Alzheimer Disease–Associated Peptide, Amyloid β40, Inhibits Vascular Regeneration With Induction of Endothelial Autophagy
Objective— Although the majority of cases of Alzheimer disease (AD) are known to be attributable to the sporadic (nongenetic) form of the disease, the mechanism underlying its cause and progression still remains unclear.
Methods and Results— We found that vascular β-amyloid (Aβ), Aβ40, inhibited the proliferative activity of human brain vascular endothelial cells (HBECs) without toxic effects on them. This peptide also inhibited tube formation and migration of HBECs. Moreover, Aβ40 inhibited ex vivo hippocampal revascularization, reendothelialization, and the differentiation of adult endothelial progenitor cells. Importantly, Aβ40 suppressed the proliferative activity of HBECs through the induction of “self-digesting” autophagy. This induction involved the intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as Akt signaling in HBECs. Furthermore, tissue culture of murine brain sections from GFP-LC3 transgenic mice revealed that Aβ40 not only reduced the vessel density in hippocampal lesions, but also induced autophagy in neurovascular ECs.
Conclusions— Our present findings indicate that the initial progression of AD might be in part driven by Aβ40-induced endothelial autophagy and impairment of neurovascular regeneration, suggesting important implications for therapeutic approaches to AD.
Nearly 100 years ago, Alolis Alzheimer first described a possible vascular disorder as well as neuronal lesions in Alzheimer disease (AD) patients.1 AD is characterized by a progressive neuronal disorder that causes dementia and is recognized as a major cause of death in the elderly population.1–3 The number of individuals with AD is currently 4.5 million in the United States and is predicted to triple by the year 2050 in industrial countries. However, no treatment can stop AD today. Like diabetes, 2 distinct conditions exist in AD: rare early-onset inherited familial cases, which account for only about 5% of cases, and nongenetic sporadic cases, which account for more than 90% of AD.4 The typical features of AD are Aβ-containing plaques and τ-containing neurofibrillary tangles in the diseased brain. Aβ42, a minor product of amyloid precursor protein (APP), has been extensively focused on in rare familial AD, not because it composes plaque but because its overproduction is associated with genetic abnormality of the APP, presenilin-1, and presenilin-2 genes. In contrast, Aβ40 (Aβ1-40), whose overproduction is not associated with presenilin mutations, predominantly exists around vessels and accumulates as a major component of vascular amyloid deposits,5 suggesting possible involvement of Aβ40 in neurovascular dysfunction in sporadic AD. However, it is still unclear how Aβ40 is involved in the pathogenesis of AD.
In the field of vascular biology, it is realized that vascular formation is essential not only for embryonic organ development but also for adult tissue regeneration.6,7 Together with the recent interpretations that sporadic AD is thought to be associated with vascular disease such as cardiovascular disease, stroke, and atherosclerosis,8 we hypothesized that Aβ40 might induce phenotypic alterations in neurovascular ECs, which then causes neurovascular dysfunction, suppresses neurovascular regeneration, and accelerates vascular neural disorder in AD.
We performed all procedures according to protocols approved by Animal Care Committees of Gifu University Graduate School of Medicine and Osaka University Graduate School of Medicine.
Aβ40 (Aβ1-40), Aβ40-1, and Aβ42 were obtained from Peptide Institute. Rapamycin and 3-methyladenin (3-MA) were purchased from Sigma. Please see methods in detail regarding Aβ peptides in the supplemental materials (available online at http://atvb.ahajournals.org).
HBECs, human aortic ECs, human umbilical vein ECs, and VSMCs were obtained from Applied Cell Biology Research Institute. We cultured ECs in Modified MCDB131 medium supplemented with 2% FBS and an endothelial growth supplement kit (Clonetics), so-called EGM, and used them for experiments between passages 3 to 5. Vascular smooth muscle cells were cultured in DMEM supplemented with 10% FBS. Cells that had reached 80% confluence were used for the following experiments.
ECs or VSMCs cultured on 12-well culture plates were preincubated in Modified MCDB131 medium with 5% FBS for 24 hours, and stimulated with Aβ peptides for 72 hours. The cell number in each group was manually counted with a hemocytometer. Proliferative activity of vascular cells was also confirmed by BrdU-ELISA according to the manufacturer’s instructions (Roche).
