Cochlear Pericyte Responses to Acoustic Trauma and the Involvement of Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.080739
ISSN1525-2191
Autores Tópico(s)Cardiovascular and Diving-Related Complications
ResumoThis study explored the effect of acoustic trauma on cochlear pericytes. Transmission electron microscopy revealed that pericytes on capillaries of the stria vascularis were closely associated with the endothelium in both control guinea pigs and mice. Pericyte foot processes were tightly positioned adjacent to endothelial cells. Exposure to wide-band noise at a level of 120 dB for 3 hours per day for 2 consecutive days produced a significant hearing threshold shift and structurally damaged blood vessels in the stria vascularis. Additionally, the serum protein, IgG, was observed to leak from capillaries of the stria vascularis, and pericytes lost their tight association with endothelial cells. Levels of the pericyte structural protein, desmin, substantially increased after noise exposure in both guinea pigs and mice with a corresponding increase in pericyte coverage of vessels. Increased expression levels of desmin were associated with the induction of hypoxia inducible factor (HIF)-1α and the up-regulation of vascular endothelial growth factor (VEGF). Inhibition of HIF-1α activity caused a decrease in VEGF expression levels in stria vascularis vessels. Blockade of VEGF activity with SU1498, a VEGF receptor inhibitor, significantly attenuated the expression of desmin in pericytes. These data demonstrate that cochlear pericytes are markedly affected by acoustic trauma and display an abnormal morphology. HIF-1α activation and VEGF up-regulation are important factors for the alteration of the pericyte structural protein desmin. This study explored the effect of acoustic trauma on cochlear pericytes. Transmission electron microscopy revealed that pericytes on capillaries of the stria vascularis were closely associated with the endothelium in both control guinea pigs and mice. Pericyte foot processes were tightly positioned adjacent to endothelial cells. Exposure to wide-band noise at a level of 120 dB for 3 hours per day for 2 consecutive days produced a significant hearing threshold shift and structurally damaged blood vessels in the stria vascularis. Additionally, the serum protein, IgG, was observed to leak from capillaries of the stria vascularis, and pericytes lost their tight association with endothelial cells. Levels of the pericyte structural protein, desmin, substantially increased after noise exposure in both guinea pigs and mice with a corresponding increase in pericyte coverage of vessels. Increased expression levels of desmin were associated with the induction of hypoxia inducible factor (HIF)-1α and the up-regulation of vascular endothelial growth factor (VEGF). Inhibition of HIF-1α activity caused a decrease in VEGF expression levels in stria vascularis vessels. Blockade of VEGF activity with SU1498, a VEGF receptor inhibitor, significantly attenuated the expression of desmin in pericytes. These data demonstrate that cochlear pericytes are markedly affected by acoustic trauma and display an abnormal morphology. HIF-1α activation and VEGF up-regulation are important factors for the alteration of the pericyte structural protein desmin. 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Our results show that high-level noise exposure increased the expression of pericyte structural protein desmin and that excess numbers of pericytes decorate vessels resulting in increased coverage over the vessels. HIF-1α induction and VEGF up-regulation are found to be influenced by loud sound and they are important components for altering pericyte structure in response to noise stimuli. Experiments were performed on albino guinea pigs (both sexes; weight, 300 to 450 g) and 129S2/C57BL/6 mice (8 to 10 weeks old). All procedures in this study were reviewed and approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University. The animals (guinea pigs and mice) with positive Preyer reflex were divided into control and noise-exposed groups. For noise exposure, the animals were placed in wire mesh cages and exposed to broadband noise at 120 dB SPL in a sound exposure booth for 30 minutes, 1 hour, 3 hours, or for an additional 3 hours the next day. This noise exposure regime is routinely used in our laboratory and produces a permanent cochlear sensitivity loss.38Shi XR Nuttall AL Upregulated iNOS and oxidative damage