This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wagner, S. Articles by del Zoppo, G. J. Search for Related Content PubMed PubMed Citation Articles by Wagner, S. Articles by del Zoppo, G. J.

(Stroke. 1997;28:858-865.)
© 1997 American Heart Association, Inc.

Articles

Rapid Disruption of an Astrocyte Interaction With the Extracellular Matrix Mediated by Integrin ₆ß₄ During Focal Cerebral Ischemia/Reperfusion

Simone Wagner, MD; Masafumi Tagaya, MD; James A. Koziol, PhD; Vito Quaranta, MD Gregory J. del Zoppo, MD

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

Correspondence to Gregory J. del Zoppo, MD, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N Torrey Pines Rd, SBR-17, La Jolla, CA 92037. E-mail GRGDLZOP{at}RISCSM.SCRIPPS.EDU

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose Integrins participate in cerebral microvascular integrity and signaling during focal ischemia/reperfusion. The integrin subunits ₁, ₆, and ß₁ are distributed identically on normal cerebral microvessels. Studies in epithelium indicate that integrin ₆ß₄, which interacts with laminin-5 in the basal lamina/extracellular matrix, is unique. This study describes the exact location of ₆, ß₄, and ₆ß₄ and that their responses in focal cerebral ischemia are relevant to astrocyte-matrix interactions.

Methods The effect of middle cerebral artery occlusion and subsequent reperfusion on the microvascular expression of ₆ß₄ and laminin-5 in regions of cellular injury (dUTP incorporation) was examined in 15 nonhuman primates. Well-characterized antibodies against human ₆, ß₄, ₆ß₄, laminin-5 and laminin-1, endothelial CD31, and vascular markers were measured with computerized video imaging and laser confocal microscopy.

Results Integrin ₆ß₄ was localized on astrocytes where it connects with the extracellular matrix at the astrocyte-vessel interface. It represented 59.3±16.4% of ₆ antigen in cerebral microvessels <100 µm in diameter. By 2 hours of ischemia, the significant reduction in ₆ expression (2P<.001) was accompanied by decreases in ß₄/laminin-5 (0.76±0.03 to 0.20±0.09; 2P=.001) and ₆ß₄/laminin-5 (0.73±0.18 to 0.25±0.11; 2P=.001) in the region of dUTP incorporation. Parallel changes in laminin-5 and laminin-1 were less pronounced and coincided by 24 hours.

Conclusions This is the first description of a potential role of integrin ₆ß₄ in the brain, where it mediates astrocyte-matrix interactions. The dramatic disappearance of ₆ß₄ relative to its ligands reflects early loss of integrity between the astrocyte and the vessel wall in selected microvessels in response to ischemia.

Key Words: astrocytes • cell adhesion molecules • cerebral ischemia • microvascular injury • baboons

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ischemia and reperfusion cause significant alterations in the structure and integrity of the cerebral microvasculature.^{1 2} In particular, the integrity of the endothelium and the subjacent basal lamina is disturbed after MCAO/R.³ The integrity of the endothelial cell–basal lamina interface is in part related to the interaction of basal lamina components with the abluminal endothelial surface.⁴ Beneath the intima, in noncapillary brain microvessels, smooth muscle cells are ensheathed in extracellular matrix, which is contiguous with the basal lamina.⁵ Astrocytes, whose end-feet are adjacent to the extracellular matrix or the basal lamina in capillaries, provide a connection between the microvascular endothelium (smooth muscle) and the neuropil.^{6 7} During development, endothelial cells in conjunction with astrocytes contribute laminin to the extracellular matrix,⁸ while astrocytes stimulate the formation of the blood-brain barrier.^{9 10 11} The sensitivity of these structures, the nature of their interactions, and their responses to focal ischemia and reperfusion are unknown.

Integrins (heterodimeric adhesion receptors) connect endothelial cells to the components of the underlying basal lamina and therefore are structurally important. For instance, laminins, a major component of the basal lamina and extracellular matrix, are ligands for the integrin heterodimers ₁ß₁ (VLA-1), ₂ß₁ (VLA-2), ₃ß₁ (VLA-3), ₆ß₁ (VLA-6), ₇ß₁ (VLA-7),^{12 13 14 15 16} and ₆ß₄.^{17 18} In addition, certain integrins (eg, platelet _IIbß₃)¹⁹ mediate rapid activation of signal transduction pathways and play a pivotal role in active cell-cell and cell-matrix interactions.²⁰ Furthermore, in the vasculature, integrins may be actively involved in remodeling.⁴

The responses of extracellular matrix components to ischemia may also involve their integrin receptors. The cerebral microvascular distributions of laminin and of integrin subunits ₁, ₆, and ß₁ in the nonhuman primate are nearly identical.²¹ The heterodimer ₁ß₁ is a receptor for collagen and laminins, while ₆ß₁ is a receptor for laminins.²² If the ß₄ subunit is expressed in the presence of ₆ and ß₁,²³ the heterodimer ₆ß₄ is formed preferentially.²⁴ Ligands for ₆ß₄ are also laminins,^{17 25 26} but this integrin is unique because it promotes the formation of hemidesmosomes in epithelial cells,²⁵ where it interacts with laminin-5.¹⁸

The basal lamina, a specialized part of the extracellular matrix, is uniquely sensitive to focal cerebral ischemia.^{3 27 28} The gradual loss of the microvascular basal lamina antigens collagen IV, laminin, and fibronectin of cellular origin during MCAO/R signifies potential microvascular structural and functional changes of importance. Alterations in vascular permeability and basal lamina integrity lead to hemorrhagic transformation during focal ischemia.³ The role of vascular integrins in those processes is unknown. We hypothesize that certain integrins in their function as receptors for basal lamina components are present in the endothelial, smooth muscle, or astrocyte compartments of the cerebral microvasculature and are involved in the pathophysiological mechanisms of focal cerebral ischemia and reperfusion. The specific hypothesis tested here states that the fate of microvascular integrin subunit ₆ and its ß companion(s) may be determined early after MCAO and that the specific ligands suffer a similar fate. These studies point to specific interactions of astrocytes within the microvasculature.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cerebral tissues from 15 adolescent male baboons (Papio anubis/cynocephalus) were used for the MCAO and MCAO/R studies. Some of the material from these subjects has been used for other related studies. The procedures used in this study were approved by the institutional animal research committee and were performed according to standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals) and the US Department of Agriculture Animal Welfare Act. Every effort was made to ensure that the subjects were free of pain and discomfort. For all procedures a veterinarian, the primate handling staff, and the principal investigator were present.

