N.L. Flintoff-Dye and J. Welser contributed equally to this work.
Role for the α7β1 integrin in vascular development and integrity
Article first published online: 7 JUL 2005
Copyright © 2005 Wiley-Liss, Inc.
Volume 234, Issue 1, pages 11–21, September 2005
How to Cite
Flintoff-Dye, N. L., Welser, J., Rooney, J., Scowen, P., Tamowski, S., Hatton, W. and Burkin, D. J. (2005), Role for the α7β1 integrin in vascular development and integrity. Dev. Dyn., 234: 11–21. doi: 10.1002/dvdy.20462
- Issue published online: 16 AUG 2005
- Article first published online: 7 JUL 2005
- Manuscript Accepted: 10 APR 2005
- Manuscript Revised: 6 APR 2005
- Manuscript Received: 23 NOV 2004
- NIH/NCRR. Grant Numbers: P20 RR018751-01, P20 RR15581-04
- α7β1 integrin;
- LacZ knockin;
- vascular development;
- vascular smooth muscle;
- cerebral vasculature;
- vascular hemorrhaging;
- vascular integrity
The α7β1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the α7β1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the α7 integrin in these tissues during embryonic development, α7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the α7 promoter is reported by β-galactosidase. From embryonic day (ED) 11.5 to ED14.5, β-galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the α7 integrin gene resulted in partial embryonic lethality. Several α7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The α7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of α5 and α6B integrin chains was detected in the cerebral arteries of α7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the α7β1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity. Developmental Dynamics 234:11–21, 2005. © 2005 Wiley-Liss, Inc.
Integrins are a family of heterodimeric transmembrane proteins composed of αβ subunits that mediate interactions between cells and extracellular matrix proteins (Hynes, 1992). The α7β1 integrin is a laminin receptor originally identified as a marker for skeletal myogenesis (Kaufman et al., 1985). Mutations in the α7 gene (ITGA7) appear to be relatively rare and are responsible for myopathy in humans. Patients lacking the α7 integrin chain exhibit delayed developmental milestones and impaired mobility (Hayashi et al., 1998), consistent with a role for the α7β1 integrin in neuromuscular and myotendinous junction development and function. In addition, some patients with α7 gene mutations exhibit mental retardation (Hayashi et al., 1998). Secondary defects in expression of the α7β1 integrin are relatively common in a variety of human muscle diseases and may contribute to the underlying pathology (Pegoraro et al., 2002).
Mice lacking the α7 chain develop myopathy and display altered matrix deposition at the myotendinous junctions (Mayer et al., 1997; Nawrotzki et al., 2003). These mice also exhibit partial embryonic lethality, suggesting an important role for this integrin during development (Mayer et al., 1997). The underlying cause of this lethality, however, has never been determined.
The α7β1 integrin has multiple roles in the development of the limb musculature (Kaufman and Foster, 1985; Foster et al., 1987; Bozyczko et al., 1989; Kaufman et al., 1991; George-Weinstein et al., 1993; Velling et al., 1996; Nawrotzki et al., 2003). The α7β1 integrin is first detected on primary myofibers after differentiation and is expressed on proliferating and migrating secondary myoblasts (Echtermeyer et al., 1996; Yao et al., 1996b; Crawley et al., 1997). The α7β1 integrin may also participate in myoblast fusion by creating a favorable environment for cell–cell interactions (Gu et al., 1994). In skeletal muscle, the α7 integrin chain has been shown to undergo developmentally regulated RNA splicing to produce three alternative cytoplasmic domains (α7A, α7B, and α7C) and two alternative extracellular domains (α7X1 and α7X2; Collo et al., 1993; Song et al., 1993; Ziober et al., 1993). The cytoplasmic domains differ in size, sequence, and their potential for signal transduction (Song et al., 1993). The extracellular α7X1 and α7X2 integrin isoforms have different binding affinities for different laminin isoforms (Schober et al., 2000). Further extracellular domain isoforms for the α7 integrin were identified later and include α7D in adult human heart (Leung et al., 1998) and X3, X4, and X5 in skeletal muscle (Vignier et al., 1999). The role of these additional extracellular isoforms remains unknown.
In addition to its role in skeletal muscle, the α7B integrin chain is expressed in cardiac myocytes, intestinal cells, vascular smooth muscle cells, dorsal root ganglia, melanoma cells, mucosal mast cells, trophoblasts, and PC12 cells (Kramer et al., 1991a, b; Velling et al., 1996; Basora et al., 1997; Yao et al., 1997; Leung et al., 1998; Werner et al., 2000; Rosbottom et al., 2002). To date, little has been reported on the function of the α7β1 integrin in nonskeletal muscle tissues during embryonic development.
To identify the expression pattern of the α7 integrin chain during embryonic development and identify the possible cause of embryonic lethality, we have used gene targeting to generate α7 integrin chain knockout mice. Exon 1 of the α7 gene was replaced with a lacZ/neo cassette, which was used to report α7 integrin expression during development. In this study, we confirm that loss of the α7 gene results in partial embryonic lethality. Staining for β-galactosidase from embryonic day (ED) 3 to ED14.5, revealed the α7 integrin chain is expressed in the blastocyst and during the development of the vasculature, central and peripheral nervous systems, and skeletal musculature. Several α7 null embryos from ED10.5–13.5 were identified with hemorrhages, especially in the cerebral vasculature. We observed changes in the expression of other integrin α chains that may contribute to or attenuate the vascular pathology of α7 null mice. These data indicate a role for the α7β1 integrin in vascular development and integrity.
Production of α7 Integrin Null Mice
To identify the expression pattern of the α7 integrin chain in vivo, we generated α7 integrin chain null mice in which exon 1 of the mouse α7 gene was replaced with the lacZ cDNA (Fig. 1A). α7 integrin promoter activity would then drive expression of the bacterial β-galactosidase gene. Assays for β-galactosidase activity are sensitive, permitting an examination of α7 gene expression at the single cell level during embryogenesis in both heterozygous (α7+/−) and homozygous (α7−/−) mice. Transfection of the targeting construct produced 384 G418 resistant clones, which were initially screened using a forward primer (PF7) located outside the targeting region and reverse primer located in the lacZ gene. From this screen, four clones were identified that had correctly integrated at the ITGA7 locus giving a targeting efficiency of 1%. Interestingly, all four targeted ES clones were strongly positive for β-galactosidase (data not shown). Of the nine chimeras obtained, seven produced germline transmission of the targeted ES cells. Heterozygous mice from six chimeras were bred to produce α7−/− mice (Fig. 1B). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from skeletal muscle and cerebral artery revealed a lack of α7 transcript in α7−/− mice, whereas wild-type animals showed significant levels of integrin transcript (Fig. 1C), indicating the ITGA7 gene had been successfully targeted. Wild-type and α7−/− mice expressed transcripts for β1D (skeletal muscle) and β1A (cerebral vasculature and skeletal muscle) as well as the control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Fig. 1C).
Loss of the α7 Integrin Protein in Skeletal Muscle and Vasculature of α7−/− Mice
To confirm the absence of the α7 integrin chain at the protein level, Western blot analysis was performed on protein extracted from the gastrocnemii and aortae from 10-week-old wild-type, α7+/− and α7−/− mice (Fig. 2A,B). Antibodies against the α7A and α7B cytoplasmic domains were used in these studies. Western analysis revealed the expected 130-kDa α7 integrin band in protein extracts from the gastrocnemius muscle of both wild-type and α7+/− mice. The skeletal muscle of α7+/− mice showed a reduction in α7A and α7B integrin protein compared with wild-type (Fig. 2A). In contrast, mice genotyped as α7−/− lacked both α7A and α7B protein bands (Fig. 2A). To determine whether the integrin chain was also absent from the vasculature of α7−/− mice, protein was extracted from the aortae of wild-type, α7+/−, and α7−/− mice and subjected to Western analysis (Fig. 2B). Western blots showed α7+/− mice had approximately half the amount of the α7 integrin chain in vasculature compared with wild-type animals, whereas α7−/− mice completely lacked the α7 integrin chain (Fig. 2B). Wild-type, α7+/− and α7−/− mice expressed similar amounts of β1D integrin in the gastrocnemius and β1A integrin chain in the aorta (Fig. 2A,B) indicating that disruption of the α7 gene had no effect on expression of tissue-specific β-subunit partners.
Immunofluorescence analysis using antibodies that detect α7A or α7B integrin chain protein in the gastrocnemius muscle revealed normal integrin localization in the sarcolemma of muscle fibers of wild-type mice (Fig. 3A). In contrast, α7A and α7B integrin chains were absent from the sarcolemma of muscle fibers of α7−/− mice, confirming Western blot results. Immunofluorescence confirmed normal localization of β1D integrin chain in the gastrocnemius muscle of α7−/− mice (Fig. 3A).
In double-labeling experiments, smooth muscle actin–positive vascular smooth muscle cells from wild-type mice strongly expressed the α7B integrin chain (Fig. 3B). In contrast, the α7B integrin chain was absent from the vascular smooth muscle of α7−/− mice confirming the integrin chain has been targeted successfully in both skeletal muscle and vascular tissues (Fig. 3B).
