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A peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis Sandra Donnini,*,1 Raffaella Solito,*,1 Elisa Cetti,* Federico Corti,* Antonio Giachetti,* Silvia Carra,† Monica Beltrame,‡ Franco Cotelli,† and Marina Ziche*,2 *Department of Molecular Biology, University of Siena, Siena, Italy; and †Department of Biology and ‡ Department of Biomolecular Sciences and Biotechnology, Universita` degli Studi di Milano, Milan, Italy Cerebral amyloid angiopathy (CAA) caused by amyloid  (A) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic A properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that A peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild-type (WT) A and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC50 of 6.1 and 4.7 M, respectively. The 2.5 M concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. -Galactosidase staining and the cyclindependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC50 of 12.3 and 8.8 M, respectively. The 5 M concentration, devoid of acute effects on the endothelium, was applied chronically to long-term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with -galactosidase accumulation, down-regulation of telomerase reversetranscriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as A treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature A-induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.—Donnini, S., Solito, R., Cetti, E., Corti, F., Giachetti, A., Carra, S., Beltrame, M., Cotelli, F., Ziche, M. A peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. FASEB J. 24, 2385–2395 (2010). www.fasebj.org ABSTRACT
Key Words: aging 䡠 cerebral amyloid angiopathy 䡠 Alzheimer’s disease
nant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and ataxia telangiectasia, recognizes vascular dysfunction as the common underlying mechanism (1). The brain’s endothelium is heavily compromised by these pathologies, leading to the loss of important endothelial functions, such as blood-brain barrier, homeostasis, and impairment of vascular remodeling. CAA, characterized by deposition of amyloid peptides around brain vessels, recapitulates the main features of cerebrovascular pathologies, as barrier deficits, hypoperfusion, and aberrant vessels have been found in the majority of CAA patients (2). CAA, frequently coexisting with AD, is primarily an age-related disease occurring in the sixth decade of life, and therefore appears to be a suitable model for exploring the relationship between aging and the development of cerebrovascular diseases. Furthermore, numerous dominant hereditary human CAA variants, originating from genetic mutations in the amyloid precursor protein (APP), display a wide spectrum of clinical phenotypes (3, 4), differing in the localization of amyloid deposit, age of onset, disease severity, and outcomes. Among these variants, the best characterized is the Dutch mutant, known as hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), in which a point mutation at codon 693 of the APP gene yields a single amino acid substitution at position 22 (termed E22Q) of the A1– 40 peptide, originating from the proteolytic cleavage of APP (5). Despite the seemingly minor change in the peptide sequence, the Dutch variant exhibits a distinct clinical phenotype, characterized by an early onset of recurrent stroke episodes (occurring as early as in the fourth decade), and severe clinical presentation (6). The biological effects of the Dutch peptide on the endothelium have convincingly demonstrated that it disrupts endothelial functions (7, 8). Recently, we have shown that the Dutch peptide impairs the angiogenic potential of cultured brain endothelial cells, inhibiting their capacity to de1
These authors contributed equally to this work. Correspondence: Department of Molecular Biology, Via Aldo Moro 2, University of Siena, 53100 Siena, Italy. E-mail:
[email protected] doi: 10.1096/fj.09-146456 2
A heterogeneous group of cerebrovascular diseases, which includes cerebral amyloid angiopathy (CAA), Alzheimer’s disease (AD), cerebral autosomal domi0892-6638/10/0024-2385 © FASEB
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velop pseudocapillary sprouting (9). The evidence, gathered in the above and previous reports, strongly implicates a down-regulation of the fibroblast growth factor FGF-2 and its related pathway in the antiangiogenic effects exerted by the Dutch peptide (10). Given this background, the question arises whether the vascular dysfunction, typical of familial and sporadic CAAs, originates early in life during the development of the circulatory system, an injury aggravated by the accelerated vascular aging promoted by A peptides. To address this issue, we investigated the early effect of administering either the natural A1– 40 [wild-type (WT)] or the Dutch (E22Q) peptides to zebrafish during the embryonic/larval stages in which blood vessels develop rapidly, reaching maturation within a few days from fertilization. To study the long-term effect of A peptides, we utilized cultured human umbilical vein endothelial cells (HUVECs), a welldocumented in vitro model for investigating vascular senescence (11, 12). We followed the endothelial cell replication, observing whether the prolonged exposure to the peptides would influence the pattern of senescence reviewed in a recent report (13). MATERIALS AND METHODS A peptides Dissolution procedures for A peptides (WT, reverse sequence A40 –1, and Dutch A, E22Q), their aggregation properties, and their vendors have been recently described in a report from this laboratory (9). For peptides used in zebrafish studies, we evaluated their aggregation properties by incubating them in fish water in the concentration range 1–5 M for up to 7 d at 28°C. Amyloid fibril formation was determined by the thioflavin fluorescence procedure at daily intervals during the incubation (9). Cell culture HUVECs were purchased from Cambrex (East Rutherford, NJ, USA). First-passage cryopreserved HUVECs were maintained in 6-cm-diameter dishes, grown in EGM-2 containing 2% FBS, and serially passaged until they reached senescence as described previously (14) in the presence or absence of A peptides at doses