Tamoxifen, esterified estradiol, and physiologic concentrations of estradiol inhibit oxidation of low-density lipoprotein by endothelial cells

OBJECTIVE: We investigated the antioxidant effect of tamoxifen, esterified estradiol, and physiologic concentrations of 17beta-estradiol on endothelial cell-mediated oxidation of low-density lipoprotein. STUDY DESIGN: Human umbilical vein endothelial cells were preincubated with nanomolar concentrations of estradiol, estradiol stearate, and tamoxifen. Low-density lipoprotein was isolated and incubated with cells in serum-free medium. Oxidation of low-density lipoprotein was quantified after 8, 16, and 24 hours of incubation as the formation of thiobarbituric acid-reactive substances. RESULTS: Compared with control, preincubation of human umbilical vein endothelial cells with 1- or 10-nmol/L estradiol resulted in a 12% reduction in the formation of thiobarbituric acid-reactive substances at 24 hours. Preincubation of human umbilical vein endothelial cells with either 10-nmol/L estradiol 17-stearate or 10-nmol/L tamoxifen resulted in 26% and 20% decreases, respectively, in formation of thiobarbituric acid-reactive substances at 24 hours. The difference in the reduction in thiobarbituric acid-reactive substances between control and treatment wells became more pronounced over time. CONCLUSION: Under these experimental conditions, tamoxifen, esterified estradiol, and physiologic concentrations of exogenous estradiol inhibit oxidation of low-density lipoprotein by human umbilical vein endothelial cells.     

雌二醇对同型半胱氨酸诱导人脐静脉内皮细胞组织因子表达的影响

目的:探讨雌二醇(E2)对同型半胱氨酸(Hcy)诱导人脐静脉内皮细胞(HU-VEC)组织因子(TF)表达的影响及机制。方法:通过一期凝固法测总细胞促凝活性(PCA);采用RT-PCR测TF表达;用免疫组化法观察NF-κB的变化。结果:Hcy剂量依赖性促进HUVEC的PCA增加(与对照组相比r=0.969,P<0.01);E2单独作用对HUVEC的PCA没有影响(P>0.05);E2抑制Hcy诱导HUVEC表达TF(r=-0.686,P<0.01),最适浓度为1×10-7mol/L;E2可抑制Hcy引起的NF-κB核易位(P<0.01);ICI182,780对E2抑制Hcy诱导HUVEC的TF表达和核易位没有明显的拮抗作用(P>0.05)。结论:E2抑制Hcy诱导HUVEC表达TF;E2降低HUVEC的NF-κB核易位;E2抑制Hcy诱导HU-VEC表达TF作用未通过经典核受体途径。     

二氯化钴化学缺氧对人脐静脉内皮细胞增殖和血红素氧合酶表达的影响及其临床意义

目的:研究二氯化钴(CoC l2)诱发化学缺氧对培养的人脐静脉内皮细胞血红素氧合酶表达及增殖的影响。方法:采用MTT和流式细胞术检测人脐静脉内皮细胞的增殖,酶联免疫吸收法测定培养上清液中的碳氧血红蛋白(COHb),并通过RT-PCR观测CoC l2与血红素氧合酶表达间的量-效和时-效关系。结果:CoC l2可诱导人脐静脉内皮细胞中血红素氧合酶-1的表达,促进内源性一氧化碳(CO)的产生,与血红素氧合酶表达间有一定的剂量和时间依赖性。结论:CoC l2诱导慢性缺氧促进人脐静脉内皮细胞增殖并上调血红素氧合酶-1和内源性CO,HO/CO系统是保护内皮损伤的分子学基础,可能在妊娠期高血压疾病发病中起重要作用。     

