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25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner.

Nguyen VP, Chen SH, Pizzuto K, Cantarutti A, Terminesi A, Mendonca C, Dumont DJ - J Angiogenes Res (2010)

Bottom Line: These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient.The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular and Cellular Biology Research, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 3M5, Canada. dan.dumont@sri.utoronto.ca.

ABSTRACT

Background: 25-hydroxycholesterol (25-OHC) is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis.

Methods: Primary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC) were treated with 25-OHC and tested for several different cellular parameters.

Results: We found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2) expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex). Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex). These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.

Conclusions: Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

No MeSH data available.


Related in: MedlinePlus

25-OHC promotes endothelial cell proliferation. A/ Short exposure to 25-OHC promoted EC proliferation. Viable cells that excluded trypan blue were counted after 24 hours of growing cells in 25-OHC. Fold difference in the number of viable cells from seeded cells (taken as 1) was most pronounced in bmLECs compared to bmVECs and bmAECs. Mean of fold differences between treated and untreated bmAEC, bmVEC, and bmLEC from three trials analysed by t-test yielded p-values of 0.021, 0.016, and 0.0036 respectively (n = 3). B/ Temporary boost in EC proliferation due to 25-OHC exposure for 12 hours was dependent on Cox-2 activity. ECs were treated with the selective Cox-2 inhibitor celecoxib for 12 hours after 25-OHC exposure. Celecoxib (5 μM) reversed the small increase in cell number of viable cells due to 25-OHC. The number of viable bmVECs treated with celecoxib alone dropped below the number of viable vehicle-treated bmVECs. Proliferative effects of 25-OHC also applied to human colorectal carcinoma cells HCT-116. HCT-116 has previously shown to be Cox-2 deficient. For each cell type, mean of fold differences between treatments from three trials analysed by ANOVA generated p-values ≤ 0.001 (n = 3). For each treatment with celecoxib, 25-OHC, and both together, the mean fold differences between each cell type analysed by ANOVA generated p-values of 0.084, 0.011, and 0.00038, respectively (n = 3).
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Figure 2: 25-OHC promotes endothelial cell proliferation. A/ Short exposure to 25-OHC promoted EC proliferation. Viable cells that excluded trypan blue were counted after 24 hours of growing cells in 25-OHC. Fold difference in the number of viable cells from seeded cells (taken as 1) was most pronounced in bmLECs compared to bmVECs and bmAECs. Mean of fold differences between treated and untreated bmAEC, bmVEC, and bmLEC from three trials analysed by t-test yielded p-values of 0.021, 0.016, and 0.0036 respectively (n = 3). B/ Temporary boost in EC proliferation due to 25-OHC exposure for 12 hours was dependent on Cox-2 activity. ECs were treated with the selective Cox-2 inhibitor celecoxib for 12 hours after 25-OHC exposure. Celecoxib (5 μM) reversed the small increase in cell number of viable cells due to 25-OHC. The number of viable bmVECs treated with celecoxib alone dropped below the number of viable vehicle-treated bmVECs. Proliferative effects of 25-OHC also applied to human colorectal carcinoma cells HCT-116. HCT-116 has previously shown to be Cox-2 deficient. For each cell type, mean of fold differences between treatments from three trials analysed by ANOVA generated p-values ≤ 0.001 (n = 3). For each treatment with celecoxib, 25-OHC, and both together, the mean fold differences between each cell type analysed by ANOVA generated p-values of 0.084, 0.011, and 0.00038, respectively (n = 3).

Mentions: In addition to the changed morphology, we observed that plates of cells treated with 25-OHC for 24 hours or less appeared to be more tightly packed with cells than those treated with ethanol vehicle alone. We also observed some ECs loosely attached to and growing outside the monolayer (Figure 1b). Without 25-OHC treatment, all three bmECs formed a monolayer in culture with flat cells touching at all sides without squeezing tightly together. This observation led us to hypothesize that perhaps there was an increase in the number of cells packed in the monolayer. Indeed, we found this to be the case when we counted the cells after 24 hours of growing cells in 25-OHC (Figure 2a). Interestingly, the fold difference in the number of viable cells from seeded cells (taken as 1 in figure 2a) seemed most pronounced in bmLECs compared to bmVECs and bmAECs (Figure 2a).


