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Research Article
Mitochondrial aldehyde dehydrogenase-2 activation prevents β-amyloid-induced endothelial cell dysfunction and restores angiogenesis
Raffaella Solito, Federico Corti, Che-Hong Chen, Daria Mochly-Rosen, Antonio Giachetti, Marina Ziche, Sandra Donnini
Journal of Cell Science 2013 126: 1952-1961; doi: 10.1242/jcs.117184

Summary

Amyloid β peptides (Aβ1–40 and Aβ1–42) cause cerebral degeneration by impairing the activity of angiogenic factors and inducing apoptosis and senescence in the endothelium. Amyloid peptides are known to induce oxidative stress. Impairment of mitochondrial aldehyde dehydrogenase 2 (ALDH2) following oxidative stress, results in accumulation of toxic aldehydes, particularly 4-hydroxynoneal (4-HNE). We sought to determine the role of mitochondrial ALDH2 in Aβ-related impairment of angiogenesis. We hypothesized that by increasing the detoxification activity of ALDH2 we would reduce Aβ-driven endothelial injuries and restore angiogenesis. We used a selective ALDH2 activator, Alda-1, assessing its ability to repair mitochondrial dysfunction in the endothelium. Treatment of human endothelial cells with Aβ1–40 (5–50 µM) induced loss of mitochondrial membrane potential, increased cytochrome c release and ROS accumulation. These events were associated with 4-HNE accumulation and decrease in ALDH2 activity (40%), and resulted in disassembly of endothelial junctions, as evidenced by β-catenin phosphorylation, disorganization of adherens and tight junctions, and by disruption of pseudocapillary formation. Alda-1 (10–40 µM) abolished Aβ-induced 4-HNE accumulation, apoptosis and vascular leakiness, fully restoring the pro-angiogenic endothelial phenotype and responses to FGF-2. Our data document that mitochondrial ALDH2 in the endothelium is a target for the vascular effect of Aβ, including loss of barrier function and angiogenesis. ALDH2 activation, by restoring mitochondrial functions in the endothelium, prevents Aβ-induced dysfunction and anti-angiogenic effects. Thus, agents activating ALDH2 may reduce endothelial injuries including those occurring in cerebral amyloid angiopathy, preserving the angiogenic potential of the endothelium.

Introduction

A wealth of evidence indicates the mitochondrial dysfunction is a feature common to many age-related degenerative pathologies, including β-amyloid-driven diseases such as Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) (Lin and Beal, 2006; Cho et al., 2009). β-amyloids have been shown to localize mainly to the inner mitochondrial membrane, causing excessive reactive oxygen species (ROS) production (Lin and Beal, 2006). Besides the respiratory chain injuries, β-amyloids have been shown to enhance the expression of cyclophilin D, a mitochondrial protein regulating the opening of the transition pore (Du et al., 2008), and to promote S-nitrosylation of dynamin-related protein 1, a protein that governs mitochondrial dynamics (Cho et al., 2009). Β-amyloids also form a complex with the mitochondrial Aβ-peptide-binding alcohol dehydrogenase (ABAD), an interaction that leads to accelerated cell death (Yao et al., 2011).

In addition, β-amyloid-induced ROS elevation activates lipid peroxidation pathways, that generate excessive amounts of highly toxic aldehydes, particularly 4-hydroxy-2-noneal (4-HNE) (Schneider et al., 2008). Because of its electrophilic nature, 4-HNE forms stable adducts with a myriad of proteins required for mitochondrial functions (Petersen and Doorn, 2004). 4-HNE exerts toxic effects in a wide range of tissues, including neural and cardiac tissues (Neely et al., 1999; Chen et al., 2008) and endothelium (Suzuki et al., 2007).

Although metabolized by a number of detoxifying mechanisms, 4-HNE is a specific substrate of mitochondrial aldehyde dehydrogenase 2 (ALDH2) (Chen et al., 2008; Hyun et al., 2002; Mattson, 2009). In a rat myocardial infarction model, boosting the ALDH2 activity, through a novel enzyme activator, Alda-1, resulted in a 60% decrease in the ischemic damage, an effect associated with enhanced clearing of the toxic 4-HNE (Chen et al., 2008).

β-amyloid peptides, particularly the shorter vasculotropic Aβ1–40 (hereafter termed Aβ), affect brain blood vessels, altering important functions of medium and small arterioles and capillary endothelium. These derangements include impairment of vasoactive tone, vascular remodeling and barrier functions, as well as suppression of the intrinsic angiogenic properties of endothelium, such as responsiveness to fibroblast growth factor-2 (FGF-2), FGF-2 expression, its cognate receptor FGFR1, and also vascular endothelial growth factor receptor2 (VEGFR2) (Revesz et al., 2003; Patel et al., 2010; Solito et al., 2009). Collectively, these alterations contribute to CAA, a pathology characterized by Aβ1–40 deposition around cortical and leptomeningeal vessels, frequently (>80%) occurring in AD patients. Investigations from various laboratories have delineated a range of Aβ-induced biochemical and signal changes that underlie the disease. The observation that Aβ strongly activates the intrinsic apoptotic pathway is perhaps the clearest evidence that these peptides target the mitochondria to execute their toxic effects (Fossati et al., 2010).

