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(Stroke. 1998;29:1698-1707.)
© 1998 American Heart Association, Inc.

Original Contributions

Osteopontin and its Integrin Receptor _vß₃ Are Upregulated During Formation of the Glial Scar After Focal Stroke

Julie A. Ellison, PhD; James J. Velier, PhD; Patricia Spera, PhD; Zdenka L. Jonak, PhD; Xinkang Wang, PhD; Frank C. Barone, PhD; Giora Z. Feuerstein, MD

From the Departments of Cardiovascular Pharmacology and Molecular and Cellular Immunology (Z.L.J.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.

Correspondence to Giora Z. Feuerstein, MD, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, UW2523, King of Prussia, PA 19406. E-mail G_Z_Feuerstein{at}SBPHRD.COM

	Abstract

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Background and Purpose—Microglia and astrocytes in the peri-infarct region are activated in response to focal stroke. A critical function of activated glia is formation of a protective barrier that ultimately forms a new glial-limiting membrane. Osteopontin, a provisional matrix protein expressed during wound healing, is induced after focal stroke. The present study was performed to determine the spatial and temporal expression of osteopontin and its integrin receptor _vß₃ during formation of the peri-infarct gliotic barrier and subsequent formation of a new glial-limiting membrane.

Methods—Spontaneously hypertensive rats (n=19) were subjected to permanent occlusion of the middle cerebral artery and killed 3, 6, and 24 hours and 2, 5, and 15 days after occlusion. The spatial and temporal expression of osteopontin mRNA was determined by in situ hybridization, and that of osteopontin ligand and its integrin receptor _vß₃ was determined by immunohistochemistry.

Results—Osteopontin mRNA was expressed de novo in the peri-infarct region from 3 to 48 hours; by 5 days osteopontin mRNA expression was restricted to the infarct. Osteopontin protein was expressed by peri-infarct microglia beginning at 24 hours and by microglia/macrophages at 48 hours in the infarct. Integrin receptor _vß₃ was expressed in peri-infarct astrocytes at 5 and 15 days.

Conclusions—Early microglial/macrophage expression of osteopontin mRNA defines the borders and final infarct area at 24 hours. At 5 days osteopontin ligand is at a distance from the peri-infarct astrocytes expressing integrin receptor _vß₃. By 15 days astrocytes expressing integrin receptor _vß₃ are localized in an osteopontin-rich region concomitant with formation of the new glial-limiting membrane. The de novo expression and interaction of osteopontin ligand with its receptor integrin _vß₃ suggest a role in wound healing after focal stroke.

Key Words: astrocyte • cerebral ischemia • infarcts • macrophages • microglia • neuroglia

	Introduction

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Glial scar formation in the peri-infarct region after focal ischemia has been characterized as a negative event that prevents regeneration of the injured nervous system.¹ After focal ischemia, glial cells initiate a classic wound-healing response² with formation of a barrier between the injured and healthy tissue.^{3 4 5} The barrier is a cellular and molecular boundary essential for protecting the uninjured cells by confining the area of injury, thus limiting secondary injury that is often termed "bystander effect." The glial scar formed in the early stages of an injury develops into a new glial limitans reestablishing the interface of the glial-pial boundary.

Formation of the glial scar involves the transformation of quiescent astrocytes and microglia to their activated state in the peri-infarct region. Activated astrocytes undergo hypertrophy as they extend their processes to encompass the developing infarct,^{2 6} while activated microglia directly adjacent to the injured cells transform into ameboid phagocytic cells^{7 8} responsible for clearing the necrotic zone of cellular debris. The hypertrophy, hyperplasia, and migration that typify cell activation require cells to change their interaction with the extracellular matrix concomitant with rearrangements of the cytoskeletal network.

Integrins are a family of transmembrane receptors that couple intracellular cytoskeletal elements with extracellular matrix molecules.⁹ After injury to the brain, integrins and the extracellular matrix molecules are upregulated as part of the wound healing process. Chondroitin sulfate proteoglycans³ and tenascin are upregulated in astrocytes in the region adjacent to an injury.¹⁰ Two integrin receptors, _vß₃ and ₆ß₄, have been suggested to play a role in vascular integrity and remodeling after focal ischemia within the infarcted area.^{11 12} In particular, integrin _vß₃ has been demonstrated to be upregulated concomitant with an increase in one of its ligands, fibrinogen.¹¹ Integrin receptor _vß₃ can also interact with other blood vessel–associated proteins containing the arginine-glycine-aspartate (RGD) motif such as vitronectin, von Willebrand factor,¹³ thrombospondin,¹⁴ fibronectin,¹⁵ laminin,¹⁶ and osteopontin.¹⁷ The expression of these integrins and extracellular matrix molecules has been studied in the context of vascular remodeling and wound healing, but little is known regarding their potential role in nonvascular tissue remodeling associated with wound healing in the brain.

