Prostacyclin Synthase Research Articles

Homology Modeling and Molecular Dynamics Simulation Combined with X-ray Solution Scattering Defining Protein Structures of Thromboxane and Prostacyclin Synthases.

A combination of molecular dynamics (MD) simulations and X-ray scattering (SAXS) has emerged as the approach of choice for studying protein structures and dynamics in solution. This approach has potential applications for membrane proteins that neither are soluble nor form crystals easily. We explore the water-coupled dynamic structures of thromboxane synthase (TXAS) and prostacyclin synthase (PGIS) from scanning HPLC-SAXS measurements combined with MD ensemble analyses. Both proteins are heme-containing enzymes in the cytochrome P450 family, known as prostaglandin H2 (PGH2) isomerase, with counter-functions in regulation of platelet aggregation. Currently, the X-ray crystallographic structures of PGIS are available, but those for TXAS are not. The use of homology modeling of the TXAS structure with ns-μs explicit water solvation MD simulations allows much more accurate estimation of the configuration space with loop motion and origin of the protein behaviors in solution. In contrast to the stability of the conserved PGIS structure in solution, the pronounced TXAS flexibility has been revealed to have unstructured loop regions in connection with the characteristic P450 structural elements. The MD-derived and experimental-solution SAXS results are in excellent agreement. The significant protein internal motions, whole-molecule structures, and potential problems with protein folding, crystallization, and functionality are examined.

The effect of magnesium sulfate on gene expression in maternal microvascular endothelial cells

ABSTRACTPreeclampsia is a severe complication of pregnancy associated with maternal and fetal morbidity and mortality. To date, magnesium sulfate remains the preferred method of treatment used to reduce the development of eclampsia. Our aim was to investigate the effects of magnesium sulfate on the expression of genes involved with endothelial function in maternal microvascular endothelial cells from both normal and preeclamptic pregnancies. Primary cells from normal pregnancies treated with 80 mg/L magnesium sulfate for 6 h revealed an overall trend of increased expression of angiogenic and vasopressor-related factors by qPCR analyses. Primary cells from preeclamptic pregnancies revealed an overall trend of decreased expression, with significantly lowered levels for vascular endothelial growth factor receptor 2, endothelin, and vascular cell adhesion protein-1. A comparison of treated cells revealed significantly increased levels for endoglin, vascular endothelial growth factor receptor 2, soluble fms-like tyrosine kinase-1, prostacyclin synthase, tumor necrosis factor α, tumor necrosis factor receptor 1, and endothelin in normal versus preeclamptic cells following treatment. These results reveal disparate activation of overall expression activity by magnesium sulfate in maternal endothelial cells from normal pregnancies over preeclamptic pregnancies.

Endothelium-specific amyloid precursor protein deficiency causes endothelial dysfunction in cerebral arteries

The exact physiological function of amyloid-β precursor protein (APP) in endothelial cells is unknown. Endothelium-specific APP-deficient (eAPP−/−) mice were created to gain new insights into the role of APP in the control of vascular endothelial function. Endothelium-dependent relaxations to acetylcholine were significantly impaired in basilar arteries of global APP knockout (APP−/−) and eAPP−/− mice (P < 0.05). In contrast, endothelium-independent relaxations to nitric oxide (NO)-donor diethylamine-NONOate were unchanged. Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated in large cerebral arteries of APP−/− mice and eAPP−/− mice as compared to respective wild-type littermates (P < 0.05). Furthermore, basal levels of cyclic guanosine monophosphate (cGMP) were also significantly reduced in large cerebral arteries of APP-deficient mice (P < 0.05). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate (cAMP) was not affected by genetic inactivation of APP in endothelial cells. By using siRNA to knockdown APP in cultured human brain microvascular endothelial cells we also found a significant downregulation of eNOS mRNA and protein expressions in APP-deficient endothelium (P < 0.05). These findings indicate that under physiological conditions, expression of APP in cerebral vascular endothelium plays an important protective function by maintaining constitutive expression of eNOS.

