Human Uterine Microvascular Endothelial Cells Research Articles

ORIGINAL ARTICLE: Trophoblasts and Shear Stress Induce an Asymmetric Distribution of ICAM‐1 in Uterine Endothelial Cells

Problem We have used an in vitro co‐culture system consisting of early gestation macaque trophoblasts cultured on top of human uterine microvascular endothelial cells (UtMVECs) to investigate the inflammatory response of endothelial cells to trophoblasts under shear stress conditions.Method of study Uterine microvascular endothelial cells and trophoblasts were co‐cultured in a parallel plate chamber under shear stress (15 dyn/cm2) conditions. The distribution and expression of endothelial intercellular adhesion molecule‐1 (ICAM‐1) was quantified by immunofluorescence image analysis and flow cytometry. Endothelial regulated upon activation normal T‐cell expressed and secreted (RANTES) secretion was measured by enzyme‐linked immunosorbent assay and permeability was assessed using fluorescein isothiocyanate‐dextran.Results Intercellular adhesion molecule‐1, but not vascular cell adhesion molecule‐1 or platelet endothelial cell adhesion molecule‐1, was re‐distributed towards the downstream edge of endothelial cells when the cells were co‐cultured with trophoblasts under shear stress conditions. Changes in ICAM‐1 distribution were also observed when UtMVECs were co‐cultured with trophoblast‐conditioned medium under shear stress conditions. Incubation of UtMVECs with trophoblast‐conditioned medium increased endothelial permeability, RANTES secretion, and trophoblast adhesion.Conclusion These data support the idea that trophoblasts induce an inflammatory response in uterine endothelial cells that could enhance trophoblast invasion and transmigration.

Possible role of hematopoietic CD44/chondroitin sulfate interaction in extravasation of peripheral blood CD16(−) natural killer cells into human endometrium

Unique CD16(−) natural killer (NK) cells appear in the human cycling endometrium. Although their origin remains undetermined, one possible explanation is extravasation of circulating peripheral blood CD16(−) NK cells. Hematopoietic CD44 (CD44H) is an adhesion molecule expressed on leukocytes and plays a role in the initial step of leukocyte extravasation (leukocyte tethering/rolling). Recent studies have shown that CD44H binds to chondroitin sulfate (CS). To test the hypothesis that peripheral blood CD16(−) NK cells extravasate using the CD44H/CS interaction, we have compared the binding capacity of CD44H to immobilized CS among peripheral blood lymphocyte subsets, as well as determined the menstrual cycle-dependent expression of CD44H. Additionally, we have investigated the expression of the CS proteoglycan serglycin in human endometrial endothelial cells. CD44H expression on peripheral blood CD16(−) NK cells was higher compared with other lymphocyte subsets throughout the menstrual cycle. Peripheral blood CD16(−) NK cells bound preferentially to immobilized CS-A and CS-C compared with other lymphocyte subsets. The binding was significantly reduced by function-blocking anti-CD44H antibody. Serglycin expression in the human endometrial endothelial cells was greater in the secretory phase than in the proliferative phase. Progesterone (10 −7 M to 10 −8 M) significantly increased serglycin core protein expression in cultured human uterine microvascular endothelial cells, whereas 17β-estradiol had no effect. These results indicate that the interaction between CD44H on peripheral blood CD16(−) NK cells and CS on endometrial endothelial cells may play a role in extravasation of these NK cells into human endometrium. Serglycin may be a potential CS proteoglycan involved in this phenomenon.

Genes regulated by interferon-γ in human uterine microvascular endothelial cells

Interferon (IFN)-gamma plays a critical role in murine uterine spiral artery remodeling for successful pregnancy. The effect of IFN-gamma on human uterine microvasculature, however, remains poorly understood. The aim of this study was to identify the genes regulated by IFN-gamma in human uterine microvascular endothelial cells. The effect of IFN-gamma on the gene expression profile in human uterine microvascular endothelial cells was evaluated by cDNA microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction for the selected genes of interest. In vivo expression of the protein encoded by some of these genes in human uterine microvascular endothelial cells was evaluated by Western blotting and immunohistochemistry. Treatment with 10 ng/ml IFN-gamma for 4 h induced a significant > or =2-fold change in 29 genes in pooled human uterine microvascular endothelial cells; a total of 20 genes were up-regulated, whereas nine genes were down-regulated. The genes significantly up-regulated included chemokines (CXCL9, CXCL10, CCL8, IL15RA, and CCL5), enzymes (GBP5, TAP1, CYP27B1, SOD2, MX1, CASP1, and PTGES), and transcription factors (TFAP2C, IRF1, NFE2L3). The genes significantly down-regulated following IFN-gamma treatment included cytokines/cytokine receptors (CSF2, IL1R2, and SPP1), and insulin-like growth factor binding proteins (WISP2 and IGFBP3). The results of the cDNA microarray analysis were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction for the selected 17 genes of interest. The immunoreactivity for the proteins encoded by IL15RA, IFI30, and MX1 was detected in human uterine microvascular endothelial cells in vivo, whereas the immunoreactivity for CCNA1 and NQO1 was not detectable. These results suggest that IFN-gamma regulates the gene expression involved in natural killer cell recruitment, embryo and trophoblast migration, endometrial decidualization, angiogenesis, angiostasis, and anti-viral infection in human uterine microvascular endothelial cells.