of 0.05–5 M. The number of population doublings (PD) was calculated using the formula PD ⫽ (ln nch ⫺ ln ncs)/ln 2, where nch is number of cells harvested and ncs is number of cells seeded (12). For all the experimental procedures described here, cells at different cumulative population doublings (CPDs) were subcultured onto 6 cm diameter dishes and grown in parallel. To compensate for the decrease in the rate of growth that occurs as the cells acquire a senescent phenotype, seeding densities and culture times were adjusted so that the cells reached 90% confluence by the day of harvesting. The medium was changed every 3 d regardless of the confluent state. This allowed cultures at different CPDs to be simultaneously harvested and assays to be performed under the same conditions. Quantitative RT-PCR analysis of human telomerase reverse trascriptase (hTERT) and telomerase enzyme activity From HUVECs (3⫻105/6-cm plate) at CPD 6 and 18 incubated with 50 nM or 5 M of A peptides in 0.1% FBS, total 2386
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RNA was extracted using RNeasy Kit (Qiagen, Milan, Italy). Total RNA (1 g) was reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad, Milan, Italy) as described previously (15). The primers and probe for the measurement of hTERT mRNA expression were selected by the Beacon Designer 4 software (Bio-Rad). The forward primer was 5⬘-ACGGCGACATGGAGAACAA-3⬘, the reverse primer was 5⬘-CACTGTCTTCCGCAAGTTCAC-3⬘, and the fluorescent probe (FAM) was 5⬘-CTCCTGCGTTTGGTGGATGATTTCTTGTTG-3⬘. For each sample, 150 ng of cDNA was added to 25 l of PCR mix containing 300 nM of the each primers, 200 nM of the specific probe, and 2⫻ iQ Supermix. The expression of hTERT was calculated by referring to an external reference curve generated with total universal human reference RNA (from 250 ng to 3.2 pg of cDNA; Stratagene, La Jolla, CA, USA). Samples and standards were subjected to 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min in the iCycler detector (Bio-Rad). The results were expressed in terms of the equivalents of nanograms of standard RNA per 150 ng total RNA. To test telomerase enzyme activity, HUVECs were harvested by trypsinization, counted, and lysed at 4°C in 3-[3(cholamidopropyl) diethylammonio]-1-propane sulfonate (CHAPS) buffer. Aliquots of the cleared lysates equivalent to 3 ⫻ 105 cells were assayed for telomerase activity by a modified telomeric repeat amplification protocol (TRAPeze RT Telomerase Detection Kit; catalog no. S7710; Chemicon, Temecula, CA, USA) according to the vendor instructions. In the first step of the reaction, the telomerase enzyme present in the samples adds a number of telomeric repeats (TTAGGG) onto the 3⬘-end of a substrate oligonucleotide (TS). In the second step, the extended products are amplified by the Taq polymerase, using RT-PCR. The activity of each sample was detected by using fluorescence energy transfer (ET) primers generating fluorescently labeled TRAP products. This method allows detection and quantification of telomerase activity, since the fluorescence emission produced is directly proportional to the amount of TRAP products generated. p38 and p21 signals in HUVECs p38 and p21 were assayed in HUVECs at specified intervals. Cells were processed as described previously (9). Blotted membranes were incubated with anti-p21 (Upstate, Billerica, MA, USA) or anti-phospho-p38 antibodies (Cell Signaling, Boston, MA, USA). Signals were developed with the enhanced chemiluminescence detection system (Bio-Rad). In vitro angiogenesis model HUVECs at PD 6 and 12 were plated onto a thin layer (300 l) of basement membrane matrix (Matrigel; Becton Dickinson, Waltham, MA, USA) in 24-well plates at 6 ⫻ 104 cells/well in EBM (EGM-2 containing 5% FBS) and incubated at 37°C in 5% CO2 for up 18 h. Quantification of the tubular structures was performed by counting the number of complete circles produced by interlinking tubular HUVECs (16). Photomicrographs of each quadrant of individual wells were obtained using a Nikon Eclipse TE 300 inverted microscope, and the images acquisition was performed by a Nikon digital camera DS-5MC and NIS Element software (Nikon, Tokyo, Japan). Zebrafish maintenance and A peptide treatment Zebrafish were raised and maintained at 28°C on a 14/10-h light-dark cycle. Embryos were collected by natural spawning, staged according to Kimmel et al. (17) in fish water (Instant
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Ocean; Aquarium Systems, Sarrebourg, France; 0.01% methylene blue) in Petri dishes. Embryos were dechorionated with Pronase solution (100 g/ml; Roche, Basel, Switzerland) at 6 hours postfertilization (hpf), and then they were treated with 0.003% phenylthiourea to prevent pigmentation at 24 hpf. We used 2 zebrafish strains: AB (commonly referred to as WT) and tg(fli1:EGFP)y1 (17). A peptides, diluted first in DMSO and then in fish water (1, 2.5, and 5 M), were administered from the 12-somite stage (somitogenesis) until 7 days postfertilization (dpf; larval stage) of zebrafish development. Fresh aliquots were administrated every 24 h. Control embryos were maintained in 0.1% DMSO in fish water. Treated embryos/larvae were anesthetized with 0.04 mg/ml tricaine (Sigma, Milan, Italy) and observed to select images of phenotypes. Images were taken with a Leica MZFLIII epifluorescence stereomicroscope equipped with a DFC 480 R2 digital camera and LAS Leica
imaging software (Leica, Wetzlar, Germany). Images were processed using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). This imaging procedure was used for processing the in vivo data, except for plastic sections. Senescence-associated -galactosidase (-Gal) quantification in cells, in zebrafish, and in histological sections Zebrafish larvae (7 dpf) were fixed at room temperature for 2 h in 4% paraformaldehyde/PBS and then washed 3 times for 15 min in 1⫻ PBS. Zebrafish embryos were incubated for 4 h at 37°C with -Gal staining solution (KAA002; Chemicon) as recommended by the vendor. All animals were photographed under the same conditions, and -Gal activity in each animal was quantified, using a selection tool in Adobe Photoshop for blue color range, according to Kishi et al. (18).