Recent studies from our laboratory have demonstrated specific and transient induced expression of the provisional matrix protein osteopontin after focal stroke.¹⁸ Osteopontin, an integrin ligand, is an acidic, secreted phosphoprotein containing an arginine-glycine-aspartate (RGD) motif that interacts with the integrin _vß₃ to promote cell migration.¹⁹ Osteopontin and integrin _vß₃ are expressed during repair of myocardial necrosis²⁰ and restenosis.²¹ Functionally, osteopontin has chemotactic activity for smooth muscle cells²² regulating cell adhesion and migration²³ by interacting with three different integrin receptors: _vß₁, _vß₃, and _vß₅. Cell adhesion is mediated by integrin receptors _vß₁ and _vß₅,²⁴ while integrin receptor _vß₃ mediates cell migration.²⁵ Our studies demonstrated that osteopontin mRNA was maximally expressed at 5 days after ischemia by ED1+ macrophages after focal stroke. Furthermore, in a migration assay osteopontin had the capacity to induce directed migration of astrocytes.¹⁸

In the present study we extend our investigation of osteopontin expression induced by ischemic brain injury. The objective of the present study was (1) to define the spatial and temporal expression kinetics of osteopontin mRNA and protein after ischemic injury; (2) to identify the phenotype of cells expressing osteopontin ligand and integrin receptor _vß₃; and (3) to determine the timing of receptor-ligand interaction as a first step in exploring potential functional aspects of osteopontin and integrin receptor _vß₃. Our results regarding the spatial and temporal expression of osteopontin by microglia and invading monocytes and of integrin receptor _vß₃ by astrocytes demonstrate a potential role for osteopontin and _vß₃ in the formation of the peri-infarct glial scar and reestablishment of the glial limitans after focal stroke.

	Materials and Methods

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Middle Cerebral Artery Occlusion and Tissue Preparation
Adult male spontaneously hypertensive rats (weight, 250 to 350 g) were anesthetized with sodium pentobarbital (60 mg/kg IP). This rat strain was chosen for the low variability and consistency of infarct between animals. Permanent occlusion was chosen because it represents a model of infarct that most closely resembles focal stroke in humans. Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, NIH publication 85–23, revised 1985. Procedures in which laboratory animals were used were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

Focal ischemia was produced by occluding the middle cerebral artery (MCA) as previously described.²⁶ After right craniotomy and removal of the dura mater, the MCA was permanently occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein by means of electrocoagulation (Force 2 Electrosurgical Generator, Valley Laboratory Inc). During and after surgery, the rat's body temperature was maintained at 37°C. In sham-operated rats the dura was opened over the MCA, but the artery was not occluded. After permanent MCA occlusion, rats were killed at 3 (n=3), 6 (n=3), and 24 (n=3) hours and 2 (n=3), 5 (n=3), and 15 (n=2) days; sham rats (n=2) were killed 2 days after surgery. Naive control rats (n=2) were also examined. Rats were overdosed with sodium pentobarbital and perfused through the aorta with 50 mmol/L Dulbecco's PBS containing 2% paraformaldehyde for 15 minutes. The brain was then removed and postfixed in PBS containing 2% paraformaldehyde for 4 days at 4°C. Brains were cryoprotected in 20% sucrose in PBS at 4°C, then frozen in OCT (Tissuetek, Miles Inc) and stored at -70°C until sectioned.

In Vitro Transcription and In Situ Hybridization
The methods followed for transcription reactions and in situ hybridization were as previously published.^{27 33} P-UTP–labeled riboprobes were synthesized from linearized plasmids containing the entire rat osteopontin cDNA from base pairs 1 to 1435. The cloning of the rat osteopontin has been described previously.²⁸ Transcription reactions were performed at 37°C for 90 minutes in 15 µL of 40 mmol/L Tris-HCl, pH 7.5, with 6 mmol/L MgCl₂; 2 mmol/L spermidine; 0.5 mmol/L each ATP, CTP, GTP; 2 mmol/L dithiothreitol; 1 µg linearized template; 1 µL T7 or T3 polymerase; and 25 µCi ³³ P-UTP (2000 Ci/mmol; New England Nuclear). The DNA template was removed by a 10-minute incubation at 37°C with RNase-free DNase (1 U/µL). Synthesized cRNA was separated from unincorporated nucleotides by ethanol precipitation and resuspended in 10 mmol/L Tris, 1 mmol/L EDTA, pH 8.

