Investigations of Vascular Responses of Human Umbilical Vessels from Normal and Preeclamptic Pregnancies

Objective: We have investigated the isometric contractions produced by vasoactive agents in human umbilical arteries and veins from normal and preeclamptic pregnancies.

Methods: Umbilical cords were collected from 70 normotensive and 20 preeclamptic women. Ring segments of umbilical vessels were suspended in organ baths under tension. Cumulative concentration response curves were carried out to 5-hydroxytryptamine (5-HT), prostaglandin F2alpha (PGF_2(V) and endothelin 1 (ET-1).

Conclusions: There were no differences in the responsiveness of vessels from normal and preeclamptic pregnancies to 5-HT, PGF_2α and ET-1, in terms of either EC₅₀ or maximum response.

Results: In human umbilical arteries, 5-HT produced EC₅₀ (concentration producing 50% of maximum response: mean and 95% confidence limits) values of 89.1 nM (38.7-204) and 158 nM (75.3-333); PGF_2α produced EC₅₀ values of 3.3 μM (1.71-6.37) and 3.94 μM (1.72-9.02); and ET-1 produced EC₅₀ values of 12.1 nM (4.2-35.1) and 24.3 nM (7.71-76.7) in normal and preeclamptic vessels, respectively. In human umbilical veins, 5-HT produced EC₅₀ values of 257 nM (74.1-891) and 154 nM (75.9-316); PGF₂, produced EC₅₀ values of 5.86 μM (2.42-14.2) and 6.38 μM (2.48-16.4); and ET-1 produced EC₅₀ values of 14.8 nM (9.8-22.2) and 15.8 nM (7.69-32.7) in normal and preeclamptic vessels, respectively.     

Immunocytochemical Localization of Endothelin-1 in Human Placenta from Normal and Preeclamptic Pregnancies

Objective. The aim of this study was to examine the distribution of endothelin-1 (ET-1) in the human placenta at different gestational ages and to determine whether differences in ET-1 immunoreactivity occurred in preeclamptic compared with uncomplicated pregnancies.

Methods. Localization of ET-1 was investigated by the immunoperoxidase technique in first-trimester, second-trimester, and term human placentas from normal pregnancies and in placentas from preeclamptic pregnancies.

Results. In normal placentas from all gestational ages studied, endothelin-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the fetal vessels and in the syncytiotrophoblast. ET-1 IR was also expressed by the villous cytotrophoblast of first- and second-trimester normal placentas. The extravillous cytotrophoblast of the basal and chorionic plates also exhibited ET-1 IR, but with varying degrees of intensity. In preeclamptic placentas, the expression of ET-1 IR was uneven with a negative staining in all placentas from pregnancies between the 29th and 32nd weeks of gestation. The expression of ET-1 IR was most intense in some syncytiotrophoblast tissue in the terminal villi after the 33rd week of gestation. In placentas from preeclamptic pregnancies between the 35th and the 36th weeks of gestation, strong ET-1 IR expression was evident in the endothelium of fetal vessels and in the syncytiotrophoblast. Regardless of gestational age, ET-1 IR was also observed in the extravillous cytotrophoblast of the basal and chorionic plates of preeclamptic placentas.

Conclusion. This study demonstrates that ET-1 IR is widely distributed in the human placenta and provides further evidence to support the concept that ET-1 plays an important role as a modulator of vascular tone in the uteroplacental and fetoplacental units and may participate in the pathogenesis of preeclampsia.     

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     

妊高征患者血清对内皮细胞分泌NO与ET-1的影响

目的探讨妊高征患者血清中是否存在引起内皮细胞功能改变的因素以及与妊高征发生的关系.方法无菌收集妊高征及正常孕妇产前静脉血制备血清.传代培养人脐静脉内皮细胞(HUVEC),当其融合为单层时,分别加入收集的产前血清,置37℃,5%CO2培养箱中孵育24 h,收集培养液,采用Griess法和放免法测定NO及ET-1含量变化.结果①中、重度妊高征组HUVEC培养液中NO量明显降低,而轻度妊高征与正常妊高征组无差异.②中、重度妊高征HU-VEC培养液中ET-1量明显增高,而轻度妊高征与正常妊高征组无差异.结论中、重度妊高征患者产前血清可能含有某些因素,可引起血管内皮细胞功能改变,使NO、ET-1的分泌失衡,从而出现妊高征的系列表现.     