Figure 1. A) Transgenic zebrafish tg(fli1:EGFP)y1 at 72 hpf following repeated A peptide (2.5 M) treatment. Stereomicroscopic images (⫻3.2) of whole fish bodies, both brightfield (left panels) and fluorescence (right panels). Asterisks indicate disorganized intersegmental vessels. B) Transgenic zebrafish tg(fli1:EGFP)y1 at 72 hpf following A peptide treatment (2.5 M). Image represents subintestinal vein (SIV) basket in dorsal view (⫻10). Asterisks indicate protruding SIV spikes. C) Alkaline phosphatase activity in SIV basket at 72 hpf, following A peptide exposure (2.5 M). SIV basket in dorsal view (⫻10). Red arrows indicate SIV basket; black arrows indicate renal plexus. D) SIV basket (lateral view, ⫻10) at 72 hpf. White squares represent areas of quantification. Asterisk indicates SIV spikes protruding toward the yolk, together with pericardial edema overshadowing the SIV basket (E22Q). E, F) Quantification of vessel extensions and diameter in the SIV basket. *P ⬍ 0.05 vs. other groups; ANOVA. REGULATION OF VASCULAR SENESCENCE BY A AMYLOIDS
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TABLE 1. Effects of A peptide repeated treatment on AB zebrafish survival at 72 hpf Treatment
Control 1 M 2.5 M 5 M
Peptide
Survival (%)
WT E22Q WT E22Q WT E22Q
93 ⫾ 1.2 91 ⫾ 1.5 87 ⫾ 1.1 86 ⫾ 1.7 83 ⫾ 1.3 69 ⫾ 1.4 50 ⫾ 2.1
Values are means ⫾ sd (rounded to nearest decimal) for 6 measurements. AB zebrafish were treated up to 72 hpf with A peptides (1–5 m). Survival rate based on 35 embryos for each treatment.
For histological analysis, zebrafish larvae were fixed for 4 h at room temperature in 4% paraformaldehyde/PBS. Then, larvae were stained as described above and dehydrated in 30 –50-70% EtOH at room temperature. After immersion in a mixture of 2:1 LR white resin (London Resin Company, Working, UK):70% EtOH, larvae were washed and then immerged in pure LR white resin overnight on a rotary device. Embedding was performed at 50°C with LR white resin. Each larvae were sectioned on a Reichert Ultracut E (Reichert, Seefeld, Germany) at 3 m and stained with basic fuxine for light microscopy. Images were taken with a Leica microscope DM6000B equipped with a Leica DCF480 digital camera and the software LAS (Leica Application Suite). Whole-mount alkaline phosphatase staining Zebrafish embryos at 72 hpf were fixed in 4% paraformaldehyde for 2 h at room temperature and stained for endogenous alkaline phosphatase activity (19). Then, embryos were mounted in agarose-coated Petri dishes and photographed under a Leica MZFLIII epifluorescence stereomicroscope equipped with a DFC 480 R2 digital camera and LAS Leica imaging software. In situ hybridization (ISH) of p21 Whole-mount ISH was carried out as described by Thiesse et al. (20). Embryos (72 hpf) were fixed for 2 h in 4% paraformaldehyde/PBS and then rinsed with PBS-Tween, dehydrated in 100% methanol, and stored at ⫺20°C until they were processed for ISH (21). Embryos were treated 40 min with proteinase K (10 g/ml in PBS). The embryos were prehybridized at least 3 h at 62°C in hybridization buffer (50% formamide, 5⫻ SSC, 50 g/ml heparin, 500 g/ml tRNA, 0.1% Tween 20, and 1 M citric acid pH 6.0). The hybridization was done in the same buffer containing 200 ng of probe in 400 l mix overnight at 62°C (22). The p21 probe was kindly provided by Dr. M. Mione (University of Milan, Milan, Italy). Embryos were photographed as described above.
RESULTS Treatment with A peptides affects vascular development in zebrafish To study early signs of senescence and vascular degeneration in vivo, we chose the zebrafish (Danio rerio), a model organism characterized by the rapid development of the vascular system. Most studies were performed on the WT strain (AB), except for a few investigations conducted on the transgenic strain tg(fli1:EGFP)y1 for the easy imaging of blood vessels in developing living embryos (23). The influence of A peptides was examined in zebrafish starting from the 12-somite stage (⬃15 hpf). We adopted a 7-d observation period, since it covers the entire organogenesis and the larval period, enabling the evaluation of the subchronic effect of molecules on vascular development. WT and E22Q peptides were dissolved into fish water at 1, 2.5, and 5 M, renewed every 24 h, checking for fibril formation. This approach was chosen as it is widely used for testing biological active molecules in the zebrafish system (24). The range of active doses was modulated according to the different genetic background. In the AB line at 1 M, both peptides exhibited neither general toxicity (inferred from mortality) nor changes in the circulatory patterns. Embryo survival was not greatly reduced by treatment with WT and E22Q, the increase in mortality with respect to control embryos being modest at 2.5 M (survival rate ⬎80% in both groups). The general morphology was not grossly altered by this treatment, and vasculogenesis occurred normally (Fig. 1A). At the highest tested dose (5 M), the WT and E22Q peptide resulted in an increase in toxicity in the AB line (Table 1). From the data reported in this table, we calculated the IC50 for survival (6.1 M for WT and 4.7 M for E22Q). Treated AB embryos showed some phenotypic alterations as early as 48 hpf, namely, pericardial edema, increased heart frequency, alteration of trunk blood flow, and sparse cerebral microhemorrhagic foci (data not shown). The transgenic zebrafish tg(fli1:EGFP)y1 line shows a better survival rate than the AB line, when treated with A peptides at 5 M (Table 2). Although no vasculogenesis defects were found, alterations in angiogenesis TABLE 2. Effects of A peptide repeated treatment on tg(fli1: EGFP)y1 zebrafish survival at 72 hpf Treatment
Control 1 M 2.5 M
Peptide
Survival (%)
WT E22Q WT E22Q WT E22Q
90 ⫾ 2.7 86 ⫾ 1.3 85 ⫾ 1.3 84 ⫾ 1.2 79 ⫾ 1.2 75 ⫾ 1.8 64 ⫾ 2.6
Statistical analysis
5 M
When necessary, data are expressed as means ⫾ sd. An ANOVA test was used in statistical analysis for comparison, and P⬍ 0.05 was used as the criterion for statistical significance.
