Anthocyans-rich Aronia melanocarpa extract possesses ability to protect endothelial progenitor cells against angiotensin II induced dysfunction.
Arterial hypertension remains a global health problem, significantly affecting cardiovascular related mortality (Lim et al. 2012). This is partially due to the low effectiveness of the treatment for arterial hypertension, which results in an increased risk of developing atherosclerosis and the incidence of acute coronary events and cerebral stroke. They occur as a result of the instability and rupturing of atherosclerotic plaque, to which internal bleeding from microvessels associated with vasa vasorum may further contribute (van den Bouwhuijsen et al. 2012). These microvessels develop from endothelial progenitor cells (EPCs) in the process of angiogenesis (Gulati et al. 2003). It is known that Ang II, detected at high concentrations in atherosclerotic plaques (Hoshida et al. 2001), impairs the functions of EPCs and accelerates their ageing (Endtmann et al. 2011), which may lead to microvascular leaks and the accumulation of cytotoxic hemoglobin in the atherosclerotic plaque. This results in a locally increased production of free radicals created as a result of the Fenton reaction involving the [Fe.sup.2+] ions, which contribute to chronic oxidative stress and atherosclerotic plaque rupture (Winterbourn 1995).
Current epidemiological studies have demonstrated unequivocally that anthocyanins constitute a dietary factor that inhibits the development of arterial hypertension (Cassidy et al. 2011). They are present mainly in berries, but their richest source are aronia berries. Aronia melanocarpa is a shrub of North American origin, also cultivated in Central and Eastern Europe. Aronia fruits are used not only as food, but also as a medicinal plant, due to their very high levels of polyphenols, mainly anthocyanins, but also phenolic acids and flavonoids, which are very strong free radical sweepers (Oszmianski and Wojdyto 2005). Beneficial effects of aronia fruit extracts have been demonstrated both in vitro and in vivo, especially in cardiovascular diseases. Aronia fruit extract significantly inhibited TNF-ainduced expression of the ICAM-1 and VCAM-1 adhesion molecules, NF-xrB activation, and intracellular production of reactive oxygen species (ROS) in human aortic endothelial cells (Zapolska-Downar et al. 2012). It was also demonstrated that anthocyanins extracted from aronia fruits reduced production of ROS and thus prevented apoptosis of human umbilical vein endothelial cells (HUVEC) induced by 7[beta]-hydroxycholesterol (Zapolska-Downar et al. 2008). In addition, aronia fruit extracts exert a potent antiplatelet effect - they prevent platelet activation and at the same time increase the activity of platelet antioxidative enzymes, such as glutathione peroxidase, superoxide dismutase, or catalase, inhibited by hydrogen peroxide (Olas et al. 2010; Luzak et al. 2010).
As it has been demonstrated that angiotensin II induces gp91 phox expression and thus increases oxidative stress and accelerates the process of ageing of EPCs (Imanishi et al. 2005a), our objective in this study was to assess whether the extract from Aronia melanocarpa fruits, containing mainly anthocyanins with strong antioxidative properties, protected EPCs from oxidative stress induced by angiotensin II and from functional impairment. In addition, we investigated the effect of aronia fruit extract on the expression of heme oxygenase-1 (HO-1) and the activation of the Nrf2 transcription factor as a potential mechanism of action of the extract. HO-1 is the main mechanism protecting EPCs from oxidative stress and is necessary for the repairing function of EPCs. It is also known that cyanidin glucoside induces the activity of HO-1 (Sorrenti et al. 2007). Since HO-1 expression is regulated mainly by the NF-E2-related factor 2 (Nrf2), the effect of the extract on the activation of this transcription factor was also assessed.
Materials and methods
Source of Aronia melanocarpa extract
Aronia melanocarpa dried fruit extract (AME) was obtained from Adamed (Czosnow, Poland). Extract was standardized by producer on the polyphenols content (not less than 50%).
