Chow et al. 10.1073/pnas.0608251104. |
Fig. 5. Ca2+ ions are required for cerebral VSMC contraction and Ca2+ fluxes are not altered in Alzheimer's disease VSMC. (a) Time course of cerebral VSMC shortening (contraction) after 2 min of 75 mM potassium chloride (KCl) application in the presence of 2.5 mM CaCl2 followed by VSMC relaxation after removal of KCl and exposure to Krebs. (b) Relaxation of cerebral VSMC in Ca2+-free Krebs solution. (c) Lack of KCl-induced contraction in cerebral VSMC cultured in Ca2+-free Krebs medium. Mean + SEM, n = 50 cells per culture from 3 different cultures. (d) Ca2+ influx in Alzheimer's disease (AD) and age-matched control cerebral VSMC in response to 75 mM KCl. Mean + SEM, n = 3 different cultures per group.
Fig. 6. (a) Relative levels of expression of SRF and SM-calponin in AD VSMC transduced with Ad.shSRF (closed bars) and Ad.shGFP (open bars) as in Fig. 2e. Mean ± SEM from five different cultures per group. P indicates statistically significant difference in SRF and SM-calponin expression between Ad.shSRF and Ad.shGFP transduced VSMC. (b) MYOCD gene transfer to mouse aortic rings increases the levels of SM-myosin heavy chain (SM-MHC) expression. Western blot analysis of SM-MHC in Ad.MYOCD or Ad.GFP transduced vessels, and relative levels of SM-MHC expression compared to b-tubulin. Mean ± SEM from three aortic ring preparations per group. P, statistically significant difference between MYOCD-transduced vs. GFP-transduced mouse aortic rings determined by Student's t test. (Inset) Ex vivo adenoviral-mediated b-galactosidase gene expression in mouse aorta smooth muscle cells layer. (Scale bar, 100 mm.)
Fig. 7. Ab does not affect SRF expression in human cerebral VSMC. (a) Human VSMC were incubated with either normal culture medium or 20 mM Ab42 oligomers or aggregates for 8, 24, or 72 h. SRF levels were determined by Western blot analysis. (b) Relative SRF levels determined by scanning densitometry of the signal intensity of SRF vs. b-actin bands. Mean ± SEM, n = 3 different cultures per group.
Fig. 8. MYOCD gene transfer to human VSMC increases the mRNA level of Ca2+ handling proteins. (a) PCR analysis of mRNA levels of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATP2A2), casequestrin 1 (CASQ1), and myosin light chain kinase (MYLK) in Ad.MYOCD or Ad.GFP transduced cells. (b) Relative quantification of PCR products normalized to b-actin. Mean ± SEM, n = 4. P, significant differences between MYOCD-transduced compared to GFP-transduced cells determined by Student's t test. For details, see SI Text (Expression of Ca2+-related SRF-dependent genes).
Fig. 9. Effects of hypoxia on MYOCD mRNA levels in human cerebral VSMC. Increased levels of MYOCD mRNA in control human cerebral VSMC by real time PCR after 6-h exposure to hypoxia, i.e., oxygen (<1%) and glucose deprivation, a frequently used model of hypoxia in vitro. Mean ± SEM is from five different experiments.
Fig. 10. SM promoter activity in context of oxidants stress. PAC1 SMC were transfected with the indicated SM promoters (i.e., SM22a, SM a-actin, SM-calponin) linked to luciferase reporter in the absence or presence of myocardin. SMC were then stimulated with 50 mM H2O2 or equal volume of H2O for the indicated times and luciferase activity measured. The results reflect normalized luciferase units following the determination of control Renilla activity. Data represent quadruplicate samples for each condition. No statistically significant differences were seen in any condition with H2O2 treatment. For details, see SI Text (Oxidant Stress and SM promoter activity).
Movie 1. Human brain arterial smooth muscle cells were stimulated with 75 mM KCl to induce contraction. KCl was removed, and normal Krebs solution was used to induce relaxation. Images were captured at ´20 magnification by using a digital camera (Spot) driven by a Simple PCI Software package (Compix, Cranberry Township, PA).
