Enzymatic antioxidant system of endotheliocytes.

It is shown that endothelial cells from human umbilical vein have a reduced activity and gene expression of the "classic" antioxidant enzymes (Cu,Zn-superoxide dismutase, catalase, and Se-containing glutathione peroxidase). At the same time, a high expression level of peroxiredoxin genes was identified in the same endothelial cells, which obviously indicates the predominant involvement of these enzymes in protecting the endothelium from the damaging effect of free radical peroxidation.

Maternal hypercholesterolemia in pregnancy associates with umbilical vein endothelial dysfunction: role of endothelial nitric oxide synthase and arginase II.

OBJECTIVE: Human pregnancy that courses with maternal supraphysiological hypercholesterolemia (MSPH) correlates with atherosclerotic lesions in fetal arteries. It is known that hypercholesterolemia associates with endothelial dysfunction in adults, a phenomenon where nitric oxide (NO) and arginase are involved. However, nothing is reported on potential alterations in the fetoplacental endothelial function in MSPH. The aim of this study was to determine whether MSPH alters fetal vascular reactivity via endothelial arginase/urea and L-arginine transport/NO signaling pathways. APPROACH AND RESULTS: Total cholesterol <280 mg/dL was considered as maternal physiological hypercholesterolemia (n=46 women) and ≥ 280 mg/dL as MSPH (n=28 women). Maternal but not fetal total cholesterol and low-density lipoprotein-cholesterol levels were elevated in MSPH. Umbilical veins were used for vascular reactivity assays (wire myography), and primary cultures of umbilical vein endothelial cells to determine arginase, endothelial NO synthase (eNOS), and human cationic amino acid transporter 1 and human cationic amino acid transporter 2A/B expression and activity. MSPH reduced calcitonine gene-related peptide-umbilical vein relaxation and increased intima/media ratio (histochemistry), as well as reduced eNOS activity (L-citrulline synthesis from L-arginine, eNOS phosphorylation/dephosphorylation), but increased arginase activity and arginase II protein abundance. Arginase inhibition increased eNOS activity and L-arginine transport capacity without altering human cationic amino acid transporter 1 or human cationic amino acid transporter 2A/B protein abundance in maternal physiological hypercholesterolemia and MSPH. CONCLUSIONS: MSPH is a pathophysiological condition altering umbilical vein reactivity because of fetal endothelial dysfunction associated with arginase and eNOS signaling imbalance. We speculate that elevated maternal circulating cholesterol is a factor leading to fetal endothelial dysfunction, which could have serious consequences to the growing fetus.

MT1-MMP regulates MMP-2 expression and angiogenesis-related functions in human umbilical vein endothelial cells.

Membrane type 1 (MT1)-MMP is a member of matrix metalloproteinases (MMPs) that regulates extracellular matrix remodeling. In addition, MT1-MMP also serves as a multi-functional protein. However, the functional role of MT1-MMP in human endothelial cells remains unclear. In this study we use real-time PCR and Western blotting to demonstrate for the first time that MMP-2 expression is regulated by MT1-MMP in human endothelial cells. Moreover, MMP-2 activity is also modulated by MT1-MMP. In addition we found that endothelial cells, ECM adhesion and human endothelial cell tube formation, which are known to be regulated by MMP-2, are blocked by MT1-MMP siRNA. These results suggest that MT1-MMP plays an important role in regulating angiogenesis in human endothelial cells.