We sought to determine the role of mitochondrial ALDH2 in Aβ-related impairment of endothelial functions and FGF-2-mediated angiogenesis. To investigate ALDH2 in angiogenic events, and in amyloid angiopathy, we used the recently identified class of small molecular mass compounds that selectively interact with the mitochondrial enzyme increasing its catalytic activity. Specifically, Alda-1 (benzodioxyl dichlororobezamide), the prototype of compounds that activate ALDH2, inhibits the injuries produced by 4-HNE in several models of oxidative stress (Chen et al., 2008; Perez-Miller et al., 2010).

The use of this pharmacological tool enabled the determination of the role of mitochondrial ALDH2 in Aβ-impaired angiogenesis.

Results

Aβ treatment causes mitochondrial impairment in cultured endothelial cells

The effects induced by Aβ on the endothelium have been described in numerous reports (Revesz et al., 2003; Patel et al., 2010; Solito et al., 2009). Here we focused on the Aβ-induced mitochondrial dysfunction in endothelial cells, human umbilical endothelial cells (HUVEC) and human brain microvascular endothelial cells (HBMEC). To characterize the mitochondrial damage induced by Aβ, we evaluated a number of mitochondrial functions. Aβ produced a concentration-dependent loss of mitochondrial membrane potential (Δψm), as measured by JC-1 monomer levels (Fig. 1A). The reverse peptide (Aβ40–1) was used as a control in all the experiments in this study because it does not affect mitochondrial potential relative to no peptide addition (fluorescence levels: 5756±429 versus 5880±307, respectively). There was also a sevenfold increase in cytochrome c release in Aβ-treated HUVEC (Fig. 1B). Impairment of mitochondrial potential affected the integrity of the electron transport system, producing a marked rise of super oxide formation, detected by MitoSox (Fig. 1C). A similar loss of Δψm, as well as cytochrome c release and ROS overproduction occurred in HBMEC exposed to Aβ (Fig. 1D,E,F).

Fig. 1.
Fig. 1.

Aβ decreases mitochondrial membrane potential in endothelial cells. (A) Mitochondrial membrane potential in HUVEC exposed to Aβ (5–50 µM, 30 minutes), with or without Alda-1 pretreatment (20 µM, 30 minutes prior Aβ), were measured using the JC-1 probe. The fluorescence units plotted were obtained by subtracting background fluorescence (**P<0.01 and ***P<0.001 versus control, n = 3). Aβ40–1 activity (X) was used as the control. (B) Western blot analysis of cytochrome c release following Aβ treatment (50 µM, 30 minutes) with or without Alda-1 (20 µM, 30 minutes prior Aβ). A representative gels of three independent experiments is shown. (C) Mitochondrial generation of superoxide in HUVEC, detected by MitoSox fluorescence staining. Aβ (50 µM, 30 minutes), Alda-1 (20 µM, 30 minutes prior Aβ). Scale bar: 100 µm. Quantification of superoxide generation is expressed as percentage of stained cells after treatment compared with the control. (</emph>± s.e.m.; n = 3). (D) Mitochondrial membrane potential in HBMEC exposed to Aβ with or without Alda-1 (20 µM, 30 minutes prior Aβ), measured using the JC-1 probe and expressed as described in A. **P<0.01 and ***P<0.001 versus control. Data are means ± s.e.m.; n = 3–4 emission values. Aβ40–1 activity (X) was used as the control. (E) Western blot analysis of cytochrome c release in HBMEC treated as above. A representative gels of three independent experiments is shown. (F) Superoxide mitochondrial generation, detected by MitoSox, in HBMEC treated as above. Scale bar: 100 µm. Quantification of superoxide generation is expressed as percentage of stained cells after treatment compared with the control. (± s.e.m.; n = 3).

To investigate the molecular mechanism of Aβ-induced mitochondrial dysfunction, we determined whether the lipid-peroxidation-generated aldehyde, 4-HNE, accumulates following Aβ treatment. In particular, as this aldehyde is very reactive and forms adducts on macromolecules, including proteins (Petersen and Doorn, 2004), we measured 4-HNE protein adducts using the enzyme-linked immunosorbent assay (ELISA) (Fig. 2A) and immunohistochemistry (Fig. 2B) in Aβ-treated cells. There was a threefold increase in 4-HNE adduct formation (Fig. 2A,B,b), especially around the perinuclear area (Fig. 2B,b). To investigate whether 4-HNE adducts localized in mitochondria, we performed double staining of 4-HNE adduct formation in combination with an antibody against TOM 20 (a translocase of the outer mitochondrial membrane; Fig. 2C,a). We detected colocalization of 4-HNE and TOM 20 within the mitochondria (Fig. 2C,b), along with partial overlap of the two markers in the organelles. Since ALDH2 is the main enzyme that metabolizes 4-HNE, we determined whether selective activation of ALDH2 with Alda-1 decreased the levels of 4-HNE adducts. Co-treatment of HUVEC with Alda-1 and Aβ caused more than 60% decrease in 4-HNE adduct accumulation (Fig. 2A), drastically reducing the 4-HNE-associated immunofluorescence (Fig. 2B,C, compare c and b in both panels).

