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(Stroke. 2001;32:1020.)
© 2001 American Heart Association, Inc.

Original Contributions

Use of Suppression Subtractive Hybridization for Differential Gene Expression in Stroke

Discovery of CD44 Gene Expression and Localization in Permanent Focal Stroke in Rats

Hugh Wang, PhD; Yutian Zhan, MS; Lin Xu, MD; Giora Z. Feuerstein, MD Xinkang Wang, PhD

From the Departments of Cardiovascular Sciences and General Pharmacology (Y.Z.), DuPont Pharmaceuticals Company, Wilmington, Del.

Correspondence to Xinkang Wang, PhD, Department of Cardiovascular Sciences, DuPont Pharmaceuticals Company, Experimental Station, E400/3420B, Wilmington, DE 19880-0400. E-mail xinkang.wang{at}dupontpharma.com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose—CD44 is a transmembrane glycoprotein involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. The present report describes the discovery of upregulated CD44 gene expression and its spatial and temporal distribution in the brain after focal stroke.

Methods—Rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). Suppression subtractive hybridization (SSH) strategy was used to identify differentially expressed genes. Northern blotting and real-time polymerase chain reaction were used to evaluate the expression of CD44 and hyaluronan synthase 2 (HAS-2) mRNA. Western blotting and immunohistochemistry were used to examine CD44 expression and cellular distribution.

Results—CD44 upregulation after focal stroke was discovered by the SSH approach and confirmed by DNA sequencing. Northern blot using a pooled poly(A)+ RNA revealed 3 splice variants of CD44 mRNA, and their inducible expression started at 6 hours (5.3-fold increase over sham operation), peaked at 24 hours (28.6-fold increase), and persisted up to 72 hours (17.8-fold increase) after MCAO. A parallel induction profile of HAS-2 mRNA was observed in the ischemic brain tissue. The levels of CD44 were markedly elevated at 6 hours (1.8-fold increase over sham; n=3), 24 hours (2.9-fold, peak induction; P<0.01), and 72 hours (2.4-fold increase; P<0.05) after MCAO by means of Western analysis. Immunohistochemical and confocal microscopy confirmed that constitutive expression of CD44 is limited to microvessels in normal brain but is strongly induced after ischemia, where the immunoreactive signal mainly resided in endothelial cells and monocytes. Double-labeling immunohistochemistry demonstrated that a marked induction of CD44 in the ischemic lesion is dominantly located in microglia and a subset of macrophages.

Conclusions—The discovery of concomitant induction of CD44 and HAS-2 mRNA expression and the localization of CD44 in the microglia, macrophages, and microvessels of the ischemic brain tissue suggest that an active interaction between CD44 and hyaluronan may occur and play a role in the known inflammatory response and tissue remodeling after stroke.

Key Words: cerebral ischemia • cytokines • gene expression • inflammation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inflammation is one of the critical pathophysiological responses that occur after focal stroke.¹ Inflammatory cytokines, chemokines, and endothelial-leukocyte adhesion molecules are induced after focal ischemia and trauma,^{1 2} which are thought to contribute to the adhesion and migration of leukocytes into the inflammatory brain tissue. Leukocyte extravasation is a multistep process involving adhesion receptor-ligand interactions, including the E/P-selectin primary transient interaction (rolling adhesion) and intercellular adhesion molecule-1 (ICAM-1) secondary (firm) adhesion.³ Recent investigation has shown that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA), a primary ligand of CD44,^{4 5} mediates the initial adhesive interaction of these cells at the inflammatory site.⁴