25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner.

Nguyen VP, Chen SH, Pizzuto K, Cantarutti A, Terminesi A, Mendonca C, Dumont DJ - J Angiogenes Res (2010)

25-OHC promotes endothelial cell proliferation. A/ Short exposure to 25-OHC promoted EC proliferation. Viable cells that excluded trypan blue were counted after 24 hours of growing cells in 25-OHC. Fold difference in the number of viable cells from seeded cells (taken as 1) was most pronounced in bmLECs compared to bmVECs and bmAECs. Mean of fold differences between treated and untreated bmAEC, bmVEC, and bmLEC from three trials analysed by t-test yielded p-values of 0.021, 0.016, and 0.0036 respectively (n = 3). B/ Temporary boost in EC proliferation due to 25-OHC exposure for 12 hours was dependent on Cox-2 activity. ECs were treated with the selective Cox-2 inhibitor celecoxib for 12 hours after 25-OHC exposure. Celecoxib (5 μM) reversed the small increase in cell number of viable cells due to 25-OHC. The number of viable bmVECs treated with celecoxib alone dropped below the number of viable vehicle-treated bmVECs. Proliferative effects of 25-OHC also applied to human colorectal carcinoma cells HCT-116. HCT-116 has previously shown to be Cox-2 deficient. For each cell type, mean of fold differences between treatments from three trials analysed by ANOVA generated p-values ≤ 0.001 (n = 3). For each treatment with celecoxib, 25-OHC, and both together, the mean fold differences between each cell type analysed by ANOVA generated p-values of 0.084, 0.011, and 0.00038, respectively (n = 3).
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Figure 2: 25-OHC promotes endothelial cell proliferation. A/ Short exposure to 25-OHC promoted EC proliferation. Viable cells that excluded trypan blue were counted after 24 hours of growing cells in 25-OHC. Fold difference in the number of viable cells from seeded cells (taken as 1) was most pronounced in bmLECs compared to bmVECs and bmAECs. Mean of fold differences between treated and untreated bmAEC, bmVEC, and bmLEC from three trials analysed by t-test yielded p-values of 0.021, 0.016, and 0.0036 respectively (n = 3). B/ Temporary boost in EC proliferation due to 25-OHC exposure for 12 hours was dependent on Cox-2 activity. ECs were treated with the selective Cox-2 inhibitor celecoxib for 12 hours after 25-OHC exposure. Celecoxib (5 μM) reversed the small increase in cell number of viable cells due to 25-OHC. The number of viable bmVECs treated with celecoxib alone dropped below the number of viable vehicle-treated bmVECs. Proliferative effects of 25-OHC also applied to human colorectal carcinoma cells HCT-116. HCT-116 has previously shown to be Cox-2 deficient. For each cell type, mean of fold differences between treatments from three trials analysed by ANOVA generated p-values ≤ 0.001 (n = 3). For each treatment with celecoxib, 25-OHC, and both together, the mean fold differences between each cell type analysed by ANOVA generated p-values of 0.084, 0.011, and 0.00038, respectively (n = 3).
Mentions: In addition to the changed morphology, we observed that plates of cells treated with 25-OHC for 24 hours or less appeared to be more tightly packed with cells than those treated with ethanol vehicle alone. We also observed some ECs loosely attached to and growing outside the monolayer (Figure 1b). Without 25-OHC treatment, all three bmECs formed a monolayer in culture with flat cells touching at all sides without squeezing tightly together. This observation led us to hypothesize that perhaps there was an increase in the number of cells packed in the monolayer. Indeed, we found this to be the case when we counted the cells after 24 hours of growing cells in 25-OHC (Figure 2a). Interestingly, the fold difference in the number of viable cells from seeded cells (taken as 1 in figure 2a) seemed most pronounced in bmLECs compared to bmVECs and bmAECs (Figure 2a).

Bottom Line: These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient.The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular and Cellular Biology Research, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 3M5, Canada. dan.dumont@sri.utoronto.ca.

ABSTRACT

Background: 25-hydroxycholesterol (25-OHC) is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis.

Methods: Primary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC) were treated with 25-OHC and tested for several different cellular parameters.

Results: We found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2) expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex). Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex). These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.

Conclusions: Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

No MeSH data available.


Related in: MedlinePlus