Fig. 2.
Fig. 2.

Aβ increases 4-HNE adduct formation. (A) Accumulation of 4-HNE adducts in HUVEC treated as in Fig. 1A was measured by ELISA. *P<0.05 versus Ctr.; #P<0.05 versus Aβ. Values are means ± s.e.m. of three experiments. (B) 4-HNE detection by immunohistochemistry in HUVEC treated as above. (C) 4-HNE and TOM20 detection by immunohistochemistry in HUVEC treated as above. Scale bar: 10 µm.

To determine whether Aβ-induced mitochondrial dysfunction is caused by adduct accumulation, we determined the benefit of Alda-1 (10–40 µM) on mitochondrial membrane potential. The two highest Alda-1 concentrations tested provided comparable protection of mitochondrial potential, whereas the lower one was ineffective (Fig. 3A). All further experiments of this study were, therefore, conducted with 20 µM Alda-1. Confocal microscopy of HUVEC of the JC-1 stain demonstrated a clear difference between mitochondrial membrane potential (Δψm) in Aβ-treated cells and those pretreated with Alda-1. The diffuse red fluorescence in control cells denotes the presence of JC-1 aggregates typical of high Δψm (Fig. 3B, upper panels), whereas the abundant green fluorescence in Aβ-treated cells indicates low Δψm, typical of JC-1 monomers (Fig. 3B, middle panels). Note the partial reversal of Aβ-induced decline in mitochondrial potential by Alda-1, as evidenced by the colocalization of red and green fluorescence in the merge images (Fig. 3B, bottom panels). Mitochondrial damage was also observed by measuring cytochrome c levels, and apoptosis evaluated by phosphatidylserine immunostaining. Alda-1 treatment abolished Aβ-induced changes in the above mentioned parameters (Fig. 1B,E; Fig. 3C). Together these data support our hypothesis that Aβ causes mitochondrial dysfunction by inducing toxic 4-HNE accumulation, and that the resulting injuries can be counteracted by stimulating ALDH2 activity through a specific enzyme activator.

Fig. 3.
Fig. 3.

Alda-1 prevents Aβ-induced mitochondrial dysfunction. (A) Quantification of JC-1 monomer fluorescence induced by Aβ in HUVEC pretreated with Alda-1, in the concentration range 10–40 µM. Data are expressed as in Fig. 1A (means ± s.e.m., n = 3; ***P<0.001). Aβ40–1 activity (X) was used as the control. (B) Mitochondrial membrane potentials were assessed by confocal microscopy, showing the relative abundance of JC-1 aggregates (red) and monomers (green). (Upper panels) Control cells; (middle panels) Aβ-treated cells; (lower panels) cells treated with Alda-1-Aβ combined as in Fig. 1A. Scale bar: 10 µm. (C) Phosphatidylserine exposure, assessed by immunohistochemistry, in HUVEC treated for 18 hours with 50 µM Aβ with or without Alda-1. Scale bar: 100 µm.

Aβ reduces ALDH2 enzymatic activity

In view of the marked 4-HNE increase in endothelial cells following Aβ exposure, we measured ALDH2 activity (Chen et al., 2008). Application of 50 µM Aβ to HUVEC substantially reduced ALDH2 activity by 40% (Fig. 4A), exerting negligible effects on its expression (Fig. 4B). This reflects an ALDH2 inactivation by 4-HNE (Chen et al., 2008), rather than a direct interaction of Aβ with the enzyme. In fact, incubation of Aβ (10–50 µM) with recombinant ALDH2, did not reduce its enzymatic activity (Fig. 4C). As reported (Chen et al., 2008), addition of Alda-1 to the recombinant enzyme produced a significant increase of its catalytic activity (Fig. 4C). Importantly, administration of Alda-1 prior to Aβ treatment prevented the decline in ALDH2 activity in both HUVEC (Fig. 4E, P<0.01) and in HBMEC (Fig. 4F, P<0.05). Furthermore, the marked expression of ALDH2 compared with that of ALDH1 in HUVEC, suggests its pre-eminent involvement in the removal of 4-HNE in endothelial cells (Fig. 4D). To examine the possibility that reactive species other than 4-HNE were involved in ALDH2 decreased activity, we used the optimal concentration of MnTBAP, a cell permeable superoxide dismutase (SOD) mimetic (Cantara et al., 2007), and measured ALDH2 activity in HUVEC previously exposed to Aβ. The scavenger was effective in reversing Aβ-decreased ALDH2 activity, although its efficacy was lower compared with that of Alda-1 (Fig. 4E, P>0.05 versus P>0.001).

