Introduction
Cardiovascular diseases are the leading cause of mortality globally. Commonly known risk factors include high blood pressure, smoking, high cholesterol, diabetes, obesity as well as family history of cardiovascular disease. However, Intrauterine growth restriction (IUGR), affecting 7-10% of pregnancies, also seems to be a risk factor. In fact, IUGR newborns are more prone to develop higher blood pressure during their infancy, adolescence, young adulthood and later in life. This is David Barker’s theory “Developmental Origins of Health and Diseases” (DOHaD), investigating the effect of developmental programming induced by early life environment on adulthood diseases. Along with decreased nephron numbers and hypothalamic- pituitary-adrenal axis hyperactivity, alterations of the vascular system, specifically the endothelium, play an important role in the development of hypertension in infants with IUGR. The endothelium acts as a protector against thrombosis and inflammation, enhances angiogenesis and ensures adequate vascular tone and tissue perfusion. Endothelial progenitor cells (EPCs), circulating components of endothelium, play a key role in endothelial repair thanks to its proliferation and migration proprieties.
We have previously observed in a rat model of IUGR induced by low protein maternal nutrition during gestation (vs. a control (CTRL) group with normal diet), that at 6 months of age ECFCs- IUGR have a decreased number and altered functions, characterized by reduced proliferation, impaired vascular network formation and angiogenic capabilities related to oxidative stress and stress-induced premature senescence. Resveratrol, a polyphenol compound with antioxidant properties, was found to improve cardiovascular functions. However, whether resveratrol could reverse the ECFCs dysfunction observed at adulthood following
IUGR is still unknown.
Aim of the study
The aim of this study is to investigate whether the in vitro treatment of ECFCs with Resveratrol, can reverse ECFC dysfunctions observed in IUGR rat model.
Material and methods
We used two different Resveratrol treatments: 1μM and 10 μM Resveratrol for 24h.
In a first experimental trial, IUGR-ECFCs and IUGR-CTRL have been treated by 1μM Resveratrol for 48h. The proliferation was performed using BrdU incorporation, the capillary- like structure formation was evaluated on Matrigel, the superoxide anion production was measured using the oxidative fluorescent dye hydroethidine and the beta-galactosidase activity was measured with a senescence detection kit. NO production in ECFCs treated with or without Resveratrol was evaluated using DAF-2DA, as well as the expressions of angiopoietin, angiomotin, vascular endothelial growth factor, vascular endothelial growth factor receptor 2 and thrombospondin measured by immunofluorescence. Additionally, the expressions of sirtuin-1 (anti-aging protein) and eNOS (enzyme involved in NO production in the endothelium) have been quantified by immunofluorescence and western blotting.
In a second trial, to explore antioxidant defenses, IUGR-ECFCs and IUGR-CTRL have been treated by 10μM Resveratrol for 48h. The expression of Cu/Zn SOD treated with (or without) Resveratrol 10μM was measured by western blotting..
Results
The treatment with resveratrol has significantly reversed IUGR-ECFC dysfunctions. In fact, when treated with resveratrol 1μM for 48 hours, proliferation and vascular network formation were enhanced. Additionally, superoxide anion production and beta-galactosidase activity were respectively decreased by 60% and 27%. This treatment increased eNOS production by 71%. A 54% increasement of Sirtuin-1 expression level was measured by immunofluorescence and an increasement of 80% by western blot. When treated with resveratrol 10μM for 48 hours, Sirtuin-1 production measured by western blot was increased by 30% and Cu/Zn SOD by 85%. The treatment with 1μM Resveratrol for 6 hours increased NO production by 56% and for 24 hours by 98%. and has not significantly improved angiopoietin, angiomotin, vascular endothelial growth factor, vascular endothelial growth factor receptor 2 and thrombospondin expression. Resveratrol treatment has no effect on CTRL-ECFCs.
Conclusion
The treatment resveratrol has improved proliferation and vascular network formation capacity in IUGR-ECFCs. It has reduced oxidative stress by decreasing superoxide anion production and increasing Cu/Zn superoxide dismutase protein levels. Resveratrol also enhanced angiogenesis by increasing eNOS protein levels as well as NO production. Moreover, Resveratrol reversed stress- induced premature senescence of IUGR-ECFCs by improving sirtuin-1 expression and reducing β- galactosidase activity.