MAPK phosphatase-1 contributes to trichostatin A inhibition of cyclooxygenase-2 expression in human umbilical vascular endothelial cells exposed to lipopolysaccharide.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have emerged as a new class of antitumor agents because they were demonstrated to induce cell cycle arrest, promote cell apoptosis, and inhibit metastasis. Recently, HDAC inhibitors were also shown to exhibit pronounced anti-inflammatory properties. However, the underlying mechanism contributing to the suppression of inflammatory responses by HDAC inhibitors remains to be fully defined. In the present study, we explored the actions of trichostatin A (TSA), a potent HDAC inhibitor, on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 expression in human umbilical vascular endothelial cells (HUVECs). METHODS: HUVECs were exposed to LPS in the absence or presence of TSA. COX-2 expression and signaling molecules (JNK, p38MAPK and c-jun) activated by LPS were assessed. RESULTS: The LPS-induced cox-2 messenger RNA and protein were markedly suppressed by TSA. TSA inhibited JNK and p38MAPK phosphorylation in cells exposed to LPS. Treatment of cells with a JNK signaling inhibitor (JNK inhibitor II) or a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced COX-2 expression. TSA suppression of JNK and p38MAPK phosphorylation and subsequent COX-2 expression were restored by selective inhibition of MKP-1 using MKP-1 siRNA. In addition, TSA caused an increase in MKP-1 phosphatase activity in HUVECs. In conclusion, TSA may cause MKP-1 activation to dephosphorylate JNK and p38MAPK, leading to the downregulation of COX-2 in HUVECs stimulated by LPS, a proinflammatory stimulus. GENERAL SIGNIFICANCE: MKP-1 contributes to TSA's protective actions in HUVECs exposed to LPS. The present study also supports the therapeutic value of TSA in treating inflammatory vascular diseases.

HYAL1 overexpression is correlated with the malignant behavior of human breast cancer.

Extracellular matrix (ECM) is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronic acid (HA) is a component of the ECM, and hyaluronidase (HAase) is a HA-degrading endoglycosidase. Levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. In this study, we detected HYAL1 expression levels in breast cancer cells and tissues, and measured the amount HAase activity in breast cancer cells. Compared with nonmalignant breast cell line HBL-100 and normal breast tissues, HYAL1 were overexpressed in breast cancer cell lines MDA-MB-231, MCF-7, invasive duct cancer tissues and metastatic lymph nodes, respectively. Accordingly, the amount HAase activity in MDA-MB-231 and MCF-7 was higher than that in HBL-100. In addition, knockdown of HYAL1 expression in MDA-MB-231 and MCF-7 cells resulted in decreased cell growth, adhesion, invasion and angiogenesis potential. Meantime, the HYAL1 knockdown markedly inhibited breast cancer cell xenograft tumor growth and microvessel density. Further studies showed that the HYAL1, HYAL2 and HA were elevated in breast cancer, and HYAL1 could downregulate HA expression. In conclusion, HYAL1 may be a potential prognostic marker and therapeutic target in breast cancer.

Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis.

Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.

Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit.

MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases.

AIM: To investigate whether and how COS inhibited IL-8 production in LPS-induced human umbilical vein endothelial cells (HUVECs). METHODS: RT-PCR, enzyme-linked immunosorbent assays (ELISA) and Western blotting were used to study IL-8 expression and related signaling pathway. Wound healing migration assays and monocytic cell adhesion analysis were used to explore the chemotactic and adhesive activities of HUVECs. RESULTS: COS 50-200 µg/mL exerted a significant inhibitory effect on LPS 100 ng/mL-induced IL-8 expression in HUVECs at both the transcriptional and translational levels. In addition, COS 50-200 µg/mL inhibited LPS-induced HUVEC migration and U937 monocyte adhesion to HUVECs in a concentration-dependent manner. Signal transduction studies suggest that COS blocked LPS-induced activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) as well as phosphorylation of p38 mitogen-activated protein kinase (MAPK) and phosphokinase Akt. Further, the over-expression of LPS-induced IL-8 mRNA in HUVECs was suppressed by a p38 MAPK inhibitor (SB203580, 25 µmol/L) or a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002, 50 µmol/L). CONCLUSION: COS inhibited LPS-induced IL-8 expression in HUVECs through the blockade of the p38 MAPK and PI3K/Akt signaling pathways.

Angiogenin induces nitric oxide synthesis in endothelial cells through PI-3 and Akt kinases.