Fig. 4.
Fig. 4.

Aβ inhibits ALDH2 activity in endothelial cells. (A) Decline of ALDH2 activity, measured by the formation of NADH in HUVEC exposed to Aβ. Values are mean ± s.e.m. NADH production (n = 3–6). (B) Western blot analysis of ALDH2 expression in HUVEC treated with Aβ (25 µM), with or without Alda-1 (20 µM). Quantification of gels are presented as the ratio to β actin (n = 3). (C) Recombinant ALDH2 activity in the absence or presence of Aβ or Alda-1. Data are percentage NADH production over that of the control (± s.e.m. of three independent experiments). (D) Expression of ALDH2 and ALDH1 levels in HUVEC determined by western blot analysis. Known amounts of purified recombinant ALDH1 and ALDH2 (1.5–5 ng) were used for a standard curve to obtain the values given above the blots (means ± s.e.m. of three independent experiments). (E,F) Recovery of ALDH2 enzyme activity in HUVEC (E) and HMBEC (F) after Alda-1 (20 µM) or MnTbap (100 µM) treatment 30 minutes before exposure to Aβ (50 µM). **P<0.01 and ***P<0.001 versus control; ###P<0.001 and §P<0.05 versus Aβ.

ALDH2 activation prevents endothelial cell membrane disorganization and permeability caused by Aβ

We investigated whether Aβ affects the adherens and tight junctions of endothelial cells causing their functional impairment. To this end, we examined signaling molecules, i.e. VE cadherin, β-catenin and ZO-1 protein, all known to be involved in maintaining adherens and tight junction integrity.

Aβ induced cytoplasmic β-catenin phosphorylation (Fig. 5A), an event known to provoke its dissociation from VE cadherin, thus leading to disassembly of adherens junctions (Chen et al., 2012). Indeed, Aβ also abolished the distribution of VE cadherin at cell–cell contact, evaluated by immunofluorescence (Fig. 5B, compare b with a). Moreover, since adherens junctions influence tight junctions organization (Dejana, 2004), we investigated, by immunohistochemistry, the expression pattern of the tight junction protein, ZO-1. Aβ also abolished the typical distribution of ZO-1 lining the plasma membrane at the cell–cell contacts (Fig. 5B, compare e with d). Treatment with Alda-1 prevented all the above changes, hence preserving the integrity of endothelial junctions (Fig. 5A,B, compare c with b, and f with e). Taken together, these results indicate that activation of ALDH2 contributes to maintaining the integrity of endothelial cell junctions, conceivably by shielding them from the toxic insults of 4-HNE.

Fig. 5.
Fig. 5.

Aβ induces phosphorylation of β-catenin, and impairs endothelial adherens and tight junction organization and barrier function. (A) Western blot analysis of phosphorylated β-catenin at Ser33/37 and Thr41, in HUVEC treated with Aβ (50 µM) in the presence or absence of Alda-1 (20 µM). Quantifications are presented as a ratio to β actin (n = 3; **P<0.01 versus Ctr., ##P<0.01 versus Aβ treatment). (B) ZO-1 and VE-cadherin expression pattern in control cells (a,d), cells exposed to Aβ (50 µM; b,e) and cells pretreated with Alda-1 (20 µM, 30 minutes) and then exposed to Aβ (c,f). Scale bar: 50 µm (C) Increased permeability of HUVEC monolayers exposed to Aβ (50 µM) in presence or absence of Alda-1 (20 µM), detected as passage of fluorescence-conjugated FITC-dextran. Each point is the mean ± s.e.m. of 3 experiments, *P<0.05 versus Aβ.

In line with the above findings, we observed a change of permeability in HUVEC following Aβ exposure, as shown by the large paracellular flux increase of fluorescent conjugated dextran, which was more than 30% that recorded in the control. In contrast, Alda-1-pretreated cells exhibited a paracellular flux comparable with that of the control (Fig. 5C).

