a major isoform involved in endothelial permeability in HUVECs, and that PKC alpha-mediated endothelial permeability was significantly reversed by the PKC inhibitor HA.     

Anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells: G0/G1 p21-associated cell cycle arrest

The aim of this study was to examine the anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells and its possible molecular mechanism underlying. Our data showed that BJ-601 at a range of concentrations (0-40 microM) dose- and time-dependently decreased cell number in cultured human dermal microvascular endothelial cells (HDMVECs), but not human fibroblasts. The BJ-601-induced growth inhibition in HDMVECs was reversible. [3H]thymidine incorporation demonstrated that BJ-601 arrested the HDMVECs at the G0/G1 phase of the cell cycle. Western blot analysis revealed that BJ-601 (0-40 microM) dose-dependently increased the levels of the protein p21, but not of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase 2 (CDK2), and CDK4 in HDMVECs. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not CDK2-p27, CDK4-p21 and CDK4-p27 complexes, was increased in the BJ-601-treated HDMVECs. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in a dose-dependent manner in the BJ-601-treated HDMVECs. Pretreatment of HDMVECs with a p21 antisense oligonucleotide, which blocked the expression of p21 protein, reversed the BJ-601-induced inhibition of [3H]thymidine incorporation into HDMVECs. Moreover, cotreatment of the endothelial cells with protein kinase C (PKC) inhibitor, staurosporine, prevented the BJ-601-induced decrease of [3H]thymidine incorporation into HDMVECs. Administration of BJ-601 dose-dependently inhibited capillary-like tube formation of HDMVECs in Matrigel. In conclusion, these data suggest that BJ-601 inhibits HDMVECs proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally causes retardation of the cell cycle at the G0/G1 phase.     

Effects of TNF-alpha and curcumin on the expression of thrombomodulin and endothelial protein C receptor in human endothelial cells

The objective of this study was to elucidate the effects of tumor necrosis factor-alpha (TNF-alpha) on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human endothelial cells as well as the effect of curcumin, a spice and coloring food compound, as a potential therapeutic agent. Human umbilical vein endothelial cells (HUVECs) treated with TNF-alpha (2.0 ng/ml) showed reduced TM mRNA levels by 80%, 97%, 94%, and 97% at 3, 6, 12, and 24 h, respectively (P<0.05), by real-time PCR analysis. Dose-dependent study showed that TM mRNA levels of HUVECs were decreased by 86%, 89%, 91%, and 94% after treatment of TNF-alpha (0, 0.25, 0.5, 1, and 2 ng/ml) for 6 h, respectively (P<0.05). TM protein levels in HUVECs were significantly reduced by 69% in TNF-alpha-treated cells as compared to controls (P<0.05) by Western blot analysis. Secreted protein and activity of TM of HUVEC cultures were also significantly reduced in TNF-alpha-treated cells. In addition, EPCR mRNA levels of HUVECs were significantly reduced in TNF-alpha-treated group as compared to controls (P<0.05). Furthermore, these effects were observed in other types of endothelial cells from human coronary arteries, lung, and skin. Curcumin effectively blocked these effects of TNF-alpha on downregulation of TM and EPCR. These data demonstrate that TNF-alpha significantly decreases expression of TM and EPCR at both mRNA and protein levels in several human endothelial cells. Curcumin can effectively block TNF-alpha-induced endothelial dysfunction. This study suggests a new molecular mechanism of inflammation-induced thrombosis and a new therapeutic strategy to prevent this clinical problem.     

Anti-angiogenic activity of methanol extract of Phellinus linteus and its fractions

Aim of the study

The aim of the present study was to investigate the effects of MeOH extract of PL (PLME) and its fractions on angiogenesis.

Materials and methods

PLME and its subsequent fractions (methylene chloride, ethyl acetate, n-butanol and aqueous fractions) were evaluated in vitro. Specifically, the anti-angiogenic activities of PLME and its fractions were investigated by measuring their effects on the proliferation, migration, tube formation and phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human umbilical vein endothelial cells (HUVECs). In addition, the in vivo Matrigel plug model was applied to evaluate new vessel formation.

Results

The results revealed that PLME and its subsequent fractions, except for the aqueous fraction, led to significant inhibition of the proliferation, migration, tube formation and VEGFR-2 phosphorylation of HUVECs as well as in vivo angiogenesis.

