Patent Application
20090181911
This invention relates to at least promotion of angiogenesis, suppression of apoptosis, or increase of low density lipoprotein receptor-related protein 1 (LRP-1) mediated clearance of amyloid P peptide in neurovascular cells.
Alzheimer's disease (AD) is the major cause of dementia in the elderly population. Since the first description of neuronal and vascular lesions in this heterogenous disorder by Alzheimer1, there has been little understanding how the two lesions relate to each other and how they contribute to a chronic neurodegenerative disease process2. Recent findings on co-morbidity of cerebrovascular disorder and AD3,4, the link between atherosclerosis and AD5,6, cognitive impairment associated with amyloid angiopathy7, major brain microvascular pathology8,9, insufficient angiogenesis in AD10-12 and deficient clearance of Alzheimer neurotoxin, amyloid β-peptide (Aβ), across the blood-brain barrier (BBB)13-15, indicate that neurovascular dysfunction is a critical feature of AD and could have a major impact on the pathogenesis of a chronic neurodegenerative condition.
According to the neurovascular hypothesis2, dysfunction of the neurovascular unit suggests manifold pathogenic cascades for AD including: cerebrovascular flow dysregulation and hypoperfusion16,17, aberrant angiogenesis and vascular remodeling10-12, and faulty clearance of Aβ13-15 which all could initiate neurovascular uncoupling, vessel regression, and neurovascular inflammation, resulting in a chemical demise of the neuronal microenvironment and ultimately, synaptic and neuronal dysfunction, injury and loss. Here, we show the transcriptome profiles of human brain endothelial cells (BEC) indicate that a small subset of age-independent genes is altered in AD neurovasculature, including the homeodomain-transcription factor GAX (growth arrest-specific homeobox)18. GAX expression in the adult is restricted to the cardiovascular system and has multiple effects on the vascular phenotype19, but is low in AD neurovasculature. Restoring GAX expression in AD BEC was shown to stimulate angiogenesis, suppress AFX1 forkhead transcription factor-mediated apoptosis20, and increase the levels of a major Aβ clearance receptor at the BBB, the low density lipoprotein receptor-related protein 1 (LRP)13,14 associated with transcriptional upregulation of its receptor associated protein (RAP)21. Furthermore, partial deletion of the Gax gene in mice22 results in reductions in brain capillary density and the resting cerebral blood flow, loss of brain angiogenic response to hypoxia in vivo, and a deficient Aβ clearance from brain due to reduced LRP-1 levels at the BBB associated with low expression of RAP.
It is an object of the invention to provide an understanding of the role of GAX in neurovascular dysfunction and Alzheimer's disease.
In one embodiment, at least promotion of angiogenesis, suppression of apoptosis, increase of low density lipoprotein receptor-related protein 1 (LRP-1) mediated clearance of amyloid β peptide (Aβ), or any combination thereof is provided by a method comprising: (a) inserting a nucleic acid comprised of a GAX gene into one or more neurovascular cells and (b) expressing GAX in said neurovascular cells from said nucleic acid which is effective at least to promote angiogenesis, to suppress apoptosis, to increase LRP-1 mediated clearance of Aβ, or any combination thereof.
Further aspects of the invention will be apparent to a person skilled in the art from the following detailed description and claims, and generalizations thereto.
Methods for treating Alzheimer's disease (preventive and/or therapeutic) and use of an effective amount of a nucleic acid comprised of a GAX gene methods for manufacture of a pharmaceutical composition are provided. The amount and extent of treatment administered to a cell, tissue, or subject (any animal or human) in need of therapy or prophylaxis is effective in treating the affected cell, tissue, or subject. One or more properties/functions of neurovascular cells, vascular endothelium, and endothelial cells thereof, or the number/severity of symptoms of affected subjects, may be improved, reduced, normalized, ameliorated, or otherwise treated. GAX expression is directed by choice of transcriptional regulatory region, replication of nucleic acid, and delivery by a carrier of nucleic acid. A pharmaceutical composition comprised of an effective amount of nucleic acid comprised of a GAX gene and a physiologically-acceptable vehicle, which is packaged in an aseptic container, is also provided.
Such methods may be used alone or in combination with other known methods. Instructions for performing these methods, reference values, and controls (i.e., positive/negative) may also be used. Mammals (e.g., humans and rodent or primate models of disease) may be treated. Thus, both veterinary and medical methods are contemplated.
Preparations of endothelial cells, isolated endothelium, neurovascular cells, and in vitro cell cultures are provided from brain (e.g., microvasculature) or other organs (e.g., skin) of subjects at risk for Alzheimer's disease, affected by the disease, or not. In particular, tissues like endothelium, smooth muscle, blood vessels and capillaries of the brain, temporal and leptomeningeal arteries, or any other tissues representative of vascular endothelium can be examined for GAX expression. Blood and bone marrow cells might also be used. They can be obtained as biopsy or autopsy material; cells of interest may be isolated therefrom and then cultured. Also provided are extracts of cells; at least partially purified DNA, RNA, and protein therefrom; and methods for their isolation. These reagents can be used to establish detection limits for assays, absolute amounts of gene expression that are indicative of disease or not, ratios of gene expression that are indicative of disease or not, and the significance of differences in such values. These values for positive and/or negative controls can be measured at the time of assay, before an assay, after an assay, or any combination thereof.
Nucleotide sequences representative of the GAX gene whose expression is decreased in Alzheimer's disease may be used to identify, isolate, or detect complementary nucleotide sequences by binding assays. Similarly, one or more amino acid sequences representative of GAX which are decreased in Alzheimer's disease may be used to identify, isolate, or detect interacting proteins by binding assays. Optionally, bound complexes including interacting proteins may be identified, isolated, or detected indirectly though a specific binding molecule (e.g., antibody) for GAX.
The abundance of GAX transcript or polypeptide can be measured by techniques such as in vitro transcription, in vitro translation, Northern hybridization, nucleic acid hybridization, reverse transcription-polymerase chain reaction (RT-PCR), run-on transcription, Southern hybridization, cell surface protein labeling, metabolic protein labeling, antibody binding, immunoprecipitation (IP), enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent or histochemical staining, microscopy and digital image analysis, and fluorescence activated cell analysis or sorting (FACS).