Angiogenic phenotype and angiogenesis response disrupted by Aβ are restored by Alda-1

Beta amyloids severely impair angiogenic responses of the endothelium, as previously reported (Donnini et al., 2010; Paris et al., 2005). Here, we show that HUVEC, seeded on Matrigel, produce an organized pseudocapillary network (Fig. 6A,a and i), but they failed to organize a proper architecture in the presence of Aβ (Fig. 6A,b). The observed cell clustering and stunted growth, occurring in the presence of Aβ, was probably due to impaired cell adhesion and migration (Fig. 6A,ii). Of note, only a few endothelial cells entered apoptosis as documented by phosphatidylserine immunofluorescence (Fig. 3C) and Trypan Blue staining (data not shown). Experiments on cell migration showed a significantly reduced migration (40%, P<0.01) caused by Aβ (Fig. 7A). The severe endothelial injuries observed were invariably counteracted by prior application of Alda-1. In fact, the pseudocapillary architecture and cell migration were fully maintained in the presence of Alda-1 (Fig. 6A,c; Fig. 7A) as also shown in the graph representing quantification of pseudocapillaries (Fig. 6B).

Fig. 6.
Fig. 6.

Alda-1 preserves the ability of the endothelium to form pseudocapillaries and restores responsiveness to FGF-2. (A) Pseudocapillary formation in Matrigel by HUVEC exposed to Aβ (25 µM) with or without Alda-1 (20 µM) or FGF-2 (20 ng/ml), observed by microscopy 18 hours after cell seeding. (a) Control (Aβ40–1) cells, (b) Aβ-treated, (c) combined Aβ and Alda-1-Aβ-treated, (d) FGF-2-treated, (e) combined FGF-2 and Aβ treated, (f) combined FGF-2, Aβ and Alda-1 treated cells. Images represent three experiments. Scale bar: 20 µm. (i and iii) Representative images at 4× magnification of pseudocapillary network in control cells or after FGF-2 treatment (ii) Endothelial cell morphology after 18 hours of treatment with Aβ. Scale bars: 100 µm. (B) Quantification of pseudocapillaries. Values are means ± s.e.m.; n = 3 (***P<0.001 versus control; ###P<0.001 versus Aβ). (C), Formation of pseudocapillaries from HUVEC 2 days after seeding on cytodex microcarriers embedded in fibrin gel. Representative images of capillary formation in control condition (Aβ40–1; a), after FGF-2 stimulation (b), after FGF-2+Aβ treatment (c) and FGF-2+Aβ+Alda-1 treatment (d). Scale bar: 50 µm. (D) Quantification of pseudocapillaries. The number of grid units required to cover the entire pseudocapillary surface (means ± s.e.m.; n = 3). ###P<0.001 versus control; ***P<0.001 versus Aβ.

Fig. 7.
Fig. 7.

Alda-1 preserves endothelial cell migration and FGF-2 expression. (A) Cell migration determined using a Boyden chamber. Values are means ± s.e.m. of 3 experiments (**P<0.01 versus control; ###P<0.001 versus Aβ). (B) Western blot analysis of FGF-2 production in HUVEC treated with Aβ in the presence or absence of Alda-1. Quantification of gels are presented as the ratio to β-actin (n = 3). **P<0.001.

We and others have reported that during neovessel formation Aβ reduces the expression of major endothelial growth factors, e.g. FGF-2 and VEGF, as well as their capacity to elicit angiogenic responses (Donnini et al., 2010; Solito et al., 2009; Paris et al., 2005). We therefore sought to determine whether ALDH2 activation would prevent these Aβ-induced injuries. Indeed, Aβ reduced endogenous FGF-2 expression in HUVEC by ∼50%, an effect completely prevented by the activation of ALDH2 by Alda-1 (Fig. 7B). Also, Alda-1 restored responsiveness to exogenous FGF-2 (20 ng/ml) in the formation of the network of pseudocapillaries, impaired by Aβ, as documented by the quantification of pseudocapillary network formation (Fig. 6A,d–f; Fig. 6B). Similarly, the capability of endothelial cells to respond to FGF-2 was restored by Alda-1 when HUVEC were embedded in fibrin gel and exposed to Aβ, restoring their angiogenic phenotype (Fig. 6C,D).

Discussion

The mitochondrial enzyme ALDH2 exerts an important physiological protection in a variety of tissues, as it degrades highly toxic aldehydes originating from the lipid peroxidation cascade, mainly 4-HNE. Protection afforded by ALDH2 has been observed in conditions in which oxidative stress and the resulting excessive production of 4-HNE causes tissue damage, often evolving toward degenerative processes (Ohsawa et al., 2008; Wey et al., 2012). Thus, ALDH2 appears to be implicated in diverse pathologies such as Alzheimer's and Parkinson's diseases and ischemic conditions (Ohsawa et al., 2008; Wey et al., 2012). Here, we examined the role of ALDH2 in the development of endothelial dysfunction produced by the vasculotropic amyloid Aβ (Aβ1–40), known to be involved in the pathogenesis of the Alzheimer-associated cerebral amyloid angiopathy (CAA) (Donnini et al., 2010).