Effect of aqueous tobacco smoke extract and shear stress on PECAM-1 expression and cell motility in human uterine endothelial cells.

Tobacco smoke constituents have several adverse effects on endothelial cells. Exposure to tobacco smoke during pregnancy is associated with adverse effects on pregnancy outcome possibly related to endothelial dysfunction. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an important regulator of endothelial function. This study tests the idea that an aqueous extract of cigarette smoke alters the expression of PECAM-1 in uterine endothelial cells. Human uterine microvascular endothelial cells were cultured in cigarette smoke-conditioned medium (CSM) under arterial physiological flow conditions (shear or frictional stress in the range 7.5-15 dyne/cm(2)) and the expression of PECAM-1 was assessed by immunofluorescence microscopy and Western blotting. Thick reticular PECAM-1-associated bands found at cell-cell junctions in static cultures became significantly thinner or disappeared when the cells were exposed to shear stress or to CSM for 24 h. This diminution at cell junctions was accompanied by increased punctate cytoplasmic/cell surface staining. Under shear stress conditions, PECAM-1 was equally distributed between cell surface and intracellular sites. In contrast, when cells were exposed to both shear stress and CSM, PECAM-1 was predominantly localized to the cell surface. It was shown that shear stress increased endothelial cell migration and that CSM abrogated this effect. These results suggest that, under shear stress conditions, PECAM-1 is not predominantly concentrated at intercellular junctions in uterine endothelial cells. Exposure of cells to unidentified soluble components of cigarette smoke leads to alterations in PECAM-1 distribution that may cause endothelial dysfunction. If this occurs in vivo it could contribute to the adverse effects on pregnancy outcome associated with exposure to cigarette smoke.

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

Objective To investigate the effects of 17β-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°C. Main outcome measure(s) Measures of COX-2 mRNA and protein, PGE 2 production, and estrogen receptor α and β 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 β mRNA and protein were abundant in HUMEC, whereas estrogen receptor α 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.

Progesterone withdrawal causes endothelin release from cultured human uterine microvascular endothelial cells.

Current theories on the physiology of menstrual bleeding in humans offers an explanation for the shedding of the endometrium as a result of a breakdown of the extracellular matrix due to an inflammatory reaction. The link between the fall in progesterone levels and these events is not clear. Neither has an explanation been presented for the vasoconstriction in the coiled arteries occurring during menses. We have hypothesized a chain of events where the fall in progesterone levels induces an upregulation of the thrombin receptor in the small uterine arteries leading to an increased thrombin response and subsequent endothelin release. Endothelial cells from human umbilical cord (HUVECs) and from human small uterine arteries (UtMVECs) were cultured under conditions partly resembling the female hormonal cycle with progesterone withdrawal. Following progesterone increase and subsequent withdrawal, we found an increased production of thrombin receptor and an increased release of endothelin from UtMVECs compared with HUVECs. Endothelin release in response to progesterone withdrawal in UtMVECs can offer an explanation for the vasoconstriction seen in the coiled arteries during menses in humans.

Levamisole induced apoptosis in cultured vascular endothelial cells.

To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5 - 2 mmol l(-1)) alone or in combination with antioxidants (10 mmol l(-1) glutathione or 5 mmol l(-1) N-Acetylcysteine or 0.1 mmol l(-1) Tocopherol) were evaluated for apoptosis ((3)H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation ((3)H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (-70%), reduced expression of survival factors such as clusterin (-30%), endothelin-1 (-43%), bcl-2 (-34%), endothelial NO-synthase (-32%) and pRb (Retinoblastoma protein: -89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l(-1))-induced apoptosis was inhibited by glutathione (-50%) and N-Acetylcysteine (-36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity.