葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

Effects of fluid shear stress on apoptosis of cultured human umbilical vein endothelial cells induced by LPS

The objective of our research was to reveal the effects of shear stress on the apoptosis of cultured human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). A parallel-plate flow chamber was used to control the strength and duration of shear stress (SS), and apoptosis was measured by immunocytochemistry and radio-immunoassay. Some important conclusions were drawn. In the stationary state, apoptosis of HUVECs could be induced by LPS (50 microg/ml). An SS of 15 dyn/cm(2) could inhibit the apoptosis induced by LPS. However, an SS of 4 dyn/cm(2) had less effect on the same process. At the same time, the experiment demonstrated that the increase in IL-6 secretion by LPS can be inhibited by two different levels of shear stress. Moreover, the inhibition effect was more obvious under high level stress than under low level. We also found that the effect of shear stress on IL-8 was less effective than on IL-6. This research provides data for understanding the mechanism of the contribution of hemodynamic forces to atherosclerosis.     

Nerve growth factor promotes killing of Leishmania donovani by macrophages through the induction of hydrogen peroxide

Visceral leishmaniasis is protozoonosis that occurs worldwide and still requires effective therapies with less toxicity. In this study, we examined the antileishmanial effect of nerve growth factor (NGF) using a murine infection model. NGF blocked the infection of macrophages by Leishmania donovani, which was completely cancelled by a hydrogen peroxide inhibitor. In vivo, not only did NGF show antileishmanial effects, but combination therapy of NGF and sodium stibogluconate synergistically exhibited the activity more potently than each monotherapy. These results indicate that NGF exerts antileishmanial effect by stimulating hydrogen peroxide production in macrophages and can be a novel therapy for leishmaniasis.     

Lysophosphatidylglycerol stimulates chemotactic migration and tube formation in human umbilical vein endothelial cells

In this study, we observed that lysophosphatidylglycerol (LPG) stimulates extracellular signal-regulated kinase (ERK) in human umbilical vein endothelial cells (HUVECs). LPG-stimulated ERK activity was not inhibited by pertussis toxin (PTX), indicating PTX-sensitive G-proteins-independent manner. In terms of functional aspect, LPG induced chemotactic migration of HUVECs in a PTX-insensitive manner. Preincubation of HUVECs with an ERK inhibitor (PD98059) completely inhibited LPG-induced chemotactic migration, suggesting the crucial role of ERK in the process. LPG-induced ERK activation and chemotactic migration in HUVECs were not affected by an lysophosphatidic acid receptor-selective antagonist (Ki16425), indicating lysophosphatidic acid receptors-independency. We also found that LPG stimulated tube formation in HUVECs. Taken together we suggest that LPG stimulates HUVECs and result in chemotactic migration and tube formation, suggesting a new aspect of LPG as a modulator of endothelial cell functioning.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

解脲脲原体诱导脐静脉血管内皮细胞凋亡的研究

目的通过探讨解脲脲原体(Ureaplasma urealyticum,UU)诱导人脐静脉内皮细胞(human umbilicalvein endothelial cells;HUVEC)凋亡的情况,揭示妊娠期间UU垂直传播影响胚胎发育的可能机制。方法不同剂量血清4型UU标准菌株刺激体外培养的HUVEC,通过Annexin-V.FITC/PI双染流式细胞术和DNA Ladder实验观察细胞凋亡情况。结果对照组细胞凋亡率小于各实验组(P<0.01~0.05),其凋亡率和刺激剂量、时间之间呈现一定的剂量-时间-效应关系(P>0.05)。结论UU可诱导人脐静脉内皮细胞凋亡,提示妊娠期间UU感染可能通过诱导脐静脉内皮细胞凋亡破坏胎盘屏障而影响胚胎发育。     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Experimental study of intracellular ice growth in human umbilical vein endothelial cells

Study of the intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high resolution cryo-imaging technology, the transient intracellular ice formation and growth processes of the attached human umbilical vein endothelial cells (HUVEC) were successfully captured during freezing. It was found that the intracellular ice nucleation site was on the cell membrane closer to the nucleus. The ice growth was directional and toward the nucleus, which covered the whole nucleus before growing into the cytoplasm. The crystal growth rate in the nucleus was much larger than that in the cytoplasm, and its morphology was influenced by the cooling rate. During the thawing process, small crystals fused into larger ones inside the nucleus. Moreover, the cumulative fraction of the HUVEC with IIF was mainly dependent on the cooling rate not the confluence of the cells attached.     

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin’s effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin’s angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.     