Acute Aβ exposure of endothelial cells, HUVEC and HBMEC, representing peripheral and central nervous system vascular tissue, produced a marked mitochondrial dysfunction as shown by the dramatic loss of mitochondrial membrane potential and by the rise of cytochrome c release. Mitochondrial injuries occurred concomitantly with the surge of superoxide and with accumulation of 4-HNE adducts. These changes occurred rapidly, within 30 minutes, and are therefore likely to be caused by short oligomers of Aβ (Solito et al., 2009). A number of studies have illustrated a direct interaction between Aβ and alcohol dehydrogenase (ABAD) in neurons, which prevents NAD+ binding to the active site in ABAD, which results in oxidative stress (Yao et al., 2011). In contrast, such direct interaction of Aβ with ALDH2 does not occur (Fig. 4C). Therefore, the sharp decline of ALDH2 enzymatic activity, noted in endothelium following Aβ treatment, is clearly related to the oxidative injuries to the mitochondria, rather than a direct effect of the Aβ on ALDH2.

This is the first study that shows the decline of ALDH2 activity in endothelial cells treated with Aβ, pointing to ALDH2 activity decline as the mechanism of Aβ toxicity. Indeed, by using a specific activator of ALDH2, termed Alda-1, we found that the ALDH2 recovery fully restored mitochondrial functions, in terms of membrane potential and cytochrome c release. Alda-1 treatment reduced 4-HNE adducts and ROS formation, and rescued Aβ-impaired functions of the endothelium, supporting the hypothesis that impaired detoxification of biogenic aldehydes may be important in Aβ-induced vessel injuries. The molecular mechanism whereby Alda-1 protects ALDH2, which has been described in a report from one of our laboratories (Perez-Miller et al., 2010), illustrates how the compound reduces the accessibility of 4-HNE to key cysteine residues of the enzyme, essentially by prolonging the residence of NAD, the enzyme cofactor.

The activation of ALDH2, through Alda-1, was a remarkably efficient means of determining the Aβ-induced injuries in the endothelium. In fact, protection was evident of both basic and FGF-2-mediated endothelial functions, such as the ability of cultured endothelium to express an angiogenic phenotype (pseudocapillary formation and FGF-2 production) and to maintain the barrier function. These are important determinants of endothelial viability, as they are the basis of vessel remodeling and protection of the underlying tissues from toxic insults. Furthermore, since FGF-2 is an important pro-survival and pro-angiogenic factor, the responsiveness to FGF-2, promoted by ALDH2 activation, is a relevant determinant of endothelial angiogenic potential.

Analysis of signals involved in endothelial barrier revealed an increased phosphorylation of β-catenin, disorganization of the tight junction protein ZO-1 and of adherens junction VE cadherin in cells exposed to Aβ, indicative of intercellular gap opening, and increased permeability (Murakami and Simons, 2009; Chen et al., 2012). The neurovascular unit in the brain is a complex comprising endothelial cells, neurons, pericytes and astrocytes (Wey et al., 2012). Because components of the unit are tightly interconnected, it is plausible that damage of one component affects the functioning of other cells. Perturbations of endothelial barrier functions may expose all components to toxic insults (e.g. amyloids) and, in addition, may impair their disposal through well documented mechanisms (Quaegebeur et al., 2011; Zlokovic, 2008). Conceivably, the observed reversal by Alda-1 of enhanced permeability may be crucial for restoring the integrity of the blood–brain barrier.

The involvement of mitochondria in vascular lesions, caused by Aβ peptides or other insults inducing ROS, remains largely undefined in terms of molecular mechanisms. An earlier work reported histological evidence for vessel injuries associated with the mitochondrial dysfunction found in AD patient specimens, and in a mouse model of AD (Aliev et al., 2003). More recently, reports have described the protection of cerebral vasculature morphology and function in transgenic mice lacking the ROS-producing machinery through ablation of the Nox-2 gene (Park et al., 2008). Mitochondrial involvement in vessel injuries has been demonstrated in reports showing that TAT-linked BH4, a peptide domain of the anti-apoptotic Bcl-xL mitochondrial protein, prevented Aβ-induced damages in cultured endothelial cells and ischemic brain damage (Donnini et al., 2009; Cantara et al., 2007). Recently, a mitochondrial protein, prohibitin-1, has been shown to protect the endothelium from ROS damages (Schleicher et al., 2008). Together, these findings underscore the relevance of targeting lipid peroxidation products as a means of rescuing vessels from toxic insults.

The present work identifies a specific detoxifying mechanism, provided by activation of a mitochondrial enzyme, ALDH2, by means of the selective activator Alda-1. The protection of the vascular endothelium from injuries arising from toxic products of lipid peroxidation afforded by this activator could have implications for a wide range of diseases in which impairment of mitochondrial function and angiogenesis are the underlying pathogenetic mechanism. Our findings, demonstrating that activation of ALDH2 by Alda-1, prevents the injurious effects of Aβ on vascular endothelium and preserves angiogenic phenotype and responsiveness, may have applications for other age-related vasculopathies, including those associated with diabetes and vascular dementia.