Next 12-µm tissue sections were cut onto Fisher Superfrost Plus slides, dried on a warm plate at 37°C, and stored at -70°C until use. Slides were thawed to room temperature, then dried for 5 minutes at 60°C. With the use of Rnase-free solutions, sections were hydrated in PBS x3 for 5 minutes each. Tissue was deproteinated in 200 mmol/L HCl for 10 minutes and acetylated with 100 mmol/L triethanolamine (pH 8) with 0.25% acetic anhydride followed by a PBS rinse. Finally, slides were dehydrated in 70%, 95%, and 100% ethanol. Antisense or sense ³³ P-UTP–labeled probes (5x10⁴ cpm/µL) were applied to tissue and hybridized overnight at 60°C in a hybridization buffer containing 4x SET, 1x Denhardt's solution, 0.2% SDS, 100 mmol/L dithiothreitol, 250 µg/mL tRNA, 25 µg/mL poly A, and 25 µg/mL poly C. Posthybridization washes were as follows: 4x SSC x2 for 10 minutes each at 50°C; 20 µg/mL RNase for 30 minutes at 37°C; 5 minutes each 2x SSC, 1x SSC, and 0.5x SSC at 22°C; 0.1x SSC for 20 minutes at 55°C; and 0.1x SSC for 5 minutes at 22°C. Slides were dehydrated in ascending alcohols containing 300 mmol/L ammonium acetate with a final dehydration in 100% ethanol, then exposed to Hyperfilm ßmax (Amersham) for 2 days to estimate length of exposure time for subsequent emulsion autoradiography. For emulsion autoradiography slides were dipped in Kodak NTB2 emulsion diluted 1:1 (vol/vol) with 600 mmol/L ammonium acetate at 45°C, then dried for 2 hours in a humid chamber and exposed for 7 to 14 days at 4°C. Slides were developed in Kodak D-19 (diluted 1:1 with water) for 3 minutes, rinsed briefly in water, and cleared in Kodak Fix for 5 minutes with a final wash in water for 5 minutes. All solutions were maintained at 15°C. Slides were counterstained with hematoxylin, dehydrated, and coverslipped.

Antibodies
The following commercially available antibodies were used: polyclonal glial fibrillary acidic protein (GFAP) (DAKO Corp); monoclonal ED1 (Chemicon International Inc); and monoclonal osteopontin, MPIIIB10₁ (Developmental Studies Hybridoma Bank, University of Iowa). The antisera used to identify the integrin _vß₃, monoclonal SBJ293–346, was generated according to a modification of Kohler and Milstein.²⁹ BALB/c mice were immunized by routine protocol with purified human integrin receptor _vß₃. Hybridomas were generated by fusing spleen cells of a BALB/c mouse with the cell line SP2/0-Ag14. Positive hybridomas were subcloned by limited dilution to assure monoclonality of the hybridoma cell line. Supernatants from hybridomas were screened against a panel of human integrin receptors (_vß₃, _vß₅, _vß₁, and _IIbß₃) by ELISA. SBJ293–346 was selected on the basis of its specificity and selectivity against integrin receptors _vß₃ and _IIbß₃, presumably by cross-reacting with the ß₃ subunit, which is shared by both integrin receptors. The hybridoma secreting SBJ293–346 monoclonal antibody showed neutralizing activity against _vß₃ and _IIbß₃ receptors in an assay in which fibrinogen was used as an integrin ligand. The SBJ293–346 monoclonal antibody showed negative activity in both binding and neutralization assays against _vß₅ and _vß₁ integrin receptors.

Immunohistochemistry
Tissue was brought to room temperature and hydrated in PBS. For immunoperoxidase localization, endogenous peroxidase was quenched with either 3% H₂O₂ in PBS (integrin ß₃) or 3% H₂O₂ in methanol (GFAP, ED1). After the quench, tissue sections were blocked with 1% BSA in PBS, then incubated with the primary antibody overnight at 4°C. The sections were rinsed with PBS, and the primary antibody was detected with the use of the ABC Elite kit (Vector Labs), with diaminobenzidine (Sigma) as the chromogen, according to the manufacturer's instructions. For double immunofluorescence, the primary antibodies were localized with the use of secondary antibodies conjugated to BodipyFL or Texas Red (Molecular Probes). For specificity of SBJ293–346, the antibody (2.5 µg/mL) was adsorbed with purified integrin _vß₃ (25 µg/mL) in 1% BSA in PBS overnight at 4°C. The adsorbed antibody was then incubated with the tissue as described for the aforementioned nonadsorbed antibodies.

Combined In Situ Hybridization and Immunohistochemistry
Tissue preparation, in vitro transcription, and combined in situ hybridization and immunohistochemistry were performed as described above. Tissue sections (12 µm) were incubated with the ED1 or GFAP antibody overnight at 4°C. Immediately after antibody detection, the tissue underwent the in situ hybridization procedure described above. Double labeling of cells was visualized with Nomarski optics and epifluorescence with cross-polarized optics to enhance visualization of grains (which appear green) colocalized with immunoreactive cells.

Quantitative Analysis of In Situ Hybridization
X-ray film images of osteopontin in situ hybridization results were scanned to disk with a Hewlett Packard Deskscan program. Images were quantified with the Optimas image analysis program (Optimas Corp). Two regions were measured at 3, 6, and 24 hours and 2 and 5 days after permanent MCA occlusion. These two regions included the largest measure at 24 hours corresponding to the peri-infarct area occupied by osteopontin mRNA and that bounded by but not containing osteopontin mRNA. The area (mm²) for these two regions was analyzed by ANOVA and post hoc analysis with a Dunnett t test and was considered significant at P<0.05.

