PACAP38/PAC1 Signaling Induces Bone Marrow-Derived Cells
Homing to Ischemic Brain
Chen-Huan Lin1*, Lian Chiu2*, Hsu-Tung Lee3*, Chun-Wei Chiang1, Shih-Ping Liu1,4, Yung-Hsiang Hsu5, Shinn-Zong Lin1,6,Chung Y. Hsu7, Chia-Hung
Hsieh4,8†, Woei-Cherng Shyu1,6†
1Center for Neuropsychiatry and Translational Medicine Research Center, China
Medical University and Hospital, Taichung, Taiwan 40421
2Department of Nursing, College of Medicine and Nursing, Hungkuang University,
Taichung, Taiwan
3Department of Neurosurgery, Taichung Veterans General Hospital, Taichung,
Taiwan 40421; and Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
4Graduate Institute of Basic Science, China Medical University, Taichung, Taiwan
40421
5Department of Pathology, Buddhist Tzu-Chi General Hospital, Tzu-Chi University,
Hualien, Taiwan
6Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
7Graduate Institute of Clinical Medical Science, China Medical University, Taichung,
Taiwan 40421
8Department of Biomedical Informatics, Asia University, Taichung, Taiwan 40421
*These authors contributed equally to this article
†To whom correspondence should be addressed: C.H.H ([email protected]) and W.C.S. ([email protected])
Nonstandard abbreviations used: BMDCs, bone marrow–derived cells; PACAP38, pituitary adenylate cyclase–activating peptide 38; PAC1, PACAP type 1 receptor; HIF-1α, hypoxia-inducible factor-1α; PrPC, cellular prion protein; HSCs,
hematopoietic stem cells; CCR3, CC chemokine receptor 3; CCR4, CC chemokine receptor 3; JAM-A, junctional adhesion molecular A; JAM-C, junctional adhesion molecular A; PECAM-1, platelet/endothelial cell adhesion molecule 1; LFA-1, lymphocyte function-associated antigen 1; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; CXCR4, CXC chemokine receptor 4; hDSCs, MMP9/2, matric metalloprotease 9/2; FDG-PET, [18F]fluoro-2-deoxyglucose
positron emission tomography; GFAP, glial fibrillary acidic protein; Tuj-1, βIII-tubulin; MAP-2, microtubular associated protein-2; Neu-N, neuronal nuclear.
Understanding stem cell homing, which is governed by environmental signals from the surrounding niche, is important for developing effective stem cell-based repair strategies. The molecular mechanism by which the brain under ischemic stress recruits bone marrow–derived cells (BMDCs) to the vascular niche remains poorly characterized. Here we report that hypoxia-inducible factor-1α (HIF-1α) activation upregulates pituitary adenylate cyclase–activating peptide 38 (PACAP38), which in turn activates PACAP type 1 receptor (PAC1) under hypoxia in vitro and cerebral ischemia in vivo. BMDCs homing to endothelial cells in the ischemic brain are mediated by HIF-1α activation of the PACAP38-PAC1 signaling cascade followed by upregulation of cellular prion protein (PrPC) and α6-integrin to enhance the ability of BMDCs to bind laminin in the vascular niche. Exogenous PACAP38 confers a similar effect in facilitating BMDCs homing into the ischemic brain, resulting in reduction of ischemic brain injury. These findings suggest a novel HIF-1α-activated PACAP38-PAC1 signaling process in initiating BMDCs homing into the ischemic brain for reducing brain injury and enhancing functional recovery after ischemic stroke.
Runnning title: PACAP38 recruits BMDCs to vascular niche of stroke
Key words: BMDCs, PACAP38/PAC1 signaling, HIF-1α, vascular niche, stroke
INTRODUCTION
Cerebral ischemia creates a specific niche for stem cells in the affected brain region which is enriched with soluble factors including cytokines, chemokines, and growth factors secreted from the adjacent stroma, neurons, and peripheral blood in response
to ischemic insult Some of these factors including stromal cell-derived factor 1α (SDF-1α) and vasoactive intestinal peptide (VIP) with angiogenic actions are regulated by the oxygen-sensitive hypoxia-inducible factor-1α (HIF-1α). Pituitary adenylate cyclase–activating polypeptide (PACAP), belonging to the same neuroendocrine peptide family as VIP, exerts a multitude of actions including neuroprotection and angiogenesis by interacting with VIP/PACAP receptor 1 (VPAC1) and selective PACAP receptor 1 (PAC1). Although expression of PACAP is also induced by ischemic injury as VIP, whether HIF-1α activation involved in upregulation of PACAP expression following ischemia has not been reported.
We and others have previously reported that the ischemic microenvironment expresses signals that promote homing of BMDCs into ischemic areas The ability of stem cells to relocate and occupy niches is essential for normal stem cell biology. Though some factors that drive homing of BMDCs to vascular niches within bone marrow using several molecules under physiological conditions and following transplantation has been identified, how BMDCs home to their niche in the ischemic brain is not well known. For example in SDF-1α/CXC receptor type 4 (CXCR4) signaling system, SDF-1α also plays an important role in trafficking of circulating hematopoietic stem cells (HSCs) by interacting with it’s receptor of CXCR4 from peripheral circulation to the ischemic brain. Actin polymerization and upregulation of integrins occur in circulating HSCs, resulting in chemotaxis toward the source of SDF-1α. Regarding the impact of PACAP in cell trafficking, previous reports demonstrated that it promoted neural progenitor cells (NPCs) migration and trafficking via activation of PAC1. Accordingly, based on the crucial link that CD34+
HSCs preferentially express PAC1, we decided to pursue the idea that PACAP38/PAC1 axis plays a critical role involved in BMDCs homing to the vascular niche of ischemic brain.
Hypoxia inducible factor-1α (HIF-1α), binding to the putative hypoxic-responsive element (HRE) of gene promoters is the central transcriptional mediator of the cellular response of hypoxia and ischemia. Stem cells trafficking are modulated by the HIF-1α gradient of the vascular niche. In the present study, we hypothesize that upregulation of PACAP38 expression under hypoxia/ischemia is through HIF-1α binding putative HRE on the PACAP38 promoter. We further assume that PACAP38 actions in reducing hypoxia/ischemia injury and promoting stroke recovery are mediated by its interaction with PAC1, establishing the PACAP38-PAC1 signaling cascade to mobilize BMDCs for homing to the vascular niche in the ischemic brain.
MATERIALS AND METHODS
Immunohistochemical Analysis of Human Brain after Stroke
We investigated autopsy brain specimens from 9 cases of fatal ischemic stroke (disease duration ranged from 10 h to 7 days) treated at the Department of Neurology, China Medical University Hospital. Autopsy surgery was performed within a mean of 6 hours after death (ranging from 4-12 h). Three patients who died of a non-neurological cause served as controls (autopsies were conducted at 0.5 day, 3 days and 5 days). The study protocol was approved by the Institutional Review Board of the China Medical University Hospital. Informed consent was signed by relatives. Tissue sampling was based on individual infarct topography, which in each case was determined on the basis of cerebrovascular anatomy and the most recent MRI scan.
On autopsy, brain areas with variable degrees of infarction were identified macroscopically and ~1 cm3 cortical samples, including subcortical white matter,
were dissected and fixed with formalin prior to embedding in paraffin or frozen at -70oC until analysis, as described previously. Immunostaining of brain samples was
performed using specific antibody of PACAP38 (1:200, Santa Cruz), CD34 (1:100, BD Pharmingen), PAC1 (1: 300, Santa Cruz), Tuj-1 (1:200; Chemicon), microtubule-associated protein 2 (MAP-2, 1:200; BM). Samples from corresponding areas of the contralateral or non-infarcted hemispheres and from the control brains were processed in a similar way. The extent of PACAP38+ cell immunoreactivity was measured as the
number of cells per square millimeter (cells/mm2).