An expression vector is a recombinant polynucleotide that is in chemical form either a deoxyribonucleic acid (DNA) and/or a ribonucleic acid (RNA). The physical form of the expression vector may also vary in strandedness (e.g., single-stranded or double-stranded) and topology (e.g., linear or circular). The expression vector is preferably a double-stranded deoxyribonucleic acid (dsDNA) or is converted into a dsDNA after introduction into a cell (e.g., insertion of a retrovirus into a host genome as a provirus). The expression vector may include one or more regions from a mammalian gene expressed in the microvasculature, especially endothelial cells (e.g., angiopoietin receptors Tie-1 or Tie-2, endoglin, endothelin-1 ET1, intercellular adhesion molecule ICAM-2, vascular endothelial growth factor receptors FLT-1 or FLK-1, and vascular endothelial growth factor VEGF), or a virus (e.g., adenovirus, adeno-associated virus, cytomegalovirus, herpes simplex virus, Moloney leukemia virus, mouse mammary tumor virus, Rous sarcoma virus, SV40 virus), as well as regions suitable for gene manipulation (e.g., selectable marker, linker with multiple recognition sites for restriction endonucleases, promoter for in vitro transcription, primer annealing sites for in vitro replication). The expression vector may be associated with proteins and other nucleic acids in a carrier (e.g., packaged in a viral particle or encapsulated in a liposome).
The expression vector further comprises one or more regulatory regions for gene expression (e.g., promoter, enhancer, silencer, splice donor and acceptor sites, polyadenylation signal, cellular localization sequence). Transcription from a drug-inducible regulatory region can be activated or silenced by tetracycline or dimerized macrolides. The expression vector may be further comprised of one or more splice donor and acceptor sites within an expressed region; a Kozak consensus sequence upstream of an expressed region for initiation of translation; downstream of an expressed region, multiple stop codons in the three forward reading frames to ensure termination of translation, one or more mRNA degradation signals, a termination of transcription signal, a polyadenylation signal, and a 3′ cleavage signal. For expressed regions that do not contain an intron (e.g., a coding region from a cDNA), a pair of splice donor and acceptor sites may or may not be preferred. It would be useful, however, to include a mRNA degradation signal if it is desired to express one or more of the downstream regions only under the inducing condition. An origin of replication may be included that allows replication of the expression vector integrated in the host genome or as an autonomously replicating episome. Centromere and telomere sequences can also be included for the purposes of chromosomal segregation and protecting chromosomal ends from shortening, respectively. Random or targeted integration into the host genome is more likely to ensure maintenance of the expression vector but episomes could be maintained by selective pressure or, alternatively, may be preferred for those applications in which the expression vector is present only transiently.
An expressed region may be derived from a gene encoding GAX in operative linkage with a transcriptional regulatory region (e.g., constitutive, regulated, drug-inducible, endothelial-specific, and/or viral promoter and an optional enhancer). The expressed region may encode a translational fusion. Open reading frames of regions encoding a polypeptide and at least one heterologous domain may be ligated in register. If a reporter or selectable marker is used as the heterologous domain, then expression of the fusion protein may be readily assayed or localized.
Gene activation may be achieved by inducing an expression vector that contains a downstream region related to a GAX gene or unrelated to the GAX gene that acts to relieve suppression of gene activation (e.g., MEF2). Alternatively, the downstream expressed region may direct homologous recombination into a locus in the genome and thereby replace an endogenous transcriptional regulatory region of the gene with an expression cassette. In particular, LRP-1 expression (and transport of Aβ across the blood-brain barrier) can be induced by introduction of an exogenous GAX gene or activating an endogenous GAX gene.
An expression vector may be introduced into a host mammalian cell or non-human mammal by a transfection or transgenesis technique using, for example, chemicals (e.g., calcium phosphate, DEAE-dextran, lipids, polymers), biolistics, electroporation, naked DNA technology, microinjection, or viral infection. The introduced expression vector may integrate into the host genome of the mammalian cell or non-human mammal. Many neutral and charged lipids, sterols, and other phospholipids to make lipid carrier vehicles are known. For example, neutral lipids are dioleoyl phosphatidylcholine (DOPC) and dioleoyl phosphatidyl ethanolamine (DOPE); an anionic lipid is dioleoyl phosphatidyl serine (DOPS); cationic lipids are dioleoyl trimethyl ammonium propane (DOTAP), dioctadecyldiamidoglycyl spermine (DOGS), dioleoyltrimethyl ammonium (DOTMA), and 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamide tetraacetate (DOSPER). Dipalmitoyl phosphatidylcholine (DPPC) can be incorporated to improve the efficacy and/or stability of delivery. FUGENE 6, LIPOFECTAMINE, LIPOFECTIN, DMRIE-C, TRANSFECTAM, CELLFECTIN, PFX-1, PFX-2, PFX-3, PFX-4, PFX-5, PFX-6, PFX-7, PFX-8, TRANSFAST, TFX-10, TFX-20, TFX-50, and LIPOTAXI lipids are proprietary formulations. The polymer may be polyethylene glycol (PEG) or polyethylenimine (PEI); alternatively, polymeric materials can be formed into nanospheres or microspheres. Naked DNA technology delivers the expression vector in plasmid form to a cell, where the plasmid may or may not become integrated into the host genome, without using chemical transfecting agents (e.g., lipids, polymers) to condense the expression vector prior to introduction into the cell.
Thus, a mammalian cell may be transfected with an expression vector; also provided are transgenic nonhuman mammals. In the previously discussed alternative, a homologous region from a gene can be used to direct integration to a particular genetic locus in the host genome and thereby regulate expression of the gene at that locus. Polypeptide may be produced in vitro by culturing transfected cells; in vivo by transgenesis; or ex vivo by introducing the expression vector into allogeneic, autologous, histocompatible, or xenogeneic cells and then transplanting the transfected cells into a host organism. Special harvesting and culturing protocols will be needed for transfection and subsequent transplantation of host stem cells into a host mammal. Immunosuppression of the host mammal post-transplant or encapsulation of the host cells may be necessary to prevent rejection.