倍美力和黄体酮对人脐静脉内皮细胞tPA和PAI-1活性及其基因表达的影响

目的 :观察雌孕激素对人脐静脉内皮细胞 (HUVEC)组织型纤溶酶原激活剂(tPA)、纤溶酶原活化物抑制剂 1(PAI 1)的活性及其基因表达的影响。方法 :不同浓度的倍美力 (Premarin) (0 .0 2、0 .2 5、2 .5ng/ml)、黄体酮 (1ng/ml)、倍美力 (2 .5ng/ml)合并黄体酮 (1ng/ml)分别作用于HUVEC 12h和 2 8h ,用发色底物法测定上清液中tPA、PAI 1的活性 ,用RT PCR技术分析它们作用于HUVEC 2 8h后tPA和PAI 1mRNA表达。结果 :0 .2 5、2 .5ng/ml倍美力及 2 .5ng/ml倍美力合并 1ng/ml黄体酮作用于HUVEC 2 8h ,tPA和PAI 1mRNA表达显著减少 (P <0 .0 5 ) ,tPA活性明显升高 ,PAI 1活性明显下降 (P <0 .0 5 ) ,而 0 .0 2 5ng/ml倍美力、1ng/ml黄体酮作用 2 8h ,未见显著变化 (P >0 .0 5 )。它们作用HUVEC 12hPAI 1活性均无明显下降 (P >0 .0 5 )。结论 :(1)倍美力作用HUVEC使tPA、PAI 1mRNA表达下降 ,tPA活性增加 ,PAI 1活性降低 ,因而提高了纤溶活性 ;(2 )倍美力对HUVECPAI 1活性的影响与倍美力的剂量和作用时间有关 ;(3)黄体酮对于HUVEC可能无直接作用 ,但它与倍美力合并作用后可对抗倍美力的部分雌激素活性。     

Nicotine and cotinine affect the release of vasoactive factors by trophoblast cells and human umbilical vein endothelial cells

Objective

To examine nicotine (N) and cotinine (C) effects on trophoblast cells (TCs) and human umbilical vein endothelial cells (HUVEC) secretion of soluble fms-like tyrosine kinase (sFlt-1), soluble endoglin (sENG), placental growth factor (PlGF), transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF).

Study design

Human placentas and umbilical cords were collected from uncomplicated pregnancies at term from a total of 24 non-smoking women with a history of normal blood pressure. TCs and HUVEC were cultured for 24 h with C or N (from 10⁻¹² to 10⁻⁷ M).

Main outcome measures

sFlt-1, sENG, PlGF, TGF-beta and VEGF release and messenger RNA (mRNA) expression were evaluated by ELISA and real-time polymerase chain reaction (PCR), respectively.

Results

N and C reduced sFlt-1, sENG and PlGF release by TCs and TGF-beta release by HUVEC. Conversely, N and C increased PlGF secretion, while N alone increased sFlt-1 release by HUVEC. N and C were able to modulate VEGF mRNA expression in HUVEC.

Conclusions

Our results suggest that N and C affect the balance of some important vasoactive factors released by TCs and HUVEC. This might be one of the possible mechanism through which smoke reduces the risk of hypertensive disorders during pregnancy as well as contributes to the well known detrimental effects of smoking on fetal development.     

Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis

OBJECTIVE: To assess whether hormonal agents used in the medical treatment of endometriosis, such as danazol and GnRH agonist, exert direct regulatory action on monocyte chemotactic protein-1 (MCP-1) expression by endometrial epithelial cells. DESIGN: Primary cultures of epithelial cells isolated from human endometrium were exposed to different concentrations of cytokines and steroid hormone analogs. Expression of MCP-1 was analyzed at the levels of protein and messenger RNA. SETTING: Gynecology clinic and laboratory of endocrinology of reproduction. PATIENT(S): Women presenting for infertility or pelvic pain in whom endometriosis was diagnosed by using laparoscopy. INTERVENTION(S): Endometrial tissue biopsy performed at laparoscopy. MAIN OUTCOME MEASURE(S): Secretion of MCP-1 protein was measured by using enzyme-linked immunosorbent assay, and mRNA steady-state levels were measured by performing Northern blot analysis. RESULT(S): Buserelin acetate, a GnRH agonist (0.1-10 ng/mL), had no significant effect on MCP-1 expression, whereas danazol (10(-7)-10(-5) M), a testosterone analog, and dexamethasone, an anti-inflammatory glucocorticoid hormone (10(-12)-10(-6)M), showed a direct and a dose-dependent inhibitory effect on MCP-1 expression. This effect occurred at the level of protein and mRNA. CONCLUSION(S): The findings of the study may affect understanding of the mechanisms by which hormonal treatments act on endometriosis and influence its clinical manifestations.     