Preparation of the awake baboon MCAO/R stroke model has been described in detail in previous studies of this group.^1,3 All animals were allowed a 7-day procedure-free period after surgical implantation of the MCA balloon before the entry into the experimental protocol. The subjects were all clinically free of infection or apparent inflammation and had normal hematologic studies and neurological function before MCAO. The experimental paradigm previously reported for this awake model required inflation of the implanted 100-µL MCA balloon for 2 hours or for 3 hours with subsequent reperfusion.

In this study, 3 animals underwent MCAO for 2 hours, while 8 animals underwent MCAO for 3 hours and subsequent reperfusion for 1 hour (n=3), 4 hours (n=2), or 24 hours (n=3). The control group, which did not undergo any model preparation procedure, was composed of 3 separate animals. An additional subject underwent the implantation procedure and the 7-day recovery period but did not undergo MCAO, serving as a sham-operated control.

Preparation of tissues for immunocytochemical studies followed perfusion of the cranial structures under deep thiopental Na⁺ anesthesia with chilled (4°C) isosmotic perfusion fluid containing heparin, nitroprusside, and bovine serum albumin (Sigma) by left ventricular transcardiac puncture. Tissues were removed within 15 minutes of complete perfusion. From the excised brain, symmetrically located tissue blocks (1x1x0.2 to 0.5 cm) of the basal ganglia and parietal cortex were embedded in Tissue-Tek OCT compound (Miles, Inc) frozen in 2-methylbutane/dry ice and stored at -80°C.

For immunocytochemical studies, well-characterized murine anti-human MoAbs or polyclonal antibodies were used. The ₆ subunit was identified with the rat anti-human MoAb CLB-701 (Chemicon).²⁹ Laminin antigens were identified with a rabbit polyclonal antibody (AB949, Chemicon) ³⁰ and laminin-1 with the murine anti-human MoAb LAM 89 (Sigma) as previously described.^{31 32} Murine MoAbs against the human integrin subunit ß₄ (AA3)²³ and ₆ß₄ (S341)³³ and a rabbit anti-rat polyclonal antibody that cross-reacts with human laminin-5 (0668B)³⁴ were applied. Other murine MoAbs against the human ß₄ (3E1, Chemicon³⁵ ; 450-9D, Pharmingen³⁶ ) were used.

Preliminary immunoperoxidase experiments demonstrated identical distributions among the antibodies AA3, 3E1, and 450-9D against the ß₄ subunit. For subsequent experiments MoAb AA3 was applied.

Additional MoAbs against human smooth muscle -actin (MoAb 1A4, Sigma),³⁷ endothelial CD31 (JC/70A, Dako Corp),³⁸ and the intermediate filaments cytokeratin, vimentin, and GFAP were identified with MoAbs KL1 (Immunotech),³⁹ V9,⁴⁰ and G9269 (Sigma),⁴¹ respectively.

The cross-reactivities of the ₆, ß₄, and ₆ß₄ immunoprobes with primate were confirmed in normal baboon skin. The MoAbs for ₆, ß₄, and the heterodimer ₆ß₄ bound the basement membrane and some cutaneous vessels.²³ Laminin-5 antigen was apparent in the basement membrane, while cytokeratin stained the basal cell layer.

Immunohistochemical procedures were performed as previously described.¹ Consecutive 10-µm cryostat sections from corresponding regions of the right (ischemic) and left (nonischemic) basal ganglia were used. Afterward, sections were fixed with acetone for 10 minutes and immersed in 100 mmol/L glycine in PBS (100 mmol/L Na₂HPO₄ and 140 mmol/L NaCl, pH 7.4) for 10 minutes. This was followed by a rinse in PBS solution for 30 minutes. A subsequent 20-minute incubation with Blotto (5% hydrated nonfat dry milk in PBS) served to reduce nonspecific binding. Under humidified conditions, 100 µL of primary antibody was applied to each section followed by a 2-hour incubation period at 37°C. To reduce nonspecific binding due to plasma proteins in ischemic tissue, baboon plasma was added to the primary antibodies against ß₄ and ₆ß₄. This procedure did not affect the number of vessels stained. After the sections were washed with PBS, the biotinylated secondary antibody was incubated for 30 minutes at 37°C (Vector Laboratories). The peroxidase signal was developed with the use of the chromogen 3-amino-9-ethylcarbazole (AEC Kit, Biomeda Corp). All sections were then counterstained for 30 seconds with Mayer's hematoxylin (Biomeda Corp), blued in saturated sodium bicarbonate, and mounted in crystal mount. These procedures were modified for dual immunofluorescent studies.³ Negative and positive controls were routinely performed as described previously.¹

Nuclear DNA scission, taken as a measure of cellular injury^{42 43 44 45 46 47} after MCAO/R, was detected by TdT-mediated DIG-dUTP incorporation on 10-µm cryosections, adjacent to those used for immunohistochemistry, according to published methods.⁴⁶ The TdT-based procedure labels free 3'OH ends. Control primate cerebral tissues prepared in the manner described above demonstrated no nuclear DIG-dUTP incorporation (data not shown). The cryosections were fixed with 10% neutral-buffered formalin and immersed in ethanol/acetic acid (2:1). After sections were washed, they were treated with 2% H₂O₂ for 5 minutes and incubated with DIG-dUTP in TdT buffer at 37°C for 60 minutes. Color development followed incubation with anti–DIG-peroxidase conjugate for 30 minutes, with 0.025% DAB/0.005% H₂O₂ in 0.05 mol/L Tris buffer (pH 7.6) for 5 minutes. Positive controls were generated by incubation of nonischemic tissues with DNase I (Sigma) for 10 minutes at 20°C before DIG-dUTP development.