	Results

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In situ hybridization was used to analyze the spatial and temporal kinetics of osteopontin mRNA expression after permanent MCA occlusion. In the naive brain tissue, constitutive expression was observed only in the septal nucleus and ventral nuclei (Figure 1A, arrowheads). With sham surgery, osteopontin was expressed at the site of the surgery (Figure 1B, arrow); expression in the septal nucleus and ventral nuclei was also noted (Figure 1B, arrowheads). Induction of osteopontin mRNA was seen 3 hours after occlusion in cells in the ventromedial peri-infarct region (Figure 1C, arrow). Six hours after occlusion, expression of osteopontin mRNA at the ventromedial aspect of the infarct had increased (Figure 1D, arrow) and now extended to the dorsomedial aspect of the infarct (Figure 1D, arrowhead). At 24 hours, robust expression was seen throughout the entire peri-infarct zone (Figure 1E) as well as within the subarachnoid space (Figure 1E, arrow) clearly defining the infarcted area. At this time only a few cells within the infarct expressed osteopontin mRNA (Figure 1E, arrowhead). By 48 hours, osteopontin mRNA expression persisted in the peri-infarct region, and the numbers of osteopontin mRNA+ cells had increased within the infarct (Figure 1F). At 5 days, osteopontin mRNA was largely absent from the peri-infarct region, with robust expression in the infarct (Figure 1G). By 15 days after injury, osteopontin mRNA had declined to nearly basal level (Figure 1H). Quantitative analysis of the area bounded by osteopontin mRNA (at 24 and 48 hours after injury) and of the area containing osteopontin mRNA cells at 5 days after injury revealed that the area of the infarct at 5 days was defined by the cells expressing osteopontin mRNA at 24 hours (Figure 2).

View larger version (68K): [in this window] [in a new window]   Figure 1. Osteopontin mRNA expression after focal stroke. By in situ hybridization osteopontin mRNA expression was constitutively expressed in the septal nucleus and in ventral brain nuclei (A, arrowheads). In sham animals osteopontin mRNA was induced at the surgery site (B, arrow). At 3 hours (C) and 6 hours (D), osteopontin mRNA was induced in cells initially at the ventromedial aspect (C, arrow) and continuing to the dorsomedial aspect (D, arrowhead) of the infarct. At 24 hours, cells expressing osteopontin mRNA were still largely confined to the peri-infarct region (E), with expression at the pial surface (arrow) and by a few cells in the infarct (arrowhead). At 48 hours (F), more cells within the infarct expressed osteopontin mRNA; by 5 days (G), the majority of cells expressing osteopontin mRNA were within the infarct, with little expression in the peri-infarct region. At 15 days (H), mRNA levels returned to those found in naive rats.

View larger version (28K): [in this window] [in a new window]   Figure 2. Osteopontin (OPN) mRNA expression at 24 hours defines the area of infarct. Quantitative analysis of the region bounded by OPN mRNA and the region containing OPN mRNA revealed that the expression of OPN mRNA at 24 hours defines the final infarct area seen at 5 days. *P<0.05 different from 3-hour measure of peri-infarct OPN mRNA; **P<0.05 different from 3-hour measure of infarct OPN mRNA (ANOVA followed by Dunnett t test using 3-hour data as control).

Using a technique of combined in situ hybridization and immunohistochemistry, we determined the phenotype of the osteopontin mRNA+ cells in the peri-infarct. Osteopontin mRNA colocalized with a microglia marker, ED1 (Figure 3A, arrow); osteopontin mRNA (Figure 3B, arrow) did not colocalize with the astrocytic marker GFAP (Figure 3B, arrowheads). The majority of cells expressing osteopontin mRNA could be colocalized with ED1, but not all of the ED1+ cells expressed osteopontin mRNA (Figure 3A, arrowheads).

View larger version (129K): [in this window] [in a new window]   Figure 3. Osteopontin expression is restricted to microglia/macrophages. By combined immunohistochemistry and in situ hybridization, osteopontin mRNA (green grains) was colocalized to cells expressing ED1, a microglia marker (A, arrow). Adjacent to an ED1+ cell expressing osteopontin mRNA is an ED1+ cell that does not (A, arrowhead). Osteopontin mRNA (B, arrow, green grains) did not colocalize with GFAP+ astrocytes (B, arrowheads). Bar=20 µm for A and B.

Osteopontin protein was first detected within cells at 24 hours after injury in the peri-infarct region (Figure 4A, arrows) extending from the pial surface to the corpus callosum and at the callosal gray matter border at the medial aspect of the infarct. Cells expressing osteopontin had a characteristic ramified microglial morphology and were seen in the parenchyma (Figure 4B, arrow) and adjacent to blood vessels (Figure 4C). By 48 hours after injury, osteopontin protein was still restricted to cells in the peri-infarct region. Of these cells at 48 hours, only occasional ramified microglia expressing osteopontin could be found in the peri-infarct region (Figure 4D, arrrowhead). The majority of cells expressing osteopontin had transformed into macrophages by 48 hours, with osteopontin protein evident in a peri-nuclear position (Figure 4D, arrow). Five days after injury, osteopontin protein was abundantly expressed in the infarct and at the infarct/peri-infarct border. Intracellular expression in macrophages (Figure 4E, arrows) as well as in an extracellular distribution around macrophages (Figure 4E, arrowheads) was evident. Fifteen days after injury, osteopontin protein expression was predominantly extracellular and restricted to a thin rim directly adjacent to the newly formed glial-pial surface (Figure 4F). Omission of the primary antibody gave no positive immunoperoxidase reaction product (Figure 4G).

