Protein Synthesis In Endothelial Cells Research Articles

Assessment of the state and relationship of vasopressor and renin-angiotensin systems in patients with localized kidney cancer on the background of arterial hypertension

Background. Vasopressin, in cooperative interaction with angiotensin II, participates in cardiovascular regulation and it increases in arterial hypertension. In addition, vasopressin is involved in tumorigenesis through angiogenesis by stimulating protein synthesis in endothelial cells, induction of endothelin-1, influencing cell proliferation, and stimulating growth factors through VIA receptors in the kidneys. To assess the content of vasopressin, a measurement of copeptin, its precursor, produced in an equimolar ratio, is used.Objective: to determine the content of serum copeptin and to reveal correlations with the indices of the renin-angiotensin system in the tumor and blood serum in patients with localized kidney cancer (КС) with arterial hypertension (AH).Materials and methods. The inclusion criteria for the study were initially diagnosed localized KC T1N0M0 without special treatment, age less than 75 years, essential AH of I—II degree without treatment, controlled by angiotensin-converting enzyme inhibitors (ACEi). All patients were divided into groups: patients with KC (n = 42); KC + AH without treatment (n = 31); KC + AH + ACEi (n = 32). Serum levels of copeptin and angiotensin I, II, 1-7 and angiotensin-converting enzyme, angiotensin-converting enzyme 2 were determined by ELISA. Also, the level of components of the renin-angiotensin system was assessed in tumor tissue samples obtained by robotic-assisted kidney resection.Results and conclusion. In patients of the KC + AH group, the median of the indicator is statistically significantly higher than in the group of healthy donors (2.4 times at p <0.05). In the group KC + AH + ACEi, a decrease in the content of the studied indicator was found in comparison with the norm by 1.2 times (at p <0.05). It was found that the content of copeptin in the tumor less than 4 cm in comparison with the size of 4-7 cm is significantly lower (KC p = 0.045, KC + AH p = 0.067 and KC + AH + ACEi p = 0.036). Correlation analysis showed direct significant links between high density between the levels of copeptin and angiotensin II in the tumor and blood, and moderate tightness with tumor and serum levels of angiotensin (1-7). Multiple regression analysis revealed that the most significant factors that have a positive effect on the concentration of serum copeptin are the content of angiotensin II in the tumor and blood serum, the tumor concentration of angiotensin (1-7) and angiotensin-converting enzyme 2, as well as the level of systolic blood pressure (p <0.05).

The natural product vioprolide A exerts anti-inflammatory actions through inhibition of its cellular target NOP14 and downregulation of importin-dependent NF-ĸB p65 nuclear translocation

Chronic inflammation is characterized by persisting leukocyte infiltration of the affected tissue, which is enabled by activated endothelial cells (ECs). Chronic inflammatory diseases remain a major pharmacotherapeutic challenge, and thus the search for novel drugs and drug targets is an ongoing demand. We have identified the natural product vioprolide A (vioA) to exert anti-inflammatory actions in vivo and in ECs in vitro through inhibition of its cellular target nucleolar protein 14 (NOP14). VioA attenuated the infiltration of microglia and macrophages during laser-induced murine choroidal neovascularization and the leukocyte trafficking through the vascular endothelium in the murine cremaster muscle. Mechanistic studies revealed that vioA downregulates EC adhesion molecules and the tumor necrosis factor receptor (TNFR) 1 by decreasing the de novo protein synthesis in ECs. Most importantly, we found that inhibition of importin-dependent NF-ĸB p65 nuclear translocation is a crucial part of the action of vioA leading to reduced NF-ĸB promotor activity and inflammatory gene expression. Knockdown experiments revealed a causal link between the cellular target NOP14 and the anti-inflammatory action of vioA, classifying the natural product as unique drug lead for anti-inflammatory therapeutics.

Effect of butyrate, a bacterial by-product, on the viability and ICAM-1 expression/production of human vascular endothelial cells: Role in infectious pulpal/periapical diseases.