The absolute number and minimum transverse diameters of microvessels (<100 µm) identified by immunoperoxidase within the ischemic and nonischemic basal ganglia were defined by contiguous optical fields covering 18.3 mm² or nuclear dUTP label covering 1.51-mm² regions of interest, with the aid of computerized video-imaging microscopy at x400.^{1 3} For instance, for the ₆ antigen, each specimen was scanned in a checkerboard array of 250 image fields in a serpentine pattern covering 1000 microscopic fields (18.3 mm² total). Subsequently, for all antigens, quantitation was confined to 25 microscopic fields (1.51 mm²), centered on the respective regions of dUTP incorporation in the ischemic tissue, and applied to the nonischemic zone. The two approaches, for the ₆ subunit, produced identical temporal outcomes. The numbers of laminin-5–stained vascular structures were taken as the total number of microvascular structures per field.

Immunofluorescence colocalization studies, with the use of FITC- and TRITC-labeled secondary antibodies (Vector Laboratories), were performed with the aid of laser confocal microscopy (LSM Invert 410; Karl Zeiss) to clarify spatial relationships of specific integrin antigens to microvascular wall structures. All measurements were made with the same pinhole size (20), brightness, contract, zoom, and laser time (8 seconds).

Statistical Analysis
All data are presented as mean±SD. The distributions of relevant antigens are expressed as fractions. The fraction integrin/[control] refers to the number of microvessels expressing a given integrin for each subject at each time point relative to the mean of the number expressed in the control cohort. The fraction integrin/laminin-5 refers to the number of microvessels expressing a given integrin relative to the number of microvessels expressing laminin-5 for each subject at each time point. Two-way ANOVA and Mann-Whitney U tests were used for comparisons when appropriate. Significance was set at 2P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Localization of Integrins in Normal Brain
Integrin subunit ß₄ and heterodimer ₆ß₄ are found on microvessels associated with astrocytes (Figs 1 and 2). The subunit ₆ colocalizes with subunit ß₄ (Fig 2A). Capillaries displaying ₆ often did not express CD31, although when present both antigens were most often concentrically arranged with the endothelial CD31 antigen innermost (Fig 2B). The same relationship placed ₆ antigen external to -actin (smooth muscle) on arterioles (data not shown), where both ₆ and integrin ₆ß₄ colocalized with laminin-5 outside the myointimal layer (Fig 2C). ß₄ and ₆ß₄ were found consistently within (and frequently colocalized with) GFAP-expressing astrocyte fibers (Fig 2D). Therefore, ₆ß₄ is localized on astrocytes.

View larger version (158K): [in this window] [in a new window]   Figure 1. Localization of subunit ß4 (A) and heterodimer 6ß4 (B) to cerebral microvessels by immunoperoxidase. Bar=50 µm.

View larger version (30K): [in this window] [in a new window]   Figure 2. Colocalization of integrin antigens and vascular markers by dual immunofluorescence laser confocal microscopy. TRITC is seen as red and FITC as green in these images. Yellow indicates complete colocalization. Image width is 161.5 µm. A, Colocalization of subunits 6 (FITC) and ß4 (TRITC) on arteriole. Maximum luminal diameter is 9.8 µm. B, Concentric organization of subunit 6 antigen (FITC) outside endothelial cell CD31 (TRITC) in an arteriole. Subunit 6 antigen is unusually colocalized with CD31. Maximum luminal diameter is 42 µm. C, Distribution of subunit ß4 antigen (FITC) outside its ligand laminin-5 antigen (TRITC) in extracellular matrix of arteriole. Maximum luminal diameter is 48 µm. D, Integrin 6ß4 (FITC) colocalized with GFAP-expressing astrocytes (TRITC) in four microvascular arrangements. Luminal diameters are 5.6 to 7.0 µm.

Cytokeratin, a known cytoskeletal ligand for ß₄ within epithelial cells, has been reported on a subset of endothelial cells in noncerebral tissues⁴⁸ and in astrocytes in vitro.⁴⁹ Cytokeratin was readily identified on the basal surface of normal baboon integument, but no immunoperoxidase signal within the basal ganglia or cortex could be identified. Vimentin identified all subclasses of the cerebral microvasculature⁵⁰ and astrocytes,⁵¹ demonstrating the abundance of other intermediate filaments in cerebral microvessels (data not shown).

Microvascular Laminin Distribution
Immunocytochemical and complementary dual immunofluorescence experiments demonstrated the identical distribution of antibodies against laminin-1 (LAM 89 and AB 949) and laminin-5 (0668B). The microvascular number and diameter distributions identified among the three immunoprobes did not differ (Table 1). Because laminin-5 was found on the basal lamina and extracellular matrix of all cerebral microvascular subclasses defined by laminin-1 and is a ligand for ₆ß₄, the fate of all antigens has been referred to it here.

View this table: [in this window] [in a new window]   Table 1. Distribution of Laminin-5 and Laminin-1 in Normal Cerebral Microvessels

Microvascular Expression of ₆, ß₄, ₆ß₄, and Laminin-5
The number of microvessels that expressed ₆ antigen in both nonischemic corpora striata of control subjects (990.7±20.5 and 866.0±51.6, respectively; n=3; 2P=.066) was not different from the sham-operated control (944.0) (Table 2). Integrin subunit ₆ antigen was expressed on all microvascular diameter classes in nearly identical distribution to the laminin-5 antigen but was found on more vessels than laminin-5 in nonischemic tissue (subunit ₆/laminin-5=1.23±0.05; n=3) (Figs 3 and 4). The relative fraction of subunit ß₄ was not different from ₆ß₄ (2P=0.806); however, ₆ß₄ and ß₄ immunoreactivities were found on 59.3±16.4% and 61.9±3.7% of ₆-expressing vessels (n=3 each) (2P=.001 and 2P=.058), respectively. The mean fractions of total integrin antigen relative to laminin-5 were as follows: ₆, 1.23±0.05; ß₄, 0.76±0.03; and ₆ß₄, 0.73±0.18 (Fig 4). Subunit ß₄ and ₆ß₄ were more prominently associated with 7.5- to 30.0-µm microvessels (43.4±4.6% and 36.2±31.4%, respectively) compared with ₆ and laminin-5 (24.3±5.4% and 23.4±8.6%, respectively).