The expression vector may be used to replace the function of a gene that is down regulated or totally defective or supplement function of a partially defective gene. Thus, the cognate gene of the host may be neomorphic, hypomorphic, hypermorphic, or normal. Replacement or supplementation of function can be accomplished by the methods discussed above, and transfected mammalian cells or transgenic nonhuman mammals may be selected for high expression (e.g., assessing amount of transcribed or translated product, or physiological function of either product) of the downstream region.
Nucleic acids may be used to formulate a pharmaceutical composition with one or more of the utilities disclosed herein. Use of a physiologically acceptable vehicle and compositions which further comprise carriers for delivering a nucleic acid to a subject are known in the art. Addition of such vehicles and carriers to the composition is well within the level of skill in this art. Compositions may be administered in vitro to cells in culture, in vivo to cells in the body, or ex vivo to cells outside of the subject that may later be returned to the body of the same subject or another. Such cells may be diaggregated or provided as solid tissue.
Pharmaceutical compositions may be administered as a formulation adapted for passage through the blood-brain barrier or direct contact with the endothelium. Alternatively, pharmaceutical compositions may be added to the culture medium. In addition to the nucleic acid, such compositions may contain a physiologically-acceptable vehicle and other ingredients known to facilitate administration, condense the nucleic acid, enhance uptake, or any combination thereof (e.g., saline, dimethyl sulfoxide, lipid, polymer, affinity-based cell specific-targeting systems). The composition may be incorporated in a gel, sponge, or other permeable matrix (e.g., formed as pellets or a disk) and placed in proximity to the endothelium for sustained, local release. The composition may be administered in a single dose or in multiple doses which are administered at different times.
Pharmaceutical compositions may be administered by any known route. By way of example, the composition may be administered by a topical (e.g., epidermal, mucosal, or pulmonary) or other localized or systemic route (e.g., enteral and parenteral). The term “parenteral” includes subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intrathecal, and other injection or infusion techniques, without limitation.
Suitable choices in amounts and timing of doses, formulation, and routes of administration can be made with the goals of achieving a favorable response in the subject with Alzheimer's disease or at risk thereof (i.e., efficacy), and avoiding undue toxicity or other harm thereto (i.e., safety). Therefore, “effective” refers to such choices that involve routine manipulation of conditions to achieve a desired effect.
A bolus administered once a day is a convenient dosing schedule. Alternatively, the effective daily dose may be divided into multiple doses for administration, for example, two to twelve doses per day. Dosage levels of active ingredients in a pharmaceutical composition can also be varied so as to achieve a transient or sustained concentration of the nucleic acid in a subject, especially in and around vascular endothelium of the brain (neurovascular cells), and to result in the desired therapeutic response or protection. But it is also within the skill of the art to start doses at levels lower than required to achieve the desired effect and to gradually increase the dosage until the desired effect is achieved.
The amount of nucleic acid administered is dependent upon factors known to a person skilled in the art such as its bioactivity and bioavailability (e.g., half-life in the body, stability, and metabolism); its chemical properties (e.g., molecular weight, hydrophobicity, and solubility); route and scheduling of administration; and the like. It will also be understood that the specific dose level to be achieved for any particular subject may depend on a variety of factors, including age, gender, health, medical history, weight, combination with one or more other drugs, and severity of disease.
The term “treatment” of Alzheimer's disease refers to, inter alia, reducing or alleviating one or more symptoms in a subject, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis, and/or preventing disease in a subject who is free therefrom as well as slowing or reducing progression of existing disease. For a given subject, improvement in a symptom, its worsening, regression, or progression may be determined by an objective or subjective measure. Efficacy of treatment may be measured as an improvement in morbidity or mortality (e.g., lengthening of survival curve for a selected population). Prophylactic methods (e.g., preventing or reducing the incidence of relapse) are also considered treatment. Treatment may also involve combination with other existing modes of treatment (e.g., ARICEPT or donepezil, EXELON or rivastigmine, anti-amyloid vaccine, mental exercise or stimulation). Thus, combination treatment with one or more other drugs and one or more other medical procedures may be practiced.
The amount which is administered to a subject is preferably an amount that does not induce toxic effects which outweigh the advantages which result from its administration. Further objectives are to reduce in number, diminish in severity, and/or otherwise relieve suffering from the symptoms of the disease as compared to recognized standards of care.
Production of nucleic acids according to present regulations will be regulated for good laboratory practices (GLP) and good manufacturing practices (GMP) by governmental agencies (e.g., U.S. Food and Drug Administration). This requires accurate and complete recordkeeping, as well as monitoring of QA/QC. Oversight of patient protocols by agencies and institutional panels is also envisioned to ensure that informed consent is obtained; safety, bioactivity, appropriate dosage, and efficacy of products are studied in phases; results are statistically significant; and ethical guidelines are followed. Similar oversight of protocols using animal models, as well as the use of toxic chemicals, and compliance with regulations is required.
The following examples are merely illustrative of the invention, and are not intended to restrict or otherwise limit its practice.
