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Dinggang Liu,1,
Yan Huang,1
Ion Jovin,1
Shaw-Yung Shai,2
Themis Kyriakides,3
Robert S. Ross,4* and
Frank J. Giordano1*
Yale University School of Medicine, Section of Cardiovascular Medicine, New Haven, Connecticut 06510,1 Tulane University School of Medicine, New Orleans, Louisiana,2 Yale University, Department of Pathology, New Haven, Connecticut 06510,3 University of California San Diego and San Diego VA Healthcare, Department of Medicine, Cardiology Division, San Diego, California 921614
Received 14 March 2007/ Returned for modification 12 April 2007/ Accepted 4 October 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (24, 36-38). ICAP-1 directly binds the cytoplasmic tail of β1 integrin. Further, the ICAP-1 binding sites for both KRIT-1 and β1 integrin are similar, and both contain a crucial NPXY motif (36). It has thus been postulated that KRIT-1 and β1 integrin compete for binding to ICAP-1 and that this competition modulates β1 integrin signaling and function. Therefore, the vascular defects observed in patients with KRIT-1 gene mutations may involve abnormal β1 integrin regulation via ICAP-1.
Global homozygous deletion of the β1 integrin gene in the mouse resulted in an early embryonic lethal phenotype with a defect in implantation and failure to organize the inner embryonic mass. This phenotype prevented a definitive assessment of the role of β1 integrin in vascular development (9, 32). Murine knockouts of some known β1 binding partners have defective vascularization, further pointing to a potentially important role of β1 integrin in angiogenesis (27). Among these are 1 integrin-deficient mice which display elevated levels of angiostatin and suppressed tumor vascularization (27), 5 integrin deletion resulting in severe vascular defects and embryonic lethality (35), and 4 integrin deletion resulting in defective vascularization and abnormal distribution of pericytes (12). In order to address more specifically the role of β1 integrin in vascular development and vascular remodeling, we used a conditional Cre-lox approach to delete the β1 integrin gene in the endothelium. Here we show that there is an absolute requirement for at least one functional β1 integrin allele in endothelial cells (EC) and that homozygous β1 integrin deletion in the endothelium causes embryonic death at approximately embryonic day 9 (E9). We show that endothelial β1 integrin plays a crucial role in vascular development and patterning. Further, we show that β1 integrin effects on the vasculature are dose dependent and that heterozygous deletion of β1 integrin in the endothelium leads to abnormal patterns of flow-mediated vascular remodeling in the mature murine vasculature.
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MATERIALS AND METHODS |
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Whole-mount immunostaining and immunofluorescence. CD31 staining was carried out as previously described, with slight modification (1, 33). Embryos dissected from the yolk sac were fixed overnight in 4% paraformaldehyde at 4°C. After blocking in 5% rabbit serum, 0.1% Triton X-100, and phosphate-buffered saline for 3 h, the embryos were incubated overnight with rat anti-CD31 antibody (MEC13.3; Pharmingen) (diluted 1:100) at 4°C. After several washes, the embryos were incubated with biotinylated rabbit anti-rat antibody diluted 1:100, again washed, and developed with avidin-biotinylated horseradish peroxidase using an AEC kit (Vector Laboratories, Inc). Similar approaches were used for whole-mount NG2 (chondroitin sulfate proteoglycan 4) and β1 integrin staining (rabbit anti-NG2 antibody [Chemicon International, Temecula, CA] and goat anti-β1 integrin antibody [R&D Systems, Minneapolis, MN]). For immunofluorescence, labeled secondary antibodies were used and images were obtained by confocal microscopy in the Yale core.
Isolation and evaluation of primary murine EC. Isolation of EC was accomplished by collagenase digestion of lung, followed by magnetic bead-based separation using the MC13.3 anti-PECAM antibody as we have previously described (33). Briefly, lungs of ECβ1–/– and wild-type littermates were dissected under sterile conditions, washed, minced, and digested at 37°C in collagenase A (1 mg/ml; Worthington) for 35 to 40 min in a spinner flask. After filtering through a small-pore mesh (31 µm) to remove undigested material, the cells were washed and then incubated for 30 min at 4°C with magnetic beads to which anti-PECAM (MC13.3) antibody was attached via linked sheep anti-rat immunoglobulin G antibody (Dynal AS). The beads with attached cells were collected with a magnet and washed, and the cells were released from the beads by trypsinization. Cells were then pelleted, resuspended in EC growth medium (Clonetics), and cultured in 1% gelatin-coated 25-cm2 flasks. Characterization as EC was performed by diacetylated low-density lipoprotein uptake using a fluorescent reagent (Molecular Probes; Sorrento Valley, CA). Isolated EC were then either used acutely for gene expression studies or split 1:3 for ongoing culture.