CD44 is a widely expressed cell adhesion molecule in various tissues. The physiological role of CD44 involves cell-cell and cell-matrix adhesion by interactions with HA. Several isoforms of CD44 have been identified, and each is tightly regulated during lymphopoiesis and neoplasia.^{5 6} CD44 gene expression is induced by hyaluronic acid fragments and interleukin-1ß (IL-1ß) in cultured rat aortic smooth muscle cells and T-24 carcinoma cells.⁷ The IL-1ß modulation of CD44 gene expression in vascular smooth muscle cells is involved in the AP-1 site activation of the CD44 promoter.⁷ CD44 presented in the surface of lymphocytes is normally in an inactive form that is unable to bind HA. The conversion from the inactive to the active form of CD44 requires appropriate stimulation by antigen or cytokines,⁸ and the proinflammatory cytokine tumor necrosis factor- (TNF-) is such a factor leading to CD44 activation.⁹ In addition, the extracellular domain of CD44 is cleaved during cell migration at the membrane-proximal region that is mediated by a membrane-associated metalloprotease in cancer cells.¹⁰ The CD44 cleavage enables cells to be detached from a hyaluronate substrate and promote CD44-mediated leukocyte extravasation. Tissue inhibitor of metalloprotease-1 (TIMP-1) and metalloprotease blocker (1,10-phenanthroline) can block CD44 cleavage and thus abolish CD44-mediated cancer cell migration.¹⁰

The CD44-HA–mediated intracellular signal transduction pathways have been investigated. CD44 is associated with tyrosine kinase p56^Ick in lymphocytes. Activation of CD44 induced the tyrosine phosphorylation of ZAP-70, a substrate of p56^Ick.¹¹ CD44 can also activate transcription factor nuclear factor-B (NF-B) in T-24 (human bladder carcinoma), HeLa (human cervical carcinoma), MCF7 (breast carcinoma), and J774 (murine macrophage) cells by a novel signal transduction cascade emanating from CD44 to Ras, PKC|Gj, and IB kinase 1 and 2.¹²

Although CD44 has been implicated in lymphocyte activation and tumor cell metastasis, CD44 gene expression and activation in brain, especially after brain injury, is poorly understood. A previous study showed that low levels of CD44 mRNA were expressed in normal brain tissue and primary brain tumors, whereas high levels of the CD44 variant (CD44v) were detected in metastatic brain tumors.⁶ An additional variant, CD44v6, was highly induced in T cells infiltrated into spinal cord of patients with HAM/TSP (human T-cell lymphotropic virus type 1–associated myelopathy/tropical spastic paraparesis).¹³ CD44 upregulation was also reported in astrocytes during the early phase of canine distemper encephalitis.¹⁴ In a mouse brain stab injury model, CD44 expression was strongly activated in the area surrounding the injury within 2 days after the stab injuries and persisted for >2 months.¹⁵

In an effort to understand the molecular mechanism(s) associated with gene regulation in brain injury after focal stroke, a suppression subtractive hybridization (SSH) method¹⁶ has been applied to identify genes that are specifically regulated in focal stroke. Among a panel of differentially expressed genes discovered by this technique, one clone is identified as CD44 in this report. As an initial step to characterize the role of CD44 in brain ischemia, we have investigated the temporal and spatial distribution of CD44 transcripts and protein in rats after permanent occlusion of the middle cerebral artery (MCAO). In addition, to explore the potential involvement of HA in brain ischemia, we have also examined the mRNA expression of HA synthase 2 (HAS-2), an enzyme that is inducible and responsible for HA production during inflammation.¹⁷

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Oligonucleotide primers were synthesized by Genosys Biotechnologies. AmpliTaq DNA polymerase was from Perkin-Elmer. Mouse anti-rat CD44 monoclonal antibody (clone OX49) was from Research Diagnostics, Inc. ECL detection reagent 1 and 2 used in Western blotting was from Amersham. RNA and DNA preparation kits were from Qiagen. All other chemicals were purchased from Sigma Chemical Co.

Focal Brain Ischemia
Male Sprague-Dawley rats, aged 18 weeks and weighing 250 to 330 g, were used for our studies. Rats were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare Publication No. NIH 85-23, revised 1985). The work was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Procedures for using laboratory animal were approved by the Institutional Animal Care and Use Committee of DuPont Pharmaceuticals Company.