Values are means ⫾ sd (rounded to nearest decimal) for 4 replicative measurements. tg(fli1:EGFP)y1 zebrafish were treated for up to 72 hpf with A peptides (1–5 m). Survival rate based on 35 embryos for each treatment.
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were evident at 2.5 M of each peptide. This concentration was used throughout the zebrafish study. At this concentration, the intersegmental vessels (ISVs) were slightly disorganized (Fig. 1A) and the subintestinal vein (SIV) basket was clearly affected (Fig. 1B), showing a reduction of vessel branching with short spikes protruding toward the yolk (Fig. 1B). To better analyze angiogenic defects, we used endogenous alkaline phosphatase activity (19), focusing on the SIV basket and the adjacent renal venular plexus to examine changes occurring in the AB zebrafish vasculature (25). We found an abnormal extension of the renal plexus evident in both treated groups (Fig. 1C). The SIV basket in the WT peptide group showed a reduction of basket vessel size that was more pronounced in the E22Q group (Fig. 1C–F; n⫽30). Quantification of the SIV images provided evidence for the decreased extension and the reduction of vessel diameter in the embryonic SIV plexus throughout the dorsal ventral axis (Fig. 1E, F). A peptides induce increased -Gal activity in zebrafish -Gal activity is a marker of senescence validated in a previous work on zebrafish mutants (18). We used a -Gal assay at 7 dpf to directly analyze whether A peptides were inducing senescence in treated AB larvae. This assay reveals that staining, detectable in
control group (DMSO 0.1%, n⫽35), increased with the A treatment, reaching the highest intensity with the E22Q peptide (Fig. 2A, B; P⬍0.0001 for WT and E22Q vs. control and for E22Q vs. WT; n⫽36 for WT and n⫽41 for E22Q). Detailed histological sections were carried out to look at -Gal assay staining in specific tissues. This analysis showed staining in treated zebrafish at the level of pronephric ducts, liver, gut, and axial vessels (Fig. 2C). A peptide chronic treatment affects endothelial replicative ability Studies on isolated endothelial cells were performed on HUVECs, extensively used as model in vascular senescence. Long-term proliferation of HUVECs, cultured in 2% FBS and always passaged at 90% confluence, was followed for up to 80 –100 d, recording at each passage the extent of PDs (see Materials and Methods for details). In the experiment depicted in Fig. 3A, representative of 4 experiments with comparable results, we observed 22 CPDs. At this point, the cells ceased to proliferate, appearing flat and enlarged, displaying typical senescent phenotype. WT or E22Q peptides were used at 50 nM and 5 M, since a previous study (9) demonstrated that these concentrations (IC50 of 12.3 M for WT and IC50 of 8.8 M for E22Q) do not influence bromodeoxyuridine incorporation and are therefore devoid of acute effects on endo-
Figure 2. A) -Gal activity in whole-mount AB zebrafish at 7 dpf, following A repeated treatment at 2.5 M (images at ⫻3.2). Encircled areas (yolk) were excluded from the -Gal quantification, as recommended by Kishi et al. (18). B) -Gal quantification, expressed as stained pixel density (average n/group). ***P ⬍ 0.0001; 1-way ANOVA. C) Top panel: -Gal positive vessels in larvae trunk (transversal section, ⫻40), taken from whole mounts reported in A. Bottom panel: enlarged areas show axial vessels -Gal positive (arrows). NT, neural tube; NC, notochord; PD, pronephric ducts.
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Figure 3. A) Senescence in HUVECs under chronic treatment with A peptides. Evaluation of long-term growth curves for control (open squares), 50 nM WT (solid squares), and 50 nM E22Q (solid triangles) treatment. B) Premature onset of senescence in HUVECs under chronic treatment with A peptides (5 M). Evaluation of long-term growth curves; symbols as in A. C) Cell density of HUVECs at confluence as a function of CPD at each passage: control (open circles), WT 5 M (solid squares), and E22Q 5 M (solid triangles) treatment.