To identify novel targets in AD neurovasculature, transcriptional profiling of human BEC derived from rapid brain autopsies from the frontal pole from 36 individuals was performed. First, six AD patients with severe pathology (Braak—V-VI23, CERAD (Consortium to Establish a Registry for Alzheimer's Disease protocol)—frequent or moderate24, clinical dementia rating (CDR) score—4, age—70 yrs); six neurologically normal non-demented age-matched controls with no or sparse pathology (Braak—0 or 0-I, CERAD—negative or sparse, dementia score—0, age—70 yrs); and five young controls with no pathology (age—24 yrs) were compared. There were no differences in gender, cause of death and incidence in the vascular risk factors between AD and age-matched controls (Table 1). Comparison of the transcriptome profiles (Affymetrix U95A) by the Bayesian t-test25 indicated a small subset of 34 genes, or 0.27% of approximately 12,600 genes studied, were significantly (P<0.05) altered in AD (2-fold or more,
A subset of functionally important genes was next validated by quantitative polymerase chain reaction (QPCR) of BEC isolated from brain tissue by laser capture microdissection and of BEC in culture, and by immunostaining of microvascular endothelium in brain tissue in situ (
Total brain capillary length in AD cortical tissue used for the BEC study (
Since homeobox genes play important roles in the final transcriptional regulation of pathways mediating angiogenesis and differentiation of vascular cells19, it was hypothesized that low levels of GAX expression in AD neurovasculature (
To determine the role of GAX in brain angiogenesis, human BEC were transduced with replication-incompetent adenovirus containing a short hairpin silencing double-stranded oligonucleotide construct specific for the GAX gene (Ad.shGAX). Human BEC transduced with GAX gene specific silencer, as compared to controls (Ad.shGFP, green fluorescence protein), express 40% of GAX homeoprotein (
GAX gene silencing increased by about 2-fold the levels of the AFX1 transcription factor in BEC (
To confirm that restoring GAX levels may correct aberrant AD BEC-mediated angiogenesis, AD BEC were transduced with a human GAX gene (Ad.hGAX). Transfer of GAX gene at a low multiplicity of infection (MOI) increased VEGF-mediated brain capillary tube formation by 2.8-fold (
To further understand possible discrepancy between previous work30,31 and the current results, the dose response of AD BEC to GAX gene transfer was studied. The angiogenic effect of GAX gene transfer into AD cells followed a U-shape curve with a plateau at 50-100 MOI, whereas forced GAX expression at a higher (i.e., 500) MOI was moderately antiangiogenic (
Thus, an “effective” amount of nucleic acid comprised of a GAX gene or an “effective” amount of GAX expression is empirically determined in comparison to the GAX expression conferred in AD BEC transduced with Ad.hGAX preferably at MOI less than about 200, more preferably at MOI less than about 150, or even more preferably at MOI less than about 100. MOI more than about 50 is also preferred.
To determine whether Gax affects brain microcirculation in vivo, Gax+/− mice22, which compared to Gax+/+ mice express <50% of brain capillary Gax homeoprotein (
To establish whether normal Gax expression is required for angiogenesis in vivo, the brain response to hypoxia was determined in Gax+/− and Gax+/+ mice using an established hypoxia model in which brain angiogenesis is driven by endogenous VEGF34. After three weeks of hypoxia, Gax+/+ mice increased brain capillary length by 38%, whereas Gax+/− mice did not exhibit a significant change in brain capillary density (
Also studied was deposition of Aβ alters Gax expression in Alzheimer Tg2576 APPsw+/− mice at 18-20 months of age when significant brain and vascular Aβ and amyloid accumulations develop35,36. Brain microvascular Gax was not affected by Aβ in these Tg2576 mice (
Reduced vascular competence and possibly incomplete BEC differentiation in Gax+/− mice was studied to determine whether Aβ clearance from brain interstitial fluid (ISF) was affected, using methods as described13,14. Gax+/− mice, as compared to Gax+/+ mice, showed substantial Aβ40 brain retention (
Silencing GAX gene expression substantially reduced LRP-1 expression in BEC (
Since silencing GAX gene did not affect the proteasomal proteolytic activity (
To address possible upstream events in AD BEC leading to reduced GAX expression, expression of the myocyte-specific enhancer factor-2 (MEF2) (
Recent findings suggest that altered brain capillary-unit physiology, compromised brain microcirculation, vascular neuroinflammatory response and disruption of brain activity-mediated CBF regulation are of major importance for the pathogenesis of cognitive decline in AD2,18. The present data suggest that low expression of vascularly-restricted homebox GAX gene in AD BEC mediates an aberrant angiogenesis, activates the AFX1-dependent proapoptotic pathway20 and suppresses expression of the LRP-1 clearance receptor for Aβ13-15 at the BBB. Thus, GAX may control a major neurovascular disease pathway in AD.
Since low GAX levels in AD brains in situ was shown to correspond to the low, left end of the angiogenic AD BEC curve in vitro, one would expect that restoring GAX expression in AD will promote angiogenesis and vascular remodeling and inhibit the AFX1-mediated apoptosis20. On the other hand, forced GAX expression in neurovasculature at high MOI may activate Bax and could be antiangiogenic likely due to Bax-mediated apoptosis32. But, extremely high levels of GAX as those achieved during forced GAX expression are seen only under the experimental conditions in vitro at the right end of the U-shape angiogenic curve, and are not found normally in healthy human brain or in a disease state.
No change in Gax expression in Tg2576 mice, which do not show neuronal death36 and low GAX expression in AD patients which have neuronal loss23,24 were demonstrated. This indicates that neuronal loss and GAX loss are associated with each other, but whether neuronal loss in AD precedes or is secondary to the changes in GAX expression is not clear at present. With respect to the link with oxidative stress which kills neurons and may directly down regulate GAX through activation of redox-sensitive mitogen activated protein kinase41, Aβ is the chameleon of the two worlds and exhibits both pro-oxidant and antioxidant properties42, and therefore may not necessarily affect GAX expression. In vitro data confirmed that exposure of BEC to Aβ42 oligomers and/or aggregated forms does not suppress GAX, as found in Tg2576 mice in vivo.
There is evidence that cerebral hypoperfusion impairs neuron metabolism and compromises protein synthesis that is essential for memory formation and plasticity9,43. Cerebral protein synthesis is suppressed at the CBF reductions between 30% and 50%43, as seen in Gax+/− mice, which may suggest that neuronal function in these mice, and perhaps in AD patients with reduced CBF, could be affected even though they do not have an outright stroke.
In conclusion, the homebox GAX gene may play an important role in neurovascular dysfunction in AD relevant to AD pathology. Its low expression in AD neurovasculature and in an animal model of the Gax gene partial deletion may lead (1) to impaired angiogenesis associated with apoptosis, vessel malformation and regression ultimately resulting in reductions in brain capillary density and CBF, as seen in AD2,3,8,9,16; and (2) to a pathological BBB phenol-type with little or no Aβ clearing capability due to low levels of LRP, which may lead to Aβ accumulation13-15 as seen in AD models and AD2,15. Thus, GAX could be a potential new therapeutic target for AD neurovascular disorder.
