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The objectives of this research were to elucidate the involvement of constrictor prostanoids in the vascular reactivity to vasopressin (VP) and the role of estrogen in the regulation of the constrictor prostanoid mechanism in the female rat. Aortas obtained from male, intact (InT)-, ovariectomized (OvX)- and OvX + estrogen-replaced (OvX+Est)-female rats were studied. Contractile responses to VP were examined in the presence of nonselective and selective cyclooxygenase (COX) inhibitors. Basal and VP-stimulated release of thromboxane A2 (TxA2) and prostacyclin (PGI2) from the aortic wall were measured. Concentration-response curves to exogenous TxA2 were also obtained. To elucidate the regulatory effects of estrogen on the constrictor prostanoid pathway, the expression of COX-1, COX-2, thromboxane synthase (TxS) and thromboxane receptor (TP) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Further, immunohistochemistry was employed to determine COX-1, COX-2 and TxS protein expression in aortic endothelium and vascular smooth muscle. The major findings of this research are that: 1) The contractile responses of the female rat aorta to VP were enhanced by COX-2-mediated production of constrictor prostanoids (PGH2/TxA2), and this mechanism is potentiated by estrogen; 2) Vascular reactivity to exogenous TxA2 was higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored vascular reactivity to TxA2 in the female aorta; 3) VP-stimulated release of endogenous TxA2 and PGI2 were higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored VP-stimulated release of these endogenous prostanoids by the female aorta; and 4) The expression of COX-2 and TxS mRNA and protein, and the expression of TP mRNA were higher in InT-female than in male, and were reduced by OvX and restored by estrogen replacement therapy. In conclusion, estrogen potentiated contractile responses of the female rat aorta to VP by upregulating the expression of COX-2, TxS and TP; thereby enhancing VP-induced release of TxA2, as well as the vascular reactivity to endogenous TxA2.

The objectives of this research were to elucidate the involvement of constrictor prostanoids in the vascular reactivity to vasopressin (VP) and the role of estrogen in the regulation of the constrictor prostanoid mechanism in the female rat. Aortas obtained from male, intact (InT)-, ovariectomized (OvX)- and OvX + estrogen-replaced (OvX+Est)-female rats were studied. Contractile responses to VP were examined in the presence of nonselective and selective cyclooxygenase (COX) inhibitors. Basal and VP-stimulated release of thromboxane A2 (TxA2) and prostacyclin (PGI2) from the aortic wall were measured. Concentration-response curves to exogenous TxA2 were also obtained. To elucidate the regulatory effects of estrogen on the constrictor prostanoid pathway, the expression of COX-1, COX-2, thromboxane synthase (TxS) and thromboxane receptor (TP) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Further, immunohistochemistry was employed to determine COX-1, COX-2 and TxS protein expression in aortic endothelium and vascular smooth muscle. The major findings of this research are that: 1) The contractile responses of the female rat aorta to VP were enhanced by COX-2-mediated production of constrictor prostanoids (PGH2/TxA2), and this mechanism is potentiated by estrogen; 2) Vascular reactivity to exogenous TxA2 was higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored vascular reactivity to TxA2 in the female aorta; 3) VP-stimulated release of endogenous TxA2 and PGI2 were higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored VP-stimulated release of these endogenous prostanoids by the female aorta; and 4) The expression of COX-2 and TxS mRNA and protein, and the expression of TP mRNA were higher in InT-female than in male, and were reduced by OvX and restored by estrogen replacement therapy. In conclusion, estrogen potentiated contractile responses of the female rat aorta to VP by upregulating the expression of COX-2, TxS and TP; thereby enhancing VP-induced release of TxA2, as well as the vascular reactivity to endogenous TxA2.