Rats were anesthetized with gas inhalation composed of 30% oxygen (0.3 L/h) and 70% nitrous oxide (0.7 L/h) mixture. The gas was passed through an isoflurane vaporizer set to deliver 3% to 4% isoflurane during initial induction and 1.5% to 2% during surgery. An incision of the skin was made on top of the right common carotid artery region. The fascia was then blunt dissected until the bifurcation of the external common carotid artery and internal common carotid artery was isolated. A small incision was made on the external common carotid artery, and a 3-0 monofilament suture with a round tip was thread into the internal common carotid artery via the external common carotid artery. The suture was advanced toward the middle cerebral artery (MCA) region to create focal ischemia. For permanent MCAO, the suture was maintained in the vessel, and the wound was closed. Sham operation was performed by the same procedure except that no suture was inserted. Forebrains were removed after anesthesia at various times after permanent MCAO or sham surgery, and the ipsilateral and contralateral hemispheres were dissected and immediately frozen in liquid nitrogen and stored at -80°C for RNA and protein analysis.

The animal model was evaluated with cerebral blood flow (CBF), infarct volume, and neurological deficits. The mortality of rats was <5% observed up to 72 hours after MCAO. CBF was monitored with a laser-Doppler perfusion monitor (Moor Instruments Inc) in the area approximately 1 mm posterior and 5 mm lateral to the bregma in the ipsilateral hemisphere after thinning the skull. CBF was carefully monitored (to avoid any large vessel) before and after MCAO. More than 75% reduction in CBF was observed after MCAO. Infarct volume was evaluated with the use of 2,3,5-triphenyltetrazolium chloride staining of 2-mm-thick brain slices. The stained brain tissue was fixed in 10% formalin in phosphate-buffered saline (PBS). The image was captured with a Microtek ScanMaker 4 DUO Scanner (MicroWarehouse) within 24 hours and quantitated with Image Pro Plus 4.1 software (Media Cybernetics). Approximately 20% infarct volume versus total brain tissue was observed 24 hours after permanent MCAO. The infarct area is located in the cortical and subcortical (from caudate putamen to lat preoptic area) regions. Neurological deficits were scored according to a 5-point scale, as described by Zhang et al.¹⁸

Suppression Subtractive Hybridization
Total RNA of the ipsilateral (ischemic) or normal (nonischemic) forebrain was prepared as previously described.¹⁶ Poly(A)+ mRNA was extracted with an oligo(dT) cellulose column from total cellular RNA pooled from 25 animals at 12 hours after permanent MCAO or from normal cortex. SSH was performed with a Clontech PCR-Select cDNA Subtraction Kit according to the manufacturer’s instruction. Three micrograms poly(A)+ mRNA from ischemic cortex 12 hours after permanent MCAO (as a tester) or from normal brain cortex (as a driver) was used for the subtraction. Procedures for SSH and differential hybridization have been described in detail previously.¹⁶

Northern Blot Analysis
Northern blot analysis was performed as described in detail previously¹⁶ except that 5 µg poly(A)+ RNA (isolated from a pool of animals; n=4 for each time point) was used per lane. The rat CD44 cDNA was generated with the use of reverse transcription–polymerase chain reaction (RT-PCR) according to the published sequence.¹⁷ The forward primer (RCD44F1, 5'-ACATCATGGACAAGGTTTGGTG-3') was located in exon 1 and the reverse primer (RCD44R714: 5'-TAGGCTGTGAAGTGGGAAGGT -3') in exon 5 of CD44 gene. Ribosomal protein rpL32 was used as an internal control to normalize the mRNA loading.¹⁹

