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Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated transfer of prostaglandin H synthase cDNA.

ABSTRACT: A retroviral vector (BAG) was used to transfer human prostaglandin H synthase (PGHS-1) gene into a human endothelial cell line for enhancement of PGI2 synthesis. Cells infected with BAG containing PGHS-1 cDNA in the sense orientation relative to the retroviral promoter (PGHS(S)) expressed a 30-fold increase in mRNA but, due to a reading frame shift, did not show an increase in PGHS protein or in PGI2 synthesis, while those with PGHS-1 in reverse orientation relative to the viral promoter (PGHS(R)), produced a > 10-fold increase in PGHS mRNA over the control (169 +/- 22 vs 14.8 +/- 1.2 amol/micrograms RNA) with a concordant increase in PGHS protein (5.82 +/- 1.07 vs 0.23 +/- 0.04 ng/mg protein) and enzyme activity. Primer extension analysis of PGHS(R) revealed two transcription start sites located in the SV40 late promoter region adjacent to PGHS-1 cDNA. PGHS(R) cells produced a high basal PGI2 level which was increased by several-fold in response to stimulation by ionophore, arachidonic acid, and thrombin. Kinetic analysis revealed the PGI2 synthetic rate to be 14 ng/min-1 per million cells and t1/2 of PGI2 synthesis, 13.3 min. These findings indicate that transfer of PGHS-1 gene into vascular cells enhances PGI2 synthesis and may be a useful strategy for restoring thromboprotective property of damaged blood vessels.

SUBMITTER: Xu XM

PROVIDER: S-EPMC288168 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated transfer of prostaglandin H synthase cDNA.

Xu X M XM Ohashi K K Sanduja S K SK Ruan K H KH Wang L H LH Wu K K KK

The Journal of clinical investigation 19930401 4

A retroviral vector (BAG) was used to transfer human prostaglandin H synthase (PGHS-1) gene into a human endothelial cell line for enhancement of PGI2 synthesis. Cells infected with BAG containing PGHS-1 cDNA in the sense orientation relative to the retroviral promoter (PGHS(S)) expressed a 30-fold increase in mRNA but, due to a reading frame shift, did not show an increase in PGHS protein or in PGI2 synthesis, while those with PGHS-1 in reverse orientation relative to the viral promoter (PGHS(R ...[more]

PMID: 8473524

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Project description:Study questionDoes the uterine vasculature play a localized role in promoting stromal cell decidualization in the human endometrium?Summary answerOur study demonstrated that hemodynamic forces induced secretion of specific endothelial cell-derived prostanoids that enhanced endometrial perivascular decidualization via a paracrine mechanism.What is known alreadyDifferentiation of stromal cell fibroblasts into the specialized decidua of the placenta is a progesterone-dependent process; however, histologically, it has long been noted that the first morphological signs of decidualization appear in the perivascular stroma. These observations suggest that the human endometrial vasculature plays an active role in promoting stromal differentiation.Study design, size, durationPrimary human endometrial stromal cells were co-cultured for 14 days with primary uterine microvascular endothelial cells within a microfluidic Organ-on-Chip model of the endometrium.Participants/materials, setting, methodsCultures were maintained with estradiol and a progestin, with or without continuous laminar perfusion to mimic hemodynamic forces derived from the blood flow. Some cultures additionally received exogenous agonist-mediated challenges. Decidualization in the microfluidic model was assessed morphologically and biochemically. ELISA was used to examine the culture effluent for expression of decidualization markers and prostaglandins. Immunofluorescence was used to monitor cyclooxygenase-2 expression in association with decidualization.Main results and the role of chanceA significantly enhanced stromal decidualization response was observed in the co-cultures when the endothelial cells were stimulated with hemodynamic forces (e.g. laminar shear stress) derived from controlled microfluidic perfusion (<0.001). Furthermore, the enhanced progestin-driven stromal differentiation was mediated via cyclooxygenase-2 and the paracrine action of prostaglandin E2 and prostacyclin. Altogether, these translational findings indicate that the vascular endothelium plays a key physiologic role during the early events of perivascular decidualization in the human endometrium.Large scale dataN/A.Limitations, reasons for cautionThis report is largely an in vitro study. Although we were able to experimentally mimic hemodynamic forces in our microfluidic model, we have not yet determined the contribution of additional cell types to the decidualization process or determined the precise physiological rates of shear stress that the microvasculature of the endometrium undergoes in vivo.Wider implications of the findingsIdentification of specific endothelial-derived prostaglandins and their role during endometrial reproductive processes may have clinical utility as therapeutic targets for reproductive disorders such as infertility, endometriosis, adenomyosis, pre-eclampsia and poor pregnancy outcomes.Study funding/competing interest(s)This work was supported by the Veterans Affairs (I01 BX002853), the Bill and Melinda Gates Foundation Grand Challenges Exploration (OPP1159411), the Environmental Toxicology Training Grant (NIH T32 ES007028) and the Environmental Protection Agency STAR Center Grant (83573601).Conflict of interestThe authors report no conflicts of interest.Trial registration numberN/A.

Project description:Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses.Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFR?, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction.Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFR? and FGFR2, respectively.A novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

Dataset Information

Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated transfer of prostaglandin H synthase cDNA.

Publications

Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated transfer of prostaglandin H synthase cDNA.

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