To investigate the effects of butyric acid (BA), a metabolic product generated by pulp and root canal pathogens, on the viability and intercellular adhesion molecule-1 (ICAM-1) production of endothelial cells, which are crucial to angiogenesis and pulpal/periapical wound healing. Endothelial cells were exposed to butyrate with/without inhibitors. Cell viability, apoptosis and reactive oxygen species (ROS) were evaluated using an MTT assay, PI/annexin V and DCF fluorescence flow cytometry respectively. RNA and protein expression was determined using a polymerase chain reaction assay and Western blotting or immunofluorescent staining. Soluble ICAM-1 (sICAM-1) was measured using an enzyme-linked immunosorbent assay. The quantitative results were expressed as mean±standard error (SE) of the mean. The data were analysed using a paired Student's t-test where necessary. A p-value ≤0.05 was considered to indicate a statistically significant difference between groups. Butyrate (>4mM) inhibited cell viability and induced cellular apoptosis and necrosis. It inhibited cyclin B1 but stimulated p21 and p27 expression. Butyrate stimulated ROS production and hemeoxygenase-1 (HO-1) expression as well as activated the Ac-H3, p-ATM, p-ATR, p-Chk1, p-Chk2, p-p38 and p-Akt expression of endothelial cells. Butyrate stimulated ICAM-1mRNA/protein expression and significant sICAM-1 production (p<.05). Superoxide dismutase, 5z-7oxozeaenol, SB203580 and compound C (p<.05), but not ZnPP, CGK733, AZD7762 or LY294002, attenuated butyrate cytotoxicity to endothelial cells. Notably, little effect on butyrate-stimulated sICAM-1secretion was found. Valproic acid, phenylbutyrate and trichostatin (three histone deacetylase inhibitors) significantly induced sICAM-1 production (p<.05). Butyric acid inhibited proliferation, induced apoptosis, stimulated ROS and HO-1 production and increased ICAM-1mRNA expression and protein synthesis in endothelial cells. Cell viability affected by BA was diminished by some inhibitors; however, the increased sICAM-1secretion by BA was not affected by any of the tested inhibitors. These results facilitate understanding of the pathogenesis, prevention and treatment of pulpal/periapical diseases.

Xenocoumacin 2 reduces protein biosynthesis and inhibits inflammatory and angiogenesis-related processes in endothelial cells

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase β (IKKβ). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.

Narciclasine inhibits angiogenic processes by activation of Rho kinase and by downregulation of the VEGF receptor 2

The process of angiogenesis is involved in several pathological conditions, such as tumor growth or age-related macular degeneration. Although the available anti-angiogenic drugs have improved the therapy of these diseases, major drawbacks, such as unwanted side effects and resistances, still exist. Consequently, the search for new anti-angiogenic substances is still ongoing. Narciclasine, a plant alkaloid from different members of the Amaryllidaceae family, has extensively been characterized as anti-tumor compound. Beyond the field of cancer, the compound has recently been shown to possess anti-inflammatory properties. Surprisingly, potential actions of narciclasine on endothelial cells in the context of angiogenesis have been neglected so far. Thus, we aimed to analyze the effects of narciclasine on angiogenic processes in vitro and in vivo and to elucidate the underlying mechanism. Narciclasine (100–300 nM) effectively inhibited the proliferation, undirected and directed migration, network formation and angiogenic sprouting of human primary endothelial cells. Moreover, narciclasine (1 mg/kg/day) strongly reduced the VEGF-triggered angiogenesis in vivo (Matrigel plug assay in mice). Narciclasine mediated its anti-angiogenic effects in part by a RhoA-independent activation of the Rho kinase ROCK. Most importantly, however, the compound reduced the de novo protein synthesis in endothelial cells by approx. 50% without exhibiting considerable cytotoxic effects. As a consequence, narciclasine diminished the presence of proteins with a short half-life, such as the VEGF receptor 2, which is the basis for its anti-angiogenic effects. Taken together, our study highlights narciclasine as an interesting anti-angiogenic compound that is worth to be further evaluated in preclinical studies.

Inhibition of cell surface expression of endothelial adhesion molecules by ursolic acid prevents intimal hyperplasia of venous bypass grafts in rats

Despite rapid progress in surgical techniques, there is still a significant lack of surgery-supportive pharmacological treatments. The aim of this study was to test the hypothesis that ursolic acid (UA) may prevent intimal hyperplasia of venous bypass grafts. The hypothesis was tested by means of primary cell isolation and culture followed by real-time polymerase chain reaction, western blotting, fluorescence microscopy and fluorescence-activated cell sorting analyses, as well as an in vivo rat model for intimal hyperplasia of venous bypass grafts and immunohistochemistry and histochemistry. The local application of UA significantly inhibited intimal hyperplasia in vivo (intimal thickness control: 25 µm, UA group: 18 µM-8 weeks after surgery). The UA treatment of grafts significantly resulted in reduced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression, reduced infiltration of the grafts vessel wall by CD45-positive cells and increased smooth muscle cell (SMC) death. In in vitro condition, it could be shown that UA inhibits VCAM-1 expression downstream of NFκB and is likely to interfere with VCAM-1 protein synthesis in endothelial cells. Quantification of cell death in vascular smooth muscle cells treated with UA indicated that UA is a potent inducer of SMC apoptosis. Our results suggest that UA-mediated inhibition of endothelial VCAM-1 expression reduces the infiltration of venous bypass grafts by CD45-positive cells and inhibits intimal hyperplasia. Apoptosis induction in SMCs may be another method in which UA reduces intimal thickening. UA may constitute a surgery-supportive pharmacon that reduces intimal hyperplasia of vein grafts.

Troglitazone acutely inhibits protein synthesis in endothelial cells via a novel mechanism involving protein phosphatase 2A-dependent p70 S6 kinase inhibition

Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, have been implicated in the inhibition of protein synthesis in a variety of cells, but the underlying mechanisms remain obscure. We report that troglitazone, the first TZD drug, acutely inhibited protein synthesis by decreasing p70 S6 kinase (p70S6K) activity in bovine aortic endothelial cells (BAEC). This inhibition was not accompanied by decreased phosphorylation status or in vitro kinase activity of mammalian target of rapamycin (mTOR). Furthermore, cotreatment with rapamycin, a specific mTOR inhibitor, and troglitazone additively inhibited both p70S6K activity and protein synthesis, suggesting that the inhibitory effects of troglitazone are not mediated by mTOR. Overexpression of the wild-type p70S6K gene significantly reversed the troglitazone-induced inhibition of protein synthesis, indicating an important role of p70S6K. Okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, partially reversed the troglitazone-induced inhibition of p70S6K activity and protein synthesis. Although troglitazone did not alter total cellular PP2A activity, it increased the physical association between p70S6K and PP2A, suggesting an underlying molecular mechanism. GW9662, a PPARgamma antagonist, did not alter any of the observed inhibitory effects. Finally, we also found that the mTOR-independent inhibitory mechanism of troglitazone holds for the TZDs ciglitazone, pioglitazone, and rosiglitazone, in BAEC and other types of endothelial cells tested. In conclusion, our data demonstrate for the first time that troglitazone (and perhaps other TZDs) acutely decreases p70S6K activity through a PP2A-dependent mechanism that is independent of mTOR and PPARgamma, leading to the inhibition of protein synthesis in endothelial cells.

Ultrastructure study on patients with spontaneous rupture of hepatocellular carcinoma

