Zohre German

Estrogen Upregulates Endothelial Nitric Oxide Synthase Gene Expression in Fetal Pulmonary Artery Endothelium

NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17beta caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10(-10) to 10(-6) mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.

Molecular Basis of Estrogen-Induced Cyclooxygenase Type 1 Upregulation in Endothelial Cells

Estrogen upregulates cyclooxygenase-1 (COX-1) expression in endothelial cells. To determine the basis of this process, studies were performed in ovine endothelial cells transfected with the human COX-1 promoter fused to luciferase. Estradiol (E2) caused activation of the COX-1 promoter with maximal stimulation at 10−8 mol/L E2, and the response was mediated by either ER&agr; or ER&bgr;. Mutagenesis revealed a primary role for a putative Sp1 binding motif at −89 (relative to the ATG codon) and lesser involvement of a consensus Sp1 site at −111. Electrophoretic mobility shift assays yielded a single complex with the site at −89, and supershift analyses implicated AP-2&agr; and ER&agr;, and not Sp1, in protein-DNA complex formation. In endothelial cells with minimal endogenous ER, the transfection of ER&agr; mutants lacking the DNA binding domain or primary nuclear localization signals caused 4-fold greater stimulation of promoter activity with E2 than wild-type ER&agr;. In contrast, mutant ER&agr; lacking the A-B domains was inactive. Thus, estrogen-mediated upregulation of COX-1 in endothelium is uniquely independent of direct ER&agr;-DNA binding and instead entails protein-DNA interaction involving AP-2&agr; and ER&agr; at a proximal regulatory element. In addition, the process may be initiated by cytoplasmic ER&agr;, and critical receptor elements reside within the amino terminus.

Establishment of an immortalized fetal intrapulmonary artery endothelial cell line.

The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218-255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs ( passage 6). eNOS was also acutely activated by estradiol to levels 197-309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-α, which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.

PULMONARY ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE EXPRESSION IS DECREASED IN A LAMB MODEL OF NEONATAL PULMONARY HYPERTENSION. • 2082

PULMONARY ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE EXPRESSION IS DECREASED IN A LAMB MODEL OF NEONATAL PULMONARY HYPERTENSION. • 2082

Amyloid burden in patients with mild cognitive impairment is associated with elevated blood pressure during sleep and altered cerebral pressure-flow dynamics

Neuropsychiatric Inventory Questionnaire (NPI-Q). Chi-square and t-tests were used for unadjusted analyses and general linear models were used for adjusted analyses. Results: Age at onset and age at death were lower among AD participants with concomitant LBs than those without LB pathology. Men more often had LB pathology. Participants with LBs more often had at least one APOE ε4 allele. After adjustment for age, gender, education, neuritic and diffuse plaque frequency, and tangle stage, scores on the NPI-Q and the mUPDRS were more impaired for participants who had comorbid LB pathology. After additional adjustment of APOE ε4 status, only the mUPDRS showed significantly higher scores among persons who had both AD and LB pathology compared to the group that had AD without LBs. Conclusions: Participants with both AD and LB pathology have a clinical phenotype which may be distinguished from AD alone. The identification of comorbidity in the absence of reliable biomarkers of alpha-synuclein pathology may eventually help in the selection of patients for specific drug therapy.

Estrogen Receptors and eNOS are Colocalized as Components of a Signaling Module on the Plasma Membrane of Endothelial Cells

Estrogen Receptors and eNOS are Colocalized as Components of a Signaling Module on the Plasma Membrane of Endothelial Cells

Establishment of an Immortalized Fetal Intrapulmonary Artery Endothelial Cell Line |[bull]| 131