thelial cell proliferation. Chronic exposure of HUVECs to either peptide (5 M) produced differential changes in the pattern of the cell cumulative replication (Fig. 3A). In fact, closer analysis of the cell proliferation plot (PD vs. days in culture) revealed distinct growth phases: an initial linear rate followed by phases in which cell proliferation was increasingly slow, with the differences between peptides being more evident. Divergences between WT and E22Q emerged at ⬃PD 12 and became larger at PD 18 and beyond, indicative of a greater decline of cell growth for E22Q relative to WT (Fig. 3A). Further indication for the E22Q aggressive action is illustrated by its effects on cell replication, evident at 50 nM (PD 18), relative to WT (50 nM) or untreated cells (Fig. 3B). The changes in the proliferation rate reflected HUVEC density. The E22Q and WT peptides (5 M) reduced cell population by ⬃1 order of magnitude, relative to control, at PD 12 (Fig. 3C). A peptides increase -Gal activity in HUVECs We evaluated the expression of -Gal activity in HUVECs at 6, 12, and 18 PD. We found time-related increases of frequency of -Gal-positive cells with either WT or E22Q (4- to 5-fold at 5 M). A representative histochemical image of cells at PD 18 is shown in Fig. 4A. Quantification of these changes, detectable at PD 12 and 18 but not at PD 6, indicates that the highest concentration of either WT and E22Q induced a significant increase (P⬍0.001) of -Gal-positive cells, relative to their respective con2390
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trol (Fig. 4B). Consistent with the reduced endothelial replication at PD 18, we also found increases of -Gal activity, with E22Q at 50 nM (P⬍0.05; Fig. 4A). A peptides reduce hTERT expression and telomerase activity We measured mRNA expression of hTERT during HUVEC incubation, precisely at PD 6 and 18, observing a reduction of mRNA hTERT expression only for the E22Q (5 M) at PD 6, whereas at PD 18 the mRNA hTERT reduction was significant for both peptides (Fig. 4C). We extended the study to the telomerase enzyme activity. Given the extremely low level of enzymes detected in HUVECs (12), measurements were limited to PD 6 and 12. At PD 6, we noted a marked decrease of enzyme activity in either WT or E22Q groups (5 M) relative to controls (Fig. 4D). Enzyme levels at PD 18 are not reported being below the assay detection limit. Senescence-associated signals in HUVECs and zebrafish Given the decline of hTERT expression and telomerase activity, we wondered whether replicative senescence involved either the p53-p21 or the ERK p38MAPK pathways. Accordingly, we analyzed these pathways at PD 6 and 12 in HUVECs exposed to A peptides (5 M).
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Figure 4. A) Photomicrographs (⫻20) of senescence-associated -Gal staining of control, WT, and E22Q (5 M) in HUVECs at PD18 obtained with an inverted microscope (Nikon Eclipse TE300). B) HUVECs expressing -Gal activity (%) at PD 12 and 18. Bars represent mean ⫾ se percentage. C) hTERT mRNA assayed by quantitative RT-PCR in HUVECs at PD 6 and 18. Data reported as equivalents of nanograms mRNA reference/150 ng of total RNA. Bars represent means ⫾ se. D) Telomerase activity in HUVECs at PD 6 and 12. Bars represent percentage activity vs. control at PD 6. E) A peptides affect p38MAPK phosphorylation, evaluated by Western blotting as described in Materials and Methods. Results were normalized vs. total p38 and the respective phospho p38/total-p38. Graph reports optical density. Gel represents 3 exhibiting similar results. F) A peptides affect p21 production in HUVECs. p21 evaluated by Western blotting. Gel represents 3 exhibiting similar results. Normalization ratio p21/actin. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs Ctr.; #P ⬍ 0.05, ##P ⬍ 0.01, ###P ⬍ 0.001vs. Ctr.; ANOVA.
Phosphorylation of ERK p38, negligible at PD 6, was evenly up-regulated in both control and treated groups at PD 12, and therefore it appears unrelated to WT or E22Q exposure (Fig. 4E). In contrast, the p21 production was enhanced in A-treated cells, marginally at PD 6, but very significantly for the E22Q over the WT at PD 12 (Fig. 4F; P⬍0.05 for WT vs. control and P⬍0.01 for E22Q vs. control). Thus, p53-p21 is the prevailing pathway involved in the A-driven senescence. We also analyzed, by whole-mount ISH, the expression of p21 in zebrafish embryos (72 hpf), finding a striking REGULATION OF VASCULAR SENESCENCE BY A AMYLOIDS
increase of signal in the rostral head region of A-peptidetreated fish (2.5 M), in sharp contrast with the absence of signal in control. The p21 expression in treated embryos was also diffused throughout the body (Fig. 5). Pseudocapillary formation in senescent endothelial cells The observed impairment of endothelial cell proliferation led us to examine their functional characteristics in terms of angiogenic potential. We compared 2391
Figure 5. p21 ISH in embryos at 72 hpf. Lateral (⫻4) view of control and treated embryos (WT and E22Q at 2.5 M), showing p21 mRNA expression in whole body, with enrichment in the head.
the ability of control HUVECs vs. chronically Apeptide-treated cells taken at PD 6 and 12 to produce capillary tubes when plated in Matrigel. HUVECs at PD 6 produced a rich meshwork of capillary-like structures invading the Matrigel in a well-organized manner (Fig. 6A). WT or E22Q peptides (both at 5 M) yielded a less dense mesh, which for E22Q was associated with evident disruption of pseudocapillary tubes (Fig. 6B, C). At PD 12, control HUVECs maintained their ability to assemble into network-like structure (Fig. 6D), while A-treated cells ceased to develop tube structures (Fig. 6E, F). Quantification of the numbers of circles formed by tubular HUVECs at PD 6 and 12 highlights the impressive decrease of capillary tubes in the presence of A peptides (Fig. 6G; P⬍0.05), reminiscent of the alterations observed in the SIV basket of zebrafish embryos.