Angiogenesis, the formation of new blood vessels, is a critical but complex phenomenon modulated by numerous physicochemical conditions. Nitric oxide (NO) is a very well known biological mediator involved in vascular physiology. This study focuses on relationships between the effect of angiogenin, a major angiogenic factor, and extracellular NO release. NO concentration was sensed electrochemically using a fibronectin-coated multiple microelectrode array. Angiogenin was shown to increase NO levels, thus triggering nitric oxide synthase (NOS) activity. The effect of angiogenin on NOS was demonstrated using l-NAME, a competitive NOS inhibitor. Dose-time dependence was investigated, showing a stimulation threshold in the 250 ng/mL-1 microg/mL range and a maximal NO release after 30 min of exposure to angiogenin. To elucidate the very complex reactive pathway of angiogenin, we have used various selective inhibitors to investigate the mechanism leading to NO production. Neomycin, an antibiotic blocking nuclear translocation, inhibited the angiogenin effect on NOS. This result demonstrates that angiogenin activates NOS by interacting with the cell nucleus. Similarly, NOS activity was stopped by blocking the PI-3/Akt kinase signaling transduction cascade, showing the importance of this pathway.

Sphingolipids of human umbilical cord vein and their alteration in preeclampsia.

Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein (UCV). We evaluate the sphingolipid composition of UCV and its alteration in preeclampsia. The veins were taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Thin layer chromatography, solid-phase extraction and high-performance liquid chromatography were employed for sphingolipid analyses. The UCV walls of newborns delivered by healthy mothers are abundant in sphingomyelins and ceramides, whereas the amounts of sphingoid bases are rather low. Preeclampsia is associated with a significant decrease in sphingomyelins and ceramides, whereas the sphingoid bases changed in uncharacteristic manner. The increase in sphinganine and sphingosine 1-phosphate was accompanied with a decrease in sphingosine, hydroxysphinganine and sphinganine 1-phosphate. Stearate is the dominating fatty acid in sphingomyelins and ceramides of both control and preeclamptic veins. Sphingolipids and some sphingoid bases are bioactive molecules which contribute to regulation of signal transduction pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of UCV wall resulting in remodelling of its composition.

Estrogen receptor-alpha and endothelial nitric oxide synthase nuclear complex regulates transcription of human telomerase.

We report that in endothelial cells, the angiogenic effect of 17beta-estradiol (E2) is inhibited by the estrogen receptor (ER) antagonist ICI or the NO synthase (NOS) inhibitor 7-nitroindazole via downregulation of hTERT, the telomerase catalytic subunit, suggesting that E2 and NO are involved in controlling hTERT transcription. Quantitative Real-Time PCR and chromatin immunoprecipitations in E2-treated human umbilical vein endothelial cells, showed recruitment of ERs on the hTERT promoter and concomitant enrichment in histone 3 methylation at Lysine 79, a modification associated with transcription-competent chromatin. Confocal microscopy and re-chromatin immunoprecipitations revealed that on E2 induction, endothelial (e)NOS rapidly localized into the nucleus and associated with ERalpha on the hTERT promoter. Transfections of a constitutively active eNOS mutant (S1177D) strongly induced the hTERT promoter, indicating a direct role of the protein in hTERT transcriptional regulation. Mutation of the estrogen response element in the promoter abolished response to both ERs and active eNOS, demonstrating that the estrogen response element integrity is required for hTERT regulation by these factors. To investigate this novel regulation in a reduced NO environment, pulmonary endothelial cells were isolated from eNOS(-/-) mice and grown with/without E2. In wild-type cells, E2 significantly increased telomerase activity. In eNOS(-/-) cells, basal telomerase activity was rescued by exogenous eNOS or an NO donor, whereas responsiveness to E2 demanded the active protein. In conclusion, we document the novel findings of a combinatorial eNOS/ERalpha complex at the hTERT estrogen response element site and that active eNOS and ligand-activated ERs cooperate in regulating hTERT expression in the endothelium.

Ruscogenin mainly inhibits nuclear factor-kappaB but not Akt and mitogen-activated protein kinase signaling pathways in human umbilical vein endothelial cells.

Our previous results suggested that ruscogenin inhibited tumor necrosis factor alpha (TNF-alpha)-induced leukocyte adhesion, which correlated with its suppression of intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. In the present studies, we further examined its effects on the main signaling pathways involved in upregulation of ICAM-1 induced by TNF-alpha in human umbilical vein endothelial cells (HUVECs). The results showed that ruscogenin significantly suppressed p65 phosphorylation, IkappaB-alpha phosphorylation and degradation, and inhibited IkappaB kinase alpha (IKKalpha) and IKKbeta activation induced by TNF-alpha. However, it exerted weak effects on TNF-alpha-induced phosphorylations of p38, JNK, ERK1/2, and Akt. Overall, our results indicated that downregulation of ICAM-1 expression by ruscogenin in HUVECs might be mediated by nuclear factor-kappaB (NF-kappaB), but not by mitogen-activated protein kinase (MAPK) and Akt signaling pathways.

Endothelial nitric oxide synthase immunreactivity and the ultrastructure of endothelial cells of umbilical artery in normal and preeclamptic pregnancies.

Our objective was to investigate the endothelial nitric oxide synthase (eNOS) immuno-reactivity and the ultrastructure of endothelial cells of a human umbilical artery in both normal and preeclamptic pregnancies. The umbilical cords from normal and preeclamptic pregnancies were collected immediately after vaginal and abdominal deliveries. Umbilical arteries were isolated and fixed in 10% neutral formaline solution, embedded in paraffin, and then stained with hematoxylin and eosin (H&E) for the histologic investigation, and eNOS activation were examined in samples by streptavidine-biotine immunohistochemical methods. The arterial sections were also fixed in phosphate-buffered 2.5% glutaraldehyde solution (pH 7.2) for 3 h and post-fixed with 1% osmium tetroxide at 4°C for 2 h for the investigation of the ultrastructural examination. In the umbilical artery of preeclamptic pregnancies, endothelial cells were oval, triangular, or polygonal, and were disorganized. Some endothelial cells were separated by enlarged intercellular spaces. A dilated endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some endothelial cells displayed deep invaginations and irregular outlines. Most endothelial cells had a high number of cytoplasmic vacuoles. In preeclampsia, eNOS immunoreactivity increased considerably in endothelial cells when compared to normal pregnancies. We believe that preeclampsia plays an important role in the pathogenesis of endothelial cell dysfunction and activation in the umbilical artery. However, the disturbance mechanism of endothelial cells is not known, and further studies are necessary to clarify the exact mechanism.

Propofol induces endothelial nitric oxide synthase phosphorylation and activation in human umbilical vein endothelial cells by inhibiting protein kinase C delta expression.

BACKGROUND AND OBJECTIVE: Protein kinase Cs (PKCs) are involved in the regulation of endothelial nitric oxide synthase (eNOS). The purpose of this study is to evaluate the role of PKC delta in mediating the effects of chronic treatment with propofol on eNOS activation in human umbilical venous endothelial cells. METHODS: Human umbilical venous endothelial cells were treated chronically with propofol (1-100 micromol l(-1)) to induce phosphorylation of eNOS activation, which was assessed by immunoblot and cyclic GMP accumulation assay. The involvement of the phosphoinositide 3-kinase/Akt pathway was examined by an immunoblot assay. In addition, the role of PKC delta in regulating propofol-induced activation of eNOS was explored. Finally, cells were treated with a combination of ceramide (10 nmol l(-1)) and propofol (50 micromol l(-1)), and the role of protein phosphatase 2A in mediating drug-induced eNOS activation was evaluated. RESULTS: Cells treated with propofol showed an increase in phosphorylation of eNOS at serine(1177) in a dose-dependent manner. Cyclic GMP accumulation was also increased accordingly. Akt was not phosphorylated in response to propofol (50 micromol l(-1)) at serine(473). In addition, a phosphoinositide 3-kinase inhibitor, LY294002 (10 micromol l(-1)), did not affect eNOS phosphorylation and nitric oxide production by propofol. Chronic treatment with propofol induced a downregulation of PKC delta, which was associated with eNOS activation. Moreover, ceramide inhibited the effect of propofol on eNOS activation. CONCLUSION: Propofol induces eNOS activation through a PKC delta inhibition-dependent, protein phosphatase 2A-coordinated, but phosphoinositide 3-kinase/Akt-independent pathway.

Transcription factor 8 activates R-Ras to regulate angiogenesis.

