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Inhibition of adenosine kinase induces expression of VEGF mRNA and protein in myocardial myoblasts
1Angiogenesis Research Laboratories, Department of Physiology and Biophysics, Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Abstract:
We tested whether increased endogenous adenosine produced by the adenosine kinase inhibitor GP-515 (Metabasis Therapeutics) can induce vascular endothelial growth factor (VEGF) expression in cultured rat myocardial myoblasts (RMMs). RMMs were cultured for 18 h in the absence (control) and presence of GP-515, adenosine (Ado), adenosine deaminase (ADA), or GP-515 + ADA. GP-515 (0.2-200 microM) caused a dose-related increase in VEGF protein expression (1.99-2.84 ng/mg total cell protein); control VEGF was 1.84 +/- 0.05 ng/mg. GP-515 at 2 and 20 microM also increased VEGF mRNA by 1.67- and 1. 82-fold, respectively. ADA (10 U/ml) decreased baseline VEGF protein levels by 60% and completely blocked GP-515 induction of VEGF. Ado (20 microM) and GP-515 (20 microM) caused a 59 and 39% increase in VEGF protein expression and a 98 and 33% increase in human umbilical vein endothelial cell proliferation, respectively, after 24 h of exposure. GP-515 (20 microM) had no effect on VEGF protein expression during severe hypoxia (1% O(2)) but increased VEGF by an additional 27% during mild hypoxia (10% O(2)). These results indicate that raising endogenous levels of Ado through inhibition of adenosine kinase can increase the expression of VEGF and stimulate endothelial cell proliferation during normoxic and hypoxic conditions.
Insights
Inhibiting adenosine kinase with GP-515 boosts vascular endothelial growth factor (VEGF) production in heart cells. This promotes endothelial cell growth, suggesting potential for treating conditions involving blood vessel formation.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Biochemistry
Background:
- Vascular endothelial growth factor (VEGF) is crucial for angiogenesis and tissue repair.
- Adenosine signaling plays a role in regulating cellular functions, including vascularization.
- Adenosine kinase inhibitors are being explored for therapeutic potential.
Purpose of the Study:
- To investigate if inhibiting adenosine kinase increases endogenous adenosine levels and subsequently induces VEGF expression.
- To determine the effect of GP-515 on VEGF protein and mRNA levels in rat myocardial myoblasts (RMMs).
- To assess the impact of GP-515 and adenosine on endothelial cell proliferation under normoxic and hypoxic conditions.
Main Methods:
- Culturing rat myocardial myoblasts (RMMs) and treating them with GP-515, adenosine (Ado), and adenosine deaminase (ADA).
- Quantifying VEGF protein expression using established methods.
- Measuring VEGF mRNA levels via quantitative analysis.
- Assessing human umbilical vein endothelial cell (HUVEC) proliferation.
Main Results:
- GP-515 treatment resulted in a dose-dependent increase in VEGF protein and mRNA expression in RMMs.
- Adenosine deaminase (ADA) inhibited baseline VEGF and blocked GP-515-induced VEGF.
- Both exogenous adenosine and GP-515 increased endothelial cell proliferation, with GP-515 showing effects under mild hypoxia.
Conclusions:
- Inhibition of adenosine kinase by GP-515 effectively increases endogenous adenosine levels.
- Elevated endogenous adenosine stimulates VEGF expression and promotes endothelial cell proliferation.
- This mechanism holds potential for therapeutic strategies targeting angiogenesis in cardiovascular diseases.