Gene expression analysis. Total RNA was extracted with an RNeasy kit with on-column DNase I digestion incorporated (Qiagen), according to the manufacturer's protocol. β1 integrin mRNA expression in the EC was quantified by real-time PCR. Total RNA was used to synthesize the first-strand cDNA using random primers with the StrataScript first-strand synthesis system (Stratagene). iQ SYBR green supermix (Bio-Rad) was used for real-time PC,R, and results were normalized to the expression of PECAM to ensure equal EC representation in each sample. The sequences for forward (F) and reverse (R) primers are as follows: PECAM, 5'-GAATCAAACCGTATCTCCAAAG-3' (F) and 5'-GCAGGACAGGTCCAACAACTC-3' (R); β1 integrin, 5'-AGTGCTCCCACTTCAATCTCACCA-3' (F) and 5'-TCTCCTTGCAATGGGTCACAGGAT-3' (R). All other quantitative real-time PCRs were performed as described above with primer pairs designed and previously validated in our lab. Western blotting using total extracted protein and antibodies against β1 integrin, Cre recombinase, and actin determined expression at the protein level.
External carotid artery ligation and in situ zymography. Mice were anesthetized with an intraperitoneal injection of ketamine and xylazine, and the left external carotid artery was ligated as previously described (29). Two weeks after ligation, both left and right common carotid arteries (CCAs) were harvested and embedded. Cross sections (5-µm thickness) were obtained for hematoxylin-eosin staining. Images of each carotid ring were captured digitally, and morphometric measurements (wall thickness, vessel and luminal perimeters, and lumen area) were obtained using the SCION Image, as previously described (29). In situ zymography to detect gelatinase activity (mainly matrix metalloproteinase 2 [MMP2] and MMP9) was performed as previously described on CCA sections obtained 24 h after external carotid ligation (23). Briefly, carotid artery sections were incubated with a fluorogenic gelatin substrate (DQ gelatin; Molecular Probes/Invitrogen, San Diego, CA) that gives a green fluorescent signal (530 nm) when exposed to gelatinase activity. Analysis of the fluorescent signal was done by digitalization with the program MetaMorph7.
Wound healing. Sex-matched 12-week-old mice underwent induction of uniform cutaneous wounds using a 5-mm round punch bioptome, essentially as we have described previously (28). Duplicate wounds were created on the contralateral flanks. After 7 days, wounds were excised, bisected exactly in the center, and embedded in paraffin. Serial 5-µm sections were stained with hematoxylin-eosin. Reepithelialization was assessed by measuring the distance between the leading edges of keratinocyte ingrowth on digital images. Frozen sections were immunostained for PECAM and vessel counts obtained. Similarly, sections were colabeled with the anti-NG2 and anti-PECAM antibodies (described above) for immunofluorescent analysis of pericyte recruitment.
Statistical analysis.
Statistical significance was determined by a paired t test or two-way analysis of variance where appropriate. Significance was defined as a P value of 
0.05.
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RESULTS |
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Although development proceeded more slowly in the absence of endothelial β1 integrin, the vascular abnormalities noted were not consistent with simple developmental delay. In addition to the decreased vascularization and abnormal vascular dilation described above, there was a marked reduction of secondary and tertiary vascular patterning and vascular branching in endothelial β1 integrin null embryos (Fig. 2C). Further, vascularity at later time points (E11 to E11.5) remained markedly abnormal, with no evidence of progressive development from earlier time points (Fig. 2E). Colabeling of β1 integrin and PECAM verified vascular-specific deletion of β1 integrin (Fig. 2F). Although vasculogenesis was not directly assessed and thus a defect in vasculogenesis contributing to the phenotype cannot be ruled out, PECAM staining of the yolk sac (Fig. 2G) (E9 to E9.5) demonstrated formation of vascular plexi in ECβ1–/– and control cells. However, there was markedly abnormal patterning in the ECβ1–/– yolk sac vasculature, suggestive of abnormal yolk sac angiogenesis and/or remodeling.