View this table: [in this window] [in a new window]   Table 2. Effect of Focal Ischemia and Reperfusion on Integrin Subunit 6

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of MCAO/R on subunit 6 and laminin-5. Relative decreases in microvessel associated antigens are shown. The lesion core was defined by nonvascular cells displaying dUTP incorporation (dUTP+), compared with the 18.3-mm2 region of interest encompassing the ischemic tissue (total). Note that the reduction in total 6 antigen and 6 antigen in the dUTP+ (TdT) zone exceeded the reduction in laminin-5 in the ischemic basal ganglia. The fraction integrin/[control] represents the ratio of the number of microvessels expressing a given integrin for each subject at each time point relative to the mean number of that integrin expressed in the control cohort.

View larger version (25K): [in this window] [in a new window]   Figure 4. Effect of MCAO/R on integrin subunits 6 and ß4 and the heterodimer 6ß4 relative to laminin-5 in the dUTP+ zone. The reductions in 6ß4/laminin-5 and ß4/laminin-5 are linked. No recovery in either integrin antigen relative to laminin-5 was noted. The fraction integrin/laminin-5 refers to the number of microvessels expressing a given integrin relative to the number of microvessels expressing laminin-5 for each subject at each time point.

Effect of MCAO/R on Subunit ₆
Subunit ₆ antigen decreased abruptly and significantly by 24-hour MCAO in the ischemic basal ganglia (2P<.001) compared with the nonischemic zone (2P<.001) (Fig 3). In the region of injury defined by the dUTP label (dUTP⁺), ₆ antigen decreased further than in the ischemic basal ganglia overall. Laminin-5 antigen reduction was less than that of ₆ in the dUTP⁺ region.

Effect of MCAO/R on Integrin ₆ß₄ and its Ligand Laminin-5
Since ß₄ determines the ß subunit association of ₆, its response and that of the heterodimer ₆ß₄ to laminin-5 within the dUTP⁺ region were examined during MCAO/R. While subunit ₆ was nearly as sensitive as laminin-5 to MCAO in the ischemic basal ganglia (Fig 3), a significant further equivalent reduction in the expression of subunit ß₄ and integrin ₆ß₄ relative to laminin-5 occurred by 2 hours after MCAO (2P<.001 overall and individually) but did not change substantially thereafter (Fig 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first demonstration that significant changes in the molecular relationships between astrocytes and the microvascular extracellular matrix occur early during experimental focal cerebral ischemia and reperfusion. The integrin ₆ß₄ and the subunit ß₄ were found between the myointima and the astrocyte compartments, directly associated with the basal lamina and extracellular matrix in 60% of cerebral microvessels (Fig 2D). We conclude that integrin ₆ß₄ is derived from astrocytes. MCAO produced a rapid and significant reduction in the expression of the integrin ß₄ and the heterodimer ₆ß₄ at the astrocyte-matrix interface in microvessels of the primate basal ganglia. The decrease in ₆ß₄ expression exceeded the impact of ischemia on its ligands (eg, laminin-5) even by 2 hours after MCAO in regions of severest injury, indicating that the expression of ₆ß₄ is extraordinarily sensitive to ischemia (Fig 4). These changes underscore the subtle but substantial alterations that take place at the extracellular matrix junction between the myointima and the surrounding astrocytes very soon after the onset of focal cerebral ischemia.

To confirm that the changes that took place were in the region of ischemic injury, a molecular marker of cellular injury was chosen. dUTP incorporation may be taken as an index of ischemic injury. In a separate study, nonvascular cells that display dUTP incorporation at all time points after MCAO represented the central region of injury (S.W., M.T., J.A.K., V.Q., and G.J. del Z., unpublished data, 1997).

The integrin subunits ₁, ₆, and ß₁ are expressed constitutively on normal baboon cerebral microvasculature.²¹ However, the precise relationships and heterodimeric affiliations in the brain are not known. Integrin subunit ₆ can combine with either ß₁ or ß₄ subunit, although the preferential complex is ₆ß₄.²⁴ In the brain microvessels, it appears that nonendothelial ₆ follows the common pairing with ß₄ as ₆ß₄. The heterodimer ₆ß₄ is of particular interest because it plays a major role in the structural integrity of epithelia. Integrin ₆ß₄ has a distinctly higher affinity for laminin-5 than laminin-1, the primary ligands for ₆ß₄.¹⁸

The two ₆ isoforms, _6A and _6B, which differ in their cytoplasmic tail,⁵² show no difference in ligand specificity or cytoskeletal binding behavior.⁵² The ₆ subunit is expressed throughout the microvasculature of nonischemic primate brain.²¹ In this study, ₆ expression was found on more microvessels than laminin-5 and ß₄ (2P=.01, Fig 4), consistent with its known participation in other heterodimers. An association with ß₁ (as VLA-1) would explain the differences between ₆ and ₆ß₄ seen in Figs 3 and 4. The identity of the other ß subunit(s) associated with ₆ could not be determined because of the absence of appropriate anti-heterodimer antibodies.