Conclusions

These findings indicate the potential for the use of PLME in pathological situations involving stimulated angiogenesis, such as inflammation and tumor development.     

Chrysanthemum morifolium Ramat. reduces the oxidized LDL-induced expression of intercellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells

Ethnopharmacological relevance

The flower of Chrysanthemum morifolium Ramat. (CM) with antioxidant, cardiovascular protective and anti-inflammatory functions, has been widely used in China for hundreds of years as a healthy beverage and medicine.

Aims of the study

The purpose of the present study is to investigate the effects of HCM (a hot water extract of the flower of Chrysanthemum morifolium Ramat. [CM]), ECM (an ethanol extract of CM), and the abundant flavonoids apigenin and luteolin in CM on the oxidized LDL (oxLDL)-induced expression of ICAM-1 and E-selectin in human umbilical vein endothelial cells (HUVECs). The possible mechanism of these effects was also determined.

Materials and methods

MTT assay was for cell viability. Western blot was used for ICAM-1 and E-selection protein expression, and for activation of protein kinase B (PKB) and cAMP responsive element binding protein (CREB) proteins. Fluorescence flow cytometry was for ICAM-1 and E-selectin expression on cell surface. DCF-DA flow cytometric assay was used for reactive oxygen species (ROS) production.

Results

HCM, ECM, apigenin, and luteolin dose-dependently inhibited ICAM-1 and E-selectin expression and adhesion of HL-60 by oxLDL. HCM, ECM, apigenin, and luteolin reversed the inhibition of phosphorylation of Akt and CREB by oxLDL; however, this reversion was abolished by wortmannin. In addition, wortmannin abrogated the inhibitory effects of CM extracts, apigenin and luteolin on adhesion molecule expression. The ROS scavenging capability of HCM, ECM, apigenin, and luteolin proceeded dose-dependently in the presence of oxLDL.

Conclusion

CM is a plant with cardiovascular-protective potential and the inhibitory effects of CM on ICAM-1 and E-selectin expression are, at least partially, attributed to its antioxidant activity and modulation of the PI3K/Akt signaling pathway.     

Anti-angiogenic activities of gliotoxin and its methylthio-derivative, fungal metabolites

In the search for new naturally occurring angiogenic inhibitor, we found that culture broths from two unidentified fungal strains exerted potent inhibitory activities on capillary-like tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Two active compounds were isolated by bioassay-guided separation and their structures were identified as gliotoxin (1) and its derivative methylthiogliotoxin (2) by spectroscopic analyses. These compounds significantly inhibited the migration of HUVEC assessed by in vitro wounding migration assay and exhibited at least 10 times more potent inhibition of proliferation of HUVECs as compared with that of cancer cell lines such as HeLa, MCF-7, and KB 3-1 cells. Especially, gliotoxin having disulfide group exerted more potent activities than methylthiogliotoxin, suggesting that gliotoxin could be a useful compound for further study as an anti-angiogenic agent.     

HUVECs from newborns with a strong family history of myocardial infarction overexpress adhesion molecules and react abnormally to stimulating agents

Atherosclerosis is a complex disease involved in major fatal events such as myocardial infarction and stroke. It is the result of interactions between metabolic, dietetic and environmental risk factors acting on a genetic background that could result in endothelial susceptibility. Our aim was to determine the patterns of expression of adhesion molecules and whether phosphatidylserine is translocated to the cell surface of human umbilical vein endothelial cells (HUVECs) isolated from healthy newborns born to parents with a strong family history of myocardial infarction under TNF-alpha or oxLDL stimulated conditions. Compared to control HUVECs, experimental cords showed: (a) a four-fold increase in VCAM-1 expression under basal conditions, which showed no change after stimulation with the pro-atherogenic factors; (b) a two-fold increase in basal P-selectin expression that reached a 10-fold increase with any of the pro-atherogenic factors; (c) a basal ICAM-1 expression similar to P-selectin that was not modified by the pro-atherogenic molecules; (d) a similar PECAM-1 expression. Unexpectedly, phospathidylserine expression in experimental cord HUVECs was significantly increased (211 817 versus 3354 TFU) but was not associated to apoptotic death as the percentage of dead cells induced by TNF-alpha treatment was very low (0.55 versus 9.87% in control HUVECs). The latter result was corroborated by TUNEL staining. T cell adherence to HUVECs was highly up-regulated in the genetically predisposed samples. The analysis of nonpooled HUVECs, from newborns to family predisposed myocardial-infarction individuals, might represent a useful strategy to identify phenotypical and functional alterations, and hopefully, to take early preventive actions.     

