Background: High-density lipoproteins (HDLs) can exert anti-atherogenic effects. On top of removing excess cholesterol through reverse cholesterol transport, HDLs play beneficial actions on endothelial function and integrity. In particular, HDLs are strong determinant of endothelial progenitor cells (EPCs) number and function. To gain further insights into such an effect we characterized in vitro functionality of circulating "early" EPCs obtained from 60 type 2 diabetes individuals with low HDL-cholesterol (HDL-C) and 59 with high HDL-C levels. Methods: After an overnight fast, venous blood was drawn in EDTA tubes and processed within 2-h from sampling. Peripheral blood mononuclear cells were isolated and plated on fibronectin coated culture dishes; after 3 days culture, adherent cells positive for Dil-ac-LDL/Lectin dual fluorescent staining were identified as monocytic angiogenic cells (MACs). After 5-7 days culture in EBM-2 medium, adherent cells were evaluated for viability/proliferation (MTT assay), senescence (beta-galactosidase activity detection), migration (modified Boyden chamber using VEGF as chemoattractant), adhesion capacity (on fibronectin-coated culture dishes) and ROS production (ROS-sensitive fluorescent probe CM-H2DCFDA). Results: MACs obtained from diabetic individuals with high HDL-C had 23% higher viability compared to low HDL-C (111.6 ± 32.7% vs. 90.5 ± 28.6% optical density; p = 0.002). H2O2exposure impaired MACs viability to a similar extent in both groups (109.2 ± 31.7% vs. 74.5 ± 40.8% in high HDL-C, p < 0.0001; 88.3 ± 25.5% vs. 72.3 ± 22.5% in low-HDL, p = 0.004). MACs senescence was comparable in the two groups (102.7 ± 29.8% vs. 99.2 ± 27.8%; p = 0.703) and was only slightly modified by exposure to H2O2. There was no difference in the MACs migration capacity between the two groups (91.3 ± 34.2% vs. 108.7 ± 39.5%; p = 0.111), as well as in MACs adhesion capacity (105.2 ± 32.7% vs. 94.1 ± 26.1%; p = 0.223). Finally, ROS production was slightly thought not significantly higher in MACs from type 2 diabetes individuals with low- than high-HDL. After stratification of HDL-C levels into quartiles, viability (p < 0.0001) and adhesion (p = 0.044) were higher in Q4 than in Q1-Q3. In logistic regression analysis, HDL-C was correlated to MACs viability and adhesion independently of HbA1c or BMI, respectively. Conclusions: Our data suggest that in type 2 diabetes subjects, HDL-cholesterol is an independent determinant of circulating MACs functional capacities-mainly viability, to a lesser extent adhesion-likely contributing also through this mechanism to cardiovascular protection even in type 2 diabetes.

Influence of high density lipoprotein cholesterol levels on circulating monocytic angiogenic cells functions in individuals with type 2 diabetes mellitus

Lucchesi, Daniela;Sancho, Veronica;Giusti, Laura;Garofolo, Monia;Daniele, Giuseppe;PUCCI, LAURA;Miccoli, Roberto;Penno, Giuseppe;Del Prato, Stefano
2018-01-01

Abstract

Background: High-density lipoproteins (HDLs) can exert anti-atherogenic effects. On top of removing excess cholesterol through reverse cholesterol transport, HDLs play beneficial actions on endothelial function and integrity. In particular, HDLs are strong determinant of endothelial progenitor cells (EPCs) number and function. To gain further insights into such an effect we characterized in vitro functionality of circulating "early" EPCs obtained from 60 type 2 diabetes individuals with low HDL-cholesterol (HDL-C) and 59 with high HDL-C levels. Methods: After an overnight fast, venous blood was drawn in EDTA tubes and processed within 2-h from sampling. Peripheral blood mononuclear cells were isolated and plated on fibronectin coated culture dishes; after 3 days culture, adherent cells positive for Dil-ac-LDL/Lectin dual fluorescent staining were identified as monocytic angiogenic cells (MACs). After 5-7 days culture in EBM-2 medium, adherent cells were evaluated for viability/proliferation (MTT assay), senescence (beta-galactosidase activity detection), migration (modified Boyden chamber using VEGF as chemoattractant), adhesion capacity (on fibronectin-coated culture dishes) and ROS production (ROS-sensitive fluorescent probe CM-H2DCFDA). Results: MACs obtained from diabetic individuals with high HDL-C had 23% higher viability compared to low HDL-C (111.6 ± 32.7% vs. 90.5 ± 28.6% optical density; p = 0.002). H2O2exposure impaired MACs viability to a similar extent in both groups (109.2 ± 31.7% vs. 74.5 ± 40.8% in high HDL-C, p < 0.0001; 88.3 ± 25.5% vs. 72.3 ± 22.5% in low-HDL, p = 0.004). MACs senescence was comparable in the two groups (102.7 ± 29.8% vs. 99.2 ± 27.8%; p = 0.703) and was only slightly modified by exposure to H2O2. There was no difference in the MACs migration capacity between the two groups (91.3 ± 34.2% vs. 108.7 ± 39.5%; p = 0.111), as well as in MACs adhesion capacity (105.2 ± 32.7% vs. 94.1 ± 26.1%; p = 0.223). Finally, ROS production was slightly thought not significantly higher in MACs from type 2 diabetes individuals with low- than high-HDL. After stratification of HDL-C levels into quartiles, viability (p < 0.0001) and adhesion (p = 0.044) were higher in Q4 than in Q1-Q3. In logistic regression analysis, HDL-C was correlated to MACs viability and adhesion independently of HbA1c or BMI, respectively. Conclusions: Our data suggest that in type 2 diabetes subjects, HDL-cholesterol is an independent determinant of circulating MACs functional capacities-mainly viability, to a lesser extent adhesion-likely contributing also through this mechanism to cardiovascular protection even in type 2 diabetes.
Lucchesi, Daniela; Popa, Simona Georgiana; Sancho, Veronica; Giusti, Laura; Garofolo, Monia; Daniele, Giuseppe; Pucci, Laura; Miccoli, Roberto; Penno, Giuseppe; Del Prato, Stefano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/924556
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