Nonsteroidal Anti-inflammatory Drug Interaction with Prostacyclin Synthase Protects from Miscarriage

This study evaluates the relationship between single nucleotide polymorphisms (SNPs) in nonsteroidal anti-inflammatory drug (NSAID) metabolism and related pathways and spontaneous abortion (SAB, gestation < 20 weeks) risk. Women were enrolled in Right from the Start (2004–2010) prospective cohort. Periconceptional NSAIDs reported through the sixth week of pregnancy were obtained from study interviews. We evaluated 201 SNPs in 600 European American women. Interaction analyses between NSAID use and SNPs were conducted using logistic regression, adjusted for confounders. We also evaluated prostaglandin E2 urinary metabolite (PGE-M) in an independent population for association with SNPs using linear regression. NSAID use was reported by 63% of cases and 62% controls. The most significant interaction was at prostacyclin synthase (PGIS) rs5602 (OR = 0.34, 95% CI 0.19–0.60, p = 2.45 × 10−4) and was significant after a Bonferroni correction. NSAID users were protected from SAB (OR = 0.78, 95% CI 0.56–1.10), while non-NSAID users were at increased risk (OR = 2.11, 95% CI 1.35–3.29) in rs5602 stratified analyses. rs5602 also associated with increased PGE-M levels (Beta = 0.09, 95% CI −0.002–0.19, p = 0.033). We identified an association between a PGIS variant and SAB risk that is modified by NSAIDs use during pregnancy and directly associated with increased levels of PGE metabolites. This suggests the potential use of genetic information to guide pharmaceutical intervention to prevent adverse pregnancy outcomes.

In vivo Endocrine Secretion of Prostacyclin Following Expression of a Cyclooxygenase-1/Prostacyclin Fusion Protein in the Salivary Glands of Rats Via Nonviral Gene Therapy

Pulmonary arterial hypertension (PAH) is a progressive disease that culminates in right heart failure and death. Prostacyclin (PGI2) and its derivatives are effective treatments for PAH when administered as continuous parenteral infusions. This treatment paradigm requires medical sophistication, and patients are at risk for complications from an indewelling catheter; drug interruptions may result in rebound pulmonary hypertension and death. We hypothesized that the salivary gland can be repurposed into an endogenous production site for circulating PGI2 through the expression of a fusion protein embodying cyclooxygenase-1 (Cox1) and prostacyclin synthase (PGIS) domains. We utilized ultrasound-assisted gene transfer, a nonviral gene transfer strategy that achieves robust gene transfer to the salivary gland. We initially found that Cox1-PGIS expression in livers of mice using an adenoviral vector dramatically increased circulating PGI2 relative to untreated rats or rats treated with PGIS alone. We then utilized ultrasound-assisted gene transfer to express Cox1-PGIS in the submandibular glands of rats and showed a significant elevation of circulating PGI2 that corresponded to approximately 30% of that seen in humans undergoing intravenous infusion therapy for PAH. These results suggest the feasibility of gene therapy to drive endogenous biosynthesis of PGI2 as a therapeutic strategy for the treatment of PAH.

Efficient and Functional Endothelial Repopulation of Whole Lung Organ Scaffolds.

To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44 ± 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture compared to that of the native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial-seeded lungs had CD31 gene expression of 0.074 ± 0.015 vs 0.021 ± 0.0023 for native lungs, p = 0.025, and VE-Cadherin gene expression of 0.93 ± 0.22 compared to that of the native lung at 0.13 ± 0.02, p = 0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than those of the native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had gas exchange function during mechanical ventilation on 100% O2 greater than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.