α7 Integrin Promoter Is Active in Several Tissue Types During Development
To study the activity of the α7 promoter and infer the range of tissues that express the α7 integrin chain during development, whole α7+/− embryos isolated from ED8.5 to ED14.5 were stained for β-galactosidase activity. β-Galactosidase was first detected in these embryos at ED11.5 in the somites, tail bud, forelimb, and cerebral vasculature (Fig. 4A). At ED12.5, β-galactosidase activity was dramatically up-regulated in several different tissues, including somites, midbrain, neuronal tracts within the cerebellum primordium, dorsal root ganglia, cranial ganglia, trigeminal ganglia, tongue, olfactory lobe, facial muscles, neck muscles, limb buds, and cerebral vasculature (Fig. 4A). From ED13.5 to ED14.5, there was a further increase in β-galactosidase staining in these tissues, but no new tissue staining was observed (data not shown). Although β-galactosidase was not detected in the developing heart, it was detected in adult heart (data not shown) confirming a previous report that showed the α7 integrin chain was expressed only in the postnatal heart (Belkin et al., 1996).
Embryos were sectioned to determine whether the α7 integrin was expressed in the intersomitic vasculature. β-Galactosidase was not detected in the intersomitic blood vessels at these time points; however, it was detected in the dorsal root ganglia as early as ED11.5 and was significantly increased at ED12.5 (Fig. 4B). β-Galactosidase staining was detected in the myotome component of the somite (Fig. 4B). These results show the α7 chain is transcribed in a wide variety of nonskeletal muscle tissues during development, including vascular smooth muscle and the central and peripheral nervous systems.
Lack of the α7 Integrin Chain Causes Partial Embryonic Lethality
Heterozygous matings of α7+/− mice revealed a significant deviation from the expected Mendelian distribution pattern, with only 8% α7−/− mice born instead of the expected 25% (Table 1). These results indicate that 68% of α7 null embryos die before birth. Of the 153 embryos analyzed between ED10.5 and ED14.5, 16 degenerating embryos or embryos in a state of re-absorption were observed in litters from 20 heterozygous matings. All degenerating embryos that could be genotyped were α7−/−. In addition, two pups that were born dead were genotyped as α7−/− mice. These results indicate that loss of the α7 gene results in lethality with incomplete penetrance.
Loss of the α7 Integrin Chain Results in Vascular Hemorrhaging and Reduced Vasculogenesis
To investigate the possible cause of the observed partial embryonic lethality, embryos were harvested from α7+/− heterozygous matings. Embryos were genotyped using DNA isolated from extraembryonic tissues. Embryos from ED10.5 to ED14.5 were identified that exhibited cerebrovascular hemorrhaging (Fig. 5A). The degree of hemorrhaging blood vessels appeared variable, with some embryos that showed extensive vascular trauma and others with milder vascular leakage in the brain or body wall (Fig. 5A). Of the 153 embryos analyzed between ED10.5 and ED14.5, 18 were identified with vascular hemorrhages. None of the embryos genotyped as wild-type showed any vascular hemorrhaging. Among the hemorrhaging embryos, both α7+/− and α7−/− were identified; however, vascular hemorrhaging was most severe in α7−/− embryos. These data suggest that the level of α7 protein is important to vascular integrity.
To investigate whether the loss of the α7 integrin chain affected blood vessel formation, α7−/− embryos at ED13.5 were stained for β-galactosidase activity. A comparison of α7−/− embryos that showed vascular hemorrhaging with α7−/− embryos that showed no bleeding revealed hemorrhaging had occurred near the ends of β-galactosidase–positive cerebral blood vessels (Fig. 5B). In addition, embryos with hemorrhaging blood vessels often showed reduced cerebral vascularization as detected by β-galactosidase staining (Fig. 5B, arrows). Hence, the α7β1 integrin has a role in both blood vessel formation and vascular integrity.
α7 Integrin Chain Is Expressed in Vascular Smooth Muscle and Not Endothelial Cells
To determine which cerebral vascular cell types express the α7 integrin, coimmunofluorescence experiments were performed on mouse brain cryosections (Fig. 6). In wild-type mice, the α7B integrin did not colocalize with platelet endothelial cell adhesion molecule (PECAM-1) -positive endothelial cells (Fig. 6A). In contrast, the α7B integrin chain strongly colocalized with smooth muscle actin positive cells in wild-type animals (Fig. 6B). Loss of the α7 integrin chain did not affect the detection of vascular smooth muscle or endothelial cells (Fig. 6). Together these results suggest the vascular phenotype observed in α7 null mice is due to a loss of the α7 integrin from vascular smooth muscle and not endothelial cells.