Patients and neuropathological diagnosis. BEC were isolated from rapid brain autopsies from the frontal pole (area 9/10) from 36 individuals. AD patients and age-matched controls were evaluated clinically and followed to autopsy at the AD Research Centers at the University of Southern California and the University of Rochester Medical Center. The CDR scores in AD and control individuals were 4-3 and 0, respectively. AD cases were Braak stage V-VI23 and CERAD24 frequent to moderate; controls were Braak 0 or 0-I and CERAD negative or sparse. In the first group, BEC transcriptome profiles from six AD patients, six age-matched controls, and five young controls were compared. For clinical and neuropathological characteristics see Table 1. The incidence of vascular risk factors (e.g., hypertension, atherosclerosis, etc.), the gender ratio, age, cause of death and the post-mortem interval were comparable between AD and age-matched controls. BEC from young controls (average age 23.4 years) were isolated from rapid brain autopsies of neurologically normal young individuals with no vascular risk factors autopsied after motor vehicle accidents at the Monroe Medical Examiner Center, New York. The microarray analysis of these young control BEC did not reveal significant differences in gene expression profiles compared with BEC derived from control cortical brain tissue after epilepsy surgery from young individuals of comparable age and gender. In the second group, BEC transcriptome profiles were compared in ten AD patients with severe pathology vs. nine age-matched controls with no or sparse pathology. The gender ratio, age, cause of death, the post-mortem interval and the incidence of vascular risk factors were comparable between AD and controls.
Laser capture microdissection (LCM) of BEC. Autopsy specimen of the frontal cortex (area 9/10) were snap frozen and cut (10 μm) using the Microm HM 500M cryostate. Cryosections were fixed for 5 min in ice cold acetone and air-dried. Capillary BEC were stained with biotinylated Ulex lectin (Vector 1:10) and treated with RNA SECURE (Ambion) employing ABC-peroxidase (Vector) and DAB. Contamination free LCM44 was done from dry, stained sections at 400× magnification by Zeiss AXIOVERT 200 inverted microscope equipped with PALM LCM system including a 337 nm laser and a robotic microscope table operated by the PALMROBO software. RNA was isolated from single cells or aggregates of 100-250 cells by the Zymo MINI RNA ISOLATION kit (Zymo Research #R1005). cDNA was made and two rounds linear amplification performed by the Ambion MESSAGE AMP aRNA kit. The quality and normal size distribution of cDNA fragments was controlled by Agilent 2100 Bioanalyzer using the NANO chip. The magnitude of aRNA amplification using the Ambion MESSAGE AMP aRNA kit was on the order of 105-106.
High-density oligonucleotide array hybridization. Total RNA was prepared from BEC with TRIZOL protocol (Gibco BRL). cDNA was synthesized, in vitro transcribed and hybridized to Affymetrix HG-U95A chip containing approximately 12,600 full-length cDNA from the UNIGENE cluster database. Statistical analysis was performed by the Bayesian t-test25 using the following criteria: at least 2-fold ratio of the Affymetrix signal, minimal signal of 500 (expression), and P values <0.05. Data were logarithmically transformed prior to statistical analysis. For selected genes, validation of the microarray results was performed by QPCR analysis of BEC isolated from tissue by LCM, QPCR analysis of cultured BEC (see below), and by immunostaining of BEC in tissue in situ.
Quantitative RT-PCR (QPCR). mRNA quantification was performed using TAQMAN™ chemistry with fluorescently tagged oligonucleotide probes45. Fluorescent intensity was detected by the Perkin-Elmer Applied Biosystem Sequence Detector 7700. Data were analyzed using Perkin-Elmer Sequence Detector Software version 1.6.3. Comparative analysis was performed using the delta-delta Ct approach as described by Applied Biosystems. The same cDNA was used for microarray hybridization and QPCR analysis.
Immunostaining of BEC in human tissue. Immunocytochemical analysis of selected proteins on brain microvessels in tissue was performed on paraffin sections (6 μm) of the frontal cortex (area 9/10) adjacent to the site of BEC LCM isolation. Antigen retrieval was performed by treating tissue sections with BD Retrievagen B (BD PharMingen, San Diego, Calif.). Image analysis was performed using Olympus AX70 microscope equipped with the SPOT digital camera. Ten randomly selected fields in each region from ten sections from Brodman A9/10 areas were analyzed. Monoclonal mouse antibody to human collagen IV (1:25, 75 mg/L; DAKO, A/S, Denmark) or polyclonal rabbit antibody to human Von Willebrand Factor (1:200, 5.7 mg/ml; DAKO, A/S, Denmark) were used to label microvessels, and fluorescein goat antibody to mouse IgG (1:150, 2 mg/ml; Molecular Probe, Eugene, Oreg.) was used as a secondary antibody. GAX was detected with polyclonal rabbit antibody against rat Gax which crossreacts with human GAX (1:200, gift from Dr Kenneth Walsh Boston University49) and secondary rhodamine goat antibody to rabbit IgG (1:150, 2 mg/ml); AFX1 with polyclonal rabbit antibody to human AFX1 (1:1000, 0.1 mg/ml; Sigma, St. Louis, Mo.) and rhodamine goat antibody to rabbit IgG (1:150, 2 mg/ml); ANK3 with monoclonal mouse antibody to human ankyrin G (1:100, 0.2 mg/ml; Santa Cruz Biotechnology, Santa Cruz, Calif.) and rhodamine goat antibody to mouse IgG (1:150, 2 mg/ml); PLEC1 with polyclonal goat antibody to human plectin 1 (1:100, 0.2 mg/ml; Santa Cruz Biotechnology) and rhodamine goat antibody to mouse IgG (1:150, 2 mg/ml); TGM2 with polyclonal rabbit antibody to human transglutaminase 2 (1:100,1 mg/ml; Calbiochem, San Diego, Calif.) and rhodamine goat antibody to rabbit IgG (1:150, 2 mg/ml).