Improving the regenerative potential of olfactory ensheathing cells by overexpressing prostacyclin synthetase and its application in spinal cord repair

BackgroundOlfactory ensheathing cells (OEC), specialized glia that ensheathe bundles of olfactory nerves, have been reported as a favorable substrate for axonal regeneration. Grafting OEC to injured spinal cord appears to facilitate axonal regeneration although the functional recovery is limited. In an attempt to improve the growth-promoting properties of OEC, we transduced prostacyclin synthase (PGIS) to OEC via adenoviral (Ad) gene transfer and examined the effect of OEC with enhanced prostacyclin synthesis in co-culture and in vivo. Prostacyclin is a vasodilator, platelet anti-aggregatory and cytoprotective agent.ResultsCultured OEC expressed high level of cyclooxygneases, but not PGIS. Infection of AdPGIS to OEC could selectively augument prostacyclin synthesis. When cocultured with either OEC or AdPGIS-OEC, neuronal cells were resistant to OGD-induced damage. The resulted OEC were further transplanted to the transected cavity of thoracic spinal cord injured (SCI) rats. By 6 weeks post-surgery, significant functional recovery in hind limbs occurred in OEC or AdPGIS-OEC transplanted SCI rats compared with nontreated SCI rats. At 10–12 weeks postgraft, AdPGIS-OEC transplanted SCI rats showed significantly better motor restoration than OEC transplanted SCI rats. Futhermore, regenerating fiber tracts in the distal spinal cord stump were found in 40–60% of AdPGIS-OEC transplanted SCI rats.ConclusionsEnhanced synthesis of prostacyclin in grafted OEC improved fiber tract regeneration and functional restoration in spinal cord injured rats. These results suggest an important potential of prostacyclin in stimulating OEC therapeutic properties that are relevant for neural transplant therapies.

Altered expression of eNOS, prostacyclin synthase, prostaglandin G/H synthase, and thromboxane synthase in porcine aortic endothelial cells after exposure to human serum-relevance to xenotransplantation.

Under normal conditions, the activity of platelets is stringently and precisely balanced between activation and quiescent state. This guarantees rapid hemostasis and avoids uncontrolled thrombosis. However, excessive platelet activation and resulting thrombotic microangiopathy are frequently observed in pig-to-primate xenotransplantation models. Endothelium-derived inhibitory mechanisms play an important role in regulation of platelet activation. These mainly include nitric oxide (NO), prostacyclin PGI2 , and adenosine, which are synthesized by endothelial NO synthases (eNOS), prostacyclin synthase, and CD39/CD73, respectively. We investigated whether endothelium-derived regulatory mechanisms are affected in porcine aortic endothelial cells (PAECs) after exposure to human serum. In the present study, exposure of PAECs or porcine iliac arteries to human serum suppressed gene expression of eNOS and prostacyclin synthase, while induced gene expression of prostaglandin G/H synthase and thromboxane synthase. Simultaneously, exposure to human serum reduced NO and PGI2 production in PAEC culture supernatants. Thus, human serum altered the balance of endothelium-derived inhibitory mechanisms in PAECs, which may indicate a regulatory mechanism of excessive platelet activation in pig-to-primate xenotransplantation.

Interactions among Variants in Eicosanoid Genes Increase Risk of Atherothrombotic Stroke in Chinese Populations

Eicosanoids are lipid mediators that may play a role in ischemic stroke (IS). However, the association of variants in eicosanoid genes and these interactions with IS risk has not been investigated. The aim of the present study was to investigate the association of 11 variants in eicosanoid genes with IS and to determine whether these gene-gene interactions increase the risk of IS. Eleven variants in prostaglandin H synthase-1 (PTGS1), PTGS2, thromboxane A2 synthase (TBXAS1), prostacyclin synthase (PTGIS), and prostaglandin E synthase (PTGES) genes were examined using mass spectrometry method in 297 patients with atherothrombotic stroke and 291 controls. Gene-gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) method. Platelet aggregation and platelet-leukocyte aggregates were measured on admission. There were no significant differences in the genotype distributions of the 11 variants between patients and controls. However, GMDR analysis showed a significant gene-gene interaction among rs20417, rs5602, and rs41708, which scored 10 for cross-validation consistency and 9 for the sign test (P = .014). Logistic regression analysis showed that high-risk interaction among rs20417, rs5602, and rs41708 was an independent risk factor for atherothrombotic stroke (OR = 2.45, 95% CI: 1.33-3.27, P = .019). The high-risk interactive genotypes were associated with higher platelet aggregation and platelet-leukocyte aggregates. PTGS2 rs20417, PTGIS rs5602, and TBXAS1 rs41708 three-locus interactions may confer a higher risk for atherothrombotic stroke. The combinatorial analysis used in this study may be helpful to elucidate complex genetic risk for IS.

Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.

Calpain 1 cleaves and inactivates prostacyclin synthase in mesenteric arteries from diabetic mice.

Diabetes is associated with a number of co-morbidities including an increased risk of developing cardiovascular diseases. The activation of Ca2+-activated proteases of the calpain family has been implicated in platelet activation associated with diabetes and this study aimed to determine the role of calpain activation in the development of endothelial dysfunction. Diabetes induction in miceattenuated acetylcholine-induced relaxation of mesenteric artery rings, an effect prevented in mice receiving a calpain inhibitor. A nitric oxide-independent but diclofenac-sensitive component of the relaxation-response was altered and correlated with a loss of prostacyclin (PGI2) generation and reduced vascular levels of PGI2 synthase. Calpain inhibition was also able to restore PGI2 synthase levels and PGI2 generation in arteries from diabetic animals. The effects of diabetes were reproduced in vitro by a combination of high glucose and palmitate, which elicited calpain activation, PGI2 synthase cleavage and inactivation as well as endothelial dysfunction in mesenteric arteries from wild-type mice. PGI2 cleavage was not observed in arteries from calpain 1-/- mice or mice overexpressing the endogenous calpain inhibitor calpastatin. Finally, proteomic analyses revealed that calpain 1 cleaved the C-terminal domain of PGI2 synthase close to the catalytic site of the enzyme. These data demonstrate that diabetes leads to the activation of calpain 1 in mesenteric arteries and can initiate endothelial dysfunction by cleaving and inactivating the PGI2 synthase. Given that calpain inhibition prevented diabetes-induced endothelial dysfunction in mesenteric arteries, calpains represent an interesting therapeutic target for the prevention of cardiovascular complication of diabetes.

Spatial phenotyping of the endocardial endothelium as a function of intracardiac hemodynamic shear stress

Despite substantial evidence for the central role of hemodynamic shear stress in the functional integrity of vascular endothelial cells, hemodynamic and molecular regulation of the endocardial endothelium lining the heart chambers remains understudied. We propose that regional differences in intracardiac hemodynamics influence differential endocardial gene expression leading to phenotypic heterogeneity of this cell layer. Measurement of intracardiac hemodynamics was performed using 4-dimensional flow MRI in healthy humans (n=8) and pigs (n=5). Local wall shear stress (WSS) and oscillatory shear indices (OSI) were calculated in three distinct regions of the LV – base, mid-ventricle (midV), and apex. In both the humans and pigs, WSS values were significantly lower in the apex and midV relative to the base. Additionally, both the apex and midV had greater oscillatory shear indices (OSI) than the base. To investigate regional phenotype, endocardial endothelial cells (EEC) were isolated from an additional 8 pigs and RNA sequencing was performed. A false discovery rate of 0.10 identified 1051 differentially expressed genes between the base and apex, and 321 between base and midV. Pathway analyses revealed apical upregulation of genes associated with translation initiation. Furthermore, tissue factor pathway inhibitor (TFPI; mean 50-fold) and prostacyclin synthase (PTGIS; 5-fold), genes prominently associated with antithrombotic protection, were consistently upregulated in LV apex. These spatio-temporal WSS values in defined regions of the left ventricle link local hemodynamics to regional heterogeneity in endocardial gene expression.