Antiapoptotic effect of ouabain on human umbilical vein endothelial cells

The present study investigates the effect of ouabain on caspase-3 activation in human umbilical vein endothelial cells (HUVEC). Ouabain (EC(50) 20 nM) reduced caspase-3 activity in HUVEC treated for 24h in a medium deprived of fibroblast growth factor (FGF). Incubation for 5h in the absence of both FGF and serum produced an increase in caspase-3 activity that was completely abolished by 100 nM ouabain. Pretreatment with the phosphatidylinositol 3 kinase (PI-3K) inhibitor, wortmannin, prevented the protective effect of ouabain against serum deprivation. Furthermore, Western blotting analysis revealed an increase in phosphorylation of extracellular signal-regulated kinases (ERK-1 and ERK-2) induced by 100nM ouabain in serum-deprived cells. In accord, pretreatment of HUVEC with PD98059, inhibitor of the ERK pathway, abrogated the effect of ouabain. Our results show that ouabain has an antiapoptotic effect on HUVEC through the activation of PI-3K and ERK dependent pathways.     

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-kappaB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-kappaB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Nerve growth factor stimulates the phosphorylation of a 250 kDa cytoskeletal protein in cell-free extracts of PC12 cells

Nerve growth factor (NGF) rapidly stimulates the phosphorylation of a 250 kDa cytoskeletally-associated protein (pp250) by a protein kinase which is also associated with structural elements of the cell. We have solubilized these proteins and demonstrated that NGF-stimulated phosphorylation can be observed in cell free extracts of cytoskeletons from NGF-treated PC12 cells. The pp250 substrate and the 250-kinase were solubilized from PC12 cytoskeletons by treatment with 2 M urea. Phosphorylation of pp250 was maximally stimulated following treatment of the cells for 5 min with NGF. This effect was transient, diminishing with longer exposure of the cells to hormone. The 250-kinase preferred Mn²⁺ over Mg²⁺ and was inhibited by both Na⁺ and K⁺. The phosphorylation of pp250 was not affected by Ca²⁺. Upon fractionation of the urea-soluble cytoskeletal proteins by gel filtration, the 250-kinase eluted in two peaks; one peak of enzyme activity coeluting with the pp250 substrate, and a second peak of enzyme activity eluting with an apparent Mr of approximately 60 kDa. Treatment of the PC12 cells with the phorbol ester TPA also stimulated the phosphorylation of pp250, although this effect was not as great as that produced by NGF. This cell free system should be a valuable tool in the investigation of the mechanisms of NGF action.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Induction of apoptosis in cultured endothelial cells by a cadherin antagonist peptide: involvement of fibroblast growth factor receptor-mediated signalling

Cadherins are a family of transmembrane glycoproteins mediating calcium-dependent, homophilic cell-cell adhesion. In addition, these molecules are involved in signaling events, regulating such processes as cell motility, proliferation, and apoptosis. Members of the cadherin subfamily, called either classical or type I cadherins, contain a highly conserved sequence at their homophilic binding site consisting of the three amino acids--histidine-alanine-valine (HAV). Previous studies have shown that peptides containing the HAV motif inhibit cadherin-dependent events such as cell aggregation, compaction, and neurite outgrowth. We report here that a cyclic peptide, N-Ac-CHAVC-NH2 can perturb cadherin-mediated endothelial cell interactions, resulting in a progressive apoptotic cell death. This effect depends on cell density, as it is only observed when dense cultures are treated with the peptide. Adherens junction (AJ)-associated cadherin and catenins are differentially affected by the N-Ac-CHAVC-NH2 treatment, as judged by double immunofluorescence labeling followed by immunofluorescence-ratio imaging. However, cell-cell adhesions are largely retained during the first few hours after addition of the peptide. It was also observed that following treatment, actin filaments partially lose their plasma membrane anchorage at AJs and translocate towards the cell center. Interestingly, addition of basic fibroblast growth factor to confluent, peptide-treated, endothelial cell cultures, completely blocks apoptosis and the inhibitory peptide reduce the phosphorylation of the FGF receptor target protein FRS2, suggesting that the peptide exerts its effect by inhibiting cadherin-mediated activation of fibroblast growth factor receptor signaling. We propose that cadherin-mediated signaling is essential for maintaining viability of confluent endothelial cells, and that its perturbation by N-Ac-CHAVC-NH2 drives these cells to apoptosis.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.     

Hepatocyte growth factor induces angiogenesis in injured lungs through mobilizing endothelial progenitor cells

Circulating endothelial progenitor cells (EPCs) play a pivotal role in angiogenesis. Hepatocyte growth factor (HGF) is known to induce proliferation and motility in endothelial cells, and to play a role in mitogenic and morphogenic actions. However, the role of HGF in EPC mobilization has not been clearly described yet. We investigated the effect of HGF on mobilizing EPCs and on angiogenesis in elastase-induced lung injury. HGF significantly increased the triple-positive (Sca-1(+), Flk-1(+), and c-kit(+)) fraction in peripheral mononuclear cells in mice. The bone marrow-derived cells were recruited into the injured lungs, where they differentiated to capillary endothelial cells. HGF induced proliferation of both bone marrow-derived and resident endothelial cells in the alveolar wall. In conclusion, the present study suggests that HGF induces EPC mobilization from the bone marrow and enhances the proliferation of endothelial cells in vivo. These complex effects induced by HGF orchestrate pulmonary regeneration in emphysematous lung parenchyma.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.