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M. Neu, A. Afzal, J. L. Kielczewski, S. Hazra, L. Kennedy, C. Lansing, M. B. Grant; Adenosine Modulates Endothelial Precursor Function: Implication for Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4809.
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The regulatory functions of adenosine are mediated by four G-protein coupled receptors, A1, A2A, A2B and A3. Among adenosine receptors the A2B has the lowest affinity and is believed to remain silent under normal physiological conditions when adenosine levels are low but becomes active when adenosine levels rise to micromolar concentrations, as is seen in ischemic tissue. We examined the expression of adenosine receptors on endothelial precursor cells (EPC, CD34+ cells). These cells migrate to regions of hypoxia and participate in physiological revascularization to eliminate tissue ischemia; however, under certain conditions they can contribute to pathological angiogenesis. Using EPC from individuals with severe non proliferative diabetic retinopathy (NPDR, n = 6) and controls (n= 6), we examined the effect of adenosine agonists on key steps of angiogenesis: migration, nitric oxide (NO) generation and VEGF expression.
CD34+ cells were isolated from blood of diabetics and age-matched non-diabetic controls. Adenosine receptors levels were determined by RT-PCR. Cell migration was assessed using the modified Boyden chamber assay using the adenosine analogue 5’-N-ethylcarboxamido- adenosine (NECA) as the stimulant. Following stimulation with subtype specific agonist, NO generation was quantified by labeling the cells with DAF-FM and measuring the resultant fluorescence and VEGF was quantified by RT-PCR and ELISA
Diabetic CD34+ cells expressed higher levels of the pro-angiogenic A2B receptor and reduced levels of the anti-inflammatory A1 receptor compared to non-diabetic cells. Both non-diabetic and diabetic CD34+ cells demonstrate a dose-dependent (0.01µM- 100 µM) increase in migration to the non-selective adenosine agonist, NECA (p<0.05 for all concentrations compared to control). Both non-diabetic and diabetic EPCs generated NO in response to NECA. Selective A2B activation resulted in stimulation of VEGF secretion which was markedly increase in EPCs of diabetic compared to non-diabetic origin (p<0.01).
Our study suggests that when diabetic EPCs are exposed to ischemia, as may occur in advanced stages of diabetic retinopathy, increased retinal levels of adenosine and increased A2B receptor expression may result in an increase of local VEGF levels. Both autocrine and paracrine VEGF expression by these cells could contribute to the "angiogenic switch" leading to the initiation of PDR.
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