Cyclooxygenase-2-Derived Prostaglandins Mediate Cerebral Microcirculation in a Juvenile Ischemic Rat Model.

We previously showed that the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) increases cerebral microcirculation in a juvenile ischemic rat model. We address the roles of cyclooxygenase (COX)-elaborated prostaglandins in collateral recruitment and blood supply. Fourteen-day-old rats were subjected to ischemia-reperfusion and treated with either PBS or 7-NI (25 mg/kg) at the reperfusion onset. Six-keto-prostaglandin F1α was measured using ELISA. COX-1 and COX-2 and prostaglandin terminal synthesizing enzymes were evaluated using reverse-transcriptase polymerase chain reaction and immunofluorescence. Microvascular blood flow indexes (artery diameter and capillaries number) were measured using sidestream dark-field videomicroscopy in PBS- and 7-NI-treated ischemic rats in the absence or presence of the COX-2 inhibitor NS-398 (5 mg/kg). Cell death was measured with the TUNEL (terminal transferase dUTP nick end labeling) assay and cleaved-caspase-3 immunostaining. Six-keto-prostaglandin F1α and COX-2, associated with a prostaglandin E synthase, were significantly increased in PBS- and 7-NI-treated animals 15 minutes and 1 hour after ischemia-reperfusion, respectively. In contrast and as compared with PBS, 7-NI significantly decreased prostacyclin synthase and cytosolic prostaglandins E synthase mRNA. Selective COX-2 inhibition significantly decreased blood flow indexes and significantly reversed the effects of 7-NI, including the number of TUNEL+- and cleaved-caspase-3+-nuclei. These results show that the juvenile rat brains mostly respond to ischemia by a COX-2-dependent prostaglandins production and suggest that the transcriptional responses observed under 7-NI facilitate and reorient COX-2-dependent prostaglandins production.

Oxymatrine attenuated isoproterenol-induced heart failure in rats via regulation of COX-2/PGI2 pathway.

Oxymatrine (OMT) is an active constituent of traditional Chinese herb Sophora japonica Ait which has been shown to exert potent anti-inflammatory,anti-oxidant and anti-fibrosis properties. Our previous studies have demonstrated that OMT has protective effects on isoproterenol-induced heart failure in rats through regulation of DDAH/ADMA metabolism pathway.In this study,we further investigated whether OMT could attenuate isoproterenol-induced heart failure through the regulation of COX-2/PGI2 pathway. Heart failure was induced in Sprague-Dawley rats by 5mg/kg isoproterenol subcutaneous injection for 7days. The rats were maintained on normal diet and randomly divided into five groups: control, isoproterenol, isoproterenol with OMT (50, 100mg/kg), and OMT alone groups (n=12 in each group). Serum brain natruretic peptide (BNP, a heart failure biomarker), histopathological variables, expression of Cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and Prostacyclin synthase (PGIS) were analysed. Administration of OMT significantly reduced the increased BNP in plasm of isoproterenol-induced rats, attenuated cardiac fibrosis,suppressed overexpression of myocardial COX-1 expression, up-regulated COX-2 and PGIS expression, but had no effects on isoproterenol-induced elevated protein cPLA2. And compared with control group, any indexes in sham rats treated with OMT (100mg/kg) alone were unaltered. These results demonstrated that OMT has cardioprotective effects on isoproterenol-induced heart failure in rats by regulating COX-2/PGI2 pathway.