In Vivo Ischemia/Reperfusion Brain Model
Adult male Sprague-Dawley rats (SD rat, weight 250 to 300 g) were used in this study. All surgical procedures were performed using sterile/aseptic techniques in accordance with Institutional guidelines. Rats were anesthetized with chloral hydrate (0.4 g/kg IP) and subjected to cerebral ischemia. Ligation of the right middle cerebral artery (MCA) and bilateral common carotids arteries (CCAs) was performed by methods described previously. The CCAs were clamped with non-traumatic arterial clips. The right MCA was ligated with a l0-0 nylone suture. After 90 min of ischemia, the suture on the MCA and the arterial clips on the CCAs were removed to allow
reperfusion. Core body temperature was monitored with a thermistor probe (Hewlett-Packard Model 21090A probe, Hewlett-(Hewlett-Packard Company, Andover, MA), and maintained at 37oC with a heating pad during anesthesia. After recovery from
anesthesia, rat body temperature was maintained at 37oC with a heat lamp.
In addition, conditional HIF-1α knockout mice (HIF-1α KO mice carrying a loxP-flanked allele of HIF-1α, a kind gift from Dr. Johnson).HIF-1α disruption in the HIF-1α KO mice was induced by feeding doxycycline at a dose of 2 mg/ml in 5% (wt/vol.) sucrose solution from embryonic day 15 to postnatal day 1. They were also anesthetized with chloral hydrate (0.3 g/kg, ip) and subjected to right middle cerebral artery (MCA) ligation and bilateral common carotid artery (CCAs) clamping for 120 minutes, as described above with modification.
2-Methoxyestradiol Treatment In Vivo and In Vitro
2-Methoxyestradiol (2-ME2, Sigma) was dissolved in DMSO to obtain a 10 mmol/L stock solution. For in vivo experiments, the whole procedure was as previously described. Experimental rats were treated with an intraperitoneal injection of a liposomal preparation (di-oleoyl- phosphotidylcholine; Avanti Polar Lipids) of 2-ME2 (20mg/mL) in a different concentration (50, 100 or 150 mg/kg) for 3 consecutive days pre- and after the onset of cerebral ischemia. For in vitro experiments with 2-ME2 treatment, PCC were pretreated with different concentrations of 2-ME2 (0.1 μM, 1
μM and 10 μM) for 16 h as previously described .
Total Protein Extraction for Western Blotting and ELISA
Experimental animals were decapitated at 4 h, 12 h, 3 d, and 7 d after reperfusion with 90 min MCA ligation. Three rats without MCA ligation were used as normal controls. Samples of ischemic cerebral cortex were taken from the peripheral region of infarcted brains (penumbral area). Western blot analysis was performed on these samples. Briefly, ischemic brain tissue was homogenized and lysed in the buffer containing 320 mM sucrose, 5 mM HEPES, 1 μg/mL leupeptin, and 1 μg/mL aprotinin. Lysates were centrifuged at 13,000 g for 15 min. The resulting pellet was resuspended in sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue, and 50 mM DTT) and subjected to SDS-polyacrylamide gel (4-12%) electrophoresis. Then, the gel was transferred to a Hybond-P nylon membrane. This was followed by incubation with appropriately diluted antibodies of PACAP38 (1:200; Invitrogen), HIF-1α (1:200; Novus Biologicals), HIF-2α (1:200; Novus Biologicals), PAC1 (1: 300, Santa Cruz), PrPC (1:300, Santa Cruz), α6-integrin
(1:200, Chemicon), PECAM-1 (1:200; Santa Cruz Biotechnology), selectin (1:200; Santa Cruz Biotechnology), CXCR4 (1:200; R&D Systems), CCR3 (1:200; R&D Systems), CCR4 (1:200; R&D Systems), β1-integrin (1:200; Chemicon), β2-integrin (1:200; Chemicon), JAM-A (1:200; Millipore), JAM-C (1:200; Millipore), LFA-1
(1:200; Millipore), ICAM (1:200; Millipore), VCAM-1 (1:200; Millipore), VE-cadherin (1:200; Millipore), CD99 (1:200; Millipore), FAK (1:200; Millipore), STI-1 (1:200; Santa Cruz) and β-Actin (dilution 1:2000, Santa Cruz). Expression of apoptosis-related proteins (Bcl-2, Bcl-xL, Bax and Bad) in the right cortex and striatum region was also examined. Membrane blocking, primary and secondary antibody incubations, and chemiluminescence reactions were conducted for each antibody individually according to the manufacturer’s protocol. The intensity of each band was measured using a Kodak Digital Science 1D Image Analysis System (Eastman Kodak). The ratio of band intensity of each protein in western blots in comparison with the internal control was calculated. In addition, PACAP38 levels were measured by direct ELISA using goat-polyclonal PACAP antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA) and peroxidase-labeled donkey anti-goat IgG (1:2,000, Santa Cruz Biotechnology). Optical density was measured using a spectrophotometer (Molecular Devices) and standard curves were generated with the program SOFTmax (Molecular Devices).
Measurement of HIF-1α Activity by ELISA
To measure the active HIF-1α, 50 μg nuclear extracts were incubated with biotinylated double stranded oligonucleotide containing a consensus HIF-1α binding site from Duo-set ELISA mouse active HIF-1α kit (R&D Systems) according to the
manufacturer’s instructions. The activity of HIF-1α was expressed by OD (450 nm-540 nm) as previously described. The experiments were carried out in triplicate and each experiment was repeated three times unless otherwise mentioned.
Immunohistochemical Analysis of Rat Brain
Experimental rats were re-anesthetized with chloral hydrate (0.4 g/kg IP), and were decapitated at 4 h, 12 h, 3 d, and 7 d after cerebral ischemia. Three rats without MCA ligation were used as normal controls. Rat brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde as previously described. A series of adjacent 6-μm-thick sections were cut by cryostat from each tissue block in the coronal plane, stained with H&E, and analyzed by light microscopy (Nikon, E600).
To identify the expression of cell type-specific markers in PACAP38+ cells,
double immunofluorescence was performed as previously described. Each coronal section was first stained with primary PACAP38 antibody (1:100, Santa Cruz), followed by treatment with specific antibodies: GFAP (1:400, Sigma), vWF (1:400; Sigma), Tuj-1 (1:200; Chemicon), microtubule-associated protein 2 (MAP-2, 1:200; BM), CD34 (1:100, BD Pharmingen), PAC1 (1:300, Santa Cruz), PrPC (1:300, Santa
Cruz), α6-integrin (1:200, Chemicon), Laminin (1:500, Sigma) and hypoxia inducible factor-1α (HIF-1α, 1:200; R&D Systems). The tissue sections were analyzed with a
Carl Zeiss LSM510 laser-scanning confocal microscope. FITC (green, 1:500; Jackson Immunoresearch), Cy3 (red, 1:500; Jackson Immunoresearch) and Alexa Fluor 408 (blue, 1:1000; Invitrogen) fluorochromes on the immunofluorescence-labeled slides were excited by laser beam at 488 nm, 543 nm and 680 nm, respectively. PACAP38, labeled with Cy3 (red) or FITC (green) or Alexa Fluor 680 (blue, 1:1000; Invitrogen) fluorochromes, and cell-type-specific markers of Neu-N, MAP-2, vWF, GFAP and HIF-1α, labeled with Cy3 (red) or FITC (green) fluorochromes were double immunostained in order to demonstrate their co-localization in one cell under laser-scanning confocal microscopy. For quantification, distance of cell nuclei (GFP+ cells)
from blood vessels (laminin+) was quantified with an automated system, described
previously.
Cerebral Ischemic Animal Model Treated with PACAP38
The cerebral ischemic animal model was established as described above. Experimental rats were injected intraperitoneally with different dosage of PACAP38 (0.1, 1 and 10 μg/kg, Sigma Aldrich) at 4 hours after MCA ligation for five consecutive days. In addition, two therapeutic group of PACAP38 (10 μg/kg) and vehicle control were progressed for further investigation. Core body temperature was monitored with a thermistor probe and maintained at 37oC with a heating pad during
metalloproteinase inhibitor (GM6001; Chemicon) was injected intra-peritoneally for 4 consecutive days as previously described. In addition, specific PAC1 antagonist PACAP (6-38) (Bachem, CA) 10 μg/kg was administered intraperitoneally for 4 consecutive days as previously described with modification.