Table 1. Clinical and neuropathological characteristics of Alzheimer's disease patients
Patient number | Age | Gender | PMI, hr | Cause of death | Vascular risk factors | Angiopathy | Braak | CERAD | CDR |
20 | 70 | M | 5.0 | Pneumonia | None | + | V-VI | Moderate | 4 |
41 | 80 | F | Cardiac arrest | Hypertension | + | V-VI | Frequent | 4 | |
42 | 80 | F | 5.2 | Cardiac arrest | Hypertension Atherosclerosis Myocardial Infarction | + | III-V | Moderate | 4 |
43 | 77 | M | 2.8 | Pneumonia | None | + | V-VI | Frequent | 4 |
49 | 78 | M | 5.0 | Cardiac arrest | None | + | V-VI | Frequent | 4 |
54 | 73 | M | 2.5 | Pulmonary embolism | Atherosclerosis | + | V-VI | Frequent | 5 |
122 | 99 | F | 3.5 | Cardiac arrest | Atherosclerosis | + | V-VI | Frequent | 5 |
124 | 78 | F | 3.5 | Bowel obstruction | Hypertension | + | V-VI | Frequent | 3 |
PMI, postmortem interval; CERAD, Consortium to Establish Registry for Alzheimer's Disease; CDR, Clinical Dementia Rating Score. Angiopathy indicates the presence of CAA.
Patient number | Age | Gender | PMI, hr | Cause of death | Vascular risk factors | Angiopathy | Braak | CERAD | CDR |
29 | 96 | F | 6.0 | Cardiac arrest | None | - | 0 | Negative | 0 |
38 | 58 | F | 5.5 | Pulmonary embolism | None | - | 0 | Negative | 0 |
39 | 72 | M | 4.3 | Cardiac arrest | Atherosclerosis myocardial infarcation | - | 0-І | Sparse | 0 |
40 | 73 | M | 4.7 | Myeloma | Atherosclerosis | - | I-II | Negative | 0 |
75 | 86 | F | 3.5 | Cardiac arrest | None | - | 0 | Negative | 0 |
PMI, postmortem interval; CERAD, Consortium to Establish Registry for Alzheimer's Disease; CDR, Clinical Dementia Rating Score. Angiopathy indicates the presence of CAA.
SI Text
Human VSMC culture.
Briefly, pial arterial blood vessels from postmortem human brains were dissected and then digested with 0.1% dispase and 0.1% collagenase in DMEM containing 15 mM Hepes and antibiotics (1). The minced vessels were first kept at 4°C for 2 h and then incubated at 37°C for 1.5 h followed by trituration. The cells were collected by centrifugation and cultured in DMEM containing 10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 units/ml penicillin, and 100 mg/ml streptomycin. The VSMC cultures were >99% pure. The cultured VSMC robustly expressed vascular smooth muscle cell a-actin, VSNC myosin, and SM22a. The VSMC cultures were negative for von Willbrand factor (marker for endothelial cells), glial fibrillar acidic protein (astrocytes), CD11b (microglia), and prolyl 4-hydroxylase, a marker for collagen-synthesizing fibroblasts and fibroblast-like cells (5B5 anti-human fibroblast antibody).Western blotting.
The composition of the "crack" buffer was: 50 mM Tris-HCl, pH 6.8/100 mM DTT/1 mM sodium orthovanadate/100 mg/ml PMSF/2% SDS/10% glycerol/1 mg/ml each pepstatin A, leupeptin, and aprotinin. The lysate is sheared 10´ through a 23-gauge needle, boiled for 10 min, and then spun at 4°C for 10 min at 14,000 ´ g. The supernatant was collected, quantitated with a protein assay kit (Pierce), and analyzed on a Coomassie-stained polyacrylamide gel for integrity and relative loading. Typically, 10% denaturing polyacrylamide gels (BioRad miniprotean) are loaded with 20-50 mg per lane of protein and electrophoresed for 1-2 h at 150 V. The gel is transferred to nitrocellulose and then processed for Western blot analysis by established methods. Primary antisera and their dilution include SRF (1:1,000, Santa Cruz, sc-335), SM-calponin (1:10,000, hCP, Sigma), MYOCD (1:100, Santa Cruz, sc-21561), smooth muscle myosin heavy chain (SM-MHC, 1:500, Santa Cruz, sc-6956), SM a -actin (1:1,000, Sigma, A-2547), SM22a (1:2,000, gift from Dr. Julian Solway, University of Chicago), b -tubulin (1:1,000, PharMingen 556321), and b-actin (1:1000, Santa Cruz, sc-1616). Following incubation with appropriate secondary antisera, immunoreactive products were detected with a chemiluminescent kit (Pierce). The relative levels of immunoreactive product were calculated with a laser densitometer (Molecular Dynamics) and normalized to the b-tubulin or b-actin control antibody.VSMC contractile competence assay.
The composition of the culture medium was DMEM containing 10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 units/ml penicillin, and 100 mg/ml streptomycin until 50-60% confluence. We typically see the peak in contractile activity within 5-10 min of KCl stimulation (see SI Movie 1). To determine whether calcium is needed for VSMC contraction, 2.5 mM CaCl2 was left out of the KCl-free and -containing Krebs solutions, and the assay was performed as described above. We used the same assay to evaluate the effects of adenoviral-mediated MYOCD gene transfer and SRF gene silencing.Measurement of [Ca2+]i in single VSMC.