Endometrial prostaglandin synthases, ovarian steroids, and oxytocin receptors in mares with oxytocin-induced luteal maintenance

Oxytocin (OXT) has been used to prolong the luteal phase in mares, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of chronic exogenous OXT administration to mid-luteal phase mares on luteal maintenance. Also, endometrial expression of prostaglandin endoperoxide synthase 2 (PTGS2), prostaglandin F2α, E2 and I2 synthases (AKR1C3, PTGES, and PTGIS), oxytocin receptor (OXTR), progesterone receptor (PGR), and estrogen receptors 1 (ESR1) and 2 (ESR2) were assessed in mares experiencing luteal maintenance 2 weeks after chronic exogenous OXT administration. Control mares (n = 5; C group) received 6 mL of saline im, whereas OXT (60 units/mare) was administered im (n = 6; OXT group), every 12 hours, on days 7 to 14 postovulation. After endometrial biopsy in groups C (Day 10) and OXT (Day 24), luteolysis occurred within 3 or 6 days, respectively. Luteal maintenance took place in 4 of 6 (67%) of OXT-treated mares. Progesterone in C group was the highest on biopsy day (P < 0.05). In OXT mares, PTGS2, ESR1 (P < 0.05), PTGES, PTGIS, PGR, and ESR2 (P < 0.01) gene transcription decreased, whereas OXTR increased (P < 0.05) in comparison with the C group. In OXT-treated mares, endometrial ESR2 protein expression decreased (P < 0.05), but OXTR increased (P < 0.05) compared with control animals. In both experimental groups, PTGS2 was mainly immunolocalized in surface epithelium, whereas AKR1C3, PTGES, PTGIS, and PGR were in surface and glandular epithelia. ESR1 and ESR2 were found in glandular epithelium and OXTR in stromal cells. High immunolabeling for PTGES, PTGIS, PGR, and OXTR and low for ESR2 was detected in endometrium of OXT-group mares with extended diestrus. Prolonged luteal function associated with chronic OXT treatment may be related to different spatial expression of OXTR and PGR in the endometrium. The observed reduction of endometrial ESR2 may be responsible for the maintenance of PGR in luminal and glandular epithelium. Also, ESR2 may attenuate the transcriptional activity of ESR1 in mare endometrium. This study offers new knowledge on the endometrial expression of ovarian steroids and OXT receptors in OXT pharmacologically induced luteal maintenance in the mare.

Abstract P339: Endothelium-specific Deletion of Amyloid Precursor Protein Causes Endothelial Dysfunction of Cerebral Arteries

Amyloid-β precursor protein (APP) is an integral membrane protein expressed abundantly in the endothelium of cerebral arteries. However, the exact physiological function of APP in endothelial cells is unknown. Mice with vascular endothelium-specific deletion of APP gene (eAPP-KO) were generated using loxP/Cre technology to test the hypothesis that APP plays a key role in the control of vascular endothelial nitric oxide (NO) function. Vasoreactivity of isolated basilar arteries were studied in vitro under pressurized conditions and compared to control littermates. eAPP-KO mice were normotensive (118±2 mmHg vs. control mice: 113±2 mmHg; n=5) and plasma cholesterol and blood glucose levels were not affected by endothelium-specific genetic inactivation of APP. Endothelium-dependent relaxations to acetylcholine were significantly impaired in eAPP-KO mice (maximal relaxation: 30±4% vs. 45±5% for control littermates; P&lt;0.05; n=7-8) while endothelium-independent relaxations to NO-donor diethylamine-NONOate were unchanged (n=5-6). Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated by 31% in cerebral arteries of eAPP-KO mice (P&lt;0.05 vs. littermates; n=6). Furthermore, basal levels of cyclic guanosine monophosphate were also significantly reduced in cerebral arteries of eAPP-KO mice (0.64±0.09 pmol/mg; P&lt;0.05 vs. littermates: 0.97±0.11 pmol/mg; n=11). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate were not affected by genetic inactivation of APP in endothelial cells (n=5-7). Moreover, superoxide anion levels as determined by HPLC analysis of 2-hydroxyethidium were unaltered in eAPP-KO mice cerebral arteries (1.4±0.2 nmol/mg vs. littermates: 1.2±0.1 nmol/mg; P=n.s.; n=7). Our results demonstrate that impaired endothelium-dependent relaxations to acetylcholine in eAPP-KO mice are caused by the reduced expression and function of eNOS. These findings indicate that under physiological conditions APP expression in cerebral vascular endothelium plays an important role in control of endothelial function.