Brain capillary length in human tissue. The paraffin-embedded coronal sections (6 μm thickness; adjacent to the BEC LCM isolation site) of each individual were cut and sampled in a systematic uniform random manner for each AD patient or control subject. Sections were immunostained for human Von Willebrand Factor in order to label vessels (10 sections per subject). Total brain capillary length was determined using IMAGEPRO PLUS software, similar as reported8.
Human BEC cultures. Primary human BEC were isolated from the frontal pole (area 9/10) adjacent to the site of BEC LCM isolation and immunostaining. BEC were sorted by flow activated cell sorting with Dil-Ac-LDL and characterized as reported46. Cells were cultured (10% fetal calf serum, 10% Nuserum, endothelial cell growth factors, nonessential amino acids, vitamins and penicillin/streptomycin in RPMI 1640) in 5% CO2 at 37° C. BEC were >98% positive for endothelial markers Factor VIII and CD105, and negative for CD11b (monocyte/microglia), glial fibrillar acidic protein (astrocytes) and α-actin (vascular smooth muscle)48. Early passage (P2-P4) cultures were used throughout the study.
GAX silencing by RNA interference, The BLOCK-iT Adenoviral RNAi expression system (Invitrogen)47 was used. A short hairpin silencing double-stranded oligonucleotide construct for the Gax gene was designed according to the MPI algorithm (see Tuschl Lab website). A selected sequence GGAAGGAAATTAC AAGTCAGA (SEQ ID NO:1) was cloned into the BLOCK iT U6 RNAi expression entry vector. The siRNA expression cassette was recombined into the adenoviral destination vector pAD/BLOCK-iT-DEST, which was transduced into HEK 293A cells for production of recombinant replication incompetent adenovirus. After virus particle purification by VIRAKIT ADENOMINI-4 (Virapur) and determination of virus titer by ADENO-X RAPID titer kit (BD Biosciences), the viral vector (Ad.shGAX) was used to transduce primary human or mouse BEC for expression of the shRNA GAX specific silencer. pAd U6-GFP shRNA silencer of GFP (Ad.shGFP) was used as a control. Specific downregulation of GAX was confirmed by Western blot analysis.
GAX plasmid and adenoviral constructs. An adenoviral construct expressing the human homolog of GAX (Ad.hGAX) was from Dr. David Gorski (UMDNJ—Robert Wood Johnson Medical School, NJ)30. Ad.GFP was obtained from Dr. Joseph Miano (University or Rochester). Viral titers were determined by plaque assay. Prior to use of Ad.hGAX in BEC, expression of GAX mRNA and protein in transduced cells were verified by Northern and Western blot analysis.
3-D capillary morphogenesis assay. This assay has been described in detail elsewhere29. Briefly, 2×106 BEC/ml from AD and age-matched controls (Table 1) or young controls were suspended within 3-D collagen matrices at 30 μL per well in the serum-free culture Medium 199 containing VEGF165 and FGF-2 (Upstate Biotechnology, Lake Placid, N.Y.) at 40 ng/ml in 5% CO2 at 37° C. Cultures were fixed with 3% glutaraldehyde in phosphate buffer saline and stained with toluidine blue and Hoechst 33342. The formation of the intracellular vacuoles (stage I) and tubes (stage II) were studied within 24 hr. The cells were considered to be in a vacuolar stage when ≧30% of the cell surface was occupied by vacuole(s)29. Tubes were defined as elongated cells at least 15 μm in length with a lumen. Total tube length per field was measured using IMAGEPRO PLUS software. TUNEL staining was performed as described below.
Matrigel capillary tube formation assay. This was performed as previously described50. Briefly, control human primary BEC transduced with Ad.shGAX or Ad.shGFP, primary AD BEC transduced with Ad.hGAX or Ad. GFP, and primary mouse BEC derived from Gax+/− mice and littermate controls (see below) were plated on growth factor reduced Matrigel matrix (Becton Dickinson) at 2×104 cells per well in 48-well plates in RPMI1640 medium containing 0.1% FBS. After four to six hours at 37° C., VEGF165 was added to 10 ng/ml and incubation continued overnight. To quantify the tubular structures, images from four fields per well in duplicate wells were photographed at ×10 magnification with a digital camera (Spot) attached to a Nikon microscope. Total tube length per field was measured using IMAGEPRO PLUS software.
Transgenic mice. Gax+/− mice22 at 2-3-month and 10-12-month of age, Tg2576 APPsw+/− mice35 at 18-20-month of age, and AhR−/− mice38 at 2-3 month of age were used. Animal studies were performed according to the National Institutes of Health guidelines using an approved institutional protocol.
Immunostaining of BEC in mouse tissue. For Gax staining on brain microvessels in Tg2576 and control mice, 14 μm frozen acetone fixed tissue sections and double immunostaining for Gax and CD31 (endothelial marker) were used. For CD31 staining, mouse CD31-specific IgG was used as a primary antibody, and Alexa Fluor 594 donkey anti-rat IgG (1:500, Molecular Probes, Inc. Eugene, Oreg.) as a secondary antibody. Ten randomly selected fields from ten sections spanning the entire cortex from four mice per group were analyzed.
Brain capillary length in mouse tissue. To determine total brain capillary length in Gax+/− and AhR−/− and control mice hundreds paraffin-embedded coronal sections (8 μm thickness) of each mouse were cut and 1/10 of the sections were sampled in a systematic uniform random manner for each animal.
Sections were immunostained for CD31 (PECAM-1) in order to label vessels (10 sections per mouse) and total brain capillary length determined using IMAGEPRO PLUS software.