DISCUSSION The CAA sporadic form is a degeneration of brain blood vessels causing reduced blood flow of brain parenchyma, contributing to the progression of AD, with which it is often associated. CAA is primarily an age-related disease occurring in the sixth decade of life. The Dutch variant, a dominant hereditary human CAA, in which a point mutation at codon 693 of APP yields a single amino acid substitution at position 22 of the A40 peptide, exhibits a distinct clinical phenotype, characterized by an early onset of recurrent stroke episodes, occurring as early as in the 2392
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fourth decade, and severe clinical presentation. CAA pathologies are well documented in morphological terms and in their clinical presentation. Moreover, a number of concurrent mechanisms contributing to A deposition have been proposed. They include failure of enzymatic A degradation by proteases (neprilysin and insulin-degrading enzyme), (26 –27), diminished perivascular drainage, (28), and impairment of receptor-mediated adsorption into the blood via low-density lipoprotein receptor-related protein (LRP)-1 (29) and P-glycoprotein (30). The impairment of any of the above-mentioned clearance pathways results in the accumulation of A in the brain vessels. In the later stages of CAA pathology, as oligomeric and aggregated A species replace smooth muscle cells and their basement membrane, the failure of the perivascular drainage, a rate-limiting step in the overall clearance process, aggravates the pathology, producing devastating effects on all the components of the neurovascular unit (28). Here, we present evidence that the A peptides involved in neurodegeneration affect neovessel formation by impairing the angiogenesis process. In addition, we show that the endothelial dysfunction caused by the A peptides is an early event, which in the zebrafish model occurs during the embryonic stage of vascular development, and evolves to a loss of function of the vasculature associated with accelerated endothelial cell senescence. The results obtained in this study support the notion that A peptides induce premature senescence in the vascular endothelium. In vivo, we demonstrate the appearance of A-mediated endothelial senescence in zebrafish at late embryonic/early larval stages and an acceleration of senescence in isolated endothelial cells (HUVECs) cultured in the presence of A peptides. Evidence sustaining this hypothesis was gleaned in functional experiments, in morphological studies, and from the analysis of intracellular signals. The earliest signs of premature senescence were seen in A-treated zebrafish embryos in which -Gal activity, although diffused in the whole body, appeared to be particularly enriched in the trunk vessels. The progressive switch toward the senescent phenotype was also a prominent feature of endothelial cells chronically cultured in the presence of A peptides. As reported with different experimental conditions, the evidence of accelerated endothelial cells senescence was documented by the marked down-regulation of the hTERT transcript and telomerase activity (13). Measurements of p21 protein expression, and of p38 phosphorylation, showed that A treatment predominantly up-regulates p21 expression. This suggests that A-provoked senescence is driven by the telomeredependent p53/p21 pathway, which is activated in parallel to the stress-induced p38 program (31). The involvement of the p53/p21 pathway in premature senescence is underscored by the early (3 dpf) p21 expression in treated zebrafish embryos. Thus, it is plausible that p21 overexpression and the onset of senescence might be the mechanisms underlying many of the morphological changes occurring in treated
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Figure 6. In vitro tube formation by HUVECs in Matrigel, treated with A peptides (5 M). Photomicrographs of gels (representative of 4) obtained by an inverted microscope (Nikon Eclipse TE300). A) PD 6 control. B) Exposure to WT. C) Exposure to E22Q. D) PD 12 control. E) Exposure to WT. F) Expsoure to E22Q. G) Quantification of above gels. Results expressed as number of complete circles per quadrant of a 24-multiplate well (n⫽4). *P ⬍ 0.05, **P ⬍ 0.01 vs. control (Ctr.).
zebrafish and of the functional impairment found in cultured endothelial cells. Changes in the intersomitic vessels of treated embryos resulted in a disorganized vessel patterning, indicative of altered vascular development. Interestingly, the action of A on zebrafish vessels was evident at developmental stages characterized by active vessel remodeling (25). Thus, the well-documented antiangiogenic action of A on mature vessels (32) appears to be fully operant during the embryonic stage and might contribute to shape adult vessels. The acute antiangiogenic effects of A, previously described in a variety of in vitro preparations of endothelial cells, including human brain microvascular endothelium (33) and bovine coronary postcapillary venules, have been attributed to interference with the signaling pathways of proangiogenic molecules such as FGF-2 and VEGF (10, 34). Although these reports delineate an important action of A, they do not fully explain the vascular degeneration found in CAA, typically a slow progressive disease. Here, we chose to investigate neocapillary formation in HUVECs exposed to long-term (up to 20 d in culture, PD 12) A treatment in Matrigel. The results indicate that the continuous A exposure leads to irreversible modifications in the inherent angiogenic program, as the endothelium becomes REGULATION OF VASCULAR SENESCENCE BY A AMYLOIDS
progressively unresponsive to the strong stimuli (nutrients and growth factors) contained in the Matrigel assay milieu. Thus, chronic A treatment causes HUVECs to acquire typical features of senescence, such as insensitivity to mitogenic stimuli. In fact, impairment of the HUVEC angiogenic drive coincided with p21 up-regulation, reduction of HUVEC replicative capacity, and decline of hTERT mRNA expression. A peptides, besides reducing the stereotyped tubular meshwork, caused the formation of disrupted network architecture. Of note is the consistent increase of the antiangiogenic effects of Dutch mutant peptide. Since we have shown that A peptides markedly down-regulate the endothelial FGF-2 pathway (9, 10), it is also conceivable that growth factor deprivation plays a role in the disintegration of vessels noted in vitro. In fact, recent evidence clearly demonstrates that the continuous presence of FGF-2 is a prerequisite for preserving vascular integrity (35). The range of vascular alterations observed in treated zebrafish suggests a potential link to the defects found in brain microvessels of patients affected by sporadic CAA or the Dutch variant. In line with the severity of the Dutch clinical phenotype, the E22Q produced in zebrafish and cultured endothelium more aggressive and injurious effects (e.g., -Gal accumulation, decline of hTERT mRNA 2393
expression, increased p21 production, and diminished network formation). While the physiological functions of A are now being recognized (36 –38), most of the studies on this peptide focus on its accumulation leading to toxic effects and, more recently, on the specificity of its action on the endothelium (3, 33). While this report was under revision, another report (39) showed that WT A toxic effects, causing impairment of endothelial functions, occur through a mechanism of autophagy. Our results support the hypothesis that A-mediated impairment of vascular remodeling/maturation with the consequent neurovascular degeneration, typical of AD and CAA, is an early event, particularly for the mutated E22Q peptide. Consistent with this view, the reduction of vessel density and cerebral blood flow has been observed in APP transgenic models harboring mutated A peptides (40 – 42). The accelerated senescence of the endothelium, due to the continuous exposure to A peptides, contributes to progressive alterations of vessels compromised by a deficiency of angiogenic drive. The proposed mechanism provides a rationale for the widely different A-mediated neurovascular diseases, including AD and CAA. The authors thank Dr. M. Mione (University of Milan, Milan, Italy) for kindly providing the p21 probe for ISH, and Dr. E. Foglia for the technical assistance for histological analysis in zebrafish. This work was supported by TelethonProject No. GGP06148. M.B. and F.C. acknowledge financial support from the CARIPLO Foundation (grant 2006.0807) and CARIPLO N.O.B.E.L Biological and Molecular Characterization of Cancer Stem Cells, respectively.