Uterine expression of cytokines and contraction-associated genes in preterm birth of Brp-39 null mutant mice

Oxytocin (OXT) has been used to prolong the luteal phase in mares, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of chronic exogenous OXT administration to mid-luteal phase mares on luteal maintenance. Also, endometrial expression of prostaglandin endoperoxide synthase 2 (PTGS2), prostaglandin F2α, E2 and I2 synthases (AKR1C3, PTGES, and PTGIS), oxytocin receptor (OXTR), progesterone receptor (PGR), and estrogen receptors 1 (ESR1) and 2 (ESR2) were assessed in mares experiencing luteal maintenance 2 weeks after chronic exogenous OXT administration. Control mares (n = 5; C group) received 6 mL of saline im, whereas OXT (60 units/mare) was administered im (n = 6; OXT group), every 12 hours, on days 7 to 14 postovulation. After endometrial biopsy in groups C (Day 10) and OXT (Day 24), luteolysis occurred within 3 or 6 days, respectively. Luteal maintenance took place in 4 of 6 (67%) of OXT-treated mares. Progesterone in C group was the highest on biopsy day (P < 0.05). In OXT mares, PTGS2, ESR1 (P < 0.05), PTGES, PTGIS, PGR, and ESR2 (P < 0.01) gene transcription decreased, whereas OXTR increased (P < 0.05) in comparison with the C group. In OXT-treated mares, endometrial ESR2 protein expression decreased (P < 0.05), but OXTR increased (P < 0.05) compared with control animals. In both experimental groups, PTGS2 was mainly immunolocalized in surface epithelium, whereas AKR1C3, PTGES, PTGIS, and PGR were in surface and glandular epithelia. ESR1 and ESR2 were found in glandular epithelium and OXTR in stromal cells. High immunolabeling for PTGES, PTGIS, PGR, and OXTR and low for ESR2 was detected in endometrium of OXT-group mares with extended diestrus. Prolonged luteal function associated with chronic OXT treatment may be related to different spatial expression of OXTR and PGR in the endometrium. The observed reduction of endometrial ESR2 may be responsible for the maintenance of PGR in luminal and glandular epithelium. Also, ESR2 may attenuate the transcriptional activity of ESR1 in mare endometrium. This study offers new knowledge on the endometrial expression of ovarian steroids and OXT receptors in OXT pharmacologically induced luteal maintenance in the mare.

Protective effect of prostacyclin against pre-cardiac edema caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin and a thromboxane receptor agonist in developing zebrafish

The role of prostaglandin pathways has been suggested in some toxicological responses to dioxins. Cyclooxygenase type 2b (COX2b), thromboxane synthase, and the thromboxane receptor (TP) pathway have been implicated in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced pre-cardiac edema in developing zebrafish at 55 h post fertilization (hpf). Pre-cardiac edema refers to edema located in a small cavity between the heart and body wall of zebrafish eleutheroembryos. In the present study, we assessed the role of prostacyclin, which counteracts some biological effects of thromboxane, in TCDD-induced pre-cardiac edema. Pre-cardiac edema induced by TCDD exposure (0.5 and 1 ppb) beginning at 24 hpf was markedly inhibited by exposure to beraprost (5 and 10 μM), a prostacyclin receptor (IP) agonist, beginning at 33 hpf. The preventive effect of beraprost was reduced by exposure to CAY10441 (10 μM), an IP antagonist starting at 33 hpf. Knockdowns of the IP receptor (IP-KD) with two different morpholinos caused edema by themselves and enhanced pre-cardiac edema caused by the low concentration of TCDD (0.5 ppb). On the other hand, short exposure beginning at 48 hpf to U46619 (7.5–30 μM), a thromboxane receptor agonist caused pre-cardiac edema, which was inhibited by exposure beginning at 48 hpf to both ICI-192,605 (24 μM), a TP antagonist, and beraprost. Expression of prostacyclin synthase was increased from fertilization, plateaued by 48 hpf, and was maintained until at least 96 hpf. Overall, the results demonstrate a preventive effect of prostacyclin on TCDD-induced pre-cardiac edema in developing zebrafish.