Subunit ß₄ is unique because it has an exceptionally long cytoplasmic tail (>1000 residues) containing a 303–amino acid sequence, which is required for the incorporation of ₆ß₄ into epithelial hemidesmosomes.³⁶ Overexpression of a tailless ß₄ mutant leads to hemidesmosome disorganization.¹⁷ The long cytoplasmic domain of ß₄ could explain why ₆ß₄ is the only heterodimer that is directly associated with the intermediate filaments. The cytokeratins, vimentin, and GFAP belong to different classes of intermediate filaments, a family of 10-nm fibers forming the cytoskeleton in all cells (for review, see Fuchs and Weber⁵³ ). Cytokeratin, a potential candidate for ß₄ cytoplasmic tail attachment,²⁶ has been described on a subset of endothelial cells⁴⁸ and astrocytes.⁴⁹ However, it was not detected within the cerebral microvasculature and astrocytes with the methods used in this study. Vimentin, however, was readily shown in baboon cerebral endothelium⁵⁰ and astrocytes.⁵¹ Subunit ß₄⁵⁴ has also been reported in microvascular smooth muscle on a subset of human brain vessels,⁵⁵ in primate cerebral microvessels,²¹ and on human astrocytes.⁵⁶

Astrocytes may be anchored to the microvascular wall by ₆ß₄. Hemidesmosome-like structures have been described in some perivascular astrocytes, but their function has been unclear.⁵⁷ As shown here, the extracellular domains of ₆ß₄ and ß₄ (against which antibodies were directed) were embedded in the matrix as defined by antibodies against laminin-5 and other laminin antigens, and frequently colocalized with GFAP. It is intriguing that changes in astrocyte ultrastructure at the matrix during focal ischemia^{58 59} may also involve the fate of integrin ₆ß₄.

Laminin-5 antigen decreased significantly during MCAO in the ischemic core defined by cell nuclear dUTP incorporation. Its reduction was most closely paralleled by loss of ₆ antigen, which reached the same frequency (1.10±0.22) by 24 hours of reperfusion. Laminin-5 is a newly recognized member of the laminin family that is composed of one ₃, ß₂, and ₂ chain and is significantly smaller than its laminin-1 counterpart.⁶⁰ In the skin, laminin-5 is concentrated in the basement membrane underlying epithelial hemidesmosomes.⁶¹ The present study confirms its presence in all size classes of cerebral microvessels. However, in one separate study laminin-5 was not found.⁵⁵

Several integrins have been shown to bind to laminins with overlapping binding specificities (for review, see Hynes²⁰ ). Integrin ₆ß₄ may be involved in intracellular signaling. In the epithelium, interaction of ₆ß₄ with its ligand results in tyrosine phosphorylation of subunit ß₄, which is a prerequisite for assimilation with other hemidesmosome components. This suggests that signals from the extracellular matrix may be conveyed to the cell through the ß₄ subunit.⁶² Since each laminin-integrin combination may have different structural and signaling functions, it is conceivable that the linkage of cerebral microvascular ₆ß₄ to laminin-5 serves a different purpose than ₁ß₁ and laminin-1, for instance.

The implications for microvascular integrity are that ischemia disrupts the interaction between ₆ß₄ and laminin-5. The exact molecular mechanisms for the decrease in integrin ₆ß₄ antigen during MCAO/R are not evident from these studies. Changes of extracellular matrix components may reflect the fate of their corresponding integrin receptors. In the acute stage of lichen planus, the integrin ₆ß₄, laminin-5, and collagen decrease but are reexpressed during the chronic stage, possibly participating in repair functions.⁶³ Studies in epithelium offer several pathophysiological mechanisms that could explain the observed changes in ischemia.⁶⁴ During epithelial reorganization, endocytotic internalization of ₆ß₄ and ₆ß₁ may be promoted by the cytoplasmic domain of the ₆ subunit. Ischemia could lead to changes in the cytoplasm and subsequent "inside-out" signaling, leading to internalization of the heterodimers.⁶⁴

Another possible explanation derives from reduced expression of the ₆ß₄ ligand. The early loss of laminin-5 antigen could well be an "outside-in" signal leading to decreased expression of its ligand ₆ß₄. This could contribute to decreased astrocyte–basal lamina adherence with loss of microvascular integrity. Repair mechanisms may be triggered.

Cytokines, which are known to be generated during focal ischemia,^{65 66} may contribute to these processes. TNF- moderates the integrin–extracellular matrix interactions of fibroblasts in vitro in a concentration-dependent manner.⁶⁷ Endothelial cells treated with TNF- display a reduction in ₆ß₁ receptor related to downregulation of ₆ transcription alone, since ß₁ transcription is not affected.²² Transcriptional regulation of a subunit may be another avenue by which ₆ß₄ production is reduced during focal ischemia. This is hinted at by the finding of decreased expression of ß₄ protein and mRNA on Schwann cells after axotomy.⁶⁸ The rapidity of the cerebral microvascular responses of ₆ß₄ to ischemia implies the operation of regulatory mechanisms with rather short latency, as may occur with cytokine exposure.⁶⁹

The presence of ₆ß₄ at the astrocyte-matrix border suggests that specialized adhesion sites link astrocyte end-feet to the microvascular extracellular matrix and are very sensitive sites to focal ischemia. How ischemia alters ₆ß₄ is of immediate interest. The early reduction in ₆ß₄ expression seen here is consistent with the findings of astrocyte end-feet swelling after experimental MCAO.^{70 71} Since astrocytes intervene between the microvasculature and neurons and play an intermediary role in communication, structural support, and nutrient supply,^{6 7} disruption of their matrix attachment may contribute to the early neuronal (and neurological) effects of focal cerebral ischemia.

	Selected Abbreviations and Acronyms

 

DAB = 3,3'-diaminobenzidine tetrahydrochloride DIG-dUTP = digoxigenin-dUTP FITC = fluorescein isothiocyanate GFAP = glial fibrillary acidic protein MCA = middle cerebral artery MCAO = middle cerebral artery occlusion MCAO/R = middle cerebral artery occlusion and reperfusion MoAb = monoclonal antibody TdT = terminal deoxynucleotidyl transferase TNF- = tumor necrosis factor- TRITC = tetramethylrhodamine isothiocyanate VLA = very late activation (antigen)

	Acknowledgments

 
This study and its concept were supported by grant NS-26945 from the National Institutes of Health (Dr del Zoppo). Dr Wagner was supported by grant 105/1-1 from the Deutsche Forschungsgemeinschaft and is on leave from the Department of Neurology, University of Heidelberg. We are grateful to Dr Ingrid Stuiver for her expertise in the confocal microscope studies. We also wish to thank Marcia Filbert for her excellent help in preparing this manuscript.