10.
11.
12.
13. 14.
15.
16. 17. 18.
19. 20.
REFERENCES 1. 2.
3.
4. 5.
6. 7.
8.
9.
2394
Zacchigna, S., Lambrechts, D., and Carmeliet, P. (2008) Neurovascular signalling defects in neurodegeneration. Nat. Rev. Neurosci. 9, 169 –181 Revesz, T., Holton, J. L., Lashley, T., Plant, G., Frangione, B., Rostagno, A., and Ghiso, J. (2009) Genetics and molecular pathogenesis of sporadic and hereditary cerebral amyloid angiopathies. Acta Neuropathol. 118, 115–113 Revesz, T., Ghiso, J., Lashley, T., Plant, G., Rostagno, A., Frangione, B., and Holton, J. L. (2003) Cerebral amyloid angiopathies: a pathologic, biochemical, and genetic view. J. Neuropathol. Exp. Neurol. 62, 885– 898 Maia, L. F., Mackenzie, I. R., and Feldman, H. H. (2007) Clinical phenotypes of cerebral amyloid angiopathy. J. Neurol. Sci. 257, 23–30 Wisniewski, T., Ghiso, J., and Frangione, B. (1991) Peptides homologous to the amyloid protein of Alzheimer’s disease containing a glutamine for glutamic acid substitution have accelerated amyloid fibril formation. Biochem. Biophys. Res. Commun. 179, 1247–1254 Maat-Schieman, M., Roos, R., and van Duinen S. (2005) Hereditary cerebral hemorrhage with amyloidosis-Dutch type. Neuropathology 25, 288 –297 Miravalle, L., Tokuda, T., Chiarle, R., Giaccone, G., Bugiani, O., Tagliavini, F., Frangione, B., and Ghiso, J. (2000) Substitutions at codon 22 of Alzheimer’s abeta peptide induce diverse conformational changes and apoptotic effects in human cerebral endothelial cells. J. Biol. Chem. 275, 27110 –27116 Paris, D., Humphrey, J., Quadros, A., Patel, N., Crescentini, R., Crawford, F., and Mullan, M. (2003) Vasoactive effects of A beta in isolated human cerebrovessels and in a transgenic mouse model of Alzheimer’s disease: role of inflammation. Neurol. Res. 25, 642– 651 Solito, R., Corti, F., Fossati, S., Mezhericher, E., Donnini, S., Ghiso, J., Giachetti, A., Rostagno, A., and Ziche, M. (2009)
Vol. 24
July 2010
21. 22.
23. 24. 25. 26.
27.
28.
29.
Dutch and Arctic mutant peptides of beta amyloid (1– 40) differentially affect the FGF-2 pathway in brain endothelium. Exp. Cell Res. 315, 385–395 Donnini, S., Cantara, S., Morbidelli, L., Giachetti, A., and Ziche, M. (2006) FGF-2 overexpression opposes the beta amyloid toxic injuries to the vascular endothelium. Cell Death Differ. 13, 1088 – 1096 Kurz, D. J., Hong, Y., Trivier, E., Huang, H. L., Decary, S., Zang, G. H., Luscher, T. F., and Erusalimsky, J. D. (2003) Fibroblast growth factor-2, but not vascular endothelial growth factor, upregulates telomerase activity in human endothelial cells. Arterioscler. Thromb. Vasc. Biol. 23, 748 –754 Kurz, D. J., Decary, S., Hong, Y., Trivier, E., Akhmedov, A., and Erusalimsky, J. D. (2004) Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J. Cell Sci. 117, 2417–2426 Erusalimsky, J. D., and Skene, C. (2009) Mechanisms of endothelial senescence. Exp. Physiol. 94, 299 –304 Kurz, D. J., Decary, S., Hong, Y., and Erusalimsky, J. D. (2000) Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J. Cell Sci. 113, 3613–3622 Pezzatini, S., Morbidelli, L., Solito, R., Paccagnini, E., Boanini, E., Bigi, A., and Ziche, M. (2007) Nanostructured HA crystals up-regulate FGF-2 expression and activity in microvascular endothelium promoting angiogenesis. Bone 41, 523–534 Hull, M. A., Thomson, J. L., and Hawkey, C. J. (1999) Expression of cyclooxygenase 1 and 2 by human gastric endothelial cells. Gut 45, 529 –536 Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, T. F. (1995) Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 Kishi, S., Bayliss, P. E., Uchiyama, J., Koshimizu, E., Qi, J., Nanjappa, P., Imamura, S., Islam, A., Neuberg, D., Amsterdam, A., and Roberts, T. M. (2008) The identification of zebrafish mutants showing alterations in senescence-associated biomarkers. PLoS Genet. 4, e1000152 Serbedzija, G. N., Flynn, E., and Willett, C. E. (1999) Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 353–359 Thisse, C., Thisse, B., Schilling, T. F., and Postlethwait, J. H. (1993) Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 119, 1203–1215 Jowett, T., and Lettice, L. (1994) Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigeninand fluorescein-labelled probes. Trends Genet. 