to cochlear stria vascularis due to noise stress.Brain Res. 2003; 967: 1-10Crossref PubMed Scopus (90) Google Scholar Auditory brain-stem response audiometry to pure tones was used to evaluate hearing function before noise exposure and after noise exposure in five mice and five guinea pigs. For the auditory brain-stem response test, each animal was anesthetized with xylazine (10 mg/kg, i.m., IVX; Animal Health Inc., St. Joseph, MO) and ketamine (40 mg/kg, i.m.; Hospira, Inc., Lake Forest, IL), and placed on a heating pad in a sound-isolated chamber. The external ear canal and tympanic membrane were inspected using an operating microscope to ensure the ear canal was free of wax and that there was no canal deformity, no inflammation of the tympanic membrane, and no effusion in the middle ear. Needle electrodes were placed subcutaneously near the test ear, at the vertex and at the contralateral ear. Each ear was stimulated separately with a closed tube sound delivery system sealed into the ear canal. The auditory brain-stem response to a 1-ms rise-time tone burst at 4, 8, 12, 16, 24, and 32 kHz was recorded and thresholds obtained for each ear. Threshold was defined as an evoked response of 0.2 μV. This method was used to assess auditory brain-stem response both before noise exposure and immediately after noise exposure. Primary antibodies used in the experiments included monoclonal rabbit anti-desmin (catalog no. ab32362; Abcam, Cambridge, MA), polyclonal rabbit anti-VEGF (catalog no. sc-507; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal rabbit anti-HIF-1α (catalog no. sc-10790, Santa Cruz Biotechnology, Inc.), Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (catalog no. A11001; Invitrogen, Eugene, OR), Alexa Fluor 568-conjugated goat anti-guinea pig IgG (H+L) (catalog no. A11073, Invitrogen). Secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit (catalog no.11008, Invitrogen, Eugene, OR). Immunohistochemistry was performed as described previously.38Shi XR Nuttall AL Upregulated iNOS and oxidative damage to cochlear stria vascularis due to noise stress.Brain Res. 2003; 967: 1-10Crossref PubMed Scopus (90) Google Scholar Tissue sections were briefly permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, MO) for 1 hour, and immunoblocked with a solution of 10% goat serum and 1% bovine albumin in 0.02 mol/L phosphate-buffered saline (PBS) for 1 hour. The specimens were incubated overnight at 4°C with the primary antibody diluted in PBS-bovine serum albumin. After several washes in PBS, sections were incubated in secondary antibody for 1 hour at room temperature. Tissues were mounted in mounting medium (H-1000; Vector Laboratories, Inc., Burlingame, CA) and visualized under an Eclipse TE 300 inverted microscope (Nikon, Tokyo, Japan) fitted with a Bio-Rad (Richmond, CA) MRC 1024 confocal laser microscope system. Controls were prepared by replacing primary antibodies with 0.2% Triton X-100 in PBS. Desmin expression under control and noise-stimulated conditions was compared. Guinea pigs and mice from control and on the second day of noise-exposed groups were anesthetized with an overdose of ketamine hydrochloride (100 mg/kg, i.m.) and 2% xylazine hydrochloride (10 mg/kg) (Abbott Laboratories, N. Chicago, IL). The cochleae were taken after cardiovascular perfusion with PBS (pH 7.4). Each guinea pig group was comprised of six animals; each mouse group was comprised of 10 animals. The guinea pig and mouse cochleae were removed and the whole cochlear lateral walls of the guinea pigs were immediately dissected in ice-cold PBS. For the mouse cochlea, the cochlear stria vascularis was isolated from the cochlear lateral wall in cold PBS. Tissue samples were washed twice with cold PBS and immediately frozen at −80°C in 1.5-ml Eppendorf tubes. Total protein was extracted following the manufacturer's instructions (catalog no. 20-188; Upstate, Lake Placid, NY). The protein was removed and stored at −20°C. The protein concentrations were calculated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were loaded on 12% sodium dodecyl sulfate-polyacrylamide gels (90 V, room temperature for 80 minutes), electrophoresed, and transferred to nitrocellulose by electroblotting (30 V, overnight at 4°C) in 1× transfer buffer (Bio-Rad). Nitrocellulose membranes were blocked in 5% w/v nonfat dry milk and 0.1% v/v Tween 20 in PBS (pH 7.4, 0.12 mol/L) for 1 hour at 25°C before being incubated overnight at 4°C with primary antibodies [monoclonal rabbit anti-desmin 1:500 (catalog no. ab32362, Abcam, Cambridge, MA), polyclonal rabbit anti-VEGF 1:500 (catalog no. sc-507, Santa Cruz Biotechnology, Inc.)] in blocking buffer. Membranes were then incubated with a 1:3000 v/v secondary antibody (Bio-Rad) for 1 hour at room temperature. Protein bands were visualized on Kodak XAR-5 film (Eastman-Kodak, Rochester, NY) with Super Signal West Femto chemiluminescent substrate (Pierce, Rockford, IL). Equal protein loading was examined by stripping the blots and reprobing them with a 1:5000 v/v of monoclonal mouse antibody for GAPDH (Chemicon International, Temecula, CA) or monoclonal mouse antibody for α-actin (1:3000, catalog no. A1978; Sigma) followed by incubation with secondary antibody (catalog no. 170-6516, goat anti-mouse antibody; Bio-Rad) and visualization as described above. Total RNA from the cochlear lateral wall was separately extracted, with RNeasy (Qiagen, Valencia, CA), from the control, noise-exposed, and drug-pretreated noise-exposed groups. Each group was comprised of 10 mice for analysis of mRNA levels of Desmin and Vegf with quantitative real-time PCR. Two μg of total RNA and 100 ng of random hexamer were used to make 40 μl of cDNA by SuperScript II (Invitrogen) following the manufacturer's instructions. Transcript quantities were assayed by corresponding TaqMan gene expression assay: Desmin (catalog no. Mm00802455_m1; Applied Biosystems, Foster City, CA) and Vegf (catalog no. Rh02621759_m, Applied Biosystems) with a model 7300 real-time PCR system (Applied Biosystems, Foster City, CA). Thermal cycle conditions were an initial hold of 50°C for 2 minutes, 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. For assessment of mRNA level of Hif-1α, total RNA from the cochlear lateral wall was separately extracted from the control, noise exposure (30 minutes), noise exposure (1 hour), and noise exposure 3 hours in each day for 2-day groups. Each group was comprised of three mice. Two μg of total RNA sample was reverse-transcribed by using RETROscript kit (Ambion, Austin, TX). The cDNA synthesized from total RNA was diluted 10-fold with DNase-free water. Transcript quantities were assayed by corresponding TaqMan gene expression assay Hif-1α (catalog no. Mm01283758_g1, Applied Biosystems) with a StepOnePlus real-time PCR system (Applied Biosystems). Cycling conditions of real-time PCR were 95°C for 20 seconds, 40 cycles of 95°C for 1 second, 60°C for 20 seconds. Mouse Gapdh (catalog no. 4352339E, Applied Biosystems) expression was used as an endogenous control. Samples were run in triplicate for Desmin and Vegf. Samples were run six times for Hif-1α. Quantitative PCR was performed according to the guidelines provided by Applied Biosystems. The comparative cycle threshold (CT) method (ΔΔCT quantitation) was used to calculate the difference between samples. Quantitative data analysis followed the suggestions of the manufacturer. Cochlear lateral wall tissues were dissected from the control and noise-exposed animals. Segments of the cochlear lateral wall from the basal turns were fixed overnight in phosphate-buffered 3% glutaraldehyde-1.5% paraformaldehyde and postfixed in 1% osmium. Tissues were dehydrated, embedded in Araldite plastic, sectioned, stained with lead citrate and uranyl acetate, and viewed in a Phillips, CM, 100 transmission electron microscope (Eindhoven, the Netherlands). To visualize pericytes on the endothelium of the cochlear lateral wall, we double labeled lateral wall tissues with a combination of antibody for desmin (to identify pericytes) and isolectin GS-IB4 Alexa Fluor 488 (to identify vessels) (catalog no. 121411, Invitrogen). The procedure after immunohistochemical labeling for desmin was the same as described above except that 1:400 isolectin GS-IB4 was added to the medium along with primary antibody for desmin. To visualize HIF-1α translocation, we triple labeled lateral wall tissues with a combination of antibody for HIF-1α, propidium iodide (catalog no. P-3566, Molecular Probes, Eugene, OR) (to identify cell nuclei) and isolection GS-IB4 Alexa Fluor 647 (to identify vessels, catalog no. I32450, Invitrogen). The procedure after immunohistochemical labeling for HIF-1α was the same as described above except that 1:100 propidium iodide was added to the medium along with secondary antibody for HIF-1α for 1 hour. Images were analyzed using Image J (V1.38X; National Institutes of Health, West Chester, PA). The cochlear lateral