Effects of sFlt-1 and alpha 2-macroglobulin on vascular endothelial growth factor-induced endothelin-1 upregulation in human microvascular endothelial cells

Introduction

Soluble fms-like tyrosine kinase-1 (sFlt-1) is a vascular endothelial growth factor (VEGF) binding protein and potent antagonist of VEGF. Alpha 2 macroglobulin (α₂M) is another major binding protein for circulating VEGF, which is present in human plasma at higher concentration (2–4 mg/mL) than sFlt-1. This study investigated the effects of sFlt-1 and α₂M on VEGF-induced endothelin-1 (ET-1) upregulation in human microvascular endothelial cell-1 (HMEC-1).

Methods

HMEC-1 was cultured and incubated with varying concentrations of sFlt-1 and α₂M in combination with VEGF. ET-1 mRNA expression in the cells was measured by real time RT-PCR and ET-1 protein by western blot analysis.

Results

ET-1 expression in HMEC-1 incubated with VEGF significantly increased in time- and dose-dependent manners. Next, HMEC-1 was treated with the sFlt-1 (10–1000 ng/mL) or α₂M (10–10000 ng/mL) in the presence of VEGF (10 ng/mL). We found that sFlt-1 induced a significant decrease of ET-1 expression upregulated by VEGF, while α₂M did not affect the VEGF-induced ET-1 expression.

Conclusions

sFLT-1 suppressed the VEGF-induced the ET-1 expression of HMEC-1. However, α₂M did not show a significant effect on the ET-1 expression that was induced by VEGF. The results suggest that a certain proportion of the bound form α₂M-VEGF have a biological action involved in the pathophysiology of preeclampsia.     

Effect of Cytokines on Prostaglandin E2 and Prostacyclin Production in Primary Cultures of Human Myometrial Cells

The objective of this study was to characterize interleukin-1, -6, and -8 (IL-1-, IL-6-, and IL-8)-induced prostacyclin (PGI₂ as 6-keto PGF_1α) and prostaglandin E₂ (PGE₂) production in primary cultures of human myometrial cells. Prostaglandins (PGs) released into the culture media were quantitated by specific radioimmunoassays. IL-1, but not IL-6 or IL-8, caused a dose- and time-dependent increase in the production of both PGI₂ and PGE₂. Half-maximally stimulating doses (EC₅₀) of IL-1 were about 0.1 ng/ml, and maximal responses were observed at 1–10 ng/ml, amounting to 15- to 23-fold increases over unstimulated controls. The action of IL-1 was greatly potentiated by the protein kinase C-activating phorbol ester, TPA, and inhibited by actinomycin D and cycloheximide. IL-1-induced PG production was also suppressed by dexamethasone, by the natural IL-1 receptor antagonist (IL-1ra), and by transforming growth factor_1β (TGF_1β). It is concluded that IL-1 is a potent agonist of PG synthesis in human myometrial cells, acting by a mechanism dependent on the synthesis of new proteins, presumably key enzymes (phospholipase A₂ and/or cyclo-oxygenase-2). This study has added further support to the notion that the myometrium serves as a target for the inflammatory cytokine, IL-1, and thereby may be affected directly, thus promoting preterm labor associated with intrauterine infection.     