Capillary Tube Formation Assay
HBECs were seeded on 6-well culture plates coated with Matrigel Basement Membrane Matrix (BD Bioscience), and treated with Aβ40 or Aβ40-1 for 18 hours. Tube-forming ECs were photographed, and their total lengths were quantified with imaging software (Kurabo). All groups were studied in at least 4 independent experiments.
A migration assay was performed using a modified Boyden chamber (BD Bioscience). Briefly, serum-free medium with vascular endothelial growth factor (VEGF; 50 ng/mL, Peprotech Inc), Aβ40 (0.5 μmol/L), Aβ40-1 (0.5 μmol/L), or 10% FBS was put in the lower compartment of the chamber, seeded fluorescence (Calcein-AM, Molecular Probes)-labeled HBECs in the upper chamber (5×104 cells/50 μL), and they were incubated for 6 hours. Transmigrated cells were counted in 4 random high-power fields (×400). All groups were studied in at least 4 independent experiments.
Detection of Apoptosis
HBECs cultured on 12-well culture plates were stimulated with or without Aβ peptides (0.5 μmol/L) for 18 hours, and fixed with 1% paraformaldehyde (PFA) in 0.1 mol/L PBS (pH 7.4) for 20 minutes. Cells were stained with a primary antibody against cleaved caspase-3 (Cell Signaling) and a species-specific Alexa Fluor secondary antibody (Molecular Probes Inc). Advanced stages apoptotic ECs were stained with Hoechst 33342 (Sigma) after 48 hours of treatment with Aβ peptides. The number of apoptotic cells was counted and normalized with reference to the total number of cells in each field.
Cell Cytotoxicity Assay
HBECs cultured on 12-well culture plates were stimulated with or without Aβ peptides (0.5 μmol/L) for 24 hours. Released lactate dehydrogenase (LDH) in the cell culture supernatant was measured using the enzymatic reaction with an LDH cytotoxicity detection kit (Takara).
Ex Vivo Hippocampal Revascularization Assay
Please see methods in detail in the supplemental materials.
Ex Vivo Reendothelialization Assay
Please see methods in detail in the supplemental materials.
Please see methods in detail in the supplemental materials.
Detection of Autophagy
HBECs were cultured with or without Aβ peptides (0.5 μmol/L) for 18 hours. Cell sections in epon were counterstained with uranyl acetate and lead citrate and examined with a Hitachi H7600 transmission electron microscopy (TEM). Autophagic HBECs were also visualized by microtubule-associated protein light-chain3 (LC3) staining of the transduced GFP-LC3 construct as described previously.9 Please see methods in detail in the supplemental materials.
HBECs cultured in 6-well culture plates were transiently transfected with 1 μg constitutive active Akt constructs or pUSE-Amp Vector (control constructs without the Akt gene, UBI), using TransIT-LT1 reagent 48 hours before Aβ treatment. These cells were reseeded on 12-well culture plates 24 hours before Aβ treatment, and incubated with EGM for additional 24 hours. Then, cells were treated with Aβ peptides for 48 hours. Proliferative activity was examined as described above. In some case, HBECs were transfected with both active Akt construct and pEGFP-LC3. Transfection efficiency (40% to 45%) and viability of cells (85% to 90%) were initially tested at 48 hours after transfection.
Imaging of Autophagy Induction in Hippocampus
Eight-week-old male GFP-LC3 transgenic mice were used as marker of autophagy induction.10 Briefly, brain tissues from age-matched GFP-LC3 transgenic mice were vertically cut into 2-mm slices in ice-cold PBSG (PBS with 0.6% glucose). Tissue culture of these slices containing hippocampal lesions was performed with EGM and Aβ40 peptides for 48 hours. Frozen sections (6 μm) from these brain slices were postfixed with 4% PFA in 0.1 mol/L PBS for 20 minutes and stained with rat antimouse platelet endothelial cell adhesion molecule-1 (PECAM-1) primary antibody (Pharmingen), Alexa 488-conjugated goat anti-rat secondary antibody (Molecular Probe), and DAPI (Molecular Probe). Vascular density (PECAM-1) and autophagy induction (GFP-LC3) in hippocampal CA1 lesions were evaluated with a fluorescence microscope.
Ex Vivo Imaging of Autophagic Endothelium
Please see methods in detail in the supplemental materials.
Each result was obtained in at least 4 separate experiments. All values are expressed as mean±SEM. ANOVA was used to determine the significance of differences in multiple comparisons. P<0.05 was considered statistically significant.