Real-Time RT-PCR
The primers and probes (Table) used for real-time RT-PCR were designed with the use of Primer-Express 1.0 software from PE Applied Biosystems. Real-time PCR was performed basically as described in detail previously²⁰ with the following modification: 1-step RT-PCR was performed with the GIBCO BRL Platinum Taq System according to the manufacturer’s specification. Because our pilot studies revealed that only a basal level of HAS-2 mRNA was expressed in the contralateral brain tissue, the real-time PCR was performed with the use of only the ipsilateral samples. Total RNA isolated from rat ipsilateral hemisphere at 1, 6, 12, 24, and 72 hours after permanent MCAO or sham operation (12 hours) was analyzed with 1-step RT-PCR by the ABI PRISM 7700 Sequencing Detector (Perkin-Elmer). Data (n=3) were analyzed on the basis of threshold cycle (Ct) values of each sample and normalized with an internal housekeeping gene control, rpL32, with a Sequence Detector program (Perkin-Elmer, V1.6.3) and Microsoft Excel program.

View this table: [in this window] [in a new window]   Table 1. TaqMan Primers and Probes Used for Real-Time PCR

Western Blot Analysis of CD44 Protein Expression
Brain tissues stored at -80°C were thawed on ice in a lysis buffer that contained 10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 100 µg/mL phenylmethylsulfonyl fluoride, and 5 µL/ml Protease Inhibitor Cocktail Set III (CalBiochem No. 539134). The tissue was then homogenized with a Polytron homogenizer at high-speed setting for 20 seconds. The insoluble component of the tissue lysate was removed by centrifugation at 3000g for 10 minutes. Protein concentration was determined with a Bio-Rad Protein Assay kit following the manufacturer’s instruction. Protein samples (100 µg) were separated on a 10% NuPAGE Bis-Tris gel (Novex) and then transferred onto a nitrocellulose membrane using the gel blot module (Novex). After it was blocked with 5% (wt/vol) skim milk in TBS-Tween buffer (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 0.1% Tween 20) for 1 hour, the membrane was incubated with mouse anti-rat CD44 monoclonal antibody (1:2000 dilution) at room temperature for 3 hours or 4°C overnight. The membrane was washed 4 times with 1xTBS-Tween for 15 minutes each and then incubated with horseradish peroxide–conjugated anti-mouse antibody (in 1:2000 dilution) for 1 hour. After it was washed 4 times with TBS-Tween at room temperature, the membrane was incubated for 2 minutes with a fresh mixture with equal volumes of Amersham ECL detection reagent 1 and 2. CD44 signal was detected by Kodak x-ray film exposure.

Immunohistochemistry
After anesthesia, rats (6, 24, and 72 hours after permanent MCAO or 24 hours after sham operation; n=3) were perfused transcardially with saline followed by 4% paraformaldehyde in PBS (pH 7.4). The brain was removed, postfixed in the same fixative solution for 24 hours, and then stored for 1 to 2 hours in 10% dimethyl sulfoxide in PBS for cryoprotection. Serial coronal sections (40 µm) were cut on a sliding microtome and collected in PBS.

Free-floating sections were permeabilized with 0.2 Triton X-100 in PBS and blocked with 3% bovine serum albumin and 5% normal goat serum for 1 hour at room temperature. The sections were then incubated overnight at 4°C with primary antisera (diluted in 1% bovine serum albumin/PBS), including monoclonal mouse anti-rat CD44 (1:250), mouse anti-rat CD11b/c (OX42, BD PharMingen; 1:1000), mouse anti-rat monocytes/macrophages (ED1, Chemicon; 1:250), mouse anti-glial fibrillary acidic protein (GFAP, Chemicon; 1:1000), or mouse anti-neuron specific nuclear protein (NeuN, Chemicon; 1:1000). After removal of primary antisera and washing, the sections were incubated for 1 hour with biotinylated anti-mouse IgG (Vector Laboratories; 1:200), washed with PBS, and then incubated with fluorescein streptavidin (Vector Laboratories) for 1 hour at room temperature.