血红素氧化酶-1、2及内皮素-1在妊娠高血压综合征患者胎盘组织中的表达及作用

目的:通过观察妊娠高血压综合征(简称妊高征,PIH)患者胎盘组织血红素氧化酶-1、2(HO-1、2)及内皮素-1(ET-1)的表达,探讨PIH发病机制。方法:采用免疫组化SABC法,检测20例PIH患者及正常孕妇的胎盘绒毛组织中的HO-1、2及ET-1,利用免疫组化图象分析软件测量胎盘大绒毛、微绒毛合体滋养细胞及血管的平均吸光度。结果:妊高征组大绒毛、微绒毛合体滋养细胞及血管部位的HO-2表达显著低于正常孕妇组(P<0.001),HO-1在胎盘合体滋养细胞的表达显著低于正常孕妇组(P<0.001),而妊高征组大绒毛、微绒毛合体滋养细胞及血管部位的ET-1表达显著高于正常孕妇组(P<0.001);且H0-1、2在胎盘大绒毛、微绒毛合体滋养细胞的表达与平均动脉压呈负相关,而ET-1在胎盘大绒毛、微绒毛合体滋养细胞的表达与平均动脉压呈正相关。结论:妊高征患者胎盘组织血红素氧化酶的表达减少可能在妊高征的发生、发展过程中起着重要的作用。     

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。     

不同氧浓度对原代培养人早孕绒毛细胞滋养细胞血管内皮生长因子及其可溶性受体表达的影响

目的:探讨原代培养人早孕绒毛细胞滋养层细胞(CT)在缺氧时血管内皮生长因子(VEGF)和可溶性血管内皮生长因子受体-1(sFlt-1)的表达。方法:将经胰蛋白酶/DNA酶Ⅰ联合消化通过Percoll分离纯化得到的细胞滋养层细胞进行原代培养。采用ELISA法测定正常氧浓度(20%O2,A组)、低氧浓度Ⅰ组(8%O2,B组)和Ⅱ组(2%O2,C组)及低氧复氧组(2%O2/20%O2,D组)培养的不同时段细胞上清液中VEGF和sFlt-1蛋白的表达。结果:B组、C组于培养72h、96h、120h、144h时间点测得VEGF、sFlt-1的浓度明显高于A组(P<0.01);D组于培养96h、120h、144h后测得VEGF、sFlt-1的浓度较72h低,且分别与A组同期相比无明显差异(P>0.05)。结论:细胞滋养层细胞在低氧条件下VEGF、sFlt-1分泌增加,低氧复氧后VEGF和sFlt-1表达均恢复正常。     

妊高征患者血清中内皮细胞毒性因子对内皮细胞功能的影响

目的：探讨妊高征患者血清中内皮细胞毒性因子对内皮细胞功能的影响。方法：采用前瞻性随机对照研究方法。５份原代培养为妊高征患者脐静脉的内皮细胞，待细胞融合为单层时，分为３组，第１组加入妊高征患者产前血清，第２组加入正常妊娠产前血清，第３组作为对照，加入等量的细胞培养液，置于３７℃、５％ＣＯ２培养箱中孵育２４ｈ，收集培养液，用放射免疫法测定其中６－ｋｅｔｏ－ＰＧＦ１α及ＴＸＢ２的含量变化。结果：与第３组相比，第１组６－ｋｅｔｏ－ＰＧＦ１α明显下降，ＴＸＢ２保持不变，第２组６－ｋｅｔｏ－ＰＧＦ１α及ＴＸＢ２均下降。结论：妊高征患者产前血清中含有内皮细胞毒性因子，可导致血管内皮细胞功能受损，ＰＧＩ２／ＴＸＢ２比例失调，从而出现妊高征的系列表现     

Effect of estrogen and 1alpha,25(OH)2- vitamin D3 on the activity and growth of human primary osteoblast-like cells in vitro