Testosterone improves erectile function through inhibition of reactive oxygen species generation in castrated rats.

Testosterone is overwhelmingly important in regulating erectile physiology. However, the associated molecular mechanisms are poorly understood. The purpose of this study was to explore the effects and mechanisms of testosterone in erectile dysfunction (ED) in castrated rats. Forty male Sprague-Dawley rats were randomized to four groups (control, sham-operated, castration and castration-with-testosterone-replacement). Reactive oxygen species (ROS) production was measured by dihydroethidium (DHE) staining. Erectile function was assessed by the recording of intracavernous pressure (ICP) and mean arterial blood pressure (MAP). Protein expression levels were examined by western blotting. We found that castration reduced erectile function and that testosterone restored it. Nitric oxide synthase (NOS) activity was decrease in the castrated rats, and testosterone administration attenuated this decrease (each p < 0.05). The testosterone, dihydrotestosterone, cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) concentrations were lower in the castrated rats, and testosterone restored these levels (each p < 0.05). Furthermore, the cyclooxygenase-2 (COX-2) and prostacyclin synthase (PTGIS) expression levels and phospho-endothelial nitric oxide synthase (p-eNOS, Ser1177)/endothelial nitric oxide synthase (eNOS) ratio were reduced in the castrated rats compared with the controls (each p < 0.05). In addition, the p40phox and p67phox expression levels were increased in the castrated rats, and testosterone reversed these changes (each p < 0.05). Overall, our results demonstrate that testosterone ameliorates ED after castration by reducing ROS production and increasing the activity of the eNOS/cGMP and COX-2/PTGIS/cAMP signaling pathways.

Integrated Regional Cardiac Hemodynamic Imaging and RNA Sequencing Reveal Corresponding Heterogeneity of Ventricular Wall Shear Stress and Endocardial Transcriptome

BackgroundUnlike arteries, in which regionally distinct hemodynamics are associated with phenotypic heterogeneity, the relationships between endocardial endothelial cell phenotype and intraventricular flow remain largely unexplored. We investigated regional differences in left ventricular wall shear stress and their association with endocardial endothelial cell gene expression.Methods and ResultsLocal wall shear stress was calculated from 4‐dimensional flow magnetic resonance imaging in 3 distinct regions of human (n=8) and pig (n=5) left ventricle: base, adjacent to the outflow tract; midventricle; and apex. In both species, wall shear stress values were significantly lower in the apex and midventricle relative to the base; oscillatory shear index was elevated in the apex. RNA sequencing of the endocardial endothelial cell transcriptome in pig left ventricle (n=8) at a false discovery rate ≤10% identified 1051 genes differentially expressed between the base and the apex and 327 between the base and the midventricle; no differentially expressed genes were detected at this false discovery rate between the apex and the midventricle. Enrichment analyses identified apical upregulation of genes associated with translation initiation including mammalian target of rapamycin, and eukaryotic initiation factor 2 signaling. Genes of mitochondrial dysfunction and oxidative phosphorylation were also consistently upregulated in the left ventricular apex, as were tissue factor pathway inhibitor (mean 50‐fold) and prostacyclin synthase (5‐fold)—genes prominently associated with antithrombotic protection.ConclusionsWe report the first spatiotemporal measurements of wall shear stress within the left ventricle and linked regional hemodynamics to heterogeneity in ventricular endothelial gene expression, most notably to translation initiation and anticoagulation properties in the left ventricular apex, in which oscillatory shear index is increased and wall shear stress is decreased.