Racial Differences in Oxidative Stress and Inflammation: In Vitro and In Vivo

African American race is an independent risk factor for enhanced oxidative stress and inflammation. We sought to examine whether oxidative‐stress and inflammatory markers that are typically measured in humans also differ by race in cell culture. We compared levels between African American and Caucasian young adults and then separately in human umbilical vein endothelial cells (HUVECs) from both races. We found heightened oxidative stress and inflammation in the African Americans both in vitro and in vivo. African American HUVECs showed higher nitric oxide (NO) levels (10.8 ± 0.4 vs. 8.8 ± 0.7 μmol/L/mg, p = 0.03), Interleukin‐6 (IL‐6) levels (61.7 ± 4.2 vs. 23.9 ± 9.0 pg/mg, p = 0.02), and lower superoxide dismutase activity (15.6 ± 3.3 vs. 25.4 ± 2.8 U/mg, p = 0.04), and also higher protein expression (p < 0.05) of NADPH oxidase subunit p47phox, isoforms NOX2 and NOX4, endothelial nitric oxide synthase (NOS), inducible NOS, as well as IL‐6. African American adults had higher plasma protein carbonyls (1.1 ± 0.1 vs. 0.8 ± 0.1 nmol/mg, p = 0.01) and antioxidant capacity (2.3 ± 0.2 vs. 1.1 ± 0.3 mM, p = 0.01). These preliminary translational data demonstrate a racial difference in HUVECs much like that in humans, but should be interpreted with caution given its preliminary nature. It is known that racial differences exist in how humans respond to development and progression of disease, therefore these data suggest that ethnicity of cell model may be important to consider with in vitro clinical research. Clin Trans Sci 2011; Volume 4: 32–37     

大鼠肝细胞与人脐静脉内皮细胞混合共微囊化的体外研究

目的 观察人脐静脉内皮细胞(HUVECs)对共微囊化大鼠肝实质细胞的保护作用.方法 利用自制微囊发生器制备含肝细胞或肝细胞与HUVECs以10:1混合的微囊进行体外培养,同时建立非微囊化单独培养和共培养组,通过测定培养液中自蛋白、尿素的分泌量和肝细胞形态来判断肝细胞功能和活性.结果 肝细胞与HUVECs共培养或共微囊化均能提高前者的白蛋白分泌和尿素合成量(均P<0.01),存活时间延长;共微囊化组虽前7 d的白蛋白和尿素合成量较非微囊化共培养组低,但在7 d后的白蛋白和尿素合成量高于后者,且相对平稳.结论 肝细胞与HUVECs混合共微囊化能明显改善囊内肝细胞的形态、功能与寿命.     

Platelet membrane-coated nanoparticle-mediated targeting delivery of Rapamycin blocks atherosclerotic plaque development and stabilizes plaque in apolipoprotein E-deficient (ApoE−/−) mice

Although certain success has been achieved in atherosclerosis treatment, tremendous challenges remain in developing more efficient strategies to treat atherosclerosis. Platelets have inherent affinity to plaques and naturally home to atherosclerotic sites. Rapamycin features potent anti-atherosclerosis effect, but its clinical utility is limited by its low concentration at the atherosclerotic site and severe systemic toxicity. In the present study, we used platelet membrane-coated nanoparticles (PNP) as a targeted drug delivery platform to treat atherosclerosis through mimicking platelets' inherent targeting to plaques. PNP displayed 4.98-fold greater radiant efficiency than control nanoparticles in atherosclerotic arterial trees, indicating its effective homing to atherosclerotic plaques in vivo. In an atherosclerosis model established in apolipoprotein E-deficient mice, PNP encapsulating rapamycin significantly attenuated the progression of atherosclerosis and stabilized atherosclerotic plaques. These results demonstrated the perfect efficacy and pro-resolving potential of PNP as a targeted drug delivery platform for atherosclerosis treatment.