View larger version (157K): [in this window] [in a new window]   Figure 4. Osteopontin protein expression in microglia and macrophages. At 24 hours osteopontin was expressed by microglia in the peri-infarct region (A, arrows); the microglia have a ramified appearance (B and C, arrows) and do not appear activated. At 48 hours, occasional microglia (D, arrowhead) expressing osteopontin are seen, with the majority of osteopontin expressed by macrophages (D, arrow). By 5 days, osteopontin was seen in a perinuclear location intracellularly (E, arrows) and in the extracellular matrix (E, arrowheads). At 15 days, osteopontin expression was restricted to a thin area adjacent to the pial surface (F). No immunoperoxidase reaction was detected in the absence of primary antibody at any time point (G). Bar=100 µm for A; 20 µm for B and C; 40 µm for D and E; and 100 µm for F and G.

To determine the potential functional interaction between osteopontin ligand and integrin receptor _vß₃, we analyzed the expression of the _vß₃ at 5 and 15 days after injury, a time of maximal osteopontin protein expression. Earlier time points were not analyzed because the goal of the present study was to understand which cells had the potential to interact with the osteopontin localized in the extracellular matrix. By immunoperoxidase localization at 5 days after injury, expression of _vß₃ was upregulated in the gray matter adjacent to the infarct (Figure 5A) compared with the gray matter in the contralateral side (Figure 5C). The cells expressing _vß₃ appear to be hypertrophic astrocytes (Figure 5A, arrows) at the infarct/peri-infarct border (asterisks indicate border). Expression of _vß₃ by apparent hypertrophic astrocytes (Figure 5B, arrows) continued through 15 days after injury as the former boundary between the peri-infarct region and infarct was replaced by a new glial limitans composed of cells expressing _vß₃ (Figure 5B, asterisks). A notable difference between the 5- and 15-day expression of _vß₃ was the extended astrocytic processes at 15 days (Figure 5B, arrowhead). Omission of the primary antibody gave no detectable immunoperoxidase reaction product (Figure 5D). Adsorption of the SBJ293–346 antibody with purified integrin _vß₃ blocked binding of the antibody to the tissue section (Figure 6B); compare with adjacent tissue section incubated with nonadsorbed antibody in which four cells expressing _vß₃ are seen (Figure 6A).

View larger version (187K): [in this window] [in a new window]   Figure 5. Integrin vß3 expression at 5 and 15 days after occlusion. The integrin vß3 was upregulated at 5 days in the ipsilateral cortex (A) compared with the contralateral cortex (C). Integrin vß3 was expressed by cells (A, arrows) adjacent to the infarct (peri-infarct/infarct border identified by asterisks). By 15 days, cells expressing integrin vß3 (B, arrows) had elongated cell processes (B, arrowhead) and were found adjacent to and at the glial-pial boundary (identified by asterisks). Bar=100 µm for A and B; 40 µm for C and D.

View larger version (146K): [in this window] [in a new window]   Figure 6. Adsorption with integrin vß3 blocks anti-ß3 antibody binding. Adsorption with the purified integrin receptor vß3 protein blocked antibody binding to cells in the 5-day peri-infarct region (B). Compare with an adjacent section of cells labeled with the nonadsorbed antibody, which are identified by arrows (A). Bar=20 µm.

Colocalization of _vß₃ and GFAP by double immunofluorescence confirmed that the integrin receptor _vß₃-expressing cells were hypertrophic GFAP+ astrocytes. In the ipsilateral cortex, GFAP+ astrocytes (Figure 7A, arrows) expressed _vß₃ (Figure 7B, arrows). In the contralateral cortex, GFAP+ astrocytes (Figure 7C, arrow) did not express _vß₃ (Figure 7D, arrow).

View larger version (113K): [in this window] [in a new window]   Figure 7. vß3 colocalizes with GFAP+ astrocytes. Double immunofluorescence demonstrated that hypertrophic GFAP+ astrocytes (A, arrows) in the ipsilateral peri-infarct area expressed integrin vß3 (B, arrows). GFAP+ astrocytes (C, arrow) in the contralateral cortex corresponding to the peri-infarct area did not express integrin vß3 (D, arrow). Bar=40 µm.

To further explore which events of astrocyte biology osteopontin might be mediating, the expression of osteopontin ligand and integrin receptor _vß₃ were correlated with cellular and histological changes occurring at the peri-infarct/infarct border. Colocalization of GFAP+ astrocytes, which have been demonstrated to express the integrin receptor _vß₃, with osteopontin ligand expression at 5 days after injury indicated that although osteopontin (Figure 8A, arrowhead) can be demonstrated within cells adjacent to GFAP+ astrocytes (Figure 8A, arrows) in the peri-infarct region (to the left of the asterisks), extracellular osteopontin could only be detected within the infarct (Figure 8A, arrowheads to the right of the asterisks). This is in contrast to osteopontin mRNA, which is abundantly expressed in the infarct core at 5 days (Figure 1G). Ten days later, however, GFAP+ astrocytes (Figure 8B, arrows) were present within the osteopontin-rich region (Figure 8B, arrowhead) adjacent to the newly formed glial-pial boundary (Figure 8B, asterisks).