Triphenyltetrazolium Chloride (TTC) Staining
Three days after cerebral ischemia, animals were intracardially perfused with saline. The brain tissue was removed, immersed in cold saline for 5 min, and sliced into 2.0-mm-thick sections (seven slices per rat). The brain slices were incubated in 20 g/L triphenyltetrazolium chloride (TTC; Research Organics Inc), dissolved in saline for 30 min at 37oC, and then transferred into a 5% formaldehyde solution for fixation.
The area of infarction in each slice was measured with a digital scanner, as described previously.
Neurological Behavioral Measurements
Behavioral assessments were performed 3 d before cerebral ischemia, and 72 h after cerebral ischemia. The tests measured (a) body asymmetry and (b) locomotor activity. Further, grip strength was analyzed using Grip Strength Meter (TSE-Systems) as previously described with modification. The baseline-test scores were recorded in order to normalize those taken after cerebral ischemia. The elevated body swing test (EBST) was used to assess body asymmetry after MCA ligation and evaluated quantitatively as previously described. Initially, animals were examined for lateral
movement by suspending their bodies by their tails. The frequency of initial head swing contra-lateral to the ischemic side was counted in twenty continuous tests and was normalized, as follows: % recovery = [1 – (lateral swings in twenty tests – 10) / 10 x 100%. Locomotor activity: Rats were subjected to VersaMax Animal Activity monitoring (Accuscan Instruments) for about 2 h for behavioral recording. The VersaMax Animal Activity monitoring contained 16 horizontal and 8 vertical infrared sensors spaced 87 cm apart. The vertical sensors were situated 10 cm from the floor of the chamber. Motor activity was counted as the number of beams broken by a rat movement in the chamber. Three vertical parameters defined in the manufacturer’s menu option were calculated over 2 h at night: (i) vertical activity, (ii) vertical movement time, and (iii) number of vertical movements. In grip strength analysis, ratio of improvement in grip strength was measured on each forelimb separately and was calculated as the ratio between the mean strength out of 20 pulls of the side contralateral to the ischemia and the ipsilateral side. In addition, the ratio of grip strength post-treatment and baseline were also calculated and changes were presented as a percentage of baseline value.
Measurement of Infarct Size Using Magnetic Resonance Image (MRI)
MRI was performed on rats under anesthesia in an imaging system (R4, General Electronics) at 3.0 T. Brains were scanned in 6 to 8 coronal image slices, each 2 mm
thick without any gaps. T2-weighted imaging (T2WI) pulse sequences were obtained with the use of a spin-echo technique (repetition time, 4000 ms; echo time, 105 ms) and were captured sequentially for each animal at 1, 7, and 28 d after cerebral ischemia. To measure the infarction area in the right cortex, we subtracted the noninfarcted area in the right cortex from the total cortical area of the left hemisphere. The area of infarct was drawn manually from slice to slice, and the volume was then calculated by internal volume analysis software (Voxtool, General Electric).
[18F]fluoro-2-deoxyglucose Positron Emission Tomography (FDG-PET)
Examination
To assess the metabolic activity and synaptic density of brain tissue, experimental rats were examined using microPET scanning of [18F]fluoro-2- deoxyglucose (FDG) to
measure relative metabolic activity under the protocol previously described. In brief,
18F was produced by the 18O(p, n)18F nuclear reaction in a cyclotron at China Medical
University and Hospital, Taiwan, and 18F-FDG was synthesized as previously
described with an automated 18F-FDG synthesis system (Nihon Kokan). Data were
collected with a high-resolution small-animal PET (microPET, Rodent R4, Concorde Microsystems) scanner. The system parameters have been described previously by Carmichael et al.. After one week of each treatment, animals anesthetized with chloral hydrate (0.4 g/kg, ip), fixed in a customized stereotactic head holder and positioned in
the microPET scanner. Then the animals were given an intravenous bolus injection of
18F-FDG (200-250 μCi/rat) dissolved in 0.5 mL of saline. Data acquisition began at
the same time and continued for 60 min using a 3-D acquisition protocol. The image data acquired from microPET were displayed and analyzed by Interactive Data Language (IDL) ver. 5.5 (Research Systems) and ASIPro ver. 3.2 (Concorde Microsystems) software. FDG-PET images were reconstructed using a posterior-based 3-dimentional iterative algorithm and overlaid on MR templates to confirm anatomical location. Coronal sections for striatal and cortical measurements represented brain areas between 0 and +1 mm from bregma, and thalamic measurements represented brain areas between -2 and -3 mm from bregma, as estimated by visual inspection of the unlesioned side. The relative metabolic activity in regions of interest (ROI) of the striatum was expressed as a percentage deficit as previously described with modification.
Preparation of Stem Cells Culture
Bone marrow derived cells (BMDCs) were collected from rat femoral veins (5 mL) mobilized with recombinant human granulocyte colony-stimulating factor (rHuG-CSF, Kirin, Tokyo, Japan) at 50 μg/kg/day subcutaneously for 5 consecutive days. The mononuclear cells (MCs) were separated by Ficoll-Paque (1:3 dilution, StemCell Technologies). CD34+ BMDCs were separated from 2 x 106 MCs by a magnetic bead
separation method (MACS; Miltenyi Biotec, Gladbach, Germany) according the manufacturer’s instructions. Subsequently, CD34+ BMDCs (purity > 95%, 106
cell/mL) were cultured for 72 h in medium (StemSpanTM H3000 and Cytokine
Cocktail, StemCell Technologies) at 37oC in a humidified atmosphere of 5% CO 2 /
95% air and antibiotics, and prepared for experiment. In bromodeoxyuridine (Brdu) labeling and immunocytochemistry, the cells were pulsed with 10 μM BrdU for 4 hr and fixed with 4% paraformaldehyde for 20 min as previously described. In brief, DNA was denatured by treatment with 2.5 N HCl for 20 min at room temperature followed by 0.1 M boric acid treatment to neutralize the cells. Incorporated BrdU was detected with a mouse monoclonal anti-BrdU antibody (1:50, BD Biosciences) that was incubated with the cells overnight. The percentage of BrdU-positive cells was determined by counting under a phase contrast microscope and at least 500 cells per sample were scored.
Transwell Migration Assays
Enhanced migration of BMDCs by PACAP38 treatment was assessed as described previously with modifications. In brief, BMDCs treated with PACAP38 (1, 10 and 100 nM) were placed in 100 μL in the upper chamber (transwell: 6.5-mm diameter, 5.0-mm pore size) according to manufacturer’s instructions (Costar, #3421). We used SDF-1α (100 ng/mL, R&D System, positive control) in the lower chambers. For the
PAC1 receptor blocking study, PACAP (6-38) (10 μM, Bachem, Switzerland) was also added to the lower wells. The assays were conducted over a 4-hour incubation period at 37oC in a 5% CO
2 incubator. Because almost all cells stay at the lower side
of the membrane after migration, quantification can be performed by simply counting these cells. Adhered cells at the lower side of the membranes were counted under the microscopy as previously described. For assessing migrated CD34+ cells, cells were
collected from the bottom of a transwell and assessed by flow cytometry as described previously.