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Yes / Urinary prostaglandin (PG) E metabolite (PGE-M) and 11-dehydro (d)-thromboxane (TX) B2 are biomarkers of cyclooxygenase-dependent prostanoid synthesis. We investigated (1) the effect of aspirin 300 mg daily and eicosapentaenoic acid (EPA) 2000 mg daily, alone and in combination, on urinary biomarker levels and, (2) whether urinary biomarker levels predicted colorectal polyp risk, during participation in the seAFOod polyp prevention trial. Urinary PGE-M and 11-d-TXB2 were measured by liquid chromatography-tandem mass spectrometry. The relationship between urinary biomarker levels and colorectal polyp outcomes was investigated using negative binomial (polyp number) and logistic (% with one or more polyps) regression models. Despite wide temporal variability in PGE-M and 11-d-TXB2 levels within individuals, both aspirin and, to a lesser extent, EPA decreased levels of both biomarkers (74% [P ≤ .001] and 8% [P ≤ .05] reduction in median 11-d-TXB2 values, respectively). In the placebo group, a high (quartile [Q] 2-4) baseline 11-d-TXB2 level predicted increased polyp number (incidence rate ratio [IRR] [95% CI] 2.26 [1.11,4.58]) and risk (odds ratio [95% CI] 3.56 [1.09,11.63]). A low (Q1) on-treatment 11-d-TXB2 level predicted reduced colorectal polyp number compared to placebo (IRR 0.34 [0.12,0.93] for combination aspirin and EPA treatment) compared to high on-treatment 11-d-TXB2 values (0.61 [0.34,1.11]). Aspirin and EPA both inhibit PGE-M and 11-d-TXB2 synthesis in keeping with shared in vivo cyclooxygenase inhibition. Colorectal polyp risk and treatment response prediction by 11-d-TXB2 is consistent with a role for platelet activation during early colorectal carcinogenesis. The use of urinary 11-d-TXB2 measurement for a precision approach to colorectal cancer risk prediction and chemoprevention requires prospective evaluation. / Efficacy and Mechanism Evaluation Programme. Grant Number: NIHR128210. Cancer Research UK. Grant Number: C23434/A24939

Further studies were done to investigate the mechanism responsible for 1-BI-induced apoptosis in NCI-H460. It was found that 1-BI stimulated the expression of pro-apoptotic p53, Bax and cytosolic NF-kB p65 subunit but decreased pERK in NCI-H460 cells. The active forms of caspase 3 and caspase 9 were detected by Western blot, accompanied by an increase in caspase 3 activity. Reactive oxygen species (ROS) was highly generated at 24 hours after the treatment and the mitochondrial membrane potential was significantly decreased at 48 and 72 hours. The application of either N-acetyl cysteine (NAC) or glutathione (GSH) attenuated the cell growth inhibition caused by 1-BI. NCI-H460 cells pretreated with NAC showed a decrease in ROS production and p65 protein but an increase in pERK. / Taken together, these findings suggest that the inhibition of THXS suppresses lung cancer cell growth by promoting either G1 cell cycle arrest or apoptosis. The status of p53 is critical for both cell cycle arrest and apoptosis in 1-BI-mediated growth inhibition, which is evident by enhanced apoptosis detected in p53-transfected NCI-H23 and DMS 114 cells and G1 cell cycle in lung cancer cells treated with PFT-alpha. The 1-BI-induced growth-inhibitory pathway is associated with the generation of ROS, alteration of mitochondrial membrane potential, down-regulation of pERK and p65. / The result showed that THXS expressed in all of the three lung cancer cell lines (NCI-H23, DMS 114 and NCI-H460). The activity of THXS was also reflected by the presence of THXS metabolite thromobxane B2 (TXB2) in the cells, which was detected by ELISA. 1-Benzylimidazole (1-BI), a specific THXS inhibitor, suppressed the lung cancer cell proliferation measured by MTT assay. 1-BI treatment caused G1 phase arrest and enhanced the level of cyclin dependent kinase inhibitor p27 in a time-dependent manner in NCI-H23 and DMS 114 cells. It markedly increased DNA fragmentation in NCI-H460 cells. The findings suggest that 1-BI inhibits cell growth by arresting cell cycle and inducing cell death. Annexin V/PI staining revealed that the cell death induced by I-BI was mainly in the format of apoptosis. Further experiments showed that the I-BI-induced apoptosis could be enhanced by the introduction of p53 into NCI-H23 and DMS 114 cells, and such enhancement was associated with a decrease in mitochondrial membrane potential. This result suggests that the p53 may play a positive role in apoptosis induced by 1-BI through changing of the mitochondrial membrane potential. The role of p53 in I-BI-mediated apoptosis was further confirmed by the experiment of the p53 inhibition. Pifithrin-alpha hydrobromide (PFT-alpha), a p53 specific inhibitor, suppressed the 1-BI-induced p53 protein expression and increased G1 cell cycle arrest. / Thromboxane A2 (TXA2) is a potent arachidonate metabolite in the cyclooxygenase-2 (COX-2) pathway, which is produced by a member of cytochrome P450 (CYP) superfamily called thromboxane synthase (THXS). Recent studies have showed that thromboxane and THXS are associated with cancer cell migration, angiogenesis, tumor metastasis and cancer proliferation but there is limited information on their role in lung cancer development. This thesis is to test the hypothesis that inhibition of THXS could alter lung cancer cell growth. / Leung, Kin Chung. / Adviser: George G. Chen. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3319. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 130-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.