川芎嗪对妊娠高血压综合征患者脐静脉内皮细胞粘附分子表 …

目的 探讨妊娠高血压综合征（妊高征）患者血浆诱导培养的脐静脉内皮细胞（ＨＵＶＥＣ）表面细胞间粘附分子－１（ＩＣＡＭ－１）、血管细胞粘附分子－１（ＶＣＡＭ－１）的表达以及川芎嗪对其的影响。方法 采用胶原酶、胰蛋白酶混合灌注消化法，对正常妊娠妇女（正常妊娠组）、妊高征患者（妊高征组）的ＨＵＶＥＣ进行培养，待细胞融合成单层后，加或不加川芎嗪进行预处理，并以正常未妊娠妇女（对照组）作为对照。再加入上述３组     

Estrogen up-regulates cyclooxygenase-2 via estrogen receptor in human uterine microvascular endothelial cells

OBJECTIVE: To investigate the effects of 17beta-estradiol (E(2)) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in primary human uterine microvascular endothelial cells (HUMEC). DESIGN: Prospective study. SETTING: Basic research laboratory at an academic medical center. PATIENT(S): Primary HUMEC of three women donors and primary human dermal microvascular endothelial cells of three women donors (as control), purchased from a third-party source. INTERVENTION(S): The HUMEC were cultured in specific media in a humidified atmosphere with 5% CO(2) at 37 degrees C. MAIN OUTCOME MEASURE(S): Measures of COX-2 mRNA and protein, PGE(2) production, and estrogen receptor alpha and beta mRNA and protein. RESULT(S): Treatment with E(2) (10(-10) to 10(-6) M) increased COX-2 mRNA levels by 2.3-fold to 2.4-fold in HUMEC. Treatment of HUMEC with E(2) (10(-8) M) resulted in a time-dependent increase of COX-2 mRNA levels. This was accompanied by a 2.8-fold increase in COX-2 protein level and a 1.5-fold increase in PGE(2) synthesis. Pretreatment of HUMEC with a selective COX-2 inhibitor, NS-398, abolished E(2)-induced PGE(2) synthesis, suggesting that E(2) specifically up-regulates COX-2 activity. The estrogen receptor antagonist ICI 182,780 fully reversed the stimulation of COX-2 mRNA and protein levels and PGE(2) synthesis by E(2). Interestingly, estrogen receptor beta mRNA and protein were abundant in HUMEC, whereas estrogen receptor alpha mRNA or protein was barely detectable. CONCLUSION(S): We conclude that various levels of E(2) can significantly increase COX-2 expression and PGE(2) synthesis in HUMEC via the estrogen receptor.     

人脐静脉内皮细胞中肿瘤坏死因子-α诱导NOSTRIN基因表达上调的实验研究

目的:探讨肿瘤坏死因子-α(TNF-α)对人脐静脉内皮细胞(HUVEC)中内皮型一氧化氮合酶运输介导物(NOSTRIN)基因表达的诱导作用以及二硫代氨基甲酸吡咯烷(PDTC)对此诱导作用的抑制。方法:将培养的内皮细胞分为3组:第1组为无任何处理的HUVEC,第2组为子痫前期(PE)患者血清作用的HUVEC,第3组为正常晚孕妇女血清作用的HUVEC。用逆转录-聚合酶链反应(RT-PCR)检测3组细胞中NOSTRIN mR-NA的表达。酶联免疫吸附实验(ELISA)检测PE患者和正常孕妇血清中TNF-α含量。用1,3,5ng/ml3种浓度的TNF-α分别作用3组细胞6h,以及用浓度3ng/ml的TNF-α作用3组细胞分别达6h、12h、24h,RT-PCR检测NOSTRIN mRNA的表达;将PDTC(5mmol/ml)与TNF-α(3ng/ml)同时作用以及TNF-α(3ng/ml)单独作用3组细胞6h,RT-PCR检测NOSTRIN基因mRNA的表达。结果:3组细胞中都有NOSTRIN表达,第2组表达量显著高于第1组和第3组(P0.01)。ELISA结果显示,PE患者血清中TNF-α含量显著高于正常晚孕妇女(P0.01)。TNF-α作用HUVEC细胞后NOSTRIN显著高表达,呈时间和剂量依赖关系(P0.01)。PDTC和TNF-α同时作用的HUVEC与TNF-α单独作用的HUVEC相比,其NOSTRIN表达显著降低(P0.01)。结论:TNF-α可上调NOSTRIN在HUVEC中的表达。     