10, 73–74 Santoriello, C., Deflorian, G., Pezzimenti, F., Kawakami, K., Lanfrancone, L., d’Adda di Fagagna, F., and Mione, M. (2009) Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells. Dis. Model. Mech. 2, 56 – 67 Lawson, N. D., and Weinstein, B. M. (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 Zon, L. I., and Peterson, R. T. (2005) In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4, 35– 44 Isogai, S., Horiguchi, M., and Weinstein, B. M. (2001) The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev. Biol. 230, 278 –301 Farris, W., Schutz, S. G., Cirrito, J. R., Shankar, G. M., Sun, X., George A. Leissring, M. A., Walsh, D. M., Qiu, W. Q., Holtzman, D. M., and Selkoe, D. J. (2007) Loss of neprilysin function promotes amyloid plaque formation and causes cerebral amyloid angiopathy. Am. J. Pathol. 171, 241–251 Leissring, M. A., Farris, W., Chang, A. Y., Walsh, D. M., Wu, X., Sun, X., Frosch, M. P., and Selkoe, D. J. (2003) Enhanced proteolysis of beta amyloid in APP Transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 40, 1087–1093 Weller, R. O., Subash, M., Preston, S. D., Mazzanti, I., and Carare, R. O. (2008) Perivascular drainage of amyloid- peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer’s disease. Brain Pathol. 18, 253–256 Bell, R. D., and Zlokovic, B. V. (2009) Neurovascular mechanisms and blood – brain barrier disorder in Alzheimer’s disease. Acta Neuropathol. 118, 103–113
The FASEB Journal 䡠 www.fasebj.org
DONNINI ET AL.
30.
31. 32.
33.
34.
35.
Cirrito, J. R., Deane, R., Fagan, A. M., Spinner, M. L., Parsadanian, M., Finn, M. B., Jiang, H., Prior, J. L., Sagare, A., Bales, K. R., Paul, S. M., Zlokovic, B. V., Piwnica-Worms, D., and Holtzman, D. M. (2005) P-glycoprotein deficiency at the blood brain barrier increases amyloid- deposition in an Alzheimer disease mouse model. J. Clin. Invest. 115, 3285–3290 Muller, M. (2009) Cellular senescence: molecular mechanisms, in vivo significance, and redox considerations. Antioxid. Redox. Signal. 11, 59 –98 Paris, D., Townsend, K., Quadros, A., Humphrey, J., Sun, J., Brem, S., Wotoczek-Obadia, M., DelleDonne, A., Pate, N., Obregon, D. F., Crescentini, R., Abdullah, L., Coppola, D., Rojiani, A., Crawford, M. F., Sebti, S. M., and Mullan, M. (2004) Inhibition of angiogenesis by A peptides. Angiogenesis 7, 75– 85 Paris, D., Aitghezala, G., Mathura, V. S., Patel, N., Quadros, A., Laporte, V., and Mullan, M. (2005) Anti-angiogenic activity of the mutant Dutch A peptide on human brain microvascular endothelial cells. Mol. Brain Res. 136, 212–230 Patel, N. S., Mathura, V. S., Bachmeier, C., Beaulieu-Abdelahad, D., Laporte, V., Weeks, O., Mullan, M., and Paris, D. (2009) Alzheimer’s beta-amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2. J. Neurochem. 112, 66 –76 Murakami, M., Nguyen, L. T., Zhang, Z. W., Moodie, K. L., Carmeliet, P., Stan, R. V., and Simons, M. (2008) The FGF system has a key role in regulating vascular integrity. J. Clin. Invest. 118, 3355–3366
REGULATION OF VASCULAR SENESCENCE BY A AMYLOIDS
36.
37.
38. 39.
40. 41.
42.
Cantara, S., Donnini, S., Morbidelli, L., Giachetti, A., Schulz, R., Memo, M., and Ziche, M. (2004) Physiological levels of amyloid peptides stimulate the angiogenic response through FGF-2. FASEB J. 18, 1943–1945 Puzzo, D., Privitera, L., Leznik, E., Fa` M, Staniszewski, A., Palmeri, A., and Arancio, O. (2008) Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus. J. Neurosci. 28, 14537–11454 Garcia-Osta, A., and Alberini, C. M. (2009) Amyloid beta mediates memory formation. Learn. Mem. 16, 267–272 Hayashi, S., Sato, N., Yamamoto, A., Ikegame, Y., Nakashima, S., Ogihara, T., and Morishita, R. (2009) Alzheimer disease-associated peptide, amyloid beta40, inhibits vascular regeneration with induction of endothelial autophagy. Arterioscler. Throm. Vasc. Biol. 29, 1909 –1915 Iadecola, C. (2004) Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat. Rev. 5, 347–360 Miao, J., Xu, F., Davis, J., Otte-Holler, I., Verbeek, M. M., and Van Nostrand, W. (2005) Cerebral microvascular amyloid  induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid  precursor protein. Am. J. Pathol. 167, 505–515 Zipfel, G. J., Han, H., Ford, A. L., and Lee, J. M. (2009) Cerebral amyloid angiopathy: progressive disruption of the neurovascular unit. Stroke 40, S16 –S19 Received for publication September 28, 2009. Accepted for publication February 4, 2010.
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