Coenzyme Q₁₀ Protects Human Endothelial Cells from β-Amyloid Uptake and Oxidative Stress-Induced Injury

<div><p>Neuropathological symptoms of Alzheimer's disease appear in advances stages, once neuronal damage arises. Nevertheless, recent studies demonstrate that in early asymptomatic stages, ß-amyloid peptide damages the cerebral microvasculature through mechanisms that involve an increase in reactive oxygen species and calcium, which induces necrosis and apoptosis of endothelial cells, leading to cerebrovascular dysfunction. The goal of our work is to study the potential preventive effect of the lipophilic antioxidant coenzyme Q (CoQ) against ß-amyloid-induced damage on human endothelial cells. We analyzed the protective effect of CoQ against Aβ-induced injury in human umbilical vein endothelial cells (HUVECs) using fluorescence and confocal microscopy, biochemical techniques and RMN-based metabolomics. Our results show that CoQ pretreatment of HUVECs delayed Aβ incorporation into the plasma membrane and mitochondria. Moreover, CoQ reduced the influx of extracellular Ca²⁺, and Ca²⁺ release from mitochondria due to opening the mitochondrial transition pore after β-amyloid administration, in addition to decreasing O₂^.− and H₂O₂ levels. Pretreatment with CoQ also prevented ß-amyloid-induced HUVECs necrosis and apoptosis, restored their ability to proliferate, migrate and form tube-like structures <i>in vitro</i>, which is mirrored by a restoration of the cell metabolic profile to control levels. CoQ protected endothelial cells from Aβ-induced injury at physiological concentrations in human plasma after oral CoQ supplementation and thus could be a promising molecule to protect endothelial cells against amyloid angiopathy.</p></div

Primers used for quantitative RT-PCR analyses.

<p>Primers used for quantitative RT-PCR analyses.</p

CoQ impedes β-amyloid-induced mPTP opening.

<p>HUVECs were incubated for 12 h with vehicle or 5 µM CoQ and treated for additional 3–24 h with 5 µM Aβ_25–35. The effect of the treatments on mPTP opening was quantified indirectly by measuring MitoTracker Deep Red fluorescence, mitochondrial Ca²⁺ and cytochrome <i>c</i> release. After treatment with 5 µM Aβ_25–35, mitochondria were loaded with MitoTracker Deep Red <b>A</b>), Calcein-AM and CoCl₂<b>B</b>) and MitoTracker Deep Red plus Fluo-4 <b>C</b>). Fluorescence intensity for each probe was determined by fluorescence microscope in living cells (n = 3/4). Mitochondrial Ca²⁺ levels were calculated by quenching the cytosolic Calcein-AM signal with CoCl₂<b>B</b>) and by colocalization of Fluo-4 and MitoTracker and furfher image processing with ImageJ <b>C</b>). Cells were loaded with Mitotracker Deep Red and immunostained with an anti-cytochrome <i>c</i><b>D</b>). The amount of cytochrome <i>c</i> in cytosol was calculated by colocalization and image processing with ImageJ (n = 3). Results show the percentage of relative fluorescence units (RFUs) <i>vs.</i> control cells or the ratio between cytosolic/mitochondrial Fluo-4-AM or cytochrome <i>c</i> RFUs level. <i>a</i>, <i>p</i><0.05 <i>vs.</i> control; <i>b</i>, p<0.05 <i>vs.</i> Aβ_25–35.</p

The activation of Wnt/β-catenin during hepatocyte differentiation is associated with the presence of related proteins to tumoral phenotype.

<p>Relative abundance of specific proteins (DIGE analysis) in human mesenchymal stem cells undifferentiated after 21 days of culture (UC21d) and in mesenchymal stem cells differentiated into hepatocytes with conditioned medium 1 (CM1) or 2 (CM2). B) Western blot confirmation of the changes observed by DIGE analysis in the abundance of some proteins in CM1 and CM2 hepatocytes: Adenine phosphoriobosyl transferase (APT), cathepsin B precursor (CATB), L-lactate dehydrogenase β chain (LDHB), transgelin (TGL2), tropomyosin β chain (TPM2) and nuclear β-catenin. Tubulin and TFIIB were used as cytoplasm and nuclear loading control respectively.</p

CoQ delays and decreases β-amyloid incorporation into endothelial cells.

<p>HUVECs were incubated for 12 h with vehicle or 5 µM CoQ. Changes in HiLyte Fluor-labeled Aβ_25–35 were monitored by time-lapse microscopy every 30 sec in cells pretreated with PBS or 5 µM CoQ. Fluorescent peptide (5 µM) was added in min 1. Pictures show the variation of fluorescence at different time points, from 0 to 45 min. Graph shows fluorescence dynamics, expressed as normalized relative fluorescence units (RFUs), (n = 3).</p

Effects of tempol and α-tocopherol on β-amyloid-induced apoptosis, necrosis, free cytosolic Ca²⁺ and O₂^.−.