The intracellular calcium level of VSMC (SI Fig. 5) upon KCl stimulation was imaged using a calcium-sensitive fluorescent dye, Fura-2 am (Teflabs, Austin, TX), as described (2). In brief, VSMC cultured on coverslips were incubated with 4 mM Fura-2 am in DMEM for 40 min. The coverslips were transferred to a perfusion chamber fitted to a stage of an inverted Nikon Diaphot 300 microscope and superfused with normal Krebs solution for 15 min before stimulation with 75 mM KCl in Krebs solution. [Ca2+]i was measured by digital image fluorescence microscopy (objective, Fluor 40/1.3; Nikon) using the Vision 4.0 Software from T.I.L.L. Photonics (Grafelfing, Germany). The fluorescent images were collected with a charge-coupled device (CCD) camera (T.I.L.L. Photonics). Calibrated data were pooled and plotted as means ± SEM. of [Ca2+]i.Adenoviral sh.SRF and MYOCD gene constructs.
Following linearization with PacI (New England Biolabs), each adenoviral construct was transfected separately into HEK-293A cells with Lipofectamine 2000. Viral production was allowed to proceed until cell lysis was judged greater than 95% complete, at which time the supernatant was collected. A crude viral lysate was prepared from this supernatant by three freeze-thaw cycles and tested to confirm function. Subsequently, adenovirus was amplified and then purified using the AdenoMini kit from Virapur, per the manufacturer's directions. Viral titers, as measured in infectious units (IFU), were determined by using the Adeno-X Rapid Titer kit (BD CLONTECH) per the manufacturer's directions. Before recombination, a short sequence encoding the FLAG epitope was inserted in-frame at the N terminus of MYOCD. Linearization with PacI (New England Biolabs), transfection of HEK-293A cells, viral production, preparation of a crude viral lysate, amplification, and purification of adenovirus were as described.Mouse vascular contractility assay.
The resting tension was maintained at 0.5 ´ g. A lower concentration of phenylephrine was required to cause 80% maximum contraction in aortic rings transduced with Ad.MYOCD (0.75 ´ 10-7 mol/liter) compared to Ad.GFP (0.25 ´ 10-6 mol/liter). For experiments with SNP, the endothelial cells were removed by gently rubbing the luminal surface of the aorta with a glass road. The absence of endothelial function was confirmed using acetylcholine, an endothelial-dependent relaxant, on each denuded aortic ring precontracted with phenylephrine.Transduction of mouse aortic rings ex vivo.
Transduction with MYOCD gene was performed as described for ex vivo arterial preparations (3). Viral suspension (50 ml) containing 2 ´ 108 pfu of Ad.MYOCD or Ad.GFP was in human endothelial-SFM (Life Technologies) supplemented with 5´ insulin/transferrin/selenium (Sigma) and penicillin/streptomycin. After adding 100 ml of endothelial growth medium [RPMI medium 1640 containing 10% FBS, 10% Nuserum, 30 mg/ml endothelial cell growth supplements (Sigma), 5 units/ml heparin, 1 mM sodium pyruvate, 1% nonessential amino acids, 1% vitamins, 25 mM Hepes, 100 units/ml penicillin, and 100 mg/ml streptomycin], the incubation was continued overnight (20-24 h). Detection of b-galactosidase was performed as described (4, 5). After staining, arterial segments were embedded in OCT, sectioned on a cryostat at 10 mm and photographed at 4´ magnification.In vivo
transduction of cerebral arteries. The composition of mock CSF was (in mM): NaCl 125, KCl 3, NaHCO3 1.25, NaH2PO4 2.5, MgCl2 1, CaCl2 1, Glucose 10, pH 7.3.It is of note that, in our hands, this method resulted in a significant transduction of the VSMC tunica media layer in small pial arteries, as illustrated in SI Fig. 6 Inset. Although our method does not provide an exclusive VSMC expression of the target genes, and the adventitia, which represents <10% of the thickness of the arterial wall in small subrachnoid pial arteries (6), can also be transduced, the observed functional changes in arterial contractility and the CBF responses to vibrissal stimulation after gene transfer indicated that the functional levels of MYOCD have been achieved in the VSMC layer. In contrast, an earlier report suggested that the adventitia is mainly transduced in the large subdural basilar artery after injection of the viral particles into the cisterna magna (7). The differences in transduction observed between previous (7) and the present study could be attributed to different type or arteries that have been transduced, i.e., large subdural basilar vs. small subrachnoid pial, different reporter genes used, i.e., b-galactidase vs. GFP, a different CSF space route used for administration of viral particles, i.e., cisterna magna with a largest CSF volume vs. subarchnoid space with a small CSF volume, and different species, i.e., rabbit vs. mice.