View larger version (103K): [in this window] [in a new window]   Figure 8. Timing of osteopontin–integrin vß3 interaction. Double immunofluorescence at 5 (A) and 15 (B) days demonstrated that at 5 days GFAP+ astrocytes (A, arrows) were at a distance from extracellular osteopontin (A, arrowheads), which was localized to the core infarct. By 15 days GFAP+ astrocytes (B, arrows) were found within a matrix of osteopontin (B, arrowhead). The medial peri-infarct/infarct border with a large infarct region to the right (A) and the glial-pial boundary (B) are indicated by asterisks. Bar=20 µm.

	Discussion

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Focal stroke in the spontaneously hypertensive rat produces a well-defined and characteristic infarct that is consistent between animals.^{2 19} The infarct is restricted to the cortical gray matter dorsal to the corpus callosum and extends to the pial surface. Development and resolution of the infarct can be mapped over a 2-week period driven by (1) activation of glia in the peri-infarct region; (2) compartmentalization of the injured cells; (3) removal of the infarcted tissue debris by phagocytes; and (4) establishment of a new glial-pial boundary. In the present study we investigated the spatial and temporal expression of the provisional matrix protein osteopontin and its integrin receptor _vß₃, which are expressed by microglia and astrocytes, respectively, after injury.

Osteopontin mRNA is expressed very early after ischemia in the peri-infarct region. The timing of osteopontin gene expression as indicated by in situ hybridization is earlier than that seen by Northern blot analysis, in which osteopontin mRNA was first detected at 12 hours after ischemia.¹⁸ This methodological difference is consistent with the increased sensitivity of in situ hybridization compared with Northern blot analysis. This rapid induction of osteopontin gene expression suggests that osteopontin might function as a stress-response gene after focal ischemia. Within the promoter region of the osteopontin gene, an acute-phase response element has been identified.³⁰ In vitro studies have shown that cells adhered to osteopontin have enhanced expression of heat shock proteins and display a greater resistance to heat shock injury.³¹ Furthermore, Denhardt and Chambers³² have suggested that osteopontin is a protective protein that confers cellular resistance to the damaging effects of nitric oxide and oxidative burst associated with inflammation by inhibiting induction of nitric oxide synthase.³³ Though not previously considered a stress-response gene, when regarded in the context of its early gene induction pattern after injury, its specific expression in the peri-infarct region (which survives the ischemic event), and its role in cell protection, osteopontin might well be considered a stress-inducible protein with characteristics of the classic stress-response genes, such as the heat shock proteins.³⁴

Quantitative analysis of the area bounded by osteopontin mRNA+ cells at 24 hours compared with the area occupied by osteopontin mRNA+ cells at 5 days after injury indicated that the areas were not significantly different (ie, were essentially equal). Analysis of the specific cell type synthesizing osteopontin demonstrated that microglia, but not astrocytes, synthesize and secrete osteopontin. Thus, the activated microglia define the infarct border as early as 24 hours. Similar conclusions were offered by Coyle,³⁵ who found that triphenyltetrazolium chloride staining after focal stroke in the spontaneously hypertensive rat resulted in a lesion border at 24 hours that was no different than the border at 21 days. These findings indicate that the microglial activation occurs within the peri-infarct region but not the penumbra.

The early activation of microglia surrounding the infarct precedes the transformation of astrocytes from a resting to a reactive state that does not manifest fully until 2 days after injury.^{36 37} The osteopontin receptor integrin _vß₃ is expressed by astrocytes at 5 and 15 days after ischemia. These time points were chosen to identify potential cell populations that could interact with the osteopontin in the extracellular matrix. At 5 days after ischemia, astrocytes expressing integrin receptor _vß₃ are dispersed in the peri-infarct region; by 15 days these cells have reformed the glial limitans lost initially as a result of tissue injury after focal stroke. Astrocytes in vitro express _vß₃,³⁸ and radial glia of the developing nervous system have been reported to express integrin _v.³⁹ Hirsch and colleagues³⁹ suggest that this integrin might be involved in the formation and orientation of the glial fibers that extend from the ventricular zone to the cortical plate. Similarly, the astrocytes expressing _vß₃ after focal ischemia reorganize from a stellate morphology to the more bipolar morphology characteristic of astrocytes of the glial limitans.