Aβ40 Inhibits Proliferation, Capillary Tube Formation, and Migration of Human Vascular ECs
Microvascular formation initially requires several steps, including endothelial proliferation, capillary tube formation, and migration.7 As shown in Figure 1A, Aβ40 (Aβ1-40) significantly reduced proliferation of HBECs as compared to control, whereas Aβ40-1, as a control peptide with a reversed amino acid sequence, did not affect them. Aβ40 also inhibited the proliferation of human aortic ECs and human umbilical vein ECs, but did not affect proliferation of vascular smooth muscle cells (VSMCs; Figure 1A and data not shown). In contrast, Aβ42 inhibited proliferation of both human vascular ECs and VSMCs, suggesting the possibility that Aβ40 but not Aβ42 has endothelium specific effects. We also obtained consistent results that Aβ40 impaired endothelial proliferation by using a BrdU incorporation assay (data not shown). Notably, Aβ40 did not increase lactate dehydrogenase (LDH) release from HBECs after 24 hours of treatment, whereas Aβ42 markedly increased LDH release, suggesting that Aβ40 has low toxic effects on HBECs as compared to Aβ42 (Figure 1B and 1C). In addition, we showed that Aβ40 significantly reduced capillary tube formation of HBECs after 18 hours of treatment (Figure 1D). Moreover, vascular endothelial growth factor (VEGF) stimulated the migration of HBECs, whereas stimulation by VEGF was significantly reduced by Aβ40 (Figure 1E). These results indicate that Aβ40 impairs in vitro vascular regeneration through its effects on ECs.
Effects of Aβ40 on Ex Vivo Endothelial Regeneration
We next addressed the question of whether Aβ40 might affect endothelial regeneration in the setting of AD. As about 50% of patients with hippocampal hypoperfusion converted to preclinical stage AD with mild cognitive impairment,8,11 we first examined the effect of Aβ40 on endothelial regrowth in hippocampal lesions. Using our ex vivo model of hippocampal revascularization (supplemental Figure IA), we found newly formed vessels positive for PECAM-1 around hippocampal explants on day 3 (Figure 2A). In contrast, however, Aβ40 significantly impaired vessel sprouting from explants (Figure 2A and B).
We also investigated whether Aβ40 might affect the healing process of the endothelium after vascular injury. We prepared rat aortic tissue on culture dishes, made a scratched wound on the endothelial surface, and evaluated ex vivo reendothelialization after 7 days of stimulation with endothelial growth medium (EGM) and Aβ40 (supplemental Figure IB). In control vessels without Aβ40, reendothelialization was observed in around 84% of scratched endothelium, which stained negatively with Evans blue dye. Similarly, 80% of the scratched lesions were reendothelialized in vessels with Aβ 40-1. In contrast, only about 24% of scratched lesions were reendothelialized in vessels with Aβ40, indicating that Aβ40 significantly inhibited the reendothelialization after vascular injury (Figure 2C and 2D). These results were confirmed by staining for endothelium specific uptake of DiI-labeled acetylated LDL (data not shown).
Recent reports indicate that endothelial progenitor cells (EPCs) in the adult peripheral circulation, in part, contribute to revascularization in hindlimb ischemia and ischemic brain disease.12,13 As shown in Figure 2E, differentiated EPCs from isolated mononuclear cells were characterized by adherent cells double-positive for DiI acetylated-LDL uptake (red) and lectin binding (green). Interestingly, incubation of mononuclear cell with Aβ40 significantly decreased the number of EPC by about 65% compared to that in the control group, whereas Aβ40-1 did not affect the number of EPC (Figure 2E and 2F). Together, these results indicate the possibility that Aβ40 suppresses endothelial regeneration in the setting of AD.
Aβ40 Induces Endothelial Autophagy Rather Than Apoptosis in Human Neurovascular ECs
To investigate whether Aβ40 induces phenotypic alterations in neurovascular ECs, we initially performed activated-caspase-3 staining after 18 hours of treatment with Aβ peptides (0.5 μmol/L) in cultured HBECs. Immunofluorescent staining (green) revealed that Aβ40 slightly increased the number of activated-caspase-3–positive cells as compared to control cells (8% versus 2% of total cells, respectively), indicating that Aβ40 has a weak effect on the induction of apoptosis, a well-known form of programmed cell death (PCD), in neurovascular ECs. In contrast, Aβ42 significantly increased the number of apoptotic cells (22% of total cells) (Figure 3A and 3B).