	Footnotes

 
This is publication No. 10431-MEM from The Scripps Research Institute, La Jolla, Calif.

Received October 17, 1996; revision received December 12, 1996; accepted December 20, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Okada Y, Copeland BR, Fitridge R, Koziol JA, del Zoppo GJ. Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion. Stroke. 1994;25:1847-1854. [Abstract]
2. Pulsinelli W. Pathophysiology of acute ischaemic stroke. Lancet. 1992;339:533-536. [Medline] [Order article via Infotrieve]
3. Hamann GF, Okada Y, Fitridge R, del Zoppo GJ. Microvascular basal lamina antigens disappear during cerebral ischemia and reperfusion. Stroke. 1995;26:2120-2126. [Abstract/Free Full Text]
4. Luscinskas FW, Lawler J. Integrins as dynamic regulators of vascular function. FASEB J. 1994;8:929-938. [Abstract]
5. Peters A, Palay BL, Webster HD. The Fine Structure of the Nervous System: Neurons and Their Supporting Cells. 3rd ed. New York, NY: Oxford University Press; 1991.
6. Hirano A, Kwanami T, Llena JF. Electron microscopy of the blood-brain barrier in disease. Microsc Res Tech.. 1994;27:543-556. [Medline] [Order article via Infotrieve]
7. Nedergaard M. Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science. 1994;263:1768-1771. [Abstract/Free Full Text]
8. Liesi P, Risteli L. Glial cells of mammalian brain produce a variant form of laminin. Exp Neurol. 1989;105:86-92. [Medline] [Order article via Infotrieve]
9. Risau W, Hallmann R, Albrecht U, Henke-Fahle S. Brain astrocytes induce the expression of an early cell surface marker for blood-brain barrier specific endothelium. EMBO J. 1986;5:3179-3183. [Medline] [Order article via Infotrieve]
10. Lobrinus JA, Juillerat-Jeanneret L, Darekar P, Schlosshauer B, Janzer RC. Induction of the blood-brain barrier specific HT7 and neurothelin epitopes in endothelial cells of the chick chorioallantoic vessels by a soluble factor derived from astrocytes. Dev Brain Res. 1992;70:207-211. [Medline] [Order article via Infotrieve]
11. Hurwitz AA, Berman JW, Rashbaum WK, Lyman WD. Human fetal astrocytes induce the expression of blood-brain barrier specific proteins by autologous endothelial cells. Brain Res. 1993;625:238-243. [Medline] [Order article via Infotrieve]
12. Albeda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J. 1990;4:2868-2880. [Abstract]
13. Elices MJ, Hemler ME. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others. Proc Natl Acad Sci U S A. 1989;86:9906-9910. [Abstract/Free Full Text]
14. Wainer EA, Carter WG. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique and common ß subunit. J Cell Biol. 1987;105:1873-1884. [Abstract/Free Full Text]
15. Sonnenberg A, Modderman PW, Hogervost F. Laminin receptor on platelets is the integrin VLA-6. Nature. 1988;336:487-489. [Medline] [Order article via Infotrieve]
16. Gu M, Wang W, Song WK, Cooper DN, Kaufman SJ. Selective modulation of the interaction of alpha 7 beta 1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation. J Cell Sci. 1994;107:175-181. [Abstract]
17. Spinardi L, Einheber S, Cullen T, Milner TA, Giancotti FG. A recombinant tail-less integrin ß₄ subunit disrupts hemidesmosomes, but does not suppress ₆ß₄-mediated cell adhesion to laminins. J Cell Biol. 1995;129:473-487. [Abstract/Free Full Text]
18. Niessen CM, Hogervorst F, Jaspars LH, de Melker AA, Delwel GO, Hulsman EHM, Kuikman I, Sonnenberg A. The ₆ß₄ integrin is a receptor for both laminin and kalinin. Exp Cell Res. 1994;211:360-367.[Medline] [Order article via Infotrieve]
19. Clark EA, Brugge YS. Integrins and signal transduction pathways: the road taken. Science. 1995;268:233-239. [Abstract/Free Full Text]
20. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11-25.[Medline] [Order article via Infotrieve]
21. Haring H-P, Akamine P, Habermann R, Koziol JA, del Zoppo GJ. Distribution of the integrin-like immunoreactivity on primate brain microvasculature. J Neuropathol Exp Neurol. 1996;55:236-245. [Medline] [Order article via Infotrieve]
22. Defilippi P, Silengo L, Tarone G. ₆ß₁ Integrin (laminin receptor) is down-regulated by tumor necrosis factor and interleukin-1 beta in human endothelial cells. J Biol Chem. 1992;267:18303-18307. [Abstract/Free Full Text]
23. Kajiji S, Tamura RN, Quaranta V. A novel integrin (_Eß₄) from human epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J. 1989;3:673-680.
24. Giancotti FG, Stepp MA, Suzuki S, Engvall E, Ruoslahti E. Proteolytic processing of endogenous and recombinant ß₄ integrin subunit. J Cell Biol. 1992;118:951-959. [Abstract/Free Full Text]
25. Jones JCR, Kurpakus M, Cooper HM, Quaranta V. A function for the integrin ₆ß₄ in the hemidesmosome. Cell Regul. 1991;2:427-438. [Medline] [Order article via Infotrieve]
26. Stepp MA, Spurr-Michaud S, Tisdale A, Elwell J, Gipson IK. ₆ß₄ Integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci U S A. 1990;87:8970-8974. [Abstract/Free Full Text]
27. Yurchenco PD, Schittny JC. Molecular architecture of basement membranes. FASEB J. 1990;4:1577-1590. [Abstract]
28. Abrahamson DR. Recent studies on the structure and pathology of basement membranes. J Pathol. 1986;149:257-278. [Medline] [Order article via Infotrieve]
29. Hemler ME, Crouse C, Takada Y, Sonnenberg A. Multiple very late antigen (VLA) heterodimers on platelets: evidence for distinct VLA-2, VLA-5 (fibronectin receptor), and VLA-6 structures. J Biol Chem. 1988;263:7660-7665. [Abstract/Free Full Text]
30. Engvall E, Davis GE, Dickerson K, Ruoshlahti E, Varon S, Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J Cell Biol. 1986;103:2457-2465. [Abstract/Free Full Text]
31. Martin GR, Timpl R. Laminin and other basement membrane components. Annu Rev Cell Biol. 1987;3:57-85.
32. Lissitzky JC, Charpin C, Bignon C, Bouzon M, Kopp F, Delori P, Martin PM. Laminin biosynthesis in the extracellular matrix-producing cell line PFHR9 studied with monoclonal and polyclonal antibodies. Biochem J. 1988;250:843-852. [Medline] [Order article via Infotrieve]
33. Kajiji S, Davceva B, Quaranta V. Six monoclonal antibodies to human pancreatic cancer antigens. Cancer Res. 1987;47:1367-1376. [Abstract/Free Full Text]
34. Plopper G, Falk-Marzillier J, Glaser S, Fitchmun M, Giannelli G, Romano T, Jones JCR, Quaranta V. Changes in expression of monoclonal antibody epitopes on laminin-5r induced by cell contact. J Cell Sci. 1996;109:1965-1973. [Abstract]
35. Hessle H, Sakai LY, Hollister DW, Burgeson RE, Engvall E. Basement membrane diversity detected by monoclonal antibodies. Differentiation. 1984;26:49-54. [Medline] [Order article via Infotrieve]
36. Tamura RN, Rozzo C, Starr L, Chambers J, Reichardt LF, Cooper HM, Quaranta V. Epithelial integrin ₆ß₄: complete primary structure of ₆ and variant forms of ß₄. J Cell Biol. 1990;111:1593-1604. [Abstract/Free Full Text]
37. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986;103:2787-2796. [Abstract/Free Full Text]
38. Parums DV, Cordell JL, Micklem K, Heryet AR, Gatter KC, Mason DY. A new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol. 1990;43:752-757. [Abstract/Free Full Text]
39. Moll R, Franke WW, Schiller DL, Geiger B, Knepler R. The catalog of human cytokeratins, patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11-24. [Medline] [Order article via Infotrieve]
40. Osborn M, Debus E, Weber K. Monoclonal antibodies specific for vimentin. Eur J Cell Biol. 1984;34:137-143. [Medline] [Order article via Infotrieve]
41. Courel M-N, Girard N, Delpech B, Chauzy C. Specific monoclonal antibodies to glial fibrillary acidic protein (GFAP). J Neuroimmunol. 1986;11:271-276. [Medline] [Order article via Infotrieve]
42. Charriaut-Marlangue C, Margaill I, Represa A, Popovici T, Plotkine M, Ben-Ari Y. Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis. J Cereb Blood Flow Metab. 1996;16:186-194. [Medline] [Order article via Infotrieve]
43. Schmitz GG, Walter T, Seibl R, Kessler C. Nonradioactive labeling of oligonucleotide in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal Biochem. 1991;192:222-231. [Medline] [Order article via Infotrieve]
44. MacManus JP, Hill IE, Huang ZG, Rasquinha I, Xue D, Buchan AM. DNA damage consistent with apoptosis in transient focal ischaemic neocortex. Neuroreport. 1994;5:493-496. [Medline] [Order article via Infotrieve]
45. Edvinsson L, MacKenzie ET, McCulloch J. General and comparative anatomy of the cerebral circulation. In: Cerebral Blood Flow and Metabolism. New York, NY: Raven Press Publishers; 1993:3-39.
46. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994;94:1621-1628.
47. Li Y, Chopp M, Jiang N, Zhang ZG, Zaloga C. Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. Stroke. 1995;26:1252-1258. [Abstract/Free Full Text]
48. Mattey DL, Nixon N, Wynn-Jones C, Dawes PT. Demonstration of cytokeratin in endothelial cells of the synovial microvasculature in situ and in vitro. Br J Rheumatol. 1993;32:676-682. [Abstract/Free Full Text]
49. Franco MC, Gibbs CJ Jr, Rhoades DA, Gajdusek DC. Monoclonal antibody analysis of keratin expression in the central nervous system. Proc Natl Acad Sci U S A. 1987;84:3482-3485. [Abstract/Free Full Text]
50. Virgintino D, Maiorano E, Bertossi M, Pollice L, Ambrosi G, Roncali L. Vimentin- and GFAP-immunoreactivity in developing and mature neural microvessels: study in the chicken tectum and cerebellum. Eur J Histochem. 1993;37:453-462.
51. Ridet JL, Alonso G, Chauvet N, Chapron J, Koenig J, Privat A. Immunocytochemical characterization of a new marker of fibrous and reactive astrocytes. Cell Tissue Res. 1996;283:39-49. [Medline] [Order article via Infotrieve]
52. Hogervorst F, Admiraal LG, Niessen C, Kuikman I, Janssen H, Daams H, Sonnenberg A. Biochemical characterization and tissue distribution of the A and B variants of the integrin ₆ subunit. J Cell Biol. 1993;121:179-191. [Abstract/Free Full Text]
53. Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345-382. [Medline] [Order article via Infotrieve]
54. Quaranta V, Jones JCR. The internal affairs of an integrin. Trends Cell Biol. 1991;1:2-4. [Medline] [Order article via Infotrieve]
55. Cremona O, Savoia P, Marchisio PC, Gabbiani G, Chaponnier C. The ₆ and ß₄ integrin subunits are expressed by smooth muscle cells of human small vessels: a new localization in mesenchymal cells. J Histochem Cytochem. 1994;42:1221-1228. [Abstract]
56. Paulus W, Baur I, Schuppan D, Roggendorf W. Characterization of integrin receptors in normal and neoplastic human brain. Am J Pathol. 1993;143:154-163. [Abstract]
57. Wegiel J, Wisniewski HM. Rosenthal fibers, eosinophilic inclusions, and anchorage densities with desmosome-like structures in astrocytes in Alzheimer's disease. Acta Neuropathol (Berl). 1994;87:335-361.
58. Garcia JH, Yoshida Y, Chen H, Li Y, Zhang ZG, Liam J, Chen S, Chopp M. Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. Am J Pathol. 1993;142:623-635. [Abstract]
59. Petito CK, Morgello S, Felix JC, Lesser ML. The two patterns of reactive astrocytosis in postischemic rat brain. J Cereb Blood Flow Metab. 1990;10:850-859. [Medline] [Order article via Infotrieve]
60. Timpl R, Brown JC. The laminins. Matrix Biol. 1994;14:275-281. [Medline] [Order article via Infotrieve]
61. Jones JCR, Asmuth J, Baker SE, Langhofer M, Roth SI, Hopkinson SB. Hemidesmosomes: extracellular matrix/intermediate filament connectors. Exp Cell Res. 1994;213:1-11. [Medline] [Order article via Infotrieve]
62. Mainiero F, Pepe A, Wary KK, Spinardi L, Mohammadi M, Schlessinger J, Giancotti FG. Signal transduction by the ₆ß₄ integrin: distinct ß₄ subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J. 1995;14:4470-4481. [Medline] [Order article via Infotrieve]
63. Giannelli G, Brassard J, Foti C, Stetler-Stevenson WG, Falk-Marzillier J, Zambonin-Zallone A, Schiraldi O, Quaranta V. Altered expression of basement membrane proteins and theirintegrin receptors in lichen planus: possible pathogenetic role of gelatinases A and B. Lab Invest. 1996;74:1091-1104. [Medline] [Order article via Infotrieve]
64. Gaietta G, Redelmeier TE, Jackson MR, Tamura RN, Quaranta V. Quantitative measurement of ₆ß₁ and ₆ß₄ integrin internalization under cross-linking conditions: a possible role for ₆ cytoplasmic domains. J Cell Sci. 1994;107:3339-3349. [Abstract]
65. Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ. Tumor necrosis factor- expression in ischemic neurons. Stroke. 1994;25:1481-1488. [Abstract]
66. Liu T, McDonnell PC, Young PR, White RF, Siren AL, Hallenbeck JM, Barone FC, Feuerstein GZ. Interleukin-1ß mRNA expression in ischemic rat cortex. Stroke. 1993;24:1746-1751. [Abstract/Free Full Text]
67. Chou DH-I, Lee W, McCulloch CAG. TNF- inactivation of collagen receptors: implications for fibroblast function and fibrosis. J Immunol. 1996;156:4354-4362. [Abstract]
68. Feltri L, Scherer S, Nemni R, Kamholz J, Vogelbacker H, Oronzi-Scott M, Canal N, Quaranta V, Wrabetz L. ß₄ Integrin expression in myelinating Schwann cells is polarized, developmentally regulated, and axonally dependent. Development. 1994;120:1287-1301. [Abstract]
69. Limb GA, Hamblin AS, Wolstencroft RA, Dumonde DC. Rapid cytokine up-regulation of integrins, complement receptor 1 and HLA-DR on monocytes but not on lymphocytes. Immunology. 1992;77:88-94. [Medline] [Order article via Infotrieve]
70. Norenberg MD. Astrocyte responses to CNS injury. J Neuropathol Exp Neurol. 1994;53:213-220. [Medline] [Order article via Infotrieve]
71. Garcia JH, Liu K-F, Lian J, Xu J. Astrocytic and microvascular responses to the occlusion of a middle cerebral artery (Wistar rat). J Neuropathol Exp Neurol. 1993;52:288. Abstract.