Fundamentally these events require that cells change the relationship they have with the extracellular matrix from a static, fixed state to a dynamic, mobile state that allows for changes in cell attachment, shape, and locomotion. Upregulation of _vß₃ has been reported to be essential for endothelial transformation to an angiogenic phenotype⁴⁰ and for migration toward an osteopontin gradient.²⁵ The spatial-temporal interaction of extracellular osteopontin ligand and integrin receptor _vß₃ is at a distance at 5 days; however, by 15 days the astrocytes are localized within a matrix of osteopontin, suggesting that osteopontin may act as a chemotactic factor for astrocytes. Preliminary studies with human astrocytes demonstrate the capacity of osteopontin to induce directed migration of astrocytes.¹⁸

In addition to its chemoattractant role, ligation of integrin receptor _vß₃ by osteopontin ligand results in the rapid production of phosphoinositides.⁴¹ Astrocytic hypertrophy and migration are dependent on GFAP, the predominant intermediate filament expressed by these cells. The assembly/disassembly of GFAP is Ca²⁺ dependent,⁴² and inositol 1,4,5-trisphosphate–induced Ca²⁺ release in astrocytes is directed by the type 3 inositol 1,4,5-trisphosphate receptor.⁴³ Thus, osteopontin ligand binding to integrin receptor _vß₃ could stimulate release of intracellular Ca²⁺ stores, causing a subsequent reorganization of the GFAP filament network.

Ligation of integrin receptors results not only in immediate signal transduction events at the cell surface but also mediates changes in gene expression. Signaling through the fibronectin and tenascin integrin receptors upregulated synthesis of collagenase, stromelysin, gelatinase, and c-fos in fibroblasts.⁴⁴ In endothelial cells integrin ligation promoted cell survival by suppressing p53 activity and by increasing the bcl-2/bax ratio.⁴⁵ Although no data exist regarding integrin-mediated gene induction in astrocytes, the activation of astrocytes, their transformation to migratory cells, and subsequent acquisition of a pial astrocyte phenotype strongly suggest that a change in gene expression does occur.

Both integrin receptor _vß₃ and osteopontin interact with other ligands and receptors. Previous work analyzing the expression of integrin receptors after focal stroke demonstrated the interaction of fibrinogen with integrin receptor _vß₃ early in the ischemic event.¹¹ The results presented in this study demonstrate the diverse role of integrin receptors in tissue remodeling. In addition to the role in vascular remodeling seen early after focal stroke,¹¹ these findings suggest that integrin receptor _vß₃ participates in nonvascular remodeling associated with formation of a glial scar that occurs late in the resolution of the ischemic insult.

In conclusion, our data demonstrate that induction of a microglia-specific gene, osteopontin, occurs early after focal stroke. The pattern of induction in microglia is restricted to the peri-infarct region and in fact defines the final infarct area (ie, maximum size at 24 hours). Osteopontin protein is expressed by ramified microglia in the peri-infarct area at 24 hours. As the microglia transform into macrophages and the lesion develops over 5 days, macrophages in the infarcted region express osteopontin and secrete it into the extracellular matrix. The osteopontin receptor integrin _vß₃ is expressed by hypertrophic astrocytes at 5 and 15 days after stroke. These astrocytes expressing integrin receptor _vß₃ are at a distance from the osteopontin ligand in the extracellular matrix at 5 days, but by 15 days the astrocytes are localized in an osteopontin-rich region concomitant with formation of a new glial limitans. These results strongly suggest that the interaction of osteopontin ligand and integrin receptor _vß₃ may play a role in the healing of brain injury in establishing a new glial-pial boundary after stroke.

Although these data do not allow us to draw conclusions suggesting that the appearance of osteopontin mediates the expression of integrin receptor _vß₃, the data clearly demonstrate the spatial and temporal expression patterns of the two proteins, which is the first step in identifying potential receptor-ligand interactions. Further studies will be needed to address the early temporal upregulation of _vß₃ in astrocytes; the potential interaction of osteopontin with its other integrin receptors, _vß₁ and _vß₅; and the interaction of integrin receptor _vß₃ with other ligands.

	Acknowledgments

 
The authors thank Steve Trulli and Kyung Johanson for their technical expertise in preparation of the SBJ293–346 antibody. The MBPIIIB10₁ hybridoma developed by Drs Solursh and Franzen was obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences at the University of Iowa, under contract NO1-HD-7–3263.

Received January 30, 1998; revision received April 29, 1998; accepted April 30, 1998.

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Editorial Comment

Takeo Abumiya, MD, Guest Editor; Gregory J. del Zoppo, MD,Guest Editor

Department Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
In the accompanying article, Ellison and colleagues demonstrate that focal cerebral ischemia results in the early appearance of osteopontin mRNA and the late appearance of the antigen (beginning at 24 hours) together with its receptor, integrin _vß₃, in the spontaneously hypertensive rat. Integrin _vß₃ expression associated with evidence of astrocyte activation increased between 5 and 15 days, while osteopontin was apparently associated with microglial cells or macrophages. These observations are intriguing because they (1) confirm that changes in expression of selected integrins occur in the central nervous system after ischemia, (2) suggest that in vivo integrin _vß₃ antigen can appear outside blood vessels, and (3) through the appearance of osteopontin suggest the possibility of interesting receptor-ligand interactions.