NOSTRIN基因克隆及其在人脐静脉内皮细胞中的表达

目的 :构建内皮型一氧化氮合酶运输介导物 (endothelialnitricoxidesynthasetrafficinducer ,NOSTRIN)基因的真核表达载体 ,通过质粒转染人脐静脉内皮细胞 (humanumbilicalveinendothelialcell,HUVEC)获得高表达NOSTRIN基因的细胞克隆。方法 :构建真核表达载体pcDNA3.1 NOSTRIN ,采用脂质体介导将重组质粒导入体外培养的HUVEC ,Westernblot检测转染细胞和未转染细胞中NOSTRIN蛋白质的表达。结果 :成功构建了真核表达载体pcDNA3.1 NOSTRIN ,并用脂质体介导的方法获得了高稳定表达NOSTRIN的细胞克隆 ;Westernblot显示转染前后的细胞均有 5 8kD的蛋白质表达 ,转染后表达量明显增高。结论 :重组质粒pcDNA3.1 NOSTRIN经转染能在HUVEC细胞中高效表达 ,为进一步研究NOSTRIN对HUVEC的生物学影响奠定了基础     

Lack of proinflammatory effects of free fatty acids on human umbilical cord vein endothelial cells and leukocytes

AIM: To determine whether the free fatty acids (FFAs), oleic, linoleic, and palmitic acid, found elevated before 20 weeks of pregnancy in those women who later develop preeclampsia, induced changes in expression of the vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or E-selectin in cultured human umbilical cord vein endothelial cells (HUVEC), and integrin subunit CD11b, L-selectin or intracellular reactive oxygen species (ROS) in leukocytes. METHODS: The VCAM-1, ICAM-1, and E-selectin expression were measured using ELISA in HUVEC after incubation with 100 micromol of either oleic, linoleic, or palmitic acid for 6 hr and 24 hr. The co-reactivity with lipopolysaccharide (LPS), the amount of VCAM-1 mRNA in the cells, and soluble VCAM-1 in the incubation medium were measured as well. Leukocyte adhesion molecules and ROS were measured after incubation with 750 microm of either of the FFAs in a whole blood model using flow cytometry. RESULTS: No effects of the FFAs tested were found on the HUVEC or leukocyte adhesion molecule expression or intracellular ROS. The only exception to this was palmitic acid incubation, which significantly lowered the VCAM-1 expression in HUVEC after 24-hr incubation and also slowed the decay of VCAM-1 expressed after stimulation with LPS. CONCLUSIONS: The lack of significant proinflammatory changes of the FFAs tested might indicate that the elevated plasma levels of FFAs seen in preeclampsia most probably are products of the preeclamptic process rather than a causative factor.     

Role of arginase-2 and eNOS in the differential vascular reactivity and hypoxia-induced endothelial response in umbilical arteries and veins