Anthocyans content in Aronia melanocarpa fruits extract was determined by colorimetric method according to Giusti and Wrolstad (2001). Absorbance was measured using microplate reader Biotek Synergy4 (BioTek Instruments, Winooski, USA) at 528 nm. The concentration of anthocyans was calculated using cyanidin-3-glucoside standard as reference (Applichem, Darmstadt, Germany).
The analysis of the composition of Aronia melanocarpa fruits extract was performed using HPLC-DAD system (Dionex, Sunnyvale, USA) connected with the hybrid ion trap mass spectrometer (Bruker, Billerica, USA) equipped with an electrospray ionization (ESI) to perform high-resolution tandem mass spectrometry. Chromatographic separations were carried out on a Zorbax SB C18 column (150 x 2.1 mm i.d., 1.9 [micro]m, Agilent Technologies, Palo Alto, USA). The mobile phase was composed of 0.5% aqueous formic acid (A) and 0.1% formic acid in acetonitril (B), and delivered at a total flow rate of 0.2 ml/min following a gradient program 30 min, 5-30% B.
Isolation of endothelial progenitor cells and cell culture
Isolation of EPCs was performed as previously described (Parzonko et al. 2013). Buffy-coat preparations derived from healthy, non-smoking blood donors (<35 years old) were obtained from the Warsaw Blood Donation Centre (Warsaw, Poland). Peripheral blood mononuclear cells were isolated by density gradient centrifugation with LSM 1077 (PAA Laboratories, Pasching, Austria) and plated on fibronectin-coated 24-well plates, 96-well plates or 6 cm dishes (BD Biosciences, Bedford, USA). Cells were cultured in endothelial growth medium-2 (EGM-2) with 2% of fetal bovine serum (Lonza, Venders, Belgium).
Characterization of EPCs
The EPCs were identified on the basis of cell surface marker expression and direct fluorescent staining, as previously described (Parzonko et al. 2013). Quantitative FACS analysis was performed with PE-conjugated anti-human VEGFR-2 and FITC-conjugated antihuman CD-31 monoclonal antibodies (BD Biosciences) using a FACSCalibur flow cytometer (BD Biosciences) and the results were analyzed using BD CellQuest Pro software. Isolated cells were also doubly stained with acetylated low density lipoprotein labelled with l,l'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (Dil-AcLDL, Invitrogen, Eugene, USA) and FITC-labeled lectin from Ulex europaeus (FITC-UE lectin, Sigma-Aldrich, Steinheim, Germany), and afterwards observed under a fluorescent microscope Nikon Eclipse TS100F.
For each experiment, the cells were cultivated for the indicated period in EGM-2 medium in presence of human angiotensin II (Sigma) at a concentration 1.0 ptM with or without pre-treatment with several concentrations (1.0, 5.0, 10.0 and 25.0 [micro]g/ml) of Aronia melanocarpa fruits extract (AME) for 3 h. The AME for biological tests was dissolved in a PBS buffer at pH 7.4 and then an EGM2 medium was added to obtain the proper concentration of the extract in the sample. The culture medium was changed every 48 h. Senescence-associated [beta]-galactosidase (SA-[beta]-gal) activity was measured using a Senescence [beta]-Galactosidase Staining Kit (Cell Signalling, Beverly, USA). Intracellular ROS production was measured using a Total ROS/Superoxide Detection kit (Enzo Life Sciences, Plymouth Meeting, USA). A quantitative analysis of telomerase activity was then performed using a TeloTAGGG PCR [ELISA.sup.PLUS] kit, proliferative activity assay was performed using a Cell Proliferation ELISA BrdU assay (Roche Diagnostic, Mannheim, Germany). EPC migration was evaluated using QCM[TM] Endothelial Cell Migration Assay; the impact of Aronia melanocarpa extract on cell adhesion was measured using Innocyte[TM] ECM Cell Adhesion Assay; the impact of AME on angiogenesis was detected using Millicell Angiogenesis Activation Assay (Merck Millipore, Darmstadt, Germany). The quantity of heme oxygenase (HO-1) in cell lysates was measured using a Total HO-1 ELISA Kit (R&D Systems, Minneapolis, USA). The activation of Nrf2 nuclear factor was measured using Nrf2 ELISA Kit (Active Motif,). Cells were counted using inverted microscope Nikon TS100F with a fluorescence system. Absorbances were measured using a Biotek Synergy 4 microplate reader.