We next investigated whether Aβ40 induces another process of PCD with formation of autophagosomes and autolysosomes, so-called “self-digesting” autophagy.14,15 As shown in Figure 3C, the expression of microtubule-associated protein light-chain3 (LC3), as a quantification marker of the early stage of autophagic formation,9 was significantly higher in HBECs treated with Aβ40 than in control, indicating the induction of molecular alterations for autophagy. Strikingly, the present study demonstrated by transmission electron microscopy (TEM) numerous double-membrane vesicles containing cytoplasmic organelles characteristic of autophagy in about 40% of HBECs after 18 hours of Aβ40 treatment, whereas few autophagic cells were observed in control cells without Aβ40 or with Aβ 40-1 (Figure 3D, upper and middle panels, and data not shown). Alternatively, we transduced a GFP-LC3 construct into HBECs,9 and monitored GFP-LC3 spots in the cell cytosol. Immunofluorescent images showed consistent results that Aβ40 induced endothelial autophagy with enhanced LC3 spots (Figure 3D, lower right panel), whereas fewer enhanced LC3 spots were observed in the cytoplasm of GFP-LC3–transfected control cells without Aβ40 and of GFP-transfected cells with Aβ40 (Figure 3D, lower left panel, and data not shown). Notably, the number of autophagic cells with enhanced LC3 spots was markedly lower in the group with Aβ42 than in group with Aβ40 (20% versus 38%, respectively; Figure 3E).
To further investigate whether inhibition of endothelial autophagy attenuates the impairment of endothelial proliferation by Aβ40, we evaluated proliferation of HBECs after treatment with Aβ40 and 3-methyladenin (3-MA), a widely used inhibitor of autophagy as well as an inhibitor of class3 PI3K. We found that treatment of HBECs with 3-MA attenuated the impairment of endothelial proliferation by Aβ40 (Figure 3F), suggesting the possibility that inhibition of endothelial autophagy restores neurovascular regrowth, and that class 3 PI3K is in part involved in Aβ40-induced endothelial autophagy. Together, these results indicate that neurovascular ECs exposed to Aβ40 undergo autophagy rather than apoptosis.
Akt Signaling Correlates With Aβ40-Induced Endothelial Autophagy and Impairment of Endothelial Proliferation
Although Akt signaling is involved in cell survival or PCD of a wide range of cell types,16 recent reports indicated that signaling control of autophagy overlaps with class 1 PI3K-Akt signaling.17,18 As Akt signaling is also involved in VEGF-induced vascular formation and endothelial cell survival,19 the effects of Aβ40 on the Akt signaling cascade were examined in HBECs. As shown in supplemental Figure II, Aβ40 significantly suppressed the phosphorylation of Akt (Ser473-phosphorylated) induced by VEGF (50 ng/mL). Aβ40 also suppressed the active level of endothelial nitric oxide synthase (eNOS; Ser1177-phosphorylated), whose activation led to NO production responsible for vascular remodeling and vascular formation.20,21 These results suggest that Aβ40 inhibits the Akt signaling cascade in human neurovascular ECs.
To confirm whether enhancing the active Akt level may attenuate Aβ40-induced impairment of neurovascular regrowth, we transduced HBECs with the constitutive active Akt construct and evaluated their proliferative activity. Although treatment with Aβ40 (0.5 μmol/L) impaired proliferation of HBECs, transfection of the active Akt construct significantly attenuated the Aβ40-induced impairment of endothelial proliferation (Figure 4B). In vitro capillary tube formation assay showed consistent results that transfection of the active Akt construct enhanced tube formation of HBECs after treatment with Aβ40 (data no shown). To test whether Akt activation may regulate Aβ40-induced endothelial autophagy, HBECs transduced with the GFP-LC3 construct or active Akt construct were monitored after treatment with Aβ40 by fluorescence microscope. Notably, transfection of the active Akt construct significantly reduced the number of autophagic ECs with enhanced LC3 spots in their cytosol (Figure 4C and 4D). These data suggest that class 1 PI3K-Akt signaling in human neurovascular ECs is in part involved in endothelial autophagy and impairment of endothelial regrowth by Aβ40.