<p>HUVECs were incubated for 12 h with vehicle, 1 mM tempol or 100 µM α-tocopherol, and then treated for additional 3–24 h with 5 µM Aβ_25–35. <b>A</b>) Apoptosis was determined by DAPI staining and morphological analysis of nuclei. Results are expressed as the percentage of apoptotic <i>vs</i>. total nuclei (300 cells/treatment, n = 3). <b>B</b>) Necrosis was determined by staining with ethidium bromide and evaluated by qualitative fluorescence microscopy. Results are expressed as percentage of necrotic <i>vs.</i> total cells (n = 3). a, <i>p</i><0.05 vs. control; b, p<0.05 <i>vs</i>. Aβ_25–35. HUVECs were incubated for 12 h with vehicle, 1 mM tempol or 100 µM α-tocopherol and treated for additional 3 h with 5 µM Aβ_25–35. <b>C</b>) Ca²⁺ levels were determined by fluorescence microscopy with the probe Fluo-4-AM. <b>D</b>) O₂^.− levels were determined by fluorescence microscopy using the probe MitoSOX-AM. Results show the percentage of variation of fluorescence <i>vs</i>. control cells (n = 3). a, <i>p</i><0.05 <i>vs.</i> control; <i>b</i>, p<0.05 <i>vs.</i> Aβ_25–35.</p

The treatments with CM1 or CM2 increase the expression of hepatospecific genes in human mesenchymal stem cells.

<p>Relative levels of mRNA expression of A) albumin (ALB), B) α-fetoprotein (αFP), C) α1-antitrypsin (α-1-AT), D) CCAAT/enhancer-binding protein beta (C/EBP) and E) cytochrome P450 (CYP3A5) were determined in human undifferentiated mesenchymal stem cells before and after differentiation with conditioned medium 1 (CM1) or 2 (CM2) after 7, 14 and 21 days of culture; Gene expression is shown as fold-changes compared to undifferentiated cells at each time. Values are expressed as mean ± standard deviation. All genes were increased significantly respect to undifferentiated cells (UC). ^a p<0.001, ^b p<0.01 vs. CM1 or CM2.</p

CoQ lessens β-amyloid incorporation into endothelial cells mitochondria.

<p>HUVECs were incubated for 12 h with vehicle or 5 µM CoQ, loaded with Mitotracker Deep Red, and then treated with 5 µM HiLyte Fluor-labeled Aβ_25–35 for 0, 10, 20 and 40 min. Pictures were immediately acquired by confocal microscopy in living cells and show the staining with Mitotracker Deep Red, HiLyte Fluor-labeled Aβ_25–35 (green) and merges, in control (left) and CoQ incubated cells (right) at 40 min from Aβ_25–35 addition. Colocalization and image processing were performed with ImageJ. Graphs show HiLyte Fluor-labeled Aβ_25–35 relative fluorescence units (RFUs) <i>vs.</i> time in the whole cell (total; upper graph), cyotosol (middle graph) and mitochondria (bottom graph) in control (black line) and CoQ incubated cells (grey line). n = 3; a, p<0.005 <i>vs.</i> Aβ_25–35.</p

Differential stem cells markers in undifferentiated and differentiated human mesenchymal stem cells.

<p>Levels of CD13, CD49e, CD166, CD133 and VEGFR2 in undifferentiated cells (UC), CM1 and CM2-treated cells after 21 days of culture. (Conditioned medium: CM). Values are expressed as mean of percentage ± standard deviation. (a p<0.001 and b p<0.01 vs. CM1-treated cells; +++ p<0,001 and + p<0,05 vs. undifferentiated cells).</p

CoQ protects endothelial cells from β-amyloid-induced apoptosis and necrosis.

<p>HUVECs were incubated for 12 h with vehicle alone or with increasing CoQ concentrations (1 to 7.5 µM) and then treated for additional 24 h with 5 µM Aβ_25–35. <b>A</b>) Apoptosis was determined by DAPI staining and morphological analysis of nuclei. White arrows indicate typical apoptotic nuclei. Results are expressed as the percentage of apoptotic <i>vs</i>. total nuclei (300 cells/treatment, n = 3). <b>B</b>) Apoptosis was also evaluated by flow cytometry. Results are expressed as percentage of cells positive for Annexin V <i>vs.</i> total (n = 3). Viability and necrosis (<b>C, left and right, respectively</b>) were determined by cell co-staining with calcein-AM (green) and ethidium bromide (orange) and evaluated by qualitative fluorescence microscopy. Results are expressed as percentage of viable/necrotic cells <i>vs.</i> total (n = 3). a, <i>p</i><0.05 vs. control; b, p<0.05 <i>vs</i>. Aβ_25–35.</p