Gene Silencing with RNA Interference
Specific knockdown was achieved by lentiviral delivery of shRNA for PACAP38 PACAP-sh; sc-39531-V, Santa Cruz Biotechnology), shRNA for HIF-1α (LV-HIF-1α-sh; sc-35562-V, Santa Cruz Biotechnology), shRNA for HIF-2α (LV-HIF-2α-sh; sc-35316-V, Santa Cruz Biotechnology), Lenti-PrPC shRNA (LV-PrPC-sh,
sc-36318-V, Santa Cruz Biotechnology), Lenti-α6-integrin shRNA (LV-α6-integrin-sh, sc-43129-V, Santa Cruz Biotechnology) and the control scramble shRNA (LV-control-sh; sc-108080, Santa Cruz Biotechnology) under manufacture’s instruction. Lenti-viral Constructs of PACAP38, PACAP38-Flag, HIF-1α and HIF-2α
The lentiviral constructs were generated by cotransfection of human kidney derived 293T cells with three plasmids using the calcium phosphate method as previously described with modification. In the transducing vector, an expression cassette with the
Rev responsive element and the EF-1α promoter are used to direct the expression of PACAP38 (PACAP38 cDNA, SC110820, OriGene), mouse HIF-1α and HIF-2α (clone ID 4019056 and 5032291, Thermo) and GFP (GFP cDNA; Clontech). Lenti-viral vector particles were generated by transient cotransfection of 293T cells with the lentiviral shuttle plasmid from TRIP GFP plasmid vector, an HIV-1-derived packaging plasmid, and a VSV-G envelope expressing plasmid. Two days after transfection, lentiviral constructs (LV-PACAP38/-Flag, LV-HIF-1α, LV-HIF-2α or LV-GFP) were harvested in the culture medium and concentrated by ultra-centrifugation. Viral titers were quantified by using HIV-1 p24 antigen assay (Beckman Coulter) according to the manufacturer’s instructions. The p24 concentration was used to determine the vector dose (expressed in nanograms) administered in the various in vitro and in vivo experiments. The lentiviral titers were determined by infection of 293T cells seeded in six-well plates at 1 x 105 cells per
well the day before infection with serial dilution of the concentrated viral stock. After overnight incubation, the culture medium was changed and the cells incubated for two more days. GFP fluorescent cells were identified by fluorescent microscopy or by a fluorescent activated cell sorter. Titers ranged from 108 to109 infectious units
(IU)/mL.
Intracerebral administration were performed in animals under chloral hydrate anesthesia to inject with 1 x 109 viral units of LV-PACAP38-shRNA,
LV-PACAP38/-Flag, LV-GFP or control shRNA (5 μL) through a 26-gauge Hamilton syringe (Hamilton Company, USA) into three cortical areas, 3.0 to 5.0 mm below the dura. The approximate coordinates for these sites were l.0 to 2.0 mm anterior to the bregma and 2.5 to 3.0 mm lateral to the midline, 0.5 to l.5 mm posterior to the bregma and 3.5 to 4.0 mm lateral to the midline, and 3.0 to 4.0 mm posterior to the bregma and 4.5 to 5.0 mm lateral to the midline. The needle was retained in place for 5 min after each injection and a piece of bone wax was applied to the skull defects to prevent leakage of the injected solution. To assess for transgene expression after intracerebral injection of lentiviral vector, animals received intracerebral injection of lentiviral particles were then killed for histological purposes and western blot quantification of PACAP38 production in vivo. In in vitro lentiviral vectors transduction, cell culture was plated in 10-cm dishes at a density of 1 x 105 cells in 5 ml medium per dish.
Transductions were carried out in the presence of 8 μg/ml polybrene at m.o.i. of 5 or 25 for each vector. After incubation for 24 h, the transduction medium was replaced with fresh original medium for each cell.
Gel zymography (GZ). The culture supernatant containing equal amounts of protein
After electrophoresis, gels were washed in 5% Triton X-100 and then incubated in MMP assay buffer (Bio-Rad). Bands were visualized with Coomassie Brilliant Blue and destained in 30% methanol with 10% acetic acid.
Preparation of Transgenic GFP-Chimeric Mice
In order to verify the enhancement of bone marrow stem cell (BMSCs) mobilization and homing into brain, a bone marrow niche sample was removed from the long bones of adult male donor green fluorescent protein (GFP) mice as previously reported. Both ends of the femur and tibia were penetrated using a syringe with a 25-gauge needle, and the marrow was flushed out with sterile saline. Total marrow from 1 femur was diluted to 1 mL then strained through a 30-m Spectramesh (Fisher Scientific). Before bone marrow transplantation, recipient wild type (C57BL/6
mice-PAC1+/+ mice) and PAC1-/- mice [a generous gift provided by DKFZ Dr. Schutz]
underwent whole body gamma irradiation with 137Cs using a Gammacell 40 irradiator
(MDS Nordion). A total dose of 9 Gy (900 rads) was administered to ablate the whole bone marrow. The mice received rescuing bone marrow transplantations within 24 h of irradiation. Donor bone marrow was injected into the recipient animal’s tail vein as an 80 L cell suspension containing 3 x 106 cells. At 3 weeks after transplantation,
mice were anesthetized with chloral hydrate (0.3 g/kg, ip) and subjected to right middle cerebral artery (MCA) ligation and right common carotid artery (CCAs)
clamping for 120 minutes, as previously described with modification. Then, experimental mice were injected intraperitoneally with PACAP38 (50 μg/kg) and vehicle control.
Angiogenic Evaluation by FITC-dextran Perfusion and CD31
Immunohistochemistry
In order to examine the blood vessels, cerebral microcirculation was analyzed by administering the fluorescent plasma marker (FITC-dextran, Sigma) intravenously to rats and observing them under fluorescent microscopy (Carl Zeiss, Axiovert 200M), as previously described. In addition, to quantify the cerebral blood vessel density and examine the vascular remodeling by macrophage, experimental rats were anesthetized with chloral hydrate and perfused with saline. Histological sections (6 μm) were stained with specific antibody to CD-31 (1:100, BD Pharmingen) conjugated with Cy-3 or FITC (1:500, Jackson Immunoresearch PA USA). The number of blood vessels was determined as previously described.
Measurement of Cerebral Blood Flow (CBF)
Experimental rats were positioned in a stereotaxic frame and baseline local cortical blood flow (bCBF) was monitored after cerebral ischemia with a laser doppler flowmeter (LDF monitor, Moor Instrutments, Axminster, U.K.) in anesthetized state (chloral hydrate) as previously described. In brief, CBF values were calculated as percentage increase compared to the bCBF.
In Vitro Primary Cortical Cultures (PCCs) Preparation
Primary cortical cultures (PCCs) were prepared from the cerebral cortex of gestation day 17 embryos from from wild type (C57BL/6 mice-PAC1+/+ mice) and PAC1-/- mice
as described previously. PCCs were maintained under serum-free conditions in neurobasal medium (Invitrogen Corp., Carlsbad, CA, USA), supplemented with B-27 supplement (2%; Invitrogen Corp.), glutamine (0.5 mM; Sigma), glutamate (25 mM; Sigma), penicillin (100 U/ml) and streptomycin (100 mg/ml; Invitrogen Corp.). At 4 days in vitro, half of the medium was removed and replaced with fresh medium without glutamate, as indicated by the manufacturer. The cultures were maintained in a humidified incubator at 37oC with 5% CO2. At 7 days in vitro, PCCs were used for
experimentation. Hypoxia Procedure
PCCs (1 ×105/mL) cultured at 37°C in 5% CO
2-humidified incubators were treated in
normoxic (21% O2) or hypoxic conditions (1% O2) for different time point as
previously described. Hypoxic cultures were cultivated in a two-gas incubator (Jouan, Winchester, Virginia, USA) equipped with an O2 probe to regulate N2 levels. Cell
number and viability were evaluated using trypan blue exclusion assay. Immunocytochemical and Western Blot Analysis of PCCs
Following hypoxia (1% O2 for 12 hours), PCCs were collected for PACAP38
30 min at room temperature in 4% paraformaldehyde. After being washed with PBS, the fixed cultured cells were treated for 30 min with blocking solution (10 g/L BSA, 0.03% Triton X-100, and 4% serum in PBS). PCCs were incubated overnight at 4oC
with an antibody against PACAP38 (1:100, Santa Cruz Biotechnology) and then rinsed 3 times in PBS. The extent of PACAP38+ cell immunoreactivity was measured
as the number of cells per square millimeter (cells/mm2). PCCs expression of
PACAP38 were measured by western blot analyses using appropriately diluted antibodies to PACAP38 (1:100, Santa Cruz Biotechnology) as mentioned above. Terminal Deoxynucleotidyl Transferase-Mediated Digoxigenin-dUTP Nick-End
Labeling (TUNEL) Histochemistry
To detect cellular apoptosis, a TUNEL staining Kit (DeadEndTM Fluorimetric TUNEL
system, Promega) was utilized for the TUNEL assay. Twenty-four hours after OGD, the cells were fixed with 4% paraformaldehyde in PBS for 20 min at 4°C and subjected to permeabilization for 20 min at room temperature with 0.1% sodium citrate containing 0.1% Triton X-100. The fixed and permeabilized PCCs were labeled with the TUNEL reaction mixture for 60 min at 37°C. The nuclei of these PCCs were counter-stained with DAPI. The percentage of TUNEL labeling was expressed as the number of TUNEL-positive nuclei divided by the total number of nuclei stained with DAPI.