Altered Vascular Smooth Muscle in α7 Null Embryos and Mice
To determine whether loss of the α7 integrin resulted in an altered vascular smooth muscle phenotype in α7 null embryos, the heads from wild-type and α7−/− ED13.5 embryos were cryosectioned and subjected to immunofluorescence to detect smooth muscle actin (Fig 7A). In wild-type mice, strong smooth muscle actin immunofluorescence was detected in cerebral vascular smooth muscle cells (Fig. 7A). In contrast, smooth muscle actin immunofluorescence appeared weaker in the cerebral arteries from α7−/− mice, and the actin cytoskeleton appeared disrupted. Vascular smooth muscle cells in α7−/− mice appeared larger than in wild-type, suggesting these cells may have undergone hypertrophy (Fig. 7A, arrows).
Nuclei within cells positive for smooth muscle actin were counted to determine the number of vascular smooth muscle cells. A 1.8-fold reduction (P < 0.05; N = 5) in the number of smooth muscle actin–positive cells was observed in nonhemorrhaging α7 null embryos compared with wild-type (Fig. 7B). In contrast, hemorrhaging α7 null embryos showed a 4.4-fold reduction (P < 0.01; N = 5) in smooth muscle actin–positive cells compared with wild-type (Fig. 7B). These results show that α7 null embryos have fewer vascular smooth muscle cells, and this deficiency can contribute to reduced vascular integrity and lead to vascular hemorrhaging.
Because defects in the vascular integrity of α7−/− mice are likely to contribute to embryonic lethality, we hypothesized that those α7−/− mice that survive to birth might still exhibit vascular abnormalities. To test this hypothesis, the aortae and brains from 5-week wild-type and α7−/− mice were cryosectioned and subjected to immunofluorescence to detect smooth muscle actin. Nuclei within cells positive for smooth muscle actin were counted to determine the number of smooth muscle cells (Fig. 7C). Results indicate a 1.8-fold increase (P < 0.05; N = 5) in the number of smooth muscle cells in the cerebral arteries of mice that lack the α7 integrin chain and survive to birth compared with wild-type littermates (Fig. 7C). A 2.1-fold increase (P < 0.05; N = 6) in the number of smooth muscle cells was observed in the aortae of mice that lack the α7 integrin chain compared with wild-type littermates (data not shown). Therefore, although α7 null embryos exhibit vascular smooth muscle hypoplasia, those α7 null mice that survive to birth show vascular smooth muscle cell hyperplasia. Vascular smooth muscle hyperplasia may provide increased vascular strength and integrity and help compensate for the absence of the integrin in these cells.
Loss of the α7 Integrin Chain Alters Expression of Other Integrin Chains in the Cerebral Vasculature
To investigate if loss of the α7 integrin affects expression of other integrin chains, semiquantitative RT-PCR was used. Levels of transcripts encoding α3A, α3B, αv, α5, α6A, α6B, and β1A integrin chains were analyzed. These chains were chosen because α3β1 and α6β1 integrins represent other major laminin receptors expressed in vascular smooth muscle cells and mice that lack αv and α5 integrin chains exhibit vascular defects (Yang et al., 1993; Hynes and Wagner, 1996). We observed a 2.7-fold decrease (P < 0.05) in α5 integrin chain transcript in the cerebral arteries of α7 null mice (Fig. 8A). In contrast, a 2.2-fold increase (P < 0.05) in α6B integrin chain transcript was detected in the cerebral arteries of α7 null animals (Fig. 8A). All other integrin chains analyzed showed no significant change in levels compared with control animals. In addition, expression of α5 and α6B integrin chains were not altered in RNA isolated from skeletal muscle of α7 null mice (data not shown).
To confirm that changes in transcripts resulted in changes at the protein level, immunofluorescence was performed on cerebral arteries using antibodies against α5 and α6B integrin chains (Fig. 8B). Immunofluorescence confirmed a reduction in α5 integrin and an increase in α6B integrin from the cerebral vasculature of α7 null mice compared with wild-type animals (Fig. 8B). These results show that the loss of the α7 chain can alter the expression of certain vascular integrin chains, which may contribute to vascular pathology observed in α7 null mice.
The aim of this study was to define the tissue-wide expression pattern of the α7 integrin chain during embryonic development in nonskeletal muscle tissues and attempt to identify the cause of lethality observed in α7 null mice. Our analyses revealed that expression of β-galactosidase from the α7 integrin promoter could be detected as early as the blastocyst stage. From ED11.5 to ED14.5, β-galactosidase was detected in the neural tube, somites, midbrain, tracts within the cerebellum primordium, dorsal root ganglia, cranial ganglia, trigeminal ganglia, olfactory lobe, limb buds, tongue, and vasculature, indicating the α7β1 integrin is expressed in the central and peripheral nervous systems, vasculature, and skeletal muscle during development. This study shows that the α7 integrin chain is expressed in the developing cerebral vasculature from ED10.5, implicating a new role for this integrin in vascularization of the developing brain. In addition, we have identified new regions of the embryonic brain that express the α7 integrin chain. Detection of β-galactosidase in the midbrain and neuronal tracts within the cerebellum primordium suggests a potential role for this integrin chain in the development of these regions and might possibly account for the developmental delay and mental retardation observed in patients lacking the α7 integrin (Hayashi et al., 1998).