wall from the basal turn was used for the study. Images were acquired with a ×40 objective. A total of 60 images were recorded from five normal guinea pigs and a total of 80 images from seven noise-exposed guinea pigs were recorded. A total of 171 images were recorded from five normal mice and 65 images were recorded from three SU1498-treated normal mice. Seventy-five images were recorded from three sodium butyrate-treated animals. One hundred seventy-three images were recorded from seven noise-exposed mice. A total of 73 images were recorded from four noise-exposed mice treated with SU1498. Seventy-nine images were recorded from four noise-exposed mice treated with sodium butyrate and a total of ninety-one images were recorded from dimethyl sulfoxide (DMSO)-treated noise-exposed mice. To determine pericyte coverage of endothelial cells (ECs), we labeled the pericytes with an antibody for desmin and labeled the ECs with isolectin GS-IB4 conjugated to Alexa Fluor 488 as described above. Images were analyzed for overlap of blood vessels and pericytes. For analysis, endothelium (red) and pericytes (green) were displayed and both sources of images were thresholded. A region tool in the Image J software was used to define and select the outer margin of the blood vessel under consideration. A delineated area in pixels and the percentage overlap (co-localization) of endothelium and pericytes was calculated. Pericyte coverage was quantified as a ratio of desmin-labeled area to isolectin-labeled area, in concurrence with a calculation method used by others.20Braun A Xu H Hu F Kocherlakota P Siegel D Chander P Ungvari Z Csiszar A Nedergaard M Ballabh P Paucity of pericytes in germinal matrix vasculature of premature infants.J Neurosci. 2007; 27: 12012-12024Crossref PubMed Scopus (114) Google Scholar Immunostaining for VEGF was assessed and measured objectively by two independent observers (a senior researcher and a research assistant). VEGF labeling was measured with Image J (V1.38X) from a series of images obtained from the tissue segment. For each recorded image, the areas of the entire positive-labeled vessels were selected with a drawing tool, and the fluorescence intensity of the selected area was measured with the histogram function, obtaining a mean value. A background intensity was determined in a small window located away from the fluorescence of the vessels and was subtracted from the fluorescence intensity value from the vessels.9Shi X Nuttall AL Expression of adhesion molecular proteins in the cochlear lateral wall of normal and PARP-1 mutant mice.Hear Res. 2007; 224: 1-14Crossref PubMed Scopus (33) Google Scholar Fluorescence was analyzed in 43 optical section images from control animals (n = 3), 87 optical section images from noise-exposed animals (n = 6), and 48 optical section images (n = 4) from sodium butyrate-treated noise-exposed animals. To inhibit HIF-1α activity, mice were pretreated with an HIF-1α inhibitor, sodium butyrate (catalog no. 303410; Sigma-Aldrich, St. Louis, MO). Treatments were administered as acute, single-dose (intraperitoneal) injections (given in a 20-μl volume, 200 mg/kg, drugs were dissolved in saline as vehicle, Schroeder and colleagues,39Schroeder FA Lin CL Crusio WE Akbarian S Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse.Biol Psychiatry. 2007; 62: 55-64Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar) 30 minutes before the animal received the noise exposure. For inhibition of VEGF activity, the mice were pretreated with SU1498 (T4192, Sigma-Aldrich) by one periocular injection (given in a 10-μl volume, SU1498 50 mg/kg) 30 minutes before noise exposure. DMSO vehicle was the control. Dosage of SU1498 was based on studies by Saishin and colleagues,40Saishin Y Saishin Y Takahashi K Melia M Vinores SA Campochiaro PA Inhibition of protein kinase C decreases prostaglandin-induced breakdown of the blood-retinal barrier.J Cell Physiol. 2003; 195: 210-219Crossref PubMed Scopus (57) Google Scholar and Cebulla and colleagues,41Cebulla C Jockovich M Boutrid H Pina Y Ruggeri M Jiao S Bhattacharya S Feuer W Murray T Lack of effect of SU1498, an inhibitor of vascular endothelial growth factor receptor-2, in a transgenic murine model of retinoblastoma.Open Ophthalmol J. 2008; 2: 62-67Crossref PubMed Scopus (7) Google Scholar who demonstrated effects on vessel function from the injection of VEGF in C57BL/6J mice.40Saishin Y Saishin Y
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