Chromatin Immunoprecipitation (ChIP) Assay
To demonstrate the binding of HIF-1α protein to the promoter of PACAP, the ChIP assay was performed with a commercial kit (Upstate Biotechnology) using the manufacturer’s protocol with minor adjustments. The PCCs were grown and incubated in air or 1% O2 for 4 h, and formaldehyde was added directly to culture
medium to a final concentration of 1% followed by incubation for 20 min at 37°C as previously described. DNA–protein complexes were isolated on salmon sperm DNA linked to protein A agarose beads and eluted with 1% SDS, and 0.1 M NaHCO3.
Cross-linking was reversed by incubation at 65oC for 5 h. Proteins were removed with
proteinase K, and DNA was extracted with phenol/chloroform, re-dissolved and
PCR-amplified with PACAP promoter primers, sense:
5’-GAGGGACTAGGATGCTGACG-3’; and antisense: 5’-TGTTGCGCTCCG ATTTTTAT-3’.
Generation of Promoter Constructs, Transient Transfection, and Reporter Gene
Assays
A luciferase contruct containing the 5’-flanking region of the PACAP gene promoter was a kind gift from Miyata et al.. This luciferase reporter product PPR1 was further subcloned into the BamHI and SphI sites of the pGL3-basic vector (Promega) which contained one real HRE, and the generated plasmid was designated pPACAP-luc1.
One additional PACAP promoter constructs (pPACAP-luc2) using the same downstream primer as for luc1 didn’t contain the HRE. In the pPACAP-mutHRE construct, the putative HRE of pPACAP-luc1 was replaced from 5’-ACGTG-3’ to 5’-AAAAG-3’ using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All constructs were verified by DNA sequencing. 3T3 NIH cells at about 90% confluence in 24-well plates were transiently transfected with reporter plasmid (0.5 μg) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s directions. To correct for variable transfection efficiency, cells were cotransfected with the pRL-SV40 vector (0.05 μg) encoding the Renilla luciferase gene. Transfected cells were allowed to recover for 24 h in fresh medium, and then subjected to 1 % O2 for 8 hours. Cells were lysed and luciferase activity was
determined with a multiwell luminescence reader (Molecular Devices), by using the Dual-Luciferase Reporter Assay System (Promega).
Statistical Analysis
Observers were blind to the experimental conditions of each measurement. Results are expressed as mean ± SEM. The behavioral scores were evaluated for normality. We used one-way or two-way ANOVA with appropriate post hoc Newman-Keuls testing to evaluate mean differences between different groups with different treatments. A value of P < 0.05 was considered as significant.
Results
PACAP38 is Upregulated in The Ischemic Brains from Human Patients and Animals
To determine whether cerebral ischemia increases PACAP38 expression, we performed PACAP38 immunostaining in human and rat brain after stroke. Human brain samples (n = 4) from patients who died at various times after stroke and rat brains at different times (4 h to 7 d) after focal cerebral ischemia were processed for PACAP38 immunoreactivity. Increased number of PACAP38+ cells was noted in the
penumbral region in the brain samples from patients who died 1–3 days after cerebral infarction as compared with the control (Fig. 1A). PACAP38+ cells co-expressed the
neuronal marker proteins MAP-2 and Tuj-1 (Fig. 1A). Interestingly, some PACAP38+
and PAC1+ cells co-stained with CD34+–expressing cells, suggesting that circulating
CD34+-expressing cells may acquire PACAP38 and PAC1 expression upon
translocation to ischemic brain regions (Fig. 1A). Similar to the findings in stroke patients, increase in number of PACAP38+ cells were also detected in the ipsilateral
cortex near the infarct boundary in rat brains with a time-dependent manner, reaching the peak at 24 h after cerebral ischemia (Fig. 1B).
HIF-1α Induced PACAP38 Expression by Binding to the PACAP38 Promoter To determine whether HIF-1α and PACAP38 expression was increased in stroke rats, PACAP38 levels in brains (cortical region and striatum) from rats after cerebral ischemia were measured using Western blotting and ELISA and compared with non-ischemic rats. Cerebral ischemia in rat brains caused a time-dependent increase in HIF-1α and PACAP38 expression (Fig. 1C). ELISA also revealed significantly increased PACAP38 protein level in ischemic rats as compared with non-ischemic controls (Fig. 1C). To identify cell types expressing PACAP38 after cerebral
ischemia, double immunofluorescence staining under laser scanning confocal microscopy was used to observe the expression patterns of PACAP38 in colocalization with neuronal marker of Neu-N, glial marker of GFAP and endothelial marker of vWF in the ischemic brains. The PACAP38 was strongly co-expressed with Neu-N, GFAP and vWF in ischemic areas (Fig. 1D), suggesting brain ischemia upregulated PACAP38 in neurons, glia and endothelial cells. To examine the effect of hypoxia on PACAP38 expression in primary cortical cultures (PCCs), cells were placed under in vitro hypoxic conditions for 1, 3, 10, or 24 h. PACAP38 protein expression was also increased after hypoxic stress (Fig. 1, E and F).
We next examined whether PACAP38 upregulation after ischemia or hypoxia is mediated through HIF-1α activation. Cerebral ischemia increased the expression of activated nuclear HIF-1α, which was inhibited by 2-ME2 (100 mg/kg) injection (Fig. 2A). Double immunofluorescence staining in the ischemic rat cortical areas showed that increased numbers of PACAP38+ cells co-expressing with 1α as well as
HIF-1α nuclear translocation were inhibited by 2-ME2 injection (Fig. 2B). Upregulation of PACAP38 immunoreactivity after cerebral ischemia was also suppressed by 2-ME2 injection in a dose-dependent manner (Fig. 2C). Western blotting also demonstrated a dose-dependent 2-ME2 inhibition of PACAP38 expression after cerebral ischemia (Fig. 2D).Specially, ischemia-induced PACAP38 upregulation was not present in the brain of HIF-1α KO mice (Fig. 2E). In addition, HIF-1α expression in PCCs was localized in both the cytosol (including neurites) and nucleus under normoxia (Fig. 2F). HIF-1α translocation into the nucleus was noted under hypoxia (Fig. 2F). However, pretreatment of PCCs with 2-ME2 for 16 hr abolished HIF-1α translocation into the nucleus (Fig. 2F). PACAP38 expression was also suppressed when PCCs were pretreated for 16 hours with 2-ME2 (Fig. 2, F and G) or HIF-1α knockdown in PCCs via lentiviral delivery of HIF-1α shRNA (LV-HIF-1α-sh) (Fig. 2G). The 2-ME2
effect was specific to HIF-1α–induced PACAP38 expression, as it did not affect lentiviral (LV-PACAP38) infection–induced PACAP38 expression (Fig. 2G).
To prove whether the hypoxia-induced PACAP38 upregulation is only through HIF-1α, overexpression of HIF-1α or HIF-2α and knockdown of HIF-1α or HIF-2α were used to address this issue. Our results demonstrated that genetic manipulation of HIF-1α via lentiviral transduction (LV-HIF-1α or LV-HIF-1α-sh) significantly modulated the expression of PACAP38 in PCCs at one hour after hypoxia compared to genetic manipulation of HIF-2α(Fig. 2H). Then, Chromatin Immunoprecipitation (ChIP) assay was conducted to demonstrate interaction between HIF-1α and the PACAP promoter. HIF-1α was recruited to bind on the PACAP promoter in PCCs subjected to 4-h hypoxia, but not in PCCs under normoxia or PCCs treated with LV– HIF-1α–shRNA under 4-h hypoxia (Fig. 2I). Furthermore, the activity of a luciferase reporter gene construct (pPACAP-luc1) containing hypoxic-responsive element (HRE) from the PACAP gene promoter coupled to an SV40 promoter under hypoxia was much higher than a control construct (pPACAP-luc2) and an HRE-mutant construct (pPACAP-mutHRE; Fig. 2J). Taken together, these results provide direct evidence suggesting hypoxia-induced PACAP38 is through HIF-1α–dependent regulation via the direct binding to PSCAP38 promoter.