Our results confirm that mice lacking the α7 integrin chain exhibit partial embryonic lethality. We observed that 32% of α7−/− mice survive to birth, and these mice are viable and fertile. These mice exhibited myopathy, with approximately 17% of muscle fibers containing centrally located nuclei at 5 weeks of age. Therefore, the LacZ knockin mice produced in this study demonstrate a similar phenotype to α7 knockout mice produced in a previous study, which showed partial embryonic lethality and myopathy (Mayer et al., 1997).
The detection of cerebral vascular lesions in this study indicates that the loss of the α7 integrin can lead to defects in vascular integrity resulting in embryonic lethality starting as early as ED10.5. Cerebral vascularization occurs at ED10, when capillaries originating from the perineural vascular plexus invade the neuroectoderm (Marin-Padilla, 1985). During this embryonic stage, vascular endothelial cells are in close proximity to brain parenchymal cells, and cerebral microvessels become associated with pericytes (Bass et al., 1992; Janzer, 1993; Noctor et al., 2001). Several lines of recent data support the role of integrins, including αvβ3 and αvβ5, in modulating associations between endothelial cells, pericytes, and brain parenchymal cells (Milner et al., 2001; McCarty et al., 2002). In addition to these fibronectin and vitronectin receptors, our results support a role for the laminin receptor, α7β1 integrin, in early cerebral vascular development. In support of this hypothesis, we show that the α7 integrin chain is expressed in the embryonic cerebral vasculature starting around ED11.5, a time when brain vascularization is taking place. Embryos with reduced or absent α7 protein exhibit cerebral vascular hemorrhaging. Finally, those α7 null embryos exhibiting hemorrhaging often showed reduced cerebral vascularization. Together, these results indicate that the α7 integrin plays an important role in cerebral vascular development and integrity.
Coimmunofluorescence data in the cerebral vasculature of wild-type mice revealed the α7 integrin is present in vascular smooth muscle and not endothelial cells. Although the α7B transcript may be present in a subset of endothelial cells (Velling et al., 1996), colocalization data did not reveal this in the cerebral vasculature. These data are in agreement with previous studies that show high levels of α7β1 integrin expression in vascular smooth muscle (Yao et al., 1997; Martinez-Lemus et al., 2003). This finding, along with the altered vascular smooth muscle in α7 null mice, indicates the vascular phenotype in these mice is most likely due to a primary defect in vascular smooth muscle although secondary effects due to impaired cell–matrix interactions may also provide an explanation for these observations. These results suggest that incomplete vascularization and/or loss of vascular integrity can result from a failure to correctly assemble or differentiate vascular smooth muscle cells at sites of vasculogenesis.
Analysis of the cerebral vasculature from α7 null embryos revealed a decrease in vascular smooth muscle cells compared with wild-type embryos. The cerebral hemorrhaging observed in α7 null embryos appears to arise as a result of weakened cerebral blood vessels due to reduced vascular smooth muscle. In contrast, those α7 null embryos that survive to birth exhibit vascular smooth muscle hyperplasia, which may act to protect the blood vessels from rupture. These cells often appeared hypertrophic, with an altered cell cytoskeleton, which may indicate that, although there is an increase in vascular smooth muscle cells in α7 null mice, these blood vessels may exhibit altered functional responses compared with wild-type vessels. The reasons why surviving α7 null mice exhibit hyperplasia, whereas lethal α7 null embryos display hypoplasia, is unclear; however, differences in vascular smooth muscle cell growth, migration, survival, extracellular matrix deposition, and/or expression of other integrin chains may be contributing factors.
Of the 24 known integrins, at least 16 have been reported to be involved in vascular biology and at least 13 are involved in vascular smooth muscle development and function (Hynes, 1992; Hynes et al., 1999; Martinez-Lemus et al., 2003). The α7β1 integrin is strongly expressed in vascular smooth muscle, and this study shows that loss of this integrin contributes to an altered vascular smooth muscle phenotype. Because so many different integrins are found in vascular smooth muscle, altered expression of other integrin chains may contribute to the underlying vascular pathology observed in α7 null mice. Loss of the α7 integrin chain resulted in an increase in the α6B integrin chain transcript and protein in the cerebral artery of α7 null mice. Both α6B and α7B integrin isoforms are laminin receptors that show significant homology (Song et al., 1992). Up-regulation of the α6B integrin chain, therefore, may be a compensatory mechanism for the loss of the α7β1 integrin receptor in the vasculature. A decrease in α5 integrin transcript and protein in cerebral vasculature of mice that lack the α7 chain was observed. Mice that lack the α5 integrin chain exhibit mesodermal defects, muscular dystrophy and vascular defects resulting in embryonic lethality (Yang et al., 1993). Because most integrins can interact with multiple ligands or can regulate common or convergent signaling pathways, loss of a single integrin subunit often results in altered expression of other subunits (Hynes et al., 1992). Reduced α5β1 integrin in the cerebral vasculature of α7 null mice, therefore, may contribute to their vascular pathology.
Both α5β1 and αvβ3 have been shown to regulate the activity of L-type Ca2+ channels (Davis et al., 2001; Wu et al., 1998, 2001). Activation of the α5β1 integrin receptor promotes L-type Ca2+ channel opening, whereas activation of the αvβ3 integrin acts to inhibit channel activity (Davis et al., 2001). This integrin cross-talk may lead to precise regulation of the contractile phenotype of vascular smooth muscle (Davis et al., 2001). In skeletal muscle, the α7β1 integrin has been shown to regulate the activity of L-type Ca2+ channels, which may influence cell migration and differentiation (Kwon et al., 2000). If the α7β1 integrin regulates Ca2+ channels in vascular smooth muscle, loss of the α7β1 integrin and reduced α5β1 integrin may alter cell contractility, migration, or differentiation in the vasculature, and affect vascular integrity.
Integrins have also been shown to alter extracellular matrix deposition (Li et al., 2003), and changes in the expression of vascular extracellular matrix proteins may be expected to occur in α7 null mice. Remodeling of the extracellular matrix may provide an additional explanation for the loss of vascular integrity. The α7β1 integrin has been shown to bind laminin-1, -8, and -10 (Yao et al., 1996a; Klaffky et al., 2001), which are major laminin isoforms expressed in the developing and adult vasculature (Patarroyo et al., 2002). Laminin-1 surrounds developing smooth muscle cells and is concentrated in regions of vascular branching. Because laminin-1 has the ability to maintain the contractile phenotype of vascular smooth muscle cells (Patarroyo et al., 2002), the α7β1 integrin, therefore, is implicated in regulating smooth muscle cell differentiation. Laminin-5, -8, and -10 are found in the extracellular matrix of the adult vasculature (Patarroyo et al., 2002). Loss of α7β1 integrin in the vasculature of the developing embryo and in the adult, therefore, may alter the differentiation state of vascular smooth muscle cells and, thus, affect vascular integrity.
To date only a few patients have been identified with mutations in the α7 integrin gene (Hayashi et al., 1998; Pegoraro et al., 2002). While vascular problems were not reported in these patients, vascular defects observed in embryonic mice lacking the α7 integrin chain may explain why there are so few patients with α7 gene mutations. Altered expression of the α7 integrin, however, appears to be relatively common in muscle diseases and may contribute to the underlying pathology (Pegoraro et al., 2002). Recent evidence has shown that there is increased α7β1 integrin expression in the vasculature of rats with chemically induced vascular injury (Chao et al., 2004). Results from this study show for the first time that the α7β1 integrin has a role in cerebrovascular development and that loss of the α7 chain adversely affects vascular development and/or integrity. Altered α7 integrin chain expression, therefore, may contribute to the pathology of cerebrovascular and cardiovascular disease.
Restriction enzymes and molecular biology reagents were purchased from Invitrogen (La Jolla, CA) and Promega (Madison, WI). Chemicals were purchased from Sigma (St. Louis, MO) and Fisher (Pittsburgh, PA). Antibodies against the α7 cytoplasmic domains (A2 (345) and B2 (347)) were a kind gift from Dr. Stephen Kaufman (University of Illinois, Urbana, IL). Antibodies against β1D and β1A integrin chains were a gift from Dr. Woo Keun Song (Kwangju Institute for Science and Technology, Korea). The antibody against the α5 integrin chain was a gift from Dr. Maria Valencik (University of Nevada, Reno). The anti-α6B integrin antibody was purchased from Chemicon International. The lacZ/pGKneo cassette was kindly provided by Dr. Ju Chen (University of California at San Diego).