To enhance VSMC-specific gene expression, we also tried a specific VSMC promoter SM22. Unfortunately, in our hands, the expression of this promoter was associated with extremely low <10% transduction efficiency of VSMC, which was inadequate for our study. Similarly low transduction with the SMC-specific promoters have been reported by others (8).
Cerebral blood flow.
Rectal temperature was maintained at 37°C using a heated blanket (Homeothermic blanket, Harvard Apparatus). Three vibrissal stimulation trials were separated by 10-min intervals for each experimental condition.Brain analysis after in vivo gene transfer.
Adenoviral vector delivery to the brain in vivo can be associated with inflammation, which may interfere with the CBF measurements. However, our data suggest that that the CBF responses after vehicle (mock CSF) injections and control Ad.GFP or Ad.shGFP injections in wild-type mice, Dutch/Iowa/Swedish APP mutant expressing mice and APPsw+/- are not significantly different, indicating that adenoviral delivery to subarchnoid space and pial vessels did not interfere with the CBF measurements in the present study. Moreover, all CBF measurements in MYOCD or shSrf transduced mice were significantly different from their corresponding GFP controls and shGFP controls, respectively, indicating the CBF increases in response to brain activation in these experiments represent indeed the effect of SRF/MYOCD, and not the effect of adenoviral transduction. Ongoing analysis also confirmed that brains of Dutch/Iowa/Swedish APP mutant expressing mice and APPsw+/- mice express normally inflammatory markers (i.e., increased numbers of microglia, and astrocytes, increased levels of cytokines IL1b, and TNF-a) in the absence of any treatment, as reported (9-11), and these markers are not significantly altered in control adenoviral vector (i.e., GFP, shGFP)-treated mice, nor in MYOCD and shSrf-treated animals.Oxidant stress and SM promoter activity.
PAC1 SMC were grown and transfected as previously described (12). The following promoters (500 ng/well) were used to evaluate the effects of oxidant stress on Myocardin-dependent transactivation: a -505-bp promoter to SM22a(13), a -2,800-bp promoter to SM a-actin (14), and a -549-bp promoter to SM calponin containing the entire first intron (15), all linked to firefly luciferase. Transfections were done in quadruplicate in the absence or presence of 500 ng/well of pCDNA3-Myocardin expression plasmid (16). Fifty ng/well of a tk-driven Renilla plasmid (Promega) was included as an internal control for transfection efficiency. The following day, fresh DMEM containing 1% FBS was added to each well in the absence or presence of 50 mM H2O2 (we found in separate dose-response studies that 50 mM H2O2 was well tolerated by PAC1 SMC for periods up to 24 h). Cell lysates were prepared 6 or 24 h following 50 mM H2O2 treatment for dual luciferase assays as described (15). Raw luciferase data were normalized to Renilla bioluminescence and the H2O2 data expressed as fold changes from control. Data were analyzed with GraphPad Prism software, including Mixed ANOVA and Tukey's posthoc test for ascertaining statistical significance (P < 0.05).Expression of Ca2+-related SRF-dependent genes.
Control human VSMC were infected with Ad.MYOCD or Ad.GFP to overexpress the respective gene and protein. In all experiments, 100 multiplicity of infection (moi) of virus was used. After 48 h of infection, total RNA was isolated using RNeasy mini kit (Cat No. 74106, Qiagen, Valencia, CA). First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Cat. No. 18064-014, Invitrogen, Carlsbad, CA). PCR amplification of target genes was performed using Platinum TaqDNA Polymerase (Cat. No. 10966-026, Invitrogen) using the following primers: ATP2A2 (289 bp): sense 5'-TCCACCGAGTTACCGGCTGAAGAA-3', antisense 5'-TGTCCTGTCGATACACTTTGCCCA-3'; MYLK (302 bp): sense 5'-GCTTGGTCAGCCTGTTGTTTCCAA-3', antisense 5'-ACACTCCCACGTCATCTTGGTTGA-3'; CASQ1 (211 bp) sense 5'-TGACCATCCCAGACAAGCCCAATA-3', antisense 5'-TTATCTTGGGCCACAGCCTTGAGA-3'. b-Actin (294 bp) was used as control primer (Cat. No. AM1720, Ambion, Austin, TX). After 25-28 cycles (leaner amplification phase) in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA), the PCR products were ran on a 1% agarose gel, and analyzed using AlphaImager 2220. Comparisons of the PCR products were made between Ad.MYOCD vs. Ad.GFP groups. Experiments were performed in four independent cell cultures.1. Park L, Anrather J, Zhou P, Frys K, Pitstick R, Younkin S, Carlson GA, Iadecola C (2005) J Neurosci 25:1769-1777.
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