PACAP38/PAC1 Signaling Promoted BMDC Proliferation and Trafficking Next, we investigated the role of PACAP38 in cell proliferation and migration in
vitro, we measured cell numbers and BrdU incorporation and tested the ability of
CD34+ BMDCs to migrate in response to increasing PACAP38 concentrations. Cell
index were increased following PACAP38 administration in a dose-dependent manner (Fig. 3A). The migration of CD34+ BMDCs treated with PACAP38 occurred in a
concentration-dependent manner (Fig. 3B). PACAP (6-38), a PAC1 antagonist, inhibited PACAP38-induced BMDC migration (Fig. 3B). To explore molecular mechanism of PACAP38 induced-cell migration and adhesion in vitro, we analyzed the protein expression of known target proteins that are involved in cell adhesion and migration in PACAP38-treated BMDCs: cellular prion protein (PrPC), CCR3, CCR4,
α6-integrin, β1-integrin, β2-integrin, p-selectin, CD99, JAM-A, JAM-C, PECAM-1, LFA-1, ICAM, VCAM, VE-cadherin, CXCR4, FAK and STI-1 . Ten to twelve hours after treatment, PACAP38 upregulated the expression of the adhesion molecules PrPC
and α6-integrin in a dose-dependent manner (Fig. 3C). In addition, PACAP38 also enhanced the expression and activity of matrix metalloproteases (MMP) 2 and 9 in a dose-dependent manner (Fig. 3, D and E). PACAP (6-38) inhibited the PACAP38-induced enhancement of MMP2 and MMP9 activities (Fig. 3D), indicating PACAP38/PAC1 signaling involved in activation of MMP2 and MMP9
BMDCs Homes to the Vascular Niche of Ischemic Brain via Response to
PACAP38/PAC1 Signaling
PACAP38/PAC1 signaling in CD34+ BMDCs trafficking, we hypothesized
PACAP38/PAC1 signaling is critical pathway involved in brain ischemia-mediated recruitment of BMDCs to the vascular niche. To test this model, we first determine the association between CD34+ BMDCs homing and PACAP38/PAC1 signaling in
vivo. In a double immunofluorescence study, CD34+ BMDCs from GFP-chimeric
mice were showed co-localization of PAC1 (Fig. 4A). Moreover, significantly increased numbers of GFP+ BMDCs were found in the ischemic brains of PAC1+/+ GFP-chimeric mice as compared with PAC1−/− mice on 3 d after cerebral ischemia
(Fig. 4A). To determine whether BMDCs homed to the ischemic brain of PACAP38-treated mice, double immunohistochemical staining was conducted on brain sections on 7 d after cerebral ischemia in GFP-chimeric mice (PAC1+/+ and PAC1−/− mice). A
significantly increased number of GFP+CD34+ BMDCs were found in the right
striatum, hippocampus, and penumbral area in PACAP38-treated PAC1+/+ mice as
compared with PACAP38-treated PAC1−/− mice, saline-treated PAC1+/+ mice, and
saline-treated PAC1−/− mice (Fig. 4B). However, PACAP38-induced BMDC homing
was abolished by administration of an MMP inhibitor (GM6001) in PACAP38-treated
PAC1+/+ mice (Fig. 4B). Furthermore, the infarct volume in PACAP38-treated PAC1+/
+ mice was significantly reduced as compared with that in saline-treated PAC1+/+
To establish how BMDCs integrate into the penumbra of the ischemic brain and specifically whether they home to the vasculature, we measured the relationship of BMDCs to the closest endothelial cell surface after cerebral ischemia in GFP-chimeric mice. On three days after stroke, the penumbric region of GFP-GFP-chimeric mouse brains was stained for laminin to visualize the blood vessels (Fig. 4D). CD34+GFP+ BMDCs were predominantly observed near the penumbric vasculature
(Fig. 4D). Most CD34+GFP+ BMDCs (65 ± 4.7%) were within 5 μm of the laminin+
vasculature, 20 ± 3.1% were 5–20 μm away, and 16 ± 7.2% were >20 μm away. Thus, BMDCs migrate to the vasculature of the ischemic brain in vivo, consistent with their ability to home to this vascular niche.
We then examined the molecular mechanism that allowed BMDCs to be located near blood vessels. Because the PACAP is involved in adult NPC homing to areas of CNS injury after ischemia, we tested PACAP as a candidate for recruitment of BMDCs. PACAP38 was co-expressed with the vasculature marker laminin, and expression of the migration and adhesion markers, such as PrPC and α6-integrin, was
seen along laminin+ blood vessels in the penumbric area (Fig. 4E). Importantly, we
found that PAC1 was visible on GFP+ BMDCs associated with the vasculature after
homing to the ischemic brain of GFP-chimeric mice (Fig. 4F). Thus, the ligand, PACAP38, was enriched in the niche, and GFP+ BMDCs express the receptor, PAC1,
consistent with the hypothesis that this ligand-receptor pair is involved in BMDC homing to the stroke brain. In addition, PrPC and α6-integrin were also co-expressed
in CD34+GFP+ BMDCs (Fig. 4G). Consistent with the results from BMDC culture,
PACAP38 administration in ischemic brain significantly increased PrPC and
α6-integrin expression (Fig. 4G). Taken together, PACAP38 treatment alters the expression of key receptors associated with proliferation, adhesion, and migration of BMDCs, providing pleiotropic effects on the BMDC lineage, similar to its effects on the NPC lineage .
PACAP38/PAC1 Signaling Facilitated Stroke Recovery by BMDC Trafficking
into Brain
Our above results suggest r PACAP38 as an ischemia-inducible endogenous factor for mobilization of BMDCs into the circulation that are subsequently recruited to the ischemic brain tissue. According to this novel mechanism, we next proposed the supplement of exogenous PACAP38 as therapeutic approach may promote the homing of BMDCs to ischemic areas and neural survival. To select the most effective treatment dosage of PACAP38, TTC staining was measured in three rat groups treated with 0.1, 1, or 10 μg/kg PACAP38. The infarct volume of the rats given 10 μg/kg PACAP38 was much smaller than that in the other dosage groups at 3 days after cerebral ischemia (Fig. 5A). At 7 days after cerebral ischemia, the infarct volume and
area of the largest infarcted slice as assessed by magnetic resonance imaging were significantly reduced in PACAP38-treated mice as compared with those in the groups treated with PACAP38 + GM6001, PACAP38 + PACAP (6-38), or saline (Fig. 5B). Body asymmetry, locomotor activity tests, and grip strength measurement were used to assess neurological deficit recovery in PACAP38-treated, PACAP38+GM6001– treated, PACAP38+PACAP (6-38)–treated and control rats. PACAP38-treated rats showed better recovery in body swing tests than did rats treated with PACAP38 + GM6001, PACAP38 + PACAP (6-38), or saline control (Fig. 5C). Locomotor activities were substantially better after cerebral ischemia in rats receiving PACAP38 as compared with the other groups (Fig. 5C). In addition, comparison of forelimb grip strength before and 28 days after ischemia showed that the PACAP38-treated group had a much better grip strength ratio than did the other groups (Fig. 5C).