Targeting Construct Design
Using Pfu Turbo polymerase (Stratagene) and the primers P3 (5′-GGTGCATGCAAGACCAATGCC-3′) and R1 (5′-CAACGCTCTCCCAGCTAGTGC-3′), a 1,767-bp fragment from the α7 promoter was amplified from embryonic stem (ES) cell genomic DNA. Using the primers I1 (5′-CTGCCTCTGCATAGGATGGACC-3′) and IR1 (5′-ATGCAGTACACGAACATACACC-3′), a 2,523-bp fragment was amplified from intron 1 of the α7 integrin gene using genomic DNA isolated from ES R1 cells. These fragments were cloned into pCRscript (Stratagene), and fragment identities were confirmed by DNA sequencing. A lacZ/pGKneo cassette was subcloned into the XhoI site of pBK-RSV (Stratagene). The α7 promoter fragment was subcloned upstream of the lacZ/neo cassette using NotI and HindIII sites. The promoter-lacZ/neo fragment was subcloned into the pCRscript clone containing the 2,523-bp α7 intron 1 fragment using NotI sites. The resulting targeting construct replaced exon 1 of the α7 integrin gene with the lacZ/neo cassette to permit expression of the β-galactosidase reporter gene from the α7 promoter. The targeting construct was confirmed by DNA sequencing.
Generation of Targeted ES Cells
Primary mouse fibroblasts were mitotically inactivated by irradiation and used as feeders for ES-R1 cells. ES cells were electroporated with 20 μg of linearized targeting vector. Transfected ES cells were kept in selection medium (300 μg/ml of G418) for 10 days before colonies were isolated, expanded, and screened by PCR using the following primers: P7 (5′-TAGAACTTCAGGCAAAGCAGTC-3′) and bgalR2 (5′-GACCTGCAGGCATGCAAGC-3′). The forward primer is located outside the targeting region, and the reverse primer is located in the lacZ gene. ES cells were also stained for β-galactosidase activity.
Production of Mice Lacking α7 Integrin
The α7 null mice used in this study were produced at the Nevada Transgenic Center. ES cells carrying the correctly targeted α7 integrin allele were microinjected into blastocysts from C57BL/6J mice to generate chimeric offspring. The resulting male chimeric mice with a high percentage of agouti coat color were bred with C57BL/6J females. Resultant embryos and pups were genotyped by multiplex PCR using genomic DNA isolated from extraembryonic tissues or tail clips, respectively. The following primers: a7PF10 (5′-TGAAGGAATGAGTGCACAGTGC-3′), a7exon1R1 (5′-AGATGCCTGTGGGCAGAGTAC-3′), and bgalR2 (5′-GACCTGCAGGCATGCAAGC-3′) were used to detect the targeted and wild-type alleles. Heterozygotes were mated to generate mice lacking the α7 integrin.
RNA was isolated from gluteus muscle and cerebral artery from five 5-week-old wild-type and five α7 knockout mice using TRIzol reagent (Invitrogen). Approximately 3 μg of RNA was used to produce cDNA using a Superscript II kit (Invitrogen). The following primers were used to amplify the α7 integrin transcript: a7F (5′-CACCGGATGTCCATCAGGAC-3′) and a7R (5′-GTTGCTCAGAGATGCATCC-3′). These α7 integrin primers amplified a 366-bp fragment. The following primers were used to detect β1 integrin: b1F (5′-GGCAACAATGAAGCTATCGT-3′) and b1R (5′-CCCTCATACTTCGGATTGAC-3′). These primers amplified a 266-bp β1A integrin fragment and a 348-bp β1D integrin fragment. The following primer set was used to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH): GF (5′-GAAGGGCTCATGACCACAGTCCATG-3′), and GR (5′-TGTTGCTGTAGCCGTATTCATTGTC-3′). These primers amplified a 454-bp GAPDH fragment. Multiplex PCR reactions were performed using 100 ng of cDNA in 1.5 mM MgCl2, 0.5 μM primers, 0.4 μM dNTPs, and 0.5 U of Taq DNA polymerase (Invitrogen). Cycle parameters included an initial denaturation for 4 min at 95°C and then 30 cycles of 1 min at 95°C, 45 sec at 58°C, and 1 min at 72°C. This profile was chosen because the PCR amplification was in the linear phase for all primer sets. A final extension at 72°C for 10 min was included in the reaction. Primers and conditions for semiquantitative RT-PCR to detect α3, αv, α5, α6, and β1A integrin chains have been described elsewhere (Rosbottom et al., 2002). PCR products were run on 2.5% agarose gels and imaged by using a Bio-Rad gel imager. Integrin and GAPDH band intensities were determined using ImageQuant software. Each integrin band was normalized to GAPDH. RNA extracted from cerebral vasculature of five wild-type and five α7 integrin knockout mice were assayed, and mean value for each genotype calculated ± SD. Data were analyzed using an unpaired Student's t-test with a P < 0.05 considered significant.