Materials and Methods

Reagents

Aβ1–40 and its reversed sequence (control), synthesized at Espikem (University of Florence), were dissolved as previously described (Solito et al., 2009;). MitoSox™ Red mitochondrial superoxide indicator (M36008) was from Molecular Probes, Invitrogen, (Paisley, PA4 9RF, UK). JC-1, a mitochondrial membrane potential probe (no. 10009142) was from Cayman Chemical Company (Ann Arbor, Michigan 48108, USA). Oxiselect™ HNE–His Adduct ELISA kit (no. STA-334) was from Cell Biolabs Inc. (San Diego, CA 92126 USA). Acetaldehyde was from Sigma (St. Louis, MO, USA). MnTBAP was from Cayman Chemical Company (Ann Arbor, Michigan 48108, USA).

Cell culture

Human umbilical vein endothelial cells (HUVEC; Cambrex, East Rutherford, NJ, 07073 USA), and human brain microvascular endothelial cells (HBMEC; CellScience, Canton, MA, 02021, USA) were cultured as reported (Solito et al., 2009; Donnini et al., 2010).

Detecting mitochondrial membrane potential (ΔΨm)

Changes in ΔΨm were evaluated using JC-1, a dye that selectively enters mitochondria and changes color from green to red as the potential increases. 2×104 cells/well/100 µl cells were seeded into a 96-well plate, pretreated with Alda-1 (20 µM, 30 minutes) then exposed to Aβ (5, 25, 50 µM, 30 minutes). After treatments, JC-1 staining solution was added to each well (20 minutes), then cells were washed. Changes in ΔΨm, detectable by the presence of different JC-1 forms (either green or red fluorescence) were quantified using a fluorescence plate reader. JC1 aggregates produce a strong red fluorescence with excitation/emission at 560/595 nm, respectively. JC-1 in the monomeric form produces mostly green fluorescence at excitation/emission wavelength of 485/535 nm, respectively. The presence of JC-1 aggregates and/or the monomeric forms was evaluated by confocal microscopy on cells seeded on glass coverslips in a 24-well culture plate.

MitoSOX Red mitochondrial superoxide indicator

Cells (2.5×104) were seeded in a 24-well culture plate and treated as above. After treatments, MitoSOX reagent solution (5 µM) was added for 10 minutes. Fluorescence at excitation/emission wavelength of 510/580 nm, respectively, was observed using an inverted fluorescence microscope (Eclipse TE 300, Nikon).

OxiSelect HNE-His Adduct ELISA kit

Cells (3×105 cells) seeded in 6 cm diameter Petri dishes were treated as above, then scraped into 200 µl lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EGTA, without Triton X-100 or other detergents), sonicated, and centrifuged at 12,000 rpm for 20 minutes at 4°C. Supernatants (100 µl, 10 µg/ml protein) were adsorbed onto a 96-well plate for 2 hours at 37°C. The 4-HNE-protein adduct content, probed with an anti HNE-His antibody, was measured using an HRP-conjugated secondary antibody, and quantified by comparison with a 4-HNE-BSA standard curve.

Aldehyde dehydrogenase (ALDH) enzymatic activity

ALDH enzyme activity was determined by measuring the conversion of acetaldehyde to acetic acid, as reported (Chen et al., 2008). Briefly, cells were cultured as above, then scraped into 600 µl lysis buffer (100 mM Tris-HCl pH 8.0, 10 mM DTT, 20% glycerol, 1% Triton X-100), and centrifuged at 13,000 g for 20 minutes at 4°C. The supernatant was used to detect ALDH activity at 25°C by monitoring NADH formation from NAD+, at 340 nm in a spectrophotometer (Bio-Rad, Hercules, CA 94547, USA). The assay mixture (0.8 ml) contained 100 mM sodium pyrophosphate pH 9.0, 10 mM NAD+ and 600 µg of sample protein. The reaction was started by adding acetaldehyde (10 mM) to the cuvette. Enzyme-specific activity was expressed as nmol NADH/minute/mg protein.

The effect of Aβ on purified human recombinant ALDH2 activity was determined as reported (Chen et al., 2008). Briefly, Aβ (up to 50 µM), DMSO, or Alda 1 or MnTBAP were co-incubated with the enzyme and its activity was determined as described above.

Western blotting

Cells were scraped into a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 10 mM NaF, 1% Triton X-100 and 1% protease inhibitor cocktail. Equal amounts (60 µg) of protein were separated by SDS-PAGE on a 10% gel and transferred to a nitrocellulose membrane. The membrane was blocked (1 hour) in a solution of 5% (wt/vol) milk and then incubated overnight at 4°C with the primary antibodies (1:1000): anti-cytochrome c (Cell Signaling, Millipore, Billerica, MA, USA), anti-ALDH2 (Sigma), anti-ALDH1 (EPITOMICS, Burlingame, CA 94010-1303 USA), anti-pFAK (Tyr 861) (Sigma), pFAK (Tyr397) and p-β catenin (Ser33, 37/Thr41) (Cell Signaling), anti-ZO-1 (BD Transduction, Franklin Lakes, NJ, 07417,USA) or anti-FGF-2 (Cell Signaling, Millipore, Billerica, MA, USA). After incubation for 1 hour in a secondary antibody, anti-IgG HRP (diluted 1:2500, Promega, Madison, WI 53711 USA), the immunoreactions were revealed by chemiluminescence.