Promotion of Neural Survival and BMDC-Related Angiogensis by
PACAP38/PAC1 Signaling
To verify whether PACAP38 enhanced metabolic activity, cortical glucose metabolism was examined with 18F-FDG PET imaging at 1 week after treatment. The
microPET images of the right cortex (the site of cerebral ischemia) of the PACAP38-treated group showed a significant increase in FDG uptake, which was higher than that in the other three groups (Fig. 6A). The anti-apoptotic effects of PACAP38 in the
ischemic brain were examined with western blotting of apoptosis-related proteins. We observed significantly upregulated expression of the anti-apoptotic protein Bcl-2 in PACAP38-treated rats 24 hr after cerebral ischemia as compared with that in control rats (Fig. 6B). Cellular apoptosis in ischemic rat brain was studied with TUNEL staining. The penumbral region surrounding the ischemic core of PACAP38-treated rats contained fewer TUNEL+ cells than that of the other three groups (Fig. 6C).
To determine whether intraperitoneal PACAP38 administration induced angiogenesis by promoting the homing of BMDCs to ischemic sites FITC-dextran perfusion studies and blood vessel density assays were performed on each brain slice from each experimental rat. Visual inspection of FITC-dextran perfusion indicated that PACAP38 induced much more cerebral microvascular perfusion than did saline (Fig. 6D). Blood vessel density assays showed that ischemic rats treated with PACAP38 had better neovascularization in the penumbral area than did the other groups (Fig. 6D). We next used laser Doppler flowmetry (LDF) to examine whether the increased blood vessel density enhanced functional cerebral blood flow (CBF) in the ischemic brains. One week after cerebral ischemia, CBF in the ischemic cortex of the PACAP38-treated rats was significantly increased as compared with that of the other three groups (Fig. 6E).
Demonstrating whether stem cells home to vascular cells is an important issue because this interaction could allow BMDCs to integrate into vascularized parenchymal regions of the ischemic brain to enhance tissue repair. Here we show that BMDCs homed toward endothelial cells under ischemic stress and provide the direct evidence for supporting PACAP38/PAC1 axis-mediated signals involved in these processes. The rationale of endothelial cells expressing high levels of PACAP38, which are required for NPC homing to the ischemic brain, let us explore the role of PACAP38 in BMDCs homing to vascular niches . Double immunofluorescence staining in ischemic stroke rat brains showed an expression pattern consistent with PACAP38 being synthesized by blood vessels and released into the nearby environment. We found that the PACAP38 receptor, PAC1, is widely expressed on BMDCs. Although PACAP38 may contribute to the build-up of neural lineage cells around blood vessels in the long term, it is unlikely that cells preferentially survive or proliferate near blood vessels. Pursuing the hypothesis that homing was responsible, we demonstrated that blood vessel endothelial cells secrete factors of PACAP38 that elicit BMDC chemotaxis when provided as a gradient. Furthermore, PACAP38 upregulates α6-integrin and PrPC expression on the surface of
BMDCs. This may stimulate BMDCs to move toward blood vessels and increase their binding to laminin, which is concentrated on blood vessel surfaces. Importantly,
homing of BMDCs to the penumbric area of ischemic brain was effectively blocked in PACAP (6-38)–treated and in PAC1−/− mice, demonstrating that PACAP38-PAC1
signaling is a critical component of the homing mechanism. The observation that PACAP38 level was important for BMDC homing to endothelial cells provides a satisfying parallel with the homing mechanism of NPCs; pursuing other molecular parallels in these pathways will be important.
PACAP is a potent stimulator of cAMP accumulation and gene expression. PACAP participates in immunomodulation and is related to the etiology of neuropsychiatric disease. Although some investigators have discovered PACAP upregulation following cerebral ischemia in animals and neural cell culture, we also demonstrated that increased PACAP38 expression was induced in human and rat ischemic brain. This is consistent with high PACAP expression under stress conditions. Thus, PACAP may be expressed in response to stress. In agreement with previous studies, PACAP seems to have similar functions as some trophic factors, such as the activation of endogenous protective mechanisms to enhance growth and repair of injured neural tissue. In the cerebral ischemia and hypoxia model especially, PACAP38 activation may transduce an important signal for neural adaptation to ischemia-related environmental stress.
D-aspartate (NMDA) receptor–mediated upregulation of PACAP, the cellular and molecular mechanisms of the PACAP upregulation are unclear. The novel contribution of our study is that hypoxia/ischemia-induced expression of PACAP38 was mediated by the activation and binding of HIF-1α to the HRE of the PACAP38 promoter. The level of HIF-1β did not change during the hypoxia/ischemia. Hypoxia/ischemia-induced PACAP38 upregulation was abolished in neural tissues with decreased HIF-1α activity that resulted from the injection of the HIF-1α–specific inhibitor, 2-ME2; in animals with cerebral ischemia and in a Cre-loxP–based approach to knockout HIF-1α in mice. The proximal promoter region (~0.8 kb) of the mouse PACAP gene has at least one putative HIF-1α binding site with the consensus sequence 5′-ACGTG-3′. In a reporter assay, hypoxia-induced luciferase activation was abolished after mutation of this potential HIF-1α binding site. Moreover, binding of HIF-1α to this putative HRE was demonstrated by ChIP assays in hypoxic neurons in culture.
The mobilization and homing of BMDCs are enhanced by factors such as granulocyte colony–stimulating factor (G-CSF), VEGF, SDF1-α and stem cell factor, as well as appear to involve a receptor-mediated process that includes the G-CSF receptor, VEGF receptor, CXCR4, and c-kit, respectively. Although some investigators have focused on PACAP-mediated proliferation and migration of NPCs,
few reports have studied the relationship between BMDC homing and PACAP expression. In previous studies, activation of cAMP and downstream signaling by PACAP not only enhance CD34+ cell survival and homing but also mediate G-CSF–
induced CD34+ cytoprotection. In addition, cAMP activation upregulates CXCR4
expression to augment the motility of CD34+ cells. Here we demonstrated that
endogenous PACAP38 secretion in the ischemic brain promoted BMDC mobilization, which appeared to be a PAC1-associated process. Regarding the molecular mechanism of BMDC trafficking, PACAP38 increase the expression of adhesion/migration related proteins including PrPC, α6-integrin, β1-integrin, FAK and
CXCR4, as well as enhance the activity of MMP9 and MMP2 in BMDCs to promote homing and migration. Therefore, we assume that PACAP38 induces PAC1-mediated signaling as a major pathway that regulates BMDC trafficking.
We found that the angiogenic effect played an important role in the neuroprotective outcome in the ischemic brain. Administration of PACAP38 seemed to augment angiogenesis in the penumbral area after stroke. Although some investigators have demonstrated that the angiogenic action of PACAP is mediated by VEGF stimulation, few reports have focused on direct angiogenesis by PACAP. Since one important function of HIF-1α is to promote tissue angiogenesis. HIF-1α can guide migration of endothelial precursor cells toward a hypoxic environment. Here we
discovered that PACAP38 upregulation by hypoxia/ischemia via HIF-1α activation stimulated neovascularization, thus increasing CBF in the penumbral area. Therefore, PACAP38 may be considered a new angiogenic factor that is regulated by HIF-1α induction.
CONCLUSIONS
We present experimental findings to support the contention that PACAP38 expression was upregulated by hypoxia/ischemia via activation of HIF-1α. To the best of our knowledge, HIF-1α transactivation of PACAP38 has not been previously reported. We further demonstrate that the activation of the PACAP38-PAC1 signaling cascade is pivotal for facilitating BMDC homing toward the ischemic niche in the brain to confer neuroprotective as well as angiogenic actions in reducing brain damage and in enhancing functional recovery after cerebral ischemia. The clinical significance of the PACAP38-PAC1 cascade on BMDC homing is further strengthened by the findings that exogenous PACAP38 is capable of exerting the same action as the endogenous conterpart to provide a possibly novel therapeutic intervention that may improve regeneration and repair following cerebral ischemia.
ACKNOWLEDGMENTS
We are grateful to Dr. Gunther Schutz and colleagues (German Cancer Research Center, DKFZ) for providing PAC1 cryopreserved mouse embryos. This work was supported in part by research grants from the Chen-Han Foundation for Education,
Advancement of Clinical Trials Innovation and Competitiveness (MOHW103-TDU-B-212-113002), and Taiwan’s National Science Council (NSC97-2314-B-039-036- MY3;NSC100-2314-B-039-002-MY3;NSC100-2321-B-039-006;NSC100-2321-B- 039-005;NSC100-2632-B-039-001-MY3;NSC102-2325-B-039-006;NSC101-2321-B-039-001;NSC102-2325-B-039-001).