Radioiodination of Aβ. Radioiodination of synthetic Aβ40 peptide was carried out by lactoperoxidase method as previously described14. Typically, 10 μg of Aβ40 was labeled for 18 min at room temperature with 2 mCi of Na[125I]. After radiolabeling, the preparations were subjected to reverse-phase HPLC separation using a Vydac C4 column and a 30 min linear gradient of 25% to 40% acetonitrile in 0.059% trifluoroacetic acid to separate the monoiodinated non-oxidized form of Aβ40 (which is the tracer) from diiodinated Aβ40, nonlabeled nonoxidized Aβ40, and oxidized Aβ40 species as previously reported14,33. The content of material in the peaks eluted from HPLC was determined by MALDI-TOF mass-spectrometry to ensure the purity of the radiolabeled species. For MALDI-TOF mass spectrometry Aβ peptides were labeled under identical conditions using Na[127] instead of the radioactive nuclide. The specific activity was in the range of 45 to 65 μCi/μg of peptide. For clearance studies, preparations were usually used within 24 hr of labeling that was ≧99% TCA-precipitable. If used within 72 hr of labeling, the radiolabeled peptides were stabilized in ethanol as a quenching agent. Prior to each in vitro study or infusion into animals, the tracer was purified by HPLC. The HPLC/SDS-PAGE analysis was used to confirm the monomeric state of infused radiolabeled Aβ40.
Brain clearance studies in mice. CNS clearance of 125I-labeled Aβ40 was determined simultaneously with 14C-inulin (reference marker) in Gax+/− mice and littermate controls 8-10 weeks old, using a procedure as described13,14. Calculation of clearance parameters was performed as reported14. Briefly, a stainless steel guide cannula was implanted stereotaxically into the right caudate-putamen of anesthetized mice (0.5 mg/kg ketamine and 5 mg/kg xylazine I.P.). Coordinates for tip of the cannula were 0.9 mm anterior and 1.9 mm lateral to the bregma and 2.9 mm below the surface of the brain. Animals were allowed to recover after surgery prior to radiotracer studies. The experiments were performed before substantial chronic processes have occurred, as assessed by histological analysis of tissue, i.e., negative staining for astrocytes (glial fibrillar acidic protein) and activated microglia (anti-phosphotyrosine), but allowing time for BBB repair to large molecules, typically four to six hours after the cannula insertion as reported9. Tracer fluid (0.5 μL) containing [125I]-Aβ40 and 14C-inulin was injected into brain ISF over 5 min via an ultra micropump with a MICRO4 controller (World Precision Instruments, Sarasota, Fla.). Brain and blood were sampled 30 min after tracers injection and prepared for radioactivity analysis as described14. Gamma counting was performed using Wallac VIZARD gamma counter (Perkin Elmer, Meriden, Conn.) and beta-counting using TRI-CARB 2100 liquid scintillation counter (Perkin Elmer, Conn.). Previous studies with 125I-labeled Aβ demonstrated an excellent correlation between TCA and HPLC methods. The intactness of 125I-labeled Aβ40 injected into the brain ISF was >99% by TCA/HPLC analysis. The Aβ40 standards eluted at 29.8 min. For SDS-PAGE analysis, TCA precipitated samples were resuspended in 1% SDS, vortexed and incubated at 55° C. for 5 min, then neutralized, boiled for 3 min, homogenized and analyzed by electrophoresis in 10% Tris-tricine gels followed by fluorography. Methodological details were as reported13,14.
The percentage of radioactivity remaining in the brain after microinjection was determined as % recovery in brain=100×(Nb/Ni) (1), where, Nb is the radioactivity remaining in the brain at the end of the experiment and Ni is the radioactivity injected into the brain ISF, i.e., the d.p.m. for 14C-inulin and the c.p.m. for TCA-precipitable 125I-radioactivity (intact Aβ). The percentage of Aβ cleared through the BBB was calculated using the formula [(1−Nb(Aβ)/Ni(Aβ))−(1−Nb(inulin)/Ni(inulin))]×100, using a standard time of 30 min (2).
Cerebral blood flow in mice. The CBF was studied with 14C-iodoantipyrine (14C-IAP; Amersham)33. Gax+/− and control mice were infused with 0.15 μCi of 14C-IAP and after 30 s the heads immediately immersed in liquid nitrogen. The frozen brains were sectioned at 20 μm, mounted on slides, and representative sections exposed to HYPERFILM βMAX autoradiographic film (Amersham) along with 14C standards. After a three day exposure, the film was developed and the resulting images analyzed by quantitative autoradiography on an MCID image analyzer (Imaging Research) to determine levels of 14C-IAP. The CBF was calculated using the basic equation CBF=−λ ln (1−CIN (T)/λ CPL)/T, where CIN (T) is activity in unit mass of brain at time T, CPL is the integrated concentration of 14C-IAP in arterial inflow, and λ is the distribution ratio of 14C-IAP between brain and plasma at steady state, which equals 0.8.
Hypoxia model. Male Gax+/+ and Gax+/− mice 2-3-month old were exposed to hypoxia as described34. A normobaric chamber with 10% oxygen was used on the first day, 9% on the second day, and then followed by 8% of oxygen for up to three weeks. Brains were analyzed after three weeks for capillary density and at four days for the levels of VEGF, GAX, AFX1, and BclXL.
Vascular contractility assay. The thoracic aorta, free from connective tissues, was isolated and removed from anesthetized (50 mg/kg ketamine and 5 mg/kg xylazine i.p.) Gax+/+ and Gax+/− mice. Three mm sections were used to determine contraction and relaxation using a 10 ml Radnoti organ bath system and Grass myograph (Grass-Telefactor Instruments, Warwick, R.I.). Tissue was bathe in Krebs solution, gassed continuously with 95% O2 and 5% CO2 at pH 7.4 and at 37±0.5° C. The resting tension was maintained at 0.5 g. Cumulative dose-response curves for contraction to phenylephrine and relaxation to acetylcholine following pre-contraction with 0.25×10−6 mol/l phenylephrine were determined.