Aβ40 Induces Endothelial Autophagy in Hippocampus
To further investigate whether Aβ40 in hippocampal lesions might induce endothelial autophagy, we performed tissue culture of murine hippocampal slices (2 mm) from GFP-LC3 transgenic mice, as a useful tool for detecting in vivo autophagy,10 and stimulated them with EGM and Aβ peptides for 48 hours. Frozen sections (6 μm) from these slices were used for fluorescence detection of LC3 (green) and PECAM-1 (red) in hippocampal CA1 lesions. Notably, both Aβ40 and Aβ42 significantly increased the number of autophagic cells with enhanced LC3 spots in CA1 lesions as compared to control with EGM stimulation alone (Figure 5A, GFP-LC3, and Figure 5B). In addition, as we expected from the results above (Figures 1 and 2A), both Aβ40 and Aβ42 also reduced vessel density in CA1 lesions (Figure 5A, PECAM-1). Strikingly, double-fluorescence images of LC3 and PECAM-1 revealed that Aβ40 increased the number of autophagic vessels in CA1 lesions (Figure 5A, merged, enlarged, and Figure 5C), indicating that Aβ40 induces endothelial autophagy in the hippocampus. Unexpectedly, Aβ42 induced fewer numbers of autophagic vessels in hippocampal lesions than did Aβ40, although Aβ42 showed effects on autophagy induction in nonvascular neural cells (Figure 5A, merged and enlarged, and Figure 5C). Moreover, by using tissue culture of aorta from GFP-LC3 mice, we obtained consistent results that Aβ40 rather than Aβ42 had stronger effects on autophagy induction in vascular tissue endothelium (supplemental Figure III). These results suggest that Aβ40 induces endothelial autophagy in the hippocampus in the setting of AD, and this induction associates with Aβ40-induced impairment of vascular regrowth.
It is widely believed that Aβ peptides have a fundamental role in the pathogenesis of AD, ultimately leading to neuronal degeneration and dementia. Among the Aβ fragments, Aβ42 has been extensively investigated in the field of AD research because of the following findings. First, its overproduction in rare inherited familial AD is associated with preseniline mutations and γ-secretase activity. Second, Aβ42, a minor product of APP metabolism (less than 10% of total Aβ), was characterized as being far more amyloidogenic than other major forms of Aβ, and might have a large effect on Aβ deposition. Third, several experimental findings indicated the possibility that targeting of Aβ42 production with drugs such as nonsteroidal antiinflammatory drugs, secretase inhibitors, or by immunization might improve AD symptoms.22,23 Although the clinical outcome is similar in rare familial AD and common sporadic AD, there is little evidence to support the above findings in the latter case. Additionally, those promising therapies could not stop AD and might cause serious side effects.
In contrast, Aβ40 is produced as the major form of all Aβ and is the predominant component of cerebrovascular amyloid.5 Because of its weaker amyloidgenic nature than that of Aβ42, the molecular basis of its contribution to the vascular/neural disorder in AD has remained elusive. As a growing body of evidence suggests that sporadic AD is attributable to brain dysfunction caused by impaired brain microcirculation, leading to reduce delivery of nutrients for neural activity,8 we focused on the effects of Aβ40 on vascular function to elucidate the unresolved precise mechanism. The present study demonstrated that Aβ40 impairs the critical steps of vascular regeneration, including proliferation, migration, and tube formation of human neurovascular ECs in vitro (Figure 1). Importantly, this inhibitory effect of Aβ40 on neurovascular regeneration was also confirmed by ex vivo hippocampal revascularization, reendothelialization, and differentiation of EPC (Figures 2 and 5⇑). Thus, Aβ40 might impair the capacity of ECs for brain tissue regeneration in the setting of AD. The finding that Aβ40 markedly inhibits reendothelialization is also important in anticipating the functional and structural alterations in cerebral blood vessels of the diseased brain.
One of the crucial findings of this study is that Aβ40 per se induces endothelial autophagy, a form of type2 PCD, which is characterized by the formation of double or multiple membrane-bound autophagosomes that self-digest intracellular organelles,14 but does not induce cell toxicity and apoptosis in human neurovascular ECs during our experimental time periods (Figures 1 and 3⇑). Although autophagy can be considered a temporal survival mechanism during nutrient starvation, recent studies give credence to this process as achieving cell death.14,15 Importantly, we showed that an inhibitor of autophagy, 3-MA, attenuates the inhibitory effects of Aβ40 on endothelial proliferation and hippocampal revascularization (Figure 3F and data not shown). These data suggest that induction of endothelial autophagy by Aβ40 is likely to delay the tissue response for endothelial regeneration, even if this process temporally works as a cell survival mechanism until conditions improve. The experiments using 3-MA and blot membrane with LC3B antibody also suggested that endothelial autophagy induced by Aβ40 seems to be controlled by class3 PI3K, which is involved in the early stages of autophagy.