For double immunofluorescence staining, after CD44 labeling, sections were incubated overnight at 4°C with antibodies against OX42, ED1, GFAP, or NeuN, then washed with PBS and incubated with Texas Red dye–conjugated anti-mouse IgG (Fab)2 (Jackson ImmunoResearch). Sections were washed with PBS and mounted on slides. Slides were coverslipped with the use of Vectashield antibleaching medium (Vector Laboratories) and examined with a Zeiss Axioplan fluorescence microscope. Confocal images were produced on a Leica TCS laser confocal microscope, and a plan-neofluor X40 (numerical aperture, 1.0) oil-immersion objective was used for imaging of fluorescently labeled tissues. Images were analyzed with the standard system operating software provided with Leica TCS laser confocal microscopy (version 1.6). For double-labeling studies, separate optical images of fluorescence and Texas Red were captured from the same optical section. The captured images were then pseudocolored green or red; a digital overlay was generated, and companion images were superimposed. Regions of the colocalization, reflecting the addictive effect of superimposing green and red pixels, appear in yellow.

Statistical Analysis
Data are presented as mean±SE. Statistical comparisons were made by ANOVA followed by Fisher’s protected t test. Values were considered significant at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Discovery of CD44 Gene Upregulation in Focal Stroke by SSH Strategy
To identify genes that are differentially expressed after brain ischemia compared with nonischemic tissue, mRNA isolated from rat brain tissue 12 hours after MCAO was subtracted from normal brain tissue by the SSH approach. The results in Figure 1 show that several clones were detected by ischemic probe (Figure 1A) compared with the nonischemic probe (Figure 1B). Among these ischemia-positive clones, the B4 clone (arrow) was confirmed for its upregulation in the ischemic tissue by Northern analysis (Figure 2). DNA sequence analysis and alignment with GenBank database showed that B4 clone is the rat CD44 gene.

View larger version (99K): [in this window] [in a new window]   Figure 1. Identification of CD44 gene upregulation in rat focal stroke by SSH. cDNA clones generated by SSH were blotted onto a nylon membrane with the use of a dot-blot apparatus and analyzed by Southern hybridization. The membrane was hybridized with a probe generated from ischemic (A) or nonischemic (normal) (B) brain samples. The differentially expressed clone, indicated with an arrow (B4), was confirmed to be the rat CD44 gene by DNA sequencing. Other clones (B5 and D8 for monocyte chemotactic protein-316 and heat shock protein-70,36 respectively) in the same blot were also confirmed.

View larger version (55K): [in this window] [in a new window]   Figure 2. Northern blot analysis of CD44 mRNA expression in rat brain after MCAO. A, Five micrograms poly(A)+ RNA (from a pool of n=4) per lane was used for Northern analysis. Three alternatively polyadenylated CD44 mRNA species were detected. S indicates sham controls. B, Intensity of CD44s bands that were analyzed by the PhosphorImager (Molecular Dynamics Inc). The data were quantified and illustrated as a sum of 100% after rpL32 normalization.

Time-Course Expression of CD44 mRNA in Rat Brain After Permanent MCAO
Total cellular RNA was extracted from each animal at various time points, and each time point (n=4) was pooled for poly(A)+ RNA isolation and Northern blot analysis. Figure 2 illustrates Northern blot hybridization with the use of CD44 and rpL32 cDNA probes. A low basal expression of CD44 mRNA was detected in sham-operated rats early (1 and 3 hours) after MCAO. CD44 mRNA was induced at 6 hours (5.3-fold increase over sham operation), reached a peak at 24 hours (28.6-fold increase), and persisted up to 72 hours (17.8-fold increase) in the ipsilateral brain tissues after MCAO. In contrast, a moderate CD44 mRNA increase was observed in the contralateral brain at 12 to 48 hours after MCAO. Three CD44 mRNA isoforms of 3.5, 2.5, and 1.8 kb were observed in the brain sample (Figure 2A). The induction patterns of the 3 mRNA isoforms were basically the same after MCAO, which is in agreement with the previous report that these 3 CD44 transcripts are the same gene products terminated at the different polyadenylation sites in the 3'-untranslated region.¹⁷ However, because CD44 splice variants in the coding region have been previously reported,²¹ we synthesized specific PCR primers (across exons 5 and 17) to detect the coding sequence splice variants. RT-PCR²² was applied to analyze normal and ischemic brain tissues, as well as heart, kidney, small intestine, thymus, skeletal mussel, liver, lung, and other tissues. Our result indicated that all the samples tested contained only a normal CD44 coding transcript, ie, there was no CD44 splicing variant detected (data not shown).