The main vasodilator in the placenta is nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS). Arginase-2 competes with eNOS for l-arginine, and its activity has been related with vascular dysfunction. Recently, we showed that hypoxia induces arginase-2, and decreases eNOS activity in human umbilical vein endothelial cells (HUVEC). However there is evidence that vascular responses to hypoxia are not similar throughout the placental vascular tree. We studied whether arginase-2 plays a role controlling vascular tone in human umbilical vessels, and the changes in the expression of arginase-2 and eNOS proteins by hypoxia in endothelial cells from umbilical arteries (HUAEC) and veins (HUVEC). In isolated umbilical vessels the presence of eNOS and arginase-2 was determined in the endothelium, and the NO-dependent vasoactive responses in the presence and absence of S-(2-boronoethyl)-L-cysteine (BEC, arginase inhibitor) were studied. Additionally, HUAEC and HUVEC were exposed (0-24 h) to hypoxia (2% O2) or normoxia (5% O2), and protein levels of eNOS (total and phosphorylated at serine-1177) and arginase-2 were determined. In umbilical arteries and veins arginase-2 and eNOS were detected mainly at the endothelium. BEC induced a higher concentration-dependent relaxation in umbilical arteries than veins, and these responses were NOS-dependent. In HUAEC exposed to hypoxia there were no changes in eNOS and arginase-2 levels, however there was a significant increase of p-eNOS. In contrast, HUVEC showed an increase in arginase-2 and a reduction of p-eNOS in response to hypoxia. These results show that arginases have a vascular role in placental vessels counteracting the NOS-dependent relaxation, which is differentially regulated in placental artery and vein endothelial cells.     

In vitro human decidual endothelial cell thromboxane secretion in preeclampsia Is not abnormal

Objectives: The aims of this study were to describe levels of thromboxane secretion by decidual endothelial cells from normal pregnancies and to determine whether decidual endothelial cell secretion of thromboxane, implicated in the causation of the hypertension and vasoconstriction of preeclampsia, is increased in this disorder.

Methods: We measured thromboxane generation by cultured decidual endothelial cells from 13 normal pregnancies (NDEC) and 13 pregnancies complicated by preeclampsia (PEDEC), compared with a control population of 6 normal human umbilical vein endothelial cells (HUVEC). Responses to stimulation by bacterial lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were examined.

Main Outcome Measures: Thromboxane B₂, levels in supernatants of cultured endothelial cells.

Results: The level of secretion over 24 h in culture by NDEC [14(7-26) pg/10⁶ cells] was approximately 25% that of HUVEC [63 (49-70) pg/10⁶ cells]. Levels achieved in response to all stimuli examined were consistently lower in NDEC than in HUVEC (p<0.01). Proportional stimulation by LPS and TNF-α was comparable in HUVEC and NDEC, whereas NDEC displayed a greater increase (25-fold) than HUVEC (10-fold) in response to IL-1β (p<0.01). There were no significant differences between decidual entothelial cells from normotensive and preeclamptic women in basal secretion of thromboxane or in response to the stimuli examined.

Conclusions:In vitro thromboxane secretion by decidual endothelial cells is lower than that of HUVEC, and responsiveness to specific stimuli may be quantitatively different. These findings emphasize the importance of examining endothelial cells from the involved maternal vascular bed if intrauterine vascular pathophysiological events are to be clarified. No significant differences were noted in decidual endothelial cell thromboxane secretion between normal and preeclamptic subjects.     

肿瘤坏死因子α对脐静脉血管内皮细胞增殖和细胞内游离钙浓度的影响

目的 探讨肿瘤坏死因子α（ＴＮＦ－α）对培养的人脐静脉血管内皮细胞增殖和细胞内游离钙浓度的影响,从细胞间信息传递水平探讨妊娠高血压综合征（妊高征）的发病机理。方法 用终浓度为５００、１０００、２０００Ｕ／ｍｌ的ＴＮＦ－α,将融合成单层的人脐静脉血管内皮细胞进行无血清培养２４ｈ,并以等量的０．０１ｍｏｌ／Ｌ、ｐＨ７．４的磷酸盐缓冲生理盐水作对照,用流式细胞仪测定细胞各周期细胞百分数、荧光分光光度法测定内皮细胞内游离钙浓度。结果 经ＴＮＦ－α作用后的内皮细胞被拉长,由原来的上皮型变为成纤维细胞型,细胞及核边界尚清楚、形态较规则,但在２０００Ｕ／ｍｌ浓度时,可见有细胞脱落及细胞内异常颗粒出现。内皮细胞受到ＴＮＦ－α的刺激后,由Ｇ０＋Ｇ１期进入Ｓ和Ｇ２＋Ｍ期的细胞百分比明显减少,Ｇ０＋Ｇ１期细胞百分数增多,Ｓ和Ｇ２＋Ｍ