In vitro angiogenesis model

Cells, pre-treated with Alda-1 (20 µM for 30 minutes) were exposed to Aβ (25 µM). Cells were then plated onto a thin layer (300 µl) of basement membrane matrix (growth factor reduced Matrigel; Becton Dickinson, Waltham, MA, USA) in 24-well plates at 6×104 cells/well in EBM (EGM-2 containing 2.5% FBS) and incubated at 37°C in 5% CO2 for up to 18 hours. Quantification of tubular structures and photomicroscopy were performed as previously described (Donnini et al., 2010).

Microcarrier cell culture

Gelatine-coated cytodex microcarriers (MCs) (Sigma-Aldrich, Italy) were prepared and embedded in a fibrin gel as described previously (Solito et al., 2009). Cells were stimulated with FGF-2 (20 ng/ml) either alone or together with Aβ (25 µM) and Alda-1 (20 µM). After 2 days the area occupied by capillary-like formations was evaluated using an inverted microscope (20× magnification) with an ocular grid. The sprouting area was expressed as the number of grid units required to cover the entire pseudo-capillary surface. Results are reported as mean area units ± s.e.m.

Cell migration

Cell migration assay was performed in a 48-well Boyden chemotaxis chamber (Neuroprobe Inc., Gaithersburg, USA). Briefly, trypsinized HUVEC were resuspended in culture medium with 0.1% FCS, with or without Alda-1 (20 µM, 30 minutes) and further incubated with Aβ (25 µM, 30 minutes). Cells (12.5×104) suspended in 50 µl medium were loaded into the upper compartment and were incubated for 4 hours at 37°C with 5% CO2. Migration was evaluated as number of cells migrated/well.

Immunohistochemistry

Cells were cultured on coverslips, treated with Aβ 50 µM in the presence or absence of Alda-1, and then fixed in paraformaldehyde (4%, 5 minutes), washed in PBS, and incubated with BSA (45 minutes). Cells were then incubated for 16 hours with anti-TOM20 (Sigma), anti 4-HNE (Santa Cruz), anti-phosphatidylserine (Upstate), anti-ZO-1 (BD Transduction, Millipore, Billerica, MA, USA) or anti-VE cadherin antibodies (EBioscience, San Diego, CA, USA), diluted 1∶50 in PBS containing 0.5% BSA. After incubation with the secondary antibody anti-rabbit IgG TRITC or anti-mouse IgG FITC (1 hour), cells were washed and the coverslips mounted in Mowioll ®4-88.

Permeability

Cells were seeded on collagen-coated insert membranes (Corning, Tewksbury, MA 01876, USA) with high density 0.4 µm diameter pores, and the inserts were placed in a 12-multiwell plate. Cells were seeded at 8×104/insert and cultured for 72 hours. Monolayers were treated with Aβ (50 µM, 24 hours) with or without Alda-1 (20 µM), then a 3 kDa FITC-dextran (10 µM), diluted in PBS, was added on top of cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer. The extent of permeability was determined for 60 minutes by measuring the fluorescence at 485/535 nm, excitation/emission, respectively using a plate reader (Tecan). Arbitrary values were plotted against time.

Statistical analysis

Results are expressed as means ± s.e.m. Statistical analysis was performed using Student's t-test, or analysis of variance (ANOVA). P<0.05 was considered statistically significant.

Footnotes

  • Author contributions

    R.S., F.C. and S.D. participated in research design; R.S., F.C. and C.-H.C. conducted experiments and performed data analysis; A.G., M.Z., D.M.-R. and S.D. wrote or contributed to the writing of the manuscript.

  • Funding

    This research was supported by the Italian Ministry of Instruction, University, Research, project PRIN 2008 [grant number 200875WHMR to M.Z.]; and National Institutes of Health [grant number NIAAA 11147 to D.M.-R.]. Deposited in PMC for release after 12 months.

  • Accepted February 10, 2013.

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Research Article
Mitochondrial aldehyde dehydrogenase-2 activation prevents β-amyloid-induced endothelial cell dysfunction and restores angiogenesis
Raffaella Solito, Federico Corti, Che-Hong Chen, Daria Mochly-Rosen, Antonio Giachetti, Marina Ziche, Sandra Donnini
Journal of Cell Science 2013 126: 1952-1961; doi: 10.1242/jcs.117184
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