AUTHOR CONTRIBUTIONS
W.C.S. and C.H.H designed, conducted, and supervised the experiments and contributed to manuscript preparation; C.H.L. and H.T.L. conducted experiments and contributed to BMDCs cultures and lentiviral vector preparation; S.P.L. and C.W.C. contributed to luciferase reporter cloning and animal studies; Y.H.H. contributed to human brain sample immunostating; L.C. S.Z.L. and C.Y.H. contributed to manuscript preparation.
DISCLOSURE OF POTENTIAL CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.REFERENCES
1. Shyu WC, Liu DD, Lin SZ et al. Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke. J Clin Invest. 2008;118:2482-2495.
2. Borlongan CV. Bone marrow stem cell mobilization in stroke: a 'bonehead' may be good after all! Leukemia. 2011;25:1674-1686.
3. Collado B, Sanchez-Chapado M, Prieto JC et al. Hypoxia regulation of expression and angiogenic effects of vasoactive intestinal peptide (VIP) and VIP receptors in LNCaP prostate cancer cells. Mol Cell Endocrinol. 2006;249:116-122.
4. Ribatti D, Conconi MT, Nussdorfer GG. Nonclassic endogenous novel [corrected] regulators of angiogenesis. Pharmacol Rev. 2007;59:185-205. 5. Laburthe M, Couvineau A. Molecular pharmacology and structure of VPAC
Receptors for VIP and PACAP. Regul Pept. 2002;108:165-173.
6. Stumm R, Kolodziej A, Prinz V et al. Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and
protects neurons from mild hypoxic/ischemic damage. J Neurochem. 2007;103:1666-1681.
7. Shyu WC, Lin SZ, Yang HI et al. Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells. Circulation. 2004;110:1847-1854.
8. Toth ZE, Leker RR, Shahar T et al. The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow-derived endothelial cells in brains of mice following cerebral ischemia. Blood. 2008;111:5544-5552.
9. Kaplan RN, Psaila B, Lyden D. Niche-to-niche migration of bone-marrow-derived cells. Trends Mol Med. 2007;13:72-81.
10. Peled A, Kollet O, Ponomaryov T et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood. 2000;95:3289-3296.
11. Nishimoto M, Furuta A, Aoki S et al. PACAP/PAC1 autocrine system promotes proliferation and astrogenesis in neural progenitor cells. Glia. 2007;55:317-327.
12. Zhu Y, Cao L, Su Z et al. Olfactory ensheathing cells: attractant of neural progenitor migration to olfactory bulb. Glia. 2010;58:716-729.
13. Matsuno R, Ohtaki H, Nakamachi T et al. Distribution and localization of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1R) in the rostral migratory stream of the infant mouse brain. Regul Pept. 2008;145:80-87.
14. Kawakami M, Kimura T, Kishimoto Y et al. Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells. Leukemia. 2004;18:912-921.
15. Freson K, Peeters K, De Vos R et al. PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications. Blood. 2008;111:1885-1893.
16. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3:721-732.
17. Chavez JC, LaManna JC. Activation of hypoxia-inducible factor-1 in the rat cerebral cortex after transient global ischemia: potential role of insulin-like growth factor-1. J Neurosci. 2002;22:8922-8931.
18. Bergeron M, Gidday JM, Yu AY et al. Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain. Ann Neurol.
2000;48:285-296.
19. Ceradini DJ, Kulkarni AR, Callaghan MJ et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med. 2004;10:858-864.
20. Hu CJ, Iyer S, Sataur A et al. Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol. 2006;26:3514-3526.
21. Ryan HE, Poloni M, McNulty W et al. Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth. Cancer Res. 2000;60:4010-4015.
22. Ricker JL, Chen Z, Yang XP et al. 2-methoxyestradiol inhibits hypoxia-inducible factor 1alpha, tumor growth, and angiogenesis and augments paclitaxel efficacy in head and neck squamous cell carcinoma. Clin Cancer Res. 2004;10:8665-8673.
23. Dai Y, Xu M, Wang Y et al. HIF-1alpha induced-VEGF overexpression in bone marrow stem cells protects cardiomyocytes against ischemia. J Mol Cell Cardiol. 2007;42:1036-1044.
24. Shyu WC, Lin SZ, Chiang MF et al. Secretoneurin promotes neuroprotection and neuronal plasticity via the Jak2/Stat3 pathway in murine models of stroke. J Clin Invest. 2008;118:133-148.
25. Shen Q, Wang Y, Kokovay E et al. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell. 2008;3:289-300.
26. Lee SR, Kim HY, Rogowska J et al. Involvement of matrix metalloproteinase in neuroblast cell migration from the subventricular zone after stroke. J Neurosci. 2006;26:3491-3495.
27. Boni LJ, Ploug KB, Olesen J et al. The in vivo effect of VIP, PACAP-38 and PACAP-27 and mRNA expression of their receptors in rat middle meningeal artery. Cephalalgia. 2009;29:837-847.
28. Matsumura A, Mizokawa S, Tanaka M et al. Assessment of microPET performance in analyzing the rat brain under different types of anesthesia: comparison between quantitative data obtained with microPET and ex vivo autoradiography. Neuroimage. 2003;20:2040-2050.
29. Hamacher K, Coenen HH, Stocklin G. Efficient stereospecific synthesis of no-carrier-added 2-[18F]-fluoro-2-deoxy-D-glucose using aminopolyether supported nucleophilic substitution. J Nucl Med. 1986;27:235-238.
30. Carmichael ST, Tatsukawa K, Katsman D et al. Evolution of diaschisis in a focal stroke model. Stroke. 2004;35:758-763.
activation and plasticity in the rat brain by high resolution positron emission tomography (microPET). Nat Biotechnol. 2000;18:655-660.
32. Brownell AL, Livni E, Galpern W et al. In vivo PET imaging in rat of dopamine terminals reveals functional neural transplants. Ann Neurol. 1998;43:387-390.
33. Molina-Hernandez A, Velasco I. Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors. J Neurochem. 2008;106:706-717.
34. Shalak V, Kaminska M, Mitnacht-Kraus R et al. The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component. J Biol Chem. 2001;276:23769-23776.
35. Sanchez A, Rao HV, Grammas P. PACAP38 protects rat cortical neurons against the neurotoxicity evoked by sodium nitroprusside and thrombin. Regul Pept. 2009;152:33-40.
36. Moore JP, Jr., Villafuerte BC, Unick CA et al. Developmental changes in pituitary adenylate cyclase activating polypeptide expression during the perinatal period: possible role in fetal gonadotroph regulation. Endocrinology. 2009;150:4802-4809.
37. Plett PA, Frankovitz SM, Wolber FM et al. Treatment of circulating CD34(+) cells with SDF-1alpha or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential. Exp Hematol. 2002;30:1061-1069.
38. Salmon P, Oberholzer J, Occhiodoro T et al. Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. Mol Ther. 2000;2:404-414.
39. Lin CH, Lee HT, Lee SD et al. Role of HIF-1alpha-activated Epac1 on HSC-mediated neuroplasticity in stroke model. Neurobiol Dis. 2013;58:76-91. 40. Zennou V, Petit C, Guetard D et al. HIV-1 genome nuclear import is mediated
by a central DNA flap. Cell. 2000;101:173-185.
41. Hess DC, Hill WD, Martin-Studdard A et al. Bone marrow as a source of endothelial cells and NeuN-expressing cells After stroke. Stroke. 2002;33:1362-1368.
42. Otto C, Hein L, Brede M et al. Pulmonary hypertension and right heart failure in pituitary adenylate cyclase-activating polypeptide type I receptor-deficient mice. Circulation. 2004;110:3245-3251.
43. Morris DC, Zhang Z, Davies K et al. High resolution quantitation of microvascular plasma perfusion in non-ischemic and ischemic rat brain by laser-scanning confocal microscopy. Brain Res Brain Res Protoc. 1999;4:185-191.