We also found that Aβ40 blocks activation of Akt and eNOS induced by VEGF. As an essential role of Akt and eNOS is known to be regulation of vascular formation,19 impairment of neurovascular formation by Aβ40 may be mediated by the regulation of Akt signaling in HBECs. Alternatively, we found that Aβ40 induces endothelial autophagy in part through the regulation of class1 PI3K-Akt signaling in HBECs, and this regulation seems to be involved in Aβ40-induced impairment of neurovascular regrowth (Figure 4). Further studies will be required to determine how Aβ40 modulates PI3K-Akt signaling to promote autophagy in neurovascular ECs, although recent reports show that signaling control of autophagy overlaps with phosphatase and tensin homologue (PTEN)-PI3K-Akt signaling.17,18 It is suggested that PTEN functions as a major negative regulator of PI3K-Akt signaling and is inactivated through its phosphorylation.24,25 Notably, treatment of HBECs with VEGF transiently increased the phosphorylated PTEN level (inactivated PTEN level), whereas Aβ40 significantly decreased the inactivated PTEN level (supplemental Figure II), indicating that Aβ40 might reactivate PTEN by inhibiting VEGF-induced PTEN inactivation. Further studies will be required for this mechanism.
Together, these findings suggest that Aβ40 induces endothelial autophagy rather than apoptosis through its effects on both class3 PI3K and class1 PI3K-Akt signaling.
As the initial clinical phase of AD, characterized by mild cognitive impairment, is strongly associated with hippocampal hypofusion,8,11 the present findings that Aβ40 markedly reduced vessel density in the murine hippocampus might explain this phenomenon (Figures 2A and 5⇑A). A more crucial finding is that Aβ40 increases the number of autophagic ECs in the hippocampus, and this event associates with reduced hippocampal vessel density (Figure 5). Aβ42 also reduces vessel density in the hippocampus (Figure 5A and 5C), but its effect on the autophagic pathway seems to be enhanced in neural cells rather than in ECs (Figure 5). This finding is supported by a report that Aβ42 may be involved in neuronal autophagy.26 Our series of experiments suggest the possibility that Aβ40 and Aβ42 have independent effects on the autophagic pathway in neurovascular ECs. Further studies will need to address how these differential effects of Aβ peptides on ECs occur at the molecular level, although a report suggests that the short fragment of Aβ peptides may have endothelium-specific effects.27 How Aβ peptides mediate neuronal damage in AD is also unclear. Given the recent notions that axon guidance and vascular formation often take advantage of one another to follow the same path,28 Aβ40-induced phenotypic alteration of neurovascular ECs may correlate with impaired neuronal regeneration in AD.
It is also important to note that Aβ40 may induce the transitory changes in BBB permeability and promote transendothelial migration of peripheral monocytes by regulating receptor advanced glycation end products (RAGE) in neurovascular ECs,29 suggesting that Aβ40 may have indirect effects on neurovascular endothelium apart from its direct effects on ECs.
Whatever the mechanisms are, Aβ40 is one of the critical molecules that accelerates AD pathological changes with induction of endothelial autophagy. Overall, the present study demonstrated that Aβ40 inhibited the initial steps of vascular regeneration. These mechanisms presumably involve Aβ40-induced phenotypic alteration of the neurovascular endothelium with an increase in autophagic changes. Together with advanced aging and vascular risk factors, these effects of Aβ40 might accelerate the development of AD. Our study further provides an opportunity to consider new therapeutic applications based on regulating endothelial autophagy in AD and sets a new stage for future investigation of AD.
We thank T. Yoshimori for providing the GFP-LC3 construct, N. Mizushima and Riken Bioresource Center for providing GFP-LC3 mutant mice, and laboratory members for discussion and encouragement.
Sources of Funding
This work was supported by a Dean’s grant from Gifu University (to S.-i.H.).
Received March 22, 2009; revision accepted July 22, 2009.
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