The latter point is underscored by Okada et al,¹ who showed that integrin _vß₃ was greatly upregulated very early in the myointima of noncapillary microvessels, related significantly to fibrin deposition in the same vessels, in focal brain ischemia/reperfusion in the nonhuman primate. Other recent studies have shown that selected integrins have an important role in vascular integrity and response during cerebral ischemic event. For instance, integrin ₆ß₄ is localized on astrocyte end-feet, where it connects with the basal lamina of extracellular matrix at the astrocyte-vessel interface.² In contrast to integrin _vß₃ it was downregulated very early in focal brain ischemia/reperfusion.²

Integrin _vß₃ is a promiscuous receptor in that it recognizes a wide variety of Arg-Gly-Asp (RGD)–containing ligands including fibrin(ogen), vitronectin, von Willebrand factor, thrombospondin, laminin, and collagen as well as osteopontin.³ Cell migration mediated by ligation of integrin _vß₃ to osteopontin has been demonstrated in vascular smooth muscle cells.⁴ However, other interactions associated with its vascular expression are known. A specific monoclonal antibody against integrin _vß₃ (LM609) inhibits angiogenesis in tumors and suppresses tumor growth by involution of new vasculature.⁵ It has been demonstrated that the interaction of integrin _vß₃ with vitronectin is necessary for migration of smooth muscle cells.⁶ Recently, it has been shown that blockage of integrin _vß₃ induces apoptosis in an endothelial cell culture system.⁷

Although osteopontin is distributed widely among epithelial structures in the body, the appearance of osteopontin in the brain has only been documented in gliomas and in this report of cerebral ischemia.^{8 9} The findings of Ellison et al showing that microglia and macrophages were sources of osteopontin expression in the rodent are in accord with the findings of osteopontin expression in renal ischemia.¹⁰ Therefore, osteopontin expression may be one of the common events after activation of macrophages by ischemic injury. This work implies that at least one aspect of osteopontin presentation occurs somewhat distant in time from the initial ischemic events.

It is intriguing to consider potential causes of upregulation of osteopontin and integrin _vß₃ in the ischemic parenchyma in these studies and whether they are related to each other in their expression. The upregulation of osteopontin mRNA expression may depend on the acute-phase responses of the promoter element of osteopontin gene, as the authors have suggested. However, the mechanism of upregulation of integrin _vß₃ in astrocytes remains to be confirmed in vivo and elucidated. Despite the relative spacial distributions of osteopontin and integrin _vß₃ expression in these studies, it is possible that both expressions result from independent stimuli or that integrin _vß₃ is related to one or another ligand. Studies of cultured endothelial cell preparations indicate that in those cells vascular endothelial growth factor induces integrin _vß₃ expression.¹¹ It remains to be seen how integrin _vß₃ expression is managed in the nonvascular ischemic tissue of the rodent and its relationship to the evolution of cellular injury and gliosis.

Received January 30, 1998; revision received April 29, 1998; accepted April 30, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Okada Y, Copeland BR, Hamann GF, Koziol JA, Cheresh DR, del Zoppo GJ. Integrin _vß₃ is expressed in selected microvessels following focal cerebral ischemia. Am J Pathol.. 1996;149:37–44.

2. Wagner S, Tagaya M, Koziol JA, Quaranta V, del Zoppo GJ. Rapid disruption of an astrocyte interaction with the extracellular matrix mediated by ₆ß₄ during focal cerebral ischemia/reperfusion. Stroke.. 1997;28:858–865.

3. Cheresh D. Integrins: structure, function and biological properties. Adv Mol Cell Cardiol.. 1993;6:225–252.

4. Liaw L, Skinner MP, Raines EW, Ross R, Cheresh DA, Schwartz SM, Giachelli CM. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins: role of _vß₃ in smooth muscle cell migration to osteopontin in vitro. J Clin Invest.. 1995;95:713–724.

5. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin _vß₃ antagonists promote tumor regression by promoting apoptosis of angiogenic blood vessels. Cell.. 1994;79:1157–1164.[Medline] [Order article via Infotrieve]

6. Brown SL, Lundgren CH, Nordt T, Fujii S. Stimulation of migration of human aortic muscle cells by vitronectin: implications for atherosclerosis. Cardiovasc Res.. 1994;28:1815–1820.[Abstract/Free Full Text]

7. Stromblad S, Becher JC, Yebra M, Brooks PC, Cheresh DA. Suppression of p53 activity and p21^WAF1/CIP1 expression by vascular cell integrin _vß₃ during angiogenesis. J Clin Invest.. 1996;98:426–433.

8. Saitoh Y, Kuratsu J, Takeshima H, Yamamoto S, Ushio Y. Expression of osteopontin in human glioma: its correlation with the malignancy. Lab Invest.. 1995;72:55–63.[Medline] [Order article via Infotrieve]

9. Wang X, Louden C, Yue TL, Ellison JA, Barone FC, Solleveld HA, Feuerstein GZ. Delayed expression of osteopontin after focal stroke in the rat. J Neurosci.. 1998;18:2075–2083.

10. Kleinman JG, Worcester EM, Beshensky AM, Sheridan AM, Bonventre JV, Brown D. Upregulation of osteopontin expression by ischemia in rat kidney. Ann N Y Acad Sci.. 1995;760:321–323.[Medline] [Order article via Infotrieve]

11. Senger DR, Ledbetter SR, Claffey KP, Papdopoulos-Sergiou A, Peruzzi CA, Detmar M. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the _vß₃ integrin, osteopontin, and thrombin. Am J Pathol.. 1996;149:293–305.[Abstract]

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