Skeletal, brain, and vascular tissues from wild-type, α7+/−, and α7−/− mice were harvested, embedded in OCT compound (Tissue-Tek), and frozen in liquid nitrogen cooled isopentane. Ten-micron sections were cut using a Leica CM1900 series cryostat and placed on Surgipath (Surgipath Medical Supplies, Richmond, IL) microscope slides. Sections were fixed in −20°C acetone for 1 min, rehydrated in 1× phosphate buffered saline (PBS) for 10 min, and blocked in PBS containing 5% BSA for 20 min. The α7 integrin chain was detected with rabbit polyclonal anti-α7 antibodies, B2 (347) and A2 (345) at 1/500 dilution. The β1 integrin chain was detected using rabbit polyclonal anti-β1A and anti-β1D integrin antibodies at 1/500. The α5 integrin chain was detected by using a rabbit polyclonal antibody at 1/500. The α6B integrin chain was detected by using a mouse monoclonal antibody at 1/100 and a MOM kit (Vector Laboratories, Burlingame, CA) following the manufacturer's directions. Mouse endothelial cells were detected by using rat anti-PECAM-1 (MEC13.3) monoclonal antibody (1/500) purchased from BD Pharmingen. Primary antibodies were detected by using rhodamine-labeled donkey anti-rabbit antibody, fluorescein isothiocyanate (FITC) -labeled donkey anti-rabbit antibody, FITC-labeled donkey anti-mouse, or FITC-labeled donkey anti-rat (Jackson ImmunoResearch). Smooth muscle actin was detected by using Cy3-labeled anti-smooth muscle actin antibody (1/200) from Sigma Biochemicals (St. Louis, MO).
Western Blot Analysis
Western blot analysis of protein extracted from gastrocnemii or aortae from wild-type, α7+/−, and α7−/− mice was performed as previously described (Burkin et al., 2001). Blots were probed with anti-α7 integrin antibodies A2 (345) or B2 (347) diluted 1/1,000, or anti-β1 integrin antibodies β1D or β1A diluted 1/500. Alexa 680-labeled goat anti-rabbit antibody (Molecular Probes) was used to detect the primary rabbit polyclonal antibody. Integrin bands were detected using the Odyssey Scanner (LiCor).
β-Galactosidase Staining of Mouse Embryos
Timed matings were set up to generate embryos at various stages of gestation from ED3 to ED14.5 for whole-mount β-galactosidase staining (Chu et al., 2000). Embryos were removed from the uterus, rinsed in PBS, and fixed in 4% paraformaldehyde for 2 hr. Embryos were permeabilized with a sodium deoxycholate/NP40 mixture for 3–4 hr. An X-gal solution (50 mM potassium ferrocyanide, 50 mM potassium ferricyanide, 1 M MgCl2, and 100 mg/ml X-gal) was added to the embryos, and incubated at 37°C overnight. Embryos were washed in PBS. For cross-sectional analysis, embryos were embedded in OCT and flash frozen in isopentane-cooled liquid nitrogen and cryosectioned. For whole embryo studies, embryos were post-fixed in 70% ethanol for 2 hr. Embryos were dehydrated in 95% and 100% alcohol series, cleared in methyl salicylate, and imaged using a dissecting microscope and Spot digital camera.
Smooth Muscle Cell Quantitation
Brains and aortae from 10-week wild-type and α7−/− mice were embedded in OCT and frozen in liquid nitrogen. The heads from five wild-type, five hemorrhaging, and five nonhemorrhaging α7 null ED13.5 embryos were embedded in OCT and frozen in liquid nitrogen. Using a Leica CM1900 cryostat, five 10-μm sections at least 50 μm apart were placed on microscope slides. Sections were fixed in methanol held at −20°C for 5 min, blocked with 5% BSA in PBS, incubated with Cy3-labeled anti-smooth muscle actin antibody (1/200) and washed in PBS. Sections were mounted in Vectashield containing 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI, Vector Labs). Labeled antibodies were visualized with a Zeiss Axioscope II fluorescence microscope. Care was taken to ensure sections that were analyzed were at right angles to the plane of the blood vessel. Smooth muscle actin–positive cells were identified and nuclei within positive regions counted in 10 random fields for each mouse or embryo, and data were averaged for each genotype ± SEM. Data were analyzed using an unpaired Student's t-test or analysis of variance.
The authors thank Dr. Stephen Kaufman (University of Illinois) for the anti-α7 integrin antibodies, Dr. Maria Valencik (University of Nevada) for the anti-α5 integrin antibody, Dr. Woo Keun Song (Kwangju Institute for Science and Technology, Korea) for the anti-β1 antibodies, and Dr. Ju Chen (University of California-Davis) for the lacZ/neo cassette. The authors also thank Drs. Heather Burkin and Melissa Dechert for critically reading the manuscript.
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