This article has been cited by other articles:

				B R Thanvi, S Treadwell, and T Robinson Haemorrhagic transformation in acute ischaemic stroke following thrombolysis therapy: classification, pathogenesis and risk factors Postgrad. Med. J., July 1, 2008; 84(993): 361 - 367. [Abstract] [Full Text] [PDF]

				R. Milner, S. Hung, X. Wang, G. I. Berg, M. Spatz, and G. J. del Zoppo Responses of Endothelial Cell and Astrocyte Matrix-Integrin Receptors to Ischemia Mimic Those Observed in the Neurovascular Unit Stroke, January 1, 2008; 39(1): 191 - 197. [Abstract] [Full Text] [PDF]

				G. J. del Zoppo, R. Milner, T. Mabuchi, S. Hung, X. Wang, G. I. Berg, and J. A. Koziol Microglial Activation and Matrix Protease Generation During Focal Cerebral Ischemia Stroke, February 1, 2007; 38(2): 646 - 651. [Abstract] [Full Text] [PDF]

				G. J. del Zoppo and R. Milner Integrin-Matrix Interactions in the Cerebral Microvasculature Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 1966 - 1975. [Abstract] [Full Text] [PDF]

				R. Veltkamp, D. A. Siebing, L. Sun, S. Heiland, K. Bieber, H. H. Marti, S. Nagel, S. Schwab, and M. Schwaninger Hyperbaric Oxygen Reduces Blood-Brain Barrier Damage and Edema After Transient Focal Cerebral Ischemia Stroke, August 1, 2005; 36(8): 1679 - 1683. [Abstract] [Full Text] [PDF]

B. Bosche, C. Dohmen, R. Graf, M. Neveling, F. Staub, L. Kracht, J. Sobesky, F.-G. Lehnhardt, and W.-D. Heiss Extracellular Concentrations of Non-Transmitter Amino Acids in Peri-Infarct Tissue of Patients Predict Malignant Middle Cerebral Artery Infarction Stroke, December 1, 2003; 34(12): 29