OBJECTIVE: To evaluate effects of 17beta-E(2) and 1alpha,25(OH)(2)-vitamin D(3) on human osteoblast-like (hOB) cells. DESIGN: Controlled, experimental study. SETTING: University hospital. PATIENT(S): hOB cell cultures were prepared from the upper femur of postmenopausal patients undergoing bipolar endoprosthesis arthroplasty for a fractured femoral neck. INTERVENTION(S): hOB cells were subcultured with either 17beta-E(2) or 1alpha,25(OH)(2)-vitamin D(3), or both. MAIN OUTCOME MEASURE(S): Cell proliferation and activity of alkaline phosphatase, osteocalcin, and interleukin-6. RESULT(S): 17beta-E(2) significantly reduced interleukin-6 and osteocalcin to 34% and 60% of control value but induced alkaline phosphatase and cell proliferation to 183% and 150% of control value. 1alpha,25(OH)(2)-vitamin D(3) significantly decreased cell proliferation to 88% of that of control group, but 1alpha,25(OH)(2)-vitamin D(3) plus 17beta-E(2) showed no difference from the control group. Alkaline phosphatase and osteocalcin were significantly increased by 1alpha,25(OH)(2)-vitamin D(3) alone or combined with 17beta-E(2), to 169% and 198% and to 144% and 144% of control value, respectively. 1alpha,25(OH)(2)-vitamin D(3), with or without 17beta-E(2), decreased interleukin-6 levels to 27% and 38% of control group, respectively. CONCLUSION(S): 17beta-E(2) and 1alpha,25(OH)(2)-vitamin D(3) have effects on osteoblasts. The prevention of osteoporosis by estrogen may be related not only to direct effects on osteoblastic activity and proliferation but also to indirect effects on osteoclasts by the decrease of interleukin-6 secretion.     

白血病抑制因子对人早孕滋养细胞MMP-2、MMP-9和TIMP-1表达的调节

目的:研究白血病抑制因子(LIF)对人早孕滋养细胞基质金属蛋白酶(MMP-2、MMP-9)和MMPs组织抑制物(TIMP-1)表达的影响。方法:以不同浓度的LIF作用于体外培养的细胞滋养细胞,于2h、4h、12h收集细胞。用RT-PCR方法检测MMP-2、MMP-9和TIMP-1 mRNA的表达。结果:LIF对人早孕滋养细胞MMP-2和TIMP-1 mRNA的表达无明显作用。LIF作用4h和12h,实验组MMP-9 mRNA明显上升(P<0.01),并呈明显剂量依赖关系。结论:LIF可以在一定的时间和剂量范围内促进滋养细胞MMP-9的表达。推测LIF可能通过节制性调节MMPs的表达,进而分解子宫内膜细胞外基质,介导滋养细胞的入侵。     

Effect of leonurine hydrochloride on endothelin and the endothelin receptor-mediated signal pathway in medically-induced incomplete abortion in rats

Objective

Endothelin (ET) is involved in uterine contractions. Our previous study showed that leonurine hydrochloride (LH) inhibits abnormal bleeding caused by incomplete abortion through an increase in uterine contractions in rats. The present study was conducted to show that LH treatment regulates the ET-mediated signal pathway in abortion in rats.

Study design

Early pregnancies in rats had incomplete abortions induced using mifepristone in combination with misoprostol. After the abortions, the rats were treated with LH orally for 7 days and surgery was performed. The sinistro-uterus was dissected for measurement of ET and nitric oxide (NO); the dextro-uterus was stored at −80°C for ET receptor (ET_A and ET_B) analysis. Myometrial cells from the dextro-uterus were cultured for measurement of phospholipase C (PLC) activity, intra-cellular Ca²⁺ concentration ([Ca²⁺]_i), and protein kinase C (PKC) activity.

Results

In in vivo experiments, LH treatment elevated the ET level and ET/NO ratio in rats with induced abortions and up-regulated ET_A mRNA expression (P < 0.01 vs. the model group), but there was no change in ET_B mRNA. LH significantly increased the [Ca²⁺]_i, PLC activity, and relative production of PKC protein in myometrial cells.

Conclusion

LH increased uterine contractions in rats with incomplete abortions by modulating the ET receptor-mediated signal pathway.     