The results were expressed as a mean [+ or -] SEM of the indicated number of experiments. Statistical significance of differences between means was established by ANOVA with Tukey post hoc test. P values below 0.05 were considered statistically significant. All analyses were performed using Statistica 10.
Composition of Aronia melanocarpa fruit extract
The total concentration of anthocyans in the extract was 40.51%. The main compounds present in the extract were identified by mass spectrometry according to literature data (Slimestad et al. 2005) and results are presented in Table 1. The chemical composition of the extract included cyanidin 3-O-glycosides: mostly cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, as well as small amount of cyanidin-3-O-glucoside and cyanidin-3-O-xyloside (Fig. 1). Moreover, two phenolic acids: chlorogenic and neochlorogenic were present. Small amounts of flavonoids were detected in the extract as well.
Characterization of EPCs
After 7 days in the culture, isolated cells exhibited an endothelial cell-like morphology. More than 95% of the cultured cells were positively stained both with Dil-AcLDL and FITC-UE-lectin under a fluorescence microscope and showed CD31 and VEGFR-2 expression on cell surface, as it was confirmed by FACS. Based on these findings, endothelial progenitor identity of isolated cells was confirmed, according to literature data (Timmermans et al. 2009).
Effects of AME on cell senescence
Cultivation of endothelial progenitor cells with angiotensin II significantly increased the number of [beta]-galactosidase-positive cells (52.7% vs. 16.1% in control), whereas coincubation with Aronia melanocarpa fruit extract caused a significant reduction of that effect. The percentage of [beta]-galactosidase-positive cells treated with AME and angiotensin II was 45.7%, 37.2%, 25.6% and 18.6% at 1.0 [micro]g/ml, 5.0 [micro]g/ml, 10 [micro]g/ml and 25.0 [micro]g/ml of AME, respectively (Fig. 2).
Effects of AME on intracellular ROS generation
Imanishi et al. (2005b) demonstrated that angiotensin II caused EPCs senescence through the induction of oxidative stress, therefore intracellular ROS production was measured. Incubation of EPCs with angiotensin II caused a significant increase of ROS production. The Aronia fruits extract reduced intracellular ROS production in EPCs stimulated with angiotensin II in a concentration-dependent manner. At a concentration of 25.0 [micro]g/ml of AME, ROS production was almost completely inhibited (Fig. 3).
Effects of AME on telomerase activity
Similarly to Imanishi et al. (2005b), we have demonstrated that angiotensin II significantly diminishes telomerase activity. The incubation of EPCs with angiotensin resulted in a decrease of telomerase activity by approximately 50% in comparison with untreated cells. AME strongly increased telomerase activity, which was inhibited by angiotensin II, by 17.8%, 34.2%, 53.4% and 72.1% at concentrations of 1, 5,10 and 25 [micro]g/ml, respectively, as shown in Fig. 4.
Effects of AME on cells proliferation
The incubation of EPCs with angiotensin II decreased their proliferative capacity, measured as BrdU incorporation (decrease of luminescence approximately 50%), whereas pretreatment with AME counteracted that effect in a concentration-dependent manner (Fig. 5). Cells cultivated in the presence of angiotensin II and chokeberry extract revealed an increase of proliferative activity by 81.2% at the highest concentrations of the extract, in comparison with cells that were only treated with angiotensin II alone.