We have recently reported that transcription factor 8 (TCF8) negatively regulates pathological angiogenesis by regulating endothelial invasiveness by acting as a transcriptional attenuator of matrix metalloproteinase 1. TCF8 also modulates cell-matrix and cell-cell adhesion; however molecular mechanism of this TCF8 function remains obscure. Here, we provide evidence that TCF8 activates R-Ras, another class of angiogenic regulator, to suppress angiogenesis by a mechanism other than a transcriptional attenuator. Tube formation by human umbilical vein endothelial cells (HUVECs) facilitated by TCF8 suppression was significantly inhibited by the expression of constitutive active mutant of R-Ras. When we examined the mRNA expression levels of R-Ras regulators, no significant changes were observed to explain the R-Ras activation by TCF8. Interestingly, we found that TCF8 bound to CalDAG-GEFIII, an R-Ras activator, in the cytosol, indicating that TCF8 emanates signaling for R-Ras activation from cytosol to regulate angiogenesis negatively.

Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.

Endothelial nitric oxide synthase traffic inducer in the umbilical vessels of the patients with pre-eclampsia.

The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was examined in the umbilical vessels of the patients with pre-eclampsia (PE) to explore its possible role in the pathogenesis of PE. The NOSTRIN mRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2-/NO3-, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group (P<0.01). The activity of eNOS was significantly decreased in PE group [(12.83+/-3.61) U/mg] than in normal group [(21.72+/-3.83) U/mg] (P<0.01). The level of NO2-/NO3- in PE patients (27.53+/-7.48) micromol/mg was significantly lower than that of normal group (54.27+/-9.53) micromol/mg (P<0.01). The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE (r=-0.58, P<0.01). It was concluded that the level of NOSTRIN expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.

DC-SIGN mediates adhesion and rolling of dendritic cells on primary human umbilical vein endothelial cells through LewisY antigen expressed on ICAM-2.

Immature dendritic cells (DCs) are recruited from blood into tissues to patrol for foreign antigens. After antigen uptake and processing, DCs mature and migrate to the secondary lymphoid organs where they initiate immune responses. DC-SIGN is a DC-specific C-type lectin that acts both as a pattern recognition receptor and as an adhesion molecule. As an adhesion molecule, DC-SIGN is able to mediate rolling and adhesion over endothelial cells under shear flow. In this study, we show that the binding partner of DC-SIGN on endothelial cells is the glycan epitope Lewis(Y) (Le(Y)), expressed on ICAM-2. The interaction between DC-SIGN on dendritic cells and ICAM-2 on endothelial cells is strictly glycan-specific. ICAM-2 expressed on CHO cells only served as a ligand for DC-SIGN when properly glycosylated, underscoring its function as a scaffolding protein. The expression of Le(Y) in endothelial cells is directed by the enzyme FUT1. Silencing of FUT1 results in a decrease in the rolling and adhesion of immature DCs over endothelial cells. The identification of Le(Y) as the carbohydrate ligand of DC-SIGN in endothelial cells opens new possibilities for the manipulation of DC migration.

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

Hemangiopoietin promotes endothelial cell proliferation through PI-3K/Akt pathway.

Hemangiopoietin (HAPO) is a novel human growth factor acting on the primitive cells of both hematopoietic and endothelial cell lineages. Our previous study has shown that HAPO exerts a proliferative effect on endothelial cells. However, the mechanism of this action remains unclear. Thus, we studied the signal transduction pathway whereby HAPO promotes cell proliferation in human umbilical vein endothelial cells (HUVECs). In this study, recombinant human HAPO (rhHAPO) stimulated the proliferation of HUVECs in a dose-dependent manner. The transient phosphorylation of Akt occurred after addition of rhHAPO to HUVECs. LY294002, a specific inhibitor of PI-3K, significantly inhibited Akt phosphorylation and completely abrogated HAPO-stimulated proliferation of HUVECs. rhHAPO enhanced the expression of cyclin D1, where as LY294002 inhibited the up-regulation of cyclin D1. Moreover, rhHAPO is able to selectively enhance the mitogenic activity of VEGF for vascular endothelial cells. Overall, these findings demonstrate that HAPO induces endothelial cell proliferation through the PI-3K/Akt pathway.