PKC抑制剂对子痫前期血清诱导人脐静脉内皮细胞NF-κB核转位及VCAM-1表达的影响

目的:探讨子痫前期血清诱导人脐静脉内皮细胞(HUVEC)蛋白激酶(PKC)活性改变、核因子-κB(NF-κB)核转位及内皮细胞黏附分子-1(VCAM-1)的表达以及PKC抑制剂对它们的影响。方法:用胰酶消化培养法培养正常妊娠HUVEC,传代后待细胞长满至70%~80%后,加或不加PKC抑制剂多黏菌素B(PMB)作用30m in后分别加入正常妊娠及子痫前期血清,培养2h,W estern B lot测定细胞胞浆PKC、胞膜PKC含量、胞浆核因子κB抑制因子(I-κB)、胞核NF-κBp65含量;培养48h,用MTT测定细胞活力,流式细胞学测定细胞凋亡,酶联免疫法测定HUVEC VCAM-1的表达。结果:子痫前期血清培养的HUVEC胞浆PKC及I-κB含量明显低于对照组(P<0.05),胞膜PKC含量、胞核NF-κBp65含量、VCAM-1表达、细胞凋亡率明显高于对照组(P<0.05),细胞活力明显低于对照组(P<0.05)。加PKC抑制剂预处理后,子痫前期组HUVEC胞浆PKC含量及I-κB含量明显增加(P<0.05);胞膜PKC含量、胞核NF-κBp65含量、VCAM-1表达、细胞凋亡率明显下降(P<0.05),细胞活力明显增加(P<0.05)。结论:子痫前期血清可促进HUVEC NF-κB活性及VCAM-1表达,PKC抑制剂可抑制子痫前期患者血清诱导HUVEC的NF-κB活性及VCAM-1表达,PKC、NF-κB在子痫前期内皮细胞损伤过程中可能起重要的桥梁作用。     

The impact of S- and G2-checkpoint response on the fidelity of G1-arrest by cisplatin and its comparison to a non-cross-resistant platinum(IV) analog

Objective

Cisplatin is a DNA-damaging antitumor agent that is highly effective in treating ovarian cancer. It activates the p53/p21 pathway for its cytotoxic mode of action, but it does not induce p21-dependent cell cycle arrest in G1. Therefore, we investigated this paradox, and used the model analog DAP as a positive control for p21-dependent G1-arrest.

Methods

Studies were conducted in p53-proficient ovarian A2780 tumor cells to examine Cdk activity, cell cycle distribution and DNA damage signaling after cisplatin or DAP in combination with the mitotic inhibitor nocodazole.

Results

Cisplatin consistently induced transient S-phase arrest by inhibiting Cdk2/cyclin A complex in S-phase at 12 h and then a durable G2/M-arrest by inhibiting Cdc2/cyclin B complex at 12-18 h. These inhibitions were associated with Chk1 and Chk2 activation and resultant increase in inhibitory tyrosine phosphorylation of Cdk2 and Cdc2. Cisplatin also potently inhibited G1-phase Cdk4/cyclin D1 and Cdk2/cyclin E activities at ~ 18 h. In agreement, exposure of cisplatin-treated A2780, HCT-116^p53−/− and HCT-116^p21−/− tumor cells to nocodazole revealed limited G1-arrest that was dependent on p53 and p21. In contrast, the durable G1-arrest by DAP, which failed to activate Chk1 and Chk2, was unaffected by nocodazole.

Conclusions

Cisplatin induced G1-arrest, but at an attenuated level. This was primarily due to orchestration of Cdk inhibition in S-phase first, then in G2, and finally in G1 that effectively blocked cells in G2 and prevented cells from progressing and arresting in G1. These studies demonstrate that cisplatin unequivocally activates G1-checkpoint response, but the fidelity of G1-arrest is compromised by Chk1/2 activation and checkpoint response in S- and G2/M-phase.