The endothelium plays a critical role in the growth and function of the developing pulmonary circulation. Studies of pulmonary endothelial cell gene expression and function have been limited by the requirement for early-passage primary cells due to senescence beyond passage 6-8. The purpose of the present study was to establish an immortalized fetal intrapulmonary artery endothelial cell line. As an example of a functionally-relevant phenotype, the conservation of acute endothelial nitric oxide synthase (eNOS) activation by estradiol was characterized by measuring 3H-L-arginine conversion to3 H-L-citrulline in intact cells. Ovine fetal intrapulmonary artery endothelial cells (PAEC) were sorted at passage 3 based on acetylated LDL uptake and transfected with a recombinant retroviral construct containing the E6 and E7 open reading frames of human papillomavirus type 16. These DNA viral genes inactivate host proteins involved in cell cycle control. Following selection, immortalized cells were propagated and studied at passages 18, 23, and 28, and compared to primary PAEC at passages 4-6. Acetylated LDL uptake and eNOS expression, indicative of an endothelial cell phenotype, were conserved in the immortalized cells. In addition, eNOS was acutely activated by 10-8 M estradiol in the immortalized cells, yielding NOS activity which was 346%, 279%, and 277% above basal at passage 18, 23, and 28, respectively. This level of activation was comparable to the 251% increase observed in primary cells. Acetylcholine (10-6M)-mediated eNOS activation was also demonstrable, yielding NOS activity that was 255%, 218%, and 285% above basal at passage 18, 23, and 28, respectively, compared to 221% in primary PAEC. The expression of estrogen receptor (ER) alpha, which mediates the acute response in primary PAEC, was also evident in the immortalized cells. These results indicate that fetal PAEC transfected with E6 and E7 show no signs of senescence with long-term passage, and that eNOS expression and ER-dependent acute activation of eNOS are conserved in these cells. The resulting cell line will provide an excellent model for further studies of eNOS regulation and other investigations of pulmonary endothelial cell gene expression and function.

Cell Growth Modulates Nitric Oxide Synthase Expression in Fetal Pulmonary Artery Endothelium |[bull]| 1833

Nitric oxide (NO), produced by the endothelial isoform of nitric oxide synthase (eNOS), is a critical mediator of pulmonary vascular structure and function in the developing lung, causing vasodilation and the inhibition of vascular smooth muscle growth. We have previously shown that pulmonary eNOS gene expression is developmentally regulated during late fetal and early postnatal life, suggesting the eNOS gene expression may be modulated by the state of pulmonary artery endothelial cell (PAEC) growth. We therefore studied the specific effects of cell growth on eNOS expression in cultured ovine fetal PAEC. To examine the effects of cell growth modified by two independent approaches, experiments were performed in cells at varying confluence, and in control cells versus cells stimulated with 20% ovine serum for 24-48 h. Cell proliferation was assessed by examining the incorporation of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) into replicating DNA during thymidilate synthetase inhibition with 5-flouro-2′-deoxyuridine. BrdU-positive nuclei were identified by immunocytochemistry with a specific monoclonal antibody. eNOS expression was evaluated by determinations of cell lysate NOS enzymatic activity, which is a highly sensitive measure of changes in enzyme abundance. Compared to 100% confluent cells, cell proliferation was greater at 50% confluence (3.5±0.6% vs. 9.4±0.8% BrdU-positive nuclei, respectively). Greater cell proliferation was associated with attenuated NOS activity, which was 11.9±2.4 vs. 2.3±0.1 pmol/mg prot/min at 100% vs. 50% confluence, respectively. With serum stimulation, cell proliferation increased from 2.8±0.3% to 5.9±0.4% BrdU-positive nuclei. Paralleling the findings with varying confluence, NOS activity was attenuated in the more rapidly-proliferating cells, falling from 2.0±0.3 to 0.5±0.1 pmol/mg prot/min with serum stimulation. These findings contrast with those in systemic endothelium, which exhibit enhanced eNOS expression with greater cell growth. Thus, eNOS expression is markedly attenuated with the activation of cell growth in fetal PAEC. We postulate that eNOS downregulation in rapidly-growing PAEC may facilitate concomitant smooth muscle replication, thereby coordinating the growth of the two cell types during vascularization of the developing lung.