Time-Course Expression of HAS-2 mRNA in Brain After Permanent MCAO
Because HAS-2 is a key enzyme responsible for HA production and it has been shown to be induced under inflammatory conditions,²³ in the present study we also evaluated the expression of HAS-2 mRNA in the ischemic brain tissue using real-time TaqMan RT-PCR. Only a basal level of HAS-2 mRNA expression was observed in normal or sham-operated brain tissues, but it was markedly induced after MCAO. The induction profile of HAS-2 mRNA is similar to that of CD44. It was slightly induced at 6 hours (1.7-fold increase over sham), significantly upregulated at 12 hours (3.2-fold increase; P<0.01), peaked at 24 hours (3.9-fold increase; P<0.05), and persisted up to 72 hours (2.4-fold increase) in the ischemic hemisphere after permanent MCAO (Figure 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. Real-time PCR quantification of HAS-2 mRNA expression in the brain after permanent MCAO. Total RNA isolated from rat ipsilateral hemisphere at 1, 6, 12, 24, and 72 hours after permanent MCAO or sham operation (12 hours) was analyzed with 1-step RT-PCR. Data (n=3) were analyzed on the basis of Ct values of each sample and normalized with an internal housekeeping gene control, rpL32. The relative level of HAS-2 mRNA was depicted as a sum of 100% for the complete time course. See Materials and Methods for further details.

Time-Course Expression of CD44 in Brain After Permanent MCAO
Figure 4A illustrates a representative Western blot with the use of a mouse anti-rat CD44 antibody (OX49) that recognizes the epitopes of both standard CD44 and CD44 variants (ie, CD44s and CD44v).²⁴ Two immunoreactive bands were detected by this antibody, suggesting that CD44 protein is glycosylated. The temporal expression profile of CD44 is also in agreement with that of CD44 mRNA. Sham-operated samples and/or early ischemic brain tissues only revealed the basal level of CD44 protein expression (Figure 4). Elevated expression of CD44 was observed at 6 hours (1.8-fold increase over sham, n=3), reached a peak at 24 hours (2.9-fold; P<0.01; n=3), and was sustained up to 72 hours (2.4-fold increase; P<0.05) after MCAO (Figure 4B).

View larger version (39K): [in this window] [in a new window]   Figure 4. Western analysis of CD44 expression after MCAO. A, Representative Western blot analysis of CD44 expression in rat brain tissue from sham (S) or various time points after MCAO. Western blotting was performed as described in Materials and Methods. The prestained molecular weight standards (Novex) were used to determine the molecular weight of CD44 and to monitor the efficiency of blot transfer. B, Intensity of CD44 protein signal from Western blot was analyzed with a PhosphorImager program, and data are illustrated. The relative level of CD44 protein was described as a sum of 100% for the complete time course.

Immunohistochemical Analysis of CD44 Expression in Rat Brain After MCAO
To further define the cellular components of the upregulated CD44 in response to ischemic injury, immunohistochemical techniques were applied with the mouse anti-rat CD44 monoclonal antibody. A very weak CD44-immunoreactive signal was detected in the brain of sham-operated animals, where the basal expression was primarily located in the area of microvessels (Figure 5A and 5B). Six hours after MCAO, the expression of CD44 was markedly increased but also limited to the microvessels in the ipsilateral cortical region (Figure 5C), as well as some CD44-positive cells detected in caudate putamen (Figure 5D). High-resolution confocal microscopy and double-labeling immunohistochemistry demonstrated that CD44 is mainly expressed in monocytes within the lumen and in the endothelial cells of microvessels in the ischemic zone (data not shown). A very strong induction of CD44 expression was observed throughout in the ischemic regions 24 hours after MCAO (Figure 5E and 5F). At 72 hours after MCAO, CD44 immunoreactivity was still strongly elevated in the entire ipsilateral brain region (Figure 5G and 5H). In the contralateral brain tissue, CD44 immunoreactivity was also detected in areas around vessels and in some microglia at 24 hours after MCAO.

