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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: 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).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.

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 TZDs on the induction of thymidine kinase assessed by acute [3H]-thymidine uptake and parallel estimation of [3H]-thymidine incorporation into DNA. A. Confluent serum deprived vSMCs were treated with PDGF with and without troglitazone for 20 h then cells were washed free and growth factor and drugs and the acute uptake of [3H]-thymidine over 4 min was assessed. The results show the effects of two identical experiments in duplicate. B. Shows concomitant data for routine assay of [3H]-thymidine into DNA (see methods for details).
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Figure 3: Effect of TZDs on the induction of thymidine kinase assessed by acute [3H]-thymidine uptake and parallel estimation of [3H]-thymidine incorporation into DNA. A. Confluent serum deprived vSMCs were treated with PDGF with and without troglitazone for 20 h then cells were washed free and growth factor and drugs and the acute uptake of [3H]-thymidine over 4 min was assessed. The results show the effects of two identical experiments in duplicate. B. Shows concomitant data for routine assay of [3H]-thymidine into DNA (see methods for details).

Mentions: Activation of [3H]-thymidine incorporation into DNA of proliferating cells arises from growth factor mediated activation of thymidine kinase activity and enhanced [3H]-thymidine uptake into the salvage pathway of thymidylate synthesis which provides adequate precursors for DNA synthesis in vSMC [14]. We hypothesized that troglitazone may have a growth factor like effect (gene induction) on thymidine kinase leading to enhanced uptake and consequently increased incorporation of [3H]-thymidine into DNA. Thymidine kinase activity can be assessed as the uptake by the cell of [3H]-thymidine over a period of several minutes [14]. We treated vSMCs with low (0.3 μM) and high (10 μM) concentrations of troglitazone which stimulate and inhibit, respectively, [3H]-thymidine incorporation into DNA precipitable material (Fig. 2). We undertook the acute (3 min) uptake experiment concomitant with an examination of the effect of troglitazone on incorporation of [3H]-thymidine into DNA in a parallel experiment as described above. Troglitazone caused concentration dependent inhibition of the acute uptake of [3H]-thymidine (Fig. 3A) with no evidence of stimulation. The parallel experiment for assessing [3H]-thymidine incorporation into DNA produced the expected result of enhanced [3H]-thymidine incorporation at low concentration of troglitazone (0 – 3 μM) and inhibition of incorporation at high troglitazone concentrations (10 nM) (Fig. 3B).


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 TZDs on the induction of thymidine kinase assessed by acute [3H]-thymidine uptake and parallel estimation of [3H]-thymidine incorporation into DNA. A. Confluent serum deprived vSMCs were treated with PDGF with and without troglitazone for 20 h then cells were washed free and growth factor and drugs and the acute uptake of [3H]-thymidine over 4 min was assessed. The results show the effects of two identical experiments in duplicate. B. Shows concomitant data for routine assay of [3H]-thymidine into DNA (see methods for details).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of TZDs on the induction of thymidine kinase assessed by acute [3H]-thymidine uptake and parallel estimation of [3H]-thymidine incorporation into DNA. A. Confluent serum deprived vSMCs were treated with PDGF with and without troglitazone for 20 h then cells were washed free and growth factor and drugs and the acute uptake of [3H]-thymidine over 4 min was assessed. The results show the effects of two identical experiments in duplicate. B. Shows concomitant data for routine assay of [3H]-thymidine into DNA (see methods for details).
Mentions: Activation of [3H]-thymidine incorporation into DNA of proliferating cells arises from growth factor mediated activation of thymidine kinase activity and enhanced [3H]-thymidine uptake into the salvage pathway of thymidylate synthesis which provides adequate precursors for DNA synthesis in vSMC [14]. We hypothesized that troglitazone may have a growth factor like effect (gene induction) on thymidine kinase leading to enhanced uptake and consequently increased incorporation of [3H]-thymidine into DNA. Thymidine kinase activity can be assessed as the uptake by the cell of [3H]-thymidine over a period of several minutes [14]. We treated vSMCs with low (0.3 μM) and high (10 μM) concentrations of troglitazone which stimulate and inhibit, respectively, [3H]-thymidine incorporation into DNA precipitable material (Fig. 2). We undertook the acute (3 min) uptake experiment concomitant with an examination of the effect of troglitazone on incorporation of [3H]-thymidine into DNA in a parallel experiment as described above. Troglitazone caused concentration dependent inhibition of the acute uptake of [3H]-thymidine (Fig. 3A) with no evidence of stimulation. The parallel experiment for assessing [3H]-thymidine incorporation into DNA produced the expected result of enhanced [3H]-thymidine incorporation at low concentration of troglitazone (0 – 3 μM) and inhibition of incorporation at high troglitazone concentrations (10 nM) (Fig. 3B).

Bottom Line: 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).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.

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