Effects of AME on migration of EPCs
Angiotensin II impaired the migratory capacity of EPC in comparison with the control cells (untreated cells), whereas the addition of AME enhanced cell migration in a concentration-dependent manner, up by 46.5% at a concentration of 25 [micro]g/ml, in comparison with angiotensin II treated cells (Fig. 6).
Effects of AME on adhesion of EPCs
Angiotensin II decreased the number of adhesive cells, whereas EPCs pre-treated with AME exhibited a significant increase in the number of adhesive cells after incubation in comparison with angiotensin II treated cells. The change in the number of adhesive cells was concentration-dependent with the maximal effect at 25 [micro]g/ml of AME (Fig. 7).
Effects of AME on tube formation
An in vitro vasculogenesis assay kit was used to investigate the ability of EPCs to undergo neovascularization following AME treatment. Angiotensin II impaired tube formation in vitro by EPC, whereas the tubule number increased in response to AME after 24 h of incubation, and the tubules were more complex than that those in the control cells (Fig. 8).
Effects of AME on HO-1 expression and Nrf2 activation
Aronia melanocarpa fruit extract strongly increased the expression of heme oxygenase (HO-1) diminished by angiotensin II - by 18.7%, 32.4%, 51.9% and 69.1% at concentrations 1, 5,10 and 25 [micro]g/ml, respectively, in comparison with angiotensin II treated cells (Fig. 9). The treatment of cells with AME also increased the level of Nrf2 in a concentration-dependent manner. The extract increased activation of Nrf2 approximately 4.5 times in a concentration of 25 [micro]g/ml in comparison with angiotensin II treated cells (Fig. 10).
In our studies, we demonstrated a new and previously undescribed mechanism of action of aronia fruit extract that mainly consisted of preventing the impairment of the repairing function of EPCs when induced by the pathogenic activity of angiotensin II. This may positively impact the regeneration of damage to the cardiovascular system, especially in patients with arterial hypertension. The protective effect of the extract is not only associated with antioxidative properties of the polyphenols it contains, but also with the activation of the Nrf2 transcription factor with subsequent increased HO-1 expression. This enzyme, apart from playing a major role in the degradation of heme to carbon monoxide, iron, and biliverdin, has anti-inflammatory and anti-apoptotic properties, and provides antioxidative protection to endothelial progenitor cells, by being a key enzyme in their biology (Dulak et al. 2008). The level of HO-1 in EPCs cultured in the presence of angiotensin II was significantly lower than in the control cells, and the presence of aronia extract significantly increased that level. Therefore, the protective activity of aronia fruit extract against harmful effects of reactive oxygen species is associated not only with the antioxidative activity of the polyphenols it contains; the stimulation of HO-1 expression in EPCs is equally important, especially as HO-1 expression in EPCs is necessary for their migration and induction of angiogenesis (Deshane et al. 2007). As it is known that cyanidin glucoside induces the activity of HO-1 (Sorrenti et al. 2007), it may be stated that the effect of aronia fruit extract is mainly due to the anthocyanins it contains. Our further studies showed that the elevation of HO-1 levels in EPCs is associated with a greater induction of transcription factor Nrf2 (nuclear factor erythroid 2-related factor) by extract. Nrf2 is responsible for intracellular antioxidative enzyme induction and it also protects cells against apoptosis (Levonen et al. 2007). An increase in Nrf2 activity in EPCs may be beneficial, for example in protecting these cells against oxidative stress that significantly impairs the restorative functions of EPCs. Our results show one more aspect of the favorable effects of Aronia melanocarpa on the cardiovascular system that has never been discussed before. The normal functioning of the cardiovascular system depends, among other things, on an efficient regenerative function of EPCs (Zampetaki et al. 2008). A high bioavailability of anthocyanins and the fact that they circulate in their active, not metabolized form, should not be ignored either (Wiczkowski et al. 2010).