View larger version (128K): [in this window] [in a new window]   Figure 5. Photomicrographs illustrating CD44 staining in frontal cortex (A, C, E, G) and caudate putamen (B, D, F, H) after MCAO. Weak CD44 staining was detected around blood vessels in the cortex of sham-operated animals (A, B). CD44 immunoreactivity was increased in cells around blood vessels in the cortex and caudate putamen 6 hours after MCAO (C, D). Very strong CD44 immunoreactivity was detected in microglia 24 hours after MCAO (E, F). At 72 hours, strong CD44 labeling was mainly in macrophage and microglia (G, H). The inset in panel A indicates that the capture field covered a rectangular area from the surface of the brain to the white matter of the corpus callosum; the inset in panel B covers the field of caudate putamen. Bar=100 µm in G (applies to A, C, E, and G); bar=25 µm in H (applies to B, D, F, and H).

To identify the cellular sources of CD44 immunoreactive cells, brain sections were double labeled with antibodies against CD44 and OX42, ED1, NeuN, or GFAP and analyzed by confocal microscopy. The monoclonal antibody OX42 was used to detect differentiating microglia and some tissue macrophages, and monoclonal anti-rat ED1 antibody was primarily used for monocytes/macrophages.¹⁹ NeuN immunoreactivity was used to detect neurons, and GFAP was used to detect astrocytes.²⁵ Double-labeling immunohistochemistry demonstrated that CD44 immunoreactive cells in the brain are mainly those of activated microglia and a subset of macrophages in the ischemic lesion (Figure 6). No CD44 immunoreactivity was detected in neurons or astrocytes (data not shown). CD44 immunoreactivity was initially detected in microglia in the peri-infarct cortical region at 24 hours after MCAO and later (72 hours) was located in ischemic zone. A few CD44-positive macrophages were detected in the infarct at 24 hours after MCAO. Massive CD44 immunoreactive macrophages were located throughout the ischemic area at 72 hours after MCAO. In addition, monocytes within the vasculature in the ischemic area were also noted to be CD44 positive.

View larger version (13K): [in this window] [in a new window]   Figure 6. Confocal immunofluorescence double labeling of CD44 with OX42 and ED1 in the ischemic brain tissue 24 hours after MCAO. CD44 immunoreactivity was detected with mouse anti-CD44 monoclonal antibody followed by biotinylated anti-mouse IgG incubated with fluorescein streptavidin (labeled green, A and D). OX42 or ED1 immunoreactivity was detected with mouse anti-OX42 or anti-ED1 followed by Texas Red dye–conjugated anti-mouse IgG (Fab)2 (labeled red, B and E). The merged images (C for A and B; F for D and E) illustrate regions of colocalization (yellow), demonstrating that CD44 immunopositive cells are microglia (C, ischemic cortex at 24 hours after MCAO) and a subset of macrophages (F, ischemic cortex at 72 hours after MCAO). Bar=10 µm in F (applies to A through F).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present report we described the discovery of upregulated CD44 gene expression in the brain after ischemic stroke using the SSH strategy. The Northern blot and Western blot analyses showed that the ischemia-induced expression of CD44 was time dependent in the ischemic brain tissues. A similar induction profile was also observed for HAS-2 mRNA after ischemic brain injury, suggesting the increasing biosynthesis of HA. The concomitant induction of CD44 and HAS-2 suggests that CD44 and HA may actively contribute to ischemic brain injury, possibly having a role associated with the inflammation, as has been demonstrated in a number of inflammatory diseases.^{24 26}