To investigate the ultrastructure of small artery wall in patients with spontaneous rupture of hepatocellular carcinoma (HCC). Transmission electron microscopy was used to study 11 specimens from ruptured HCC and 11 cases with non-ruptured HCC. The phenomenon of activated phagocytosis in macrophage could be found in 3 cases with ruptured HCC and 10 cases with non-ruptured HCC, respectively (P < 0.05). In 9 specimens with ruptured HCC, the evidence of vascular injury characterized as less cell junctions and larger fenestrae in endothelial cells, broken elastic lamina, proliferated and fragmented elastin and damaged structure of collagen was found in small arteries. The phenomenon of electron-dense deposit in the elastic lamina, and signs of more protein synthesis in endothelial cells were also present in these specimens. In the patients with non-ruptured HCC, the evidence of vascular injury can be found only in 2 cases (P < 0.01). Less cell junctions and larger fenestrae could increase the permeability of vascular wall. The electron-dense deposition in elastic lamina may represent the deposition of antigen-antibody complex in elastic membrane which had been found in our previous study. The vascular injury was postulated to be caused by the deposition of antigen-antibody complex in vascular wall which was identified by our previous study. The vascular wall in the patient with ruptured HCC could become stiff and weak due to the proliferated fragment elastin and damaged collagen which would make the blood vessels more prone to splitting and result in hemorrhage and the rupture of HCC. The vascular injury caused by antigen-antibody complex deposition might related to the spontaneous rupture of HCC.

Ultrastructural study on spontaneous rupture of hepatocellular carcinoma

目的 肝癌自发性破裂是一种危险的出血性并发症,其发病机制不详.我们既往的免疫组化研究已首次证实了该病人中存在血管病变现象,本实验旨在通过电子显微镜对其血管壁进行超微结构检查,以进一步证实其血管病变.方法 1995～1997年间,对11例肝癌破裂病人的手术标本与同期随机选取的11例肝癌非破裂病人手术标本,进行血管内皮细胞的透射电子显微镜检查.结果 11例肿瘤破裂的病人中,有9例病人的血管壁出现内皮细胞受损现象,包括:血管内皮细胞发生细胞连接消失、窗口直径过大及伴有高蛋白合成征象.在肿瘤非破裂的病人中,上述血管受损现象仅在2例病人中发现,二者相比有显著性差异(P<0.05).结论肝癌破裂病人中的血管内皮细胞存在通透性增加及高蛋白合成现象,其病变可能与肿瘤破裂有关。

The hepatitis C virus internal ribosome entry site facilitates efficient protein synthesis in blood vessel endothelium during tumour angiogenesis.

The development of gene delivery systems for therapeutic use involves vectors (often retrovirus or adenovirus) which typically encode one target protein, but the use of internal ribosome entry sites (IRES) can confer the ability to express more than one protein from bi- or polycistronic mRNAs. IRES elements can display tissue-specific expression, so it is necessary to determine suitable IRES for specific clinical applicability. Blood vessel endothelial cells are important clinically since many different conditions involve neo-vascularisation (angiogenesis). We have demonstrated that the viral hepatitis C IRES element is a powerful mediator of protein synthesis in angiogenesis, such as found in solid tumours. Homologous recombination was used to introduce IRES-lacZ sequences into the Lmo2 gene, which is expressed in endothelial cells. beta-Galactosidase expression was determined during vascular remodelling in mouse embryos and in sprouting endothelium during growth of solid tumours, and showed that the hepatitis C IRES is used efficiently for protein synthesis in endothelial cells. This IRES element can provide the means to express two or more therapeutic genes in blood vessel endothelium in clinical conditions, such as cancer, which depend on angiogenesis.

Ultrastructural Study of the Vascular Endothelium of Patients with Spontaneous Rupture of Hepatocellular Carcinoma

Spontaneous rupture of hepatocellular carcinoma (HCC) is common in Asia and Africa, although the mechanism is unclear. In our previous study, we found that vascular injury exemplified by collagenase synthesis and collagen degradation in small arteries was related to the HCC rupture. In this study, transmission electron microscopy was used to study 22 specimens from ruptured HCC and non-ruptured HCC. In nine specimens of ruptured HCC, there was evidence of vascular injury with fewer cell junctions and larger fenestrae in vascular endothelial cells. The phenomenon of increased endothelial protein synthesis was also present. In the specimens of non-ruptured HCC, evidence of vascular injury was found in only two cases (p < 0.01). Fewer cell junctions and larger fenestrae could increase the permeability of the vascular wall. Increased protein synthesis in endothelial cells correlates with the phenomenon that more collagenase is expressed in these cells. The resulting breakdown of collagen could render the blood vessels weak; hence, these blood vessels are more prone to splitting/breakage. We conclude from our study that this vascular injury may result in HCC rupture.