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Anti-proliferative activity of oral anti-hyperglycemic agents on human vascular smooth muscle cells: thiazolidinediones (glitazones) have enhanced activity under high glucose conditions.

Little PJ, Osman N, de Dios ST, Cemerlang N, Ballinger M, Nigro J - Cardiovasc Diabetol (2007)

Bottom Line: People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions.Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively).These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Monash University, Faculty of Medicine, Nursing and Health Sciences, Alfred Hospital, Melbourne, 3004, VIC, Australia. peter.little@baker.edu.au

ABSTRACT

Background: Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [3H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [3H]-thymidine incorporation.

Methods: VSMC proliferation was investigated by [3H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [3H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results: VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [3H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [3H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [3H]-thymidine incorporation or cell numbers.

Conclusion: TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.

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Effect of media glucose concentration on the inhibitory activity of TZDs. Panels A and B show the effect of rosiglitazone under low (5 mM) glucose (A) and high glucose (25 mM) and the data shown as a dose response curve (C). Data for pioglitazone is shown in panels D, E and F. Data represent the mean ± SEM from 3 experiments in triplicate *P < 0.05, ***P < 0.001
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Figure 1: Effect of media glucose concentration on the inhibitory activity of TZDs. Panels A and B show the effect of rosiglitazone under low (5 mM) glucose (A) and high glucose (25 mM) and the data shown as a dose response curve (C). Data for pioglitazone is shown in panels D, E and F. Data represent the mean ± SEM from 3 experiments in triplicate *P < 0.05, ***P < 0.001

Mentions: People with diabetes have blood glucose concentrations that may range at times from 2 to 30 mM and thus the effect of glucose concentration on the actions of anti-hyperglycaemic agents is clinically relevant. We identified a human vSMC preparation that showed enhanced proliferation in response to high glucose media. Under "low" glucose conditions which actually equate to normoglycemia (i.e. DMEM with 5 mM glucose) and in the presence of 5 per cent FBS, cell numbers over 3 days increased from 36,800 ± 3,800 to 91,900 ± 8500 cells/well (mean ± sem, n = 5 experiments in triplicate). Under high glucose conditions (DMEM containing 25 mM glucose) and in the presence of 5 per cent FBS, vSMC numbers increased from 54,400 ± 5500 to 250,500 ± 28,000 cells/well (mean ± sem, n = 5 experiments in triplicate). We used this cell model to assess the inhibitory activity of the two clinical TZDs, rosiglitazone and pioglitazone. Rosiglitazone was evaluated at 3–100 μM and pioglitazone 0.3–30 μM noting that pioglitazone precipitates in the culture media at concentration above 30 μM. The inhibitory effects of rosiglitazone in low and high glucose media were normalised to the control which was set as 100% (Fig. 1A and 1B, respectively). The data is presented as a dose response curve with statistical comparison of the curves (Fig. 1C). The data shows that the inhibitory effect of rosiglitazone is enhanced (P < 0.05) under high glucose condition (Fig. 1C). Similar data for the effect of pioglitazone is shown in (Figs 1D, E and 1F). Pioglitazone showed a greater inhibitory potency (P < 0.001) in high compared to low glucose DMEM.


Anti-proliferative activity of oral anti-hyperglycemic agents on human vascular smooth muscle cells: thiazolidinediones (glitazones) have enhanced activity under high glucose conditions.

Little PJ, Osman N, de Dios ST, Cemerlang N, Ballinger M, Nigro J - Cardiovasc Diabetol (2007)

Effect of media glucose concentration on the inhibitory activity of TZDs. Panels A and B show the effect of rosiglitazone under low (5 mM) glucose (A) and high glucose (25 mM) and the data shown as a dose response curve (C). Data for pioglitazone is shown in panels D, E and F. Data represent the mean ± SEM from 3 experiments in triplicate *P < 0.05, ***P < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211460&req=5

Figure 1: Effect of media glucose concentration on the inhibitory activity of TZDs. Panels A and B show the effect of rosiglitazone under low (5 mM) glucose (A) and high glucose (25 mM) and the data shown as a dose response curve (C). Data for pioglitazone is shown in panels D, E and F. Data represent the mean ± SEM from 3 experiments in triplicate *P < 0.05, ***P < 0.001
Mentions: People with diabetes have blood glucose concentrations that may range at times from 2 to 30 mM and thus the effect of glucose concentration on the actions of anti-hyperglycaemic agents is clinically relevant. We identified a human vSMC preparation that showed enhanced proliferation in response to high glucose media. Under "low" glucose conditions which actually equate to normoglycemia (i.e. DMEM with 5 mM glucose) and in the presence of 5 per cent FBS, cell numbers over 3 days increased from 36,800 ± 3,800 to 91,900 ± 8500 cells/well (mean ± sem, n = 5 experiments in triplicate). Under high glucose conditions (DMEM containing 25 mM glucose) and in the presence of 5 per cent FBS, vSMC numbers increased from 54,400 ± 5500 to 250,500 ± 28,000 cells/well (mean ± sem, n = 5 experiments in triplicate). We used this cell model to assess the inhibitory activity of the two clinical TZDs, rosiglitazone and pioglitazone. Rosiglitazone was evaluated at 3–100 μM and pioglitazone 0.3–30 μM noting that pioglitazone precipitates in the culture media at concentration above 30 μM. The inhibitory effects of rosiglitazone in low and high glucose media were normalised to the control which was set as 100% (Fig. 1A and 1B, respectively). The data is presented as a dose response curve with statistical comparison of the curves (Fig. 1C). The data shows that the inhibitory effect of rosiglitazone is enhanced (P < 0.05) under high glucose condition (Fig. 1C). Similar data for the effect of pioglitazone is shown in (Figs 1D, E and 1F). Pioglitazone showed a greater inhibitory potency (P < 0.001) in high compared to low glucose DMEM.

Bottom Line: People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions.Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively).These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Monash University, Faculty of Medicine, Nursing and Health Sciences, Alfred Hospital, Melbourne, 3004, VIC, Australia. peter.little@baker.edu.au

ABSTRACT

Background: Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [3H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [3H]-thymidine incorporation.

Methods: VSMC proliferation was investigated by [3H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [3H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results: VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [3H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [3H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [3H]-thymidine incorporation or cell numbers.

Conclusion: TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.

Show MeSH
Related in: MedlinePlus