Inflammation is one of the key pathophysiological responses after focal stroke.^{1 2} The expression of CD44 and HAS-2 correlated with the infiltration and accumulation of leukocytes in the ischemic brain tissues. Their expression profiles (though with a slight delay) are basically in parallel with a number of other inflammation-related genes, including various cytokines, chemokines, and adhesion molecules, as reported previously after brain ischemia.² Of note, TNF- and IL-1ß are key proinflammatory cytokines that are induced at 3 to 6 hours and peak at 12 hours after focal stroke.² TNF- and IL-1ß are not only able to induce CD44 and HAS-2 gene expression^{7 27} but also modulate the function of CD44.⁹ On the other hand, the HA activation of CD44 is able to directly induce TNF- and IL-1ß expression and further to indirectly (via TNF-) produce insulinlike growth factor-1 in macrophages.²⁷ The localization of CD44 expression in microglia and a subset of macrophages that are distributed along with the infarct after brain ischemia may, in part, explain its participation in the inflammatory response after ischemia injury.

The exact role of CD44 upregulation in endothelial cells of ischemic microvessels is not fully understood. The expression of CD44 in human pulmonary endothelial cells and umbilical cord vein endothelial cells has been reported.^{28 29} The endothelial expression of CD44 in the brain ischemia may be associated with cytokine production and inflammation.

In addition to its possible role in inflammation, the massive and remarkable parallel induction of CD44 and HAS-2 in ischemic lesions may reflect their role in tissue remodeling. As reported previously, the levels of HA within the extracellular matrix and cerebrospinal fluid were strictly regulated by cellular hyaluronidase and receptor-mediated endocytosis of HA.^{5 30} The extracellular matrix enriched HA coincident with periods of rapid cell proliferation, aggregation, wound healing, tissue regeneration, and remodeling.³¹ Furthermore, a previous study demonstrated that macrophages can internalize HA during lung development and may possibly play a significant role in HA removal.³² A subset of CD44-positive macrophages in the ischemic lesions may also regulate the levels of HA in situ and thus provide a final tuning of its function.

It is also possible that additional functions of upregulated CD44 after brain ischemia may be related to matrix metalloproteases (MMPs) and TIMP. Induced expressions of MMP-2 and MMP-9, as well as TIMP-1, have been previously reported after brain ischemia.^{33 34} While the effect of MMPs on CD44 function after brain ischemia is unknown, previous studies demonstrated that MMP inhibition can block the cleavage of the extracellular domain of CD44 and thus abolish CD44-mediated cancer cell migration.¹⁰

In addition to hyaluronic acid, osteopontin has also been characterized as the ligand binds to CD44.³⁵ The ischemia-induced expression of osteopontin was previously reported in a rat model of focal stroke.¹⁹ Osteopontin mRNA was expressed in the peri-infarct region from 3 to 48 hours and in the infarct by 5 days, and the peak expression of osteopontin was observed at 5 days after stroke.¹⁹ It is interesting to note that the cellular sources and distribution of osteopontin expression are strikingly similar to those of CD44 (the present report) despite some difference in their temporal expression profiles after focal stroke,¹⁹ suggesting a potential interaction between CD44 and osteopontin under this pathophysiological condition. The difference in their temporal expression profiles may reflect different animal models (Tamura’s model for osteopontin versus thread model for CD44 expression) used for these 2 studies.

In conclusion, the present study describes for the first time the discovery of CD44 gene induction in microvessels, microglia, and macrophages in brain ischemic lesions after MCAO. These data, together with previous reports on the pathophysiological role of CD44, as well as the parallel induction of HAS-2, suggest that the interaction between CD44 and HA may actively contribute to the known inflammatory response and tissue remodeling after focal stroke.

Received August 24, 2000; revision received December 14, 2000; accepted December 15, 2000.

	References

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