Favorable effects of aronia fruit extracts have also been demonstrated in vivo. The administration of aronia fruit extract in combination with statins to patients after myocardial infarction resulted in a notable reduction in the level of oxidative stress markers and inflammatory markers, reduction in expression of adhesion molecules and MCP-1 in the blood, and lowered arterial blood pressure in those patients (Naruszewicz et al. 2007). A similar effect was observed in patients with metabolic syndrome: two months of treatment with the extract resulted in a significant reduction in arterial blood pressure as well as the concentrations of total and LDL cholesterol, endothelin-1 (ET-1), and CRP (Broncel et al. 2010). A concentration-related negative effect of aronia fruit extract on the production of superoxide anions in platelets isolated from patients with risk factors of atherosclerosis as well as a potent anti-aggregation effect were also demonstrated (Ryszawa et al. 2006). Administration of aronia extract to patients with hypercholesterolaemia resulted in a 40% reduction in lipid peroxidation and increased erythrocyte membrane fluidity (Duchnowicz et al. 2012). Our present studies have significantly increased the applicability of aronia extract in primary and secondary prevention of cardiovascular diseases; however, this problem requires further clinical research.
Our results suggest that Aronia melanocarpa fruit extract, containing mainly anthocyanins and phenolic acids, may play a significant role in the prevention of the senescence of endothelial progenitor cells induced by angiotensin II. Moreover, induction of Nrf2 trancription factor in EPCs by extract, and, consequently, increase of the level of HO-1 in these cells, not only protect EPCs against oxidative stress, but also increase their ability to re-endothelize of injured arterial walls and may protect atherosclerotic plaque against destabilization.
Abbreviations: EPC, endothelial progenitor cells; Ang II, angiotensin II; Nrf2, NF-E2-related factor 2; HO-1, heme oxygenase-1; AME, Aronia melanocarpa extract.
Received 20 May 2015
Revised 16 October 2015
Accepted 23 October 2015
Conflict of interest
We wish to confirm that there are no conflicts of interests associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Andrzej Parzonko, Aleksandra Oswit, Agnieszka Bazylko, Marek Naruszewicz *
Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy. Medical University of Warsaw, Banacha 7, 02-097 Warsaw, Poland
* Corresponding author. Tel./fax: +48 22 572 09 85.
E-mail address: firstname.lastname@example.org (M. Naruszewicz).
Table 1 Chemical composition of tested Aroma melanocarpa fruit extract. No. Compound [t.sub.R] [[M-H].sup.-] [min] (m/z) 1 Neochlorogenic acid 13.8 353 2 Chlorogenic acid 17.9 353 No. Compound [t.sub.g] [[M+H].sup.*] [min] (m/z) 3 Cyanidin-3-O-galactoside 18.1 449 4 Cyanidin-3-O-gluctoside 19.1 449 5 Cyanidin-3-O-arabinoside 20 419 6 Cyanidin-3-O-xyloside 22.2 419 No. Compound [t.sub.R] [[M-H].sup.-] [min] (m/z) 7 Quercetin-3-O-rutoside 26.6 609 8 Quercetin-3-O-galactoside 27.3 463 9 Q.uercetin-3-O-glucoside 27.5 463 10 Eriodictyol-7-O-glucuronide 28 463 No. Compound Fragmentation (m/z) 1 Neochlorogenic acid 191, 179, 135 2 Chlorogenic acid 191, 179 No. Compound Fragmentation (m/z) 3 Cyanidin-3-O-galactoside 287 4 Cyanidin-3-O-gluctoside 287 5 Cyanidin-3-O-arabinoside 287 6 Cyanidin-3-O-xyloside 287 No. Compound Fragmentation (m/z) 7 Quercetin-3-O-rutoside 301, 179 8 Quercetin-3-O-galactoside 301, 179 9 Q.uercetin-3-O-glucoside 301, 179 10 Eriodictyol-7-O-glucuronide 287, 151
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|Author:||Parzonko, Andrzej; Oswit, Aleksandra; Bazylko, Agnieszka; Naruszewicz, Marek|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Dec 15, 2015|
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