Mouse BEC cultures. Primary cultures of mouse microvascular BEC were established as described49. Briefly, six to ten mice were used each time. Cerebral cortices were cut into small pieces and homogenized in MCDB131 medium containing 2% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The microvessels were isolated from the homogenate by fractionation on a 15% dextran gradient, and then digested with 0.1% collagenase/dispase (Boehringer Mannheim, Indianapolis, Ind.) in MCDB131 medium containing 2% FBS for six hours at 37° C. After centrifugation on a 45% PERCOLL gradient, the digested micro-vessels and dissociated endothelial cells in the top layer were cultured in MCDB 131 medium supplemented with 30 μg/ml ECGS (Sigma), 10% FBS, 15 U/ml heparin, 325 μg/ml glutathione, 1 μl/ml 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Sigma) on collagen 1-coated (Roche Diagnostics, Mannheim, Germany) plastic ware. BEC were further purified using rat anti-mouse CD31 antibodies (BD Pharmagen, Lexington, Ky.) and Dynabeads M-450 sheep anti-Rat IgG (Dynal Biotech, Oslo, Norway) magnetic beads.
Metabolic labeling of BEC. Human BEC (4×105) were pulsed for one hour at 37° C. with 400 μCi of [35S]-methionine (>1000 Ci/mmol; Perkin Elmer, Boston, Mass.) in methionine-free Dulbecco modified Eagle medium (Gibco BRL, New York, N.Y.) as described14. Human BEC (4×105) were pulsed for one hour at 37° C. with 400 μCi of [35S]-methionine (>1000 Ci/mmol; Perkin Elmer, Boston, Mass.) in methionine-free Dulbecco modified Eagle medium (Gibco BRL, New York, N.Y.) as described9. Cells were chased at indicated times within 48 hours. Cell lysates were immunoprecipitated with LRP-515 kDa α-chain specific IgG (8G1) on SDS-PAGE. The intensity of signal was quantified in pixels using the Storm 860 PHOSPHOIMAGER (Amersham Biosciences, Piscataway, N.J.).
Aβ treatment. Human BEC were treated for 24 hr with different concentrations of Aβ42 ranging from 0.1 to 1,000 nM. Oligomeric and aggregated forms of Aβ42 were prepared as described50.
Western blot analysis. Cell lysates were prepared for Western blot analysis as described14. Gax, rabbit polyclonal antibody to C-terminal region of the rat Gax protein that cross reacts with human GAX homeoprotein (amino acids SDHSS EHAHL, SEQ ID NO:2), 1:500 (7 mg/ml)49; polyclonal rabbit antibody to human AFX1 (1:1000, 0.1 mg/ml; Sigma, St. Louis, Mo.); polyclonal rabbit antibody to human Bcl-XL (1:200, 0.2 mg/ml; Santa Cruz Biotechnology, Santa Cruz, Calif.); monoclonal mouse antibody to human ankyrin G (1:100, 0.2 mg/ml; Santa Cruz Biotechnology); polyclonal goat antibody to human plectin (1:100, 0.2 mg/ml; Santa Cruz Biotechnology); monoclonal mouse antibody to C-terminal domain of human LRP-1 β-chain which cross reacts with mouse LRP-1 (5A6, 1:350, 5 μg/ml; EMD Biosciences, San Diego, Calif.); monoclonal mouse antibody to human LRP-1 α-chain (8G1, 1:240, 5 μg/ml; EMD Biosciences) and β-actin, goat anti-human polyclonal, 1:2,500 (0.2 mg/ml, Santa Cruz Biotechnology); polyclonal goat antibody to human MEF2 (1:500, 0.2 mg/ml; Santa Cruz Biotechnology); polyclonal rabbit antibody to human VEGF (1:100, 0.2 mg/ml; Santa Cruz Biotechnology); polyclonal rabbit antibody to human Bax (1:1000, Cell Signaling, Beverly, Mass.); mouse monoclonal antibody to hemaglutinin (HA) (1:200, 0.2 mg/ml; Santa Cruz Biotechnology); mouse monoclonal antibody to human RAP (1:500, 25 μg/ml; EMD Biosciences); monoclonal mouse antibody to human transferrin receptor (1:500, 1 μg/ml; Zymed Laboratories, South San Francisco, Calif.); and polyclonal rabbit antibody to rat Tinur which crossreacts with human TINUR (1:200, 0.2 mg/ml; Santa Cruz Biotechnology) were used.
TUNEL assay. Staining with APOPTAG kit (TUNEL) was performed according to the manufacturer's instructions (Intergen, Purchase, N.Y.).
Statistical analysis. ANOVA was used to determine statistically significant differences. P<0.05 was considered as statistically significant.
Patents, patent applications, books, and other publications cited herein are incorporated by reference in their entirety.
All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. A claim using the transition “comprising” allows the inclusion of other elements to be within the scope of the claim; the invention is also described by such claims using the transition “consisting essentially of” (i.e., allowing the inclusion of other elements to be within the scope of the claim if they do not materially affect operation of the invention) and the transition “consisting” (i.e., allowing only the elements listed in the claim other than impurities or inconsequential activities which are ordinarily associated with the invention) instead of the “comprising” term. Any of these three transitions can be used to claim the invention.
It should be understood that an element described in this specification should not be construed as a limitation of the claimed invention unless it is explicitly recited in the claims. Thus, the granted claims are the basis for determining the scope of legal protection instead of a limitation from the specification which is read into the claims. In contradistinction, the prior art is explicitly excluded from the invention to the extent of specific embodiments that would anticipate the claimed invention or destroy novelty.
Moreover, no particular relationship between or among limitations of a claim is intended unless such relationship is explicitly recited in the claim (e.g., the arrangement of components in a product claim or order of steps in a method claim is not a limitation of the claim unless explicitly stated to be so). All possible combinations and permutations of individual elements disclosed herein are considered to be aspects of the invention. Similarly, generalizations of the invention's description are considered to be part of the invention.
From the foregoing, it would be apparent to a person of skill in this art that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification.
This is a U.S. national-stage application of Int'l Appln. No. PCT/US2006/030148 under 35 U.S.C. 371, filed Aug. 3, 2006; the entire contents of which are hereby incorporated by reference in this application. This application claims priority benefit of provisional U.S. Patent Appln. No. 60/704,903, filed Aug. 3, 2005; which is also incorporated by reference.
The U.S. Government has certain rights in this invention as provided for by the terms of NIH-R37-AG023084 awarded by the Department of Health and Human Services.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/30148 | 8/3/2006 | WO | 00 | 3/18/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60704903 | Aug 2005 | US |