Heterozygous deletion of the β1 integrin gene in EC results in reduced β1 integrin protein levels and abnormal vascular remodeling in response to altered flow. Given that homozygous deficiency of endothelial β1 integrin was embryonically lethal, we hypothesized that reduced but not fully absent expression of this integrin would be involved in human disease and/or physiological adaptations of the vasculature. To address this issue, we first determined whether loss of a single allele would affect total endothelial β1 integrin expression at the protein level. Examining primary isolated EC from heterozygous β1 knockout mice or their control littermates, it was observed by Western blotting that the loss of a single β1 allele resulted in a significant reduction in β1 protein levels (Fig. 3A). A commensurate decrease in β1 integrin transcript levels was also seen (data not shown).
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To evaluate potential mechanistic differences underlying the altered flow-mediated vascular remodeling in ECβ1+/– mice, we used in situ zymography to analyze gelatinase activity in the ipsilateral and contralateral CCAs 24 h after external carotid ligation. As shown in Fig. 3F to H, there was significant induction of gelatinase activity in the ipsilateral common carotids of ECβ1+/– mice and controls at this time point (MMP2 and MMP9 are gelatinases A and B, respectively). These data demonstrate that external carotid ligation indeed initiated a remodeling process, but they also failed to demonstrate a significant difference between ECβ1+/– and control vessels. This suggests either that the differential remodeling noted is not related to MMP activation or that larger differences might be discernible at other time points. Gene expression analysis of these same vessels by quantitative real-time PCR revealed significant induced expression of monocyte chemotactic protein 1 (MCP-1) and MMP9 at 24 h in ECβ1+/– mice and controls (Fig. 3I), again documenting molecular responses to the altered flow but not detecting significant differences between these two genotypes.
Wound healing is normal in heterozygous endothelial β1 integrin mice. To evaluate the functional significance of heterozygous endothelial deletion of β1 integrin in a second, in vivo setting, we tested the effects of reduced endothelial expression of β1 integrin on cutaneous wound healing. This model was chosen since wound healing involves critical vascular responses and also since mice with homozygous deletion of β1 integrin in keratinocytes showed abnormal wound healing (13). Full-thickness circular skin wounds were created on the flanks of ECβ1+/– mice and their age and sex-matched control littermates. Wound healing and vascularization were assessed by histology/morphometry and immunohistochemical vessel counts on the wounds 5 days after the biopsy. Unlike the case for the keratinocyte-specific β1 integrin knockout mice, wound healing was not impaired in our ECβ1+/– mice (Fig. 4A), although there was a nonsignificant trend toward decreased vascularization in the peri-wound area (Fig. 4B and C). We also used this wound-healing model to assess pericyte recruitment. Coimmunofluorescent labeling of NG2 (chondroitin sulfate proteoglycan 4) and PECAM in the areas of repair and angiogenesis at the wound margins revealed no apparent differences in the investiture of vascular segments with NG2-expressing cells, suggesting that at least in the ECβ1+/– mice pericyte recruitment is not defective (Fig. 4D).
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DISCUSSION |
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vβ3 in angiogenesis, global deletion of the β3 integrin is not lethal, whereas endothelial deletion of β1 integrin alone is sufficient to induce lethality.
The development of cerebral vascular abnormalities in the ECβ1–/– embryos is particularly interesting because of the purported link between β1 integrin signaling and the CCM syndrome (36-38). As delineated in the introduction, CCM syndrome is associated with a mutation in the KRIT-1 gene, and both KRIT-1 and β1 integrin contain a binding site for ICAP-1. It has been suggested that the vascular abnormalities that occur in the CCM syndrome are due to dysregulation of β1 integrin signaling because KRIT-1 no longer competes with β1 integrin for ICAP-1 (36). Our current data suggest that this hypothesis of dysregulated β1 integrin signaling in the CCM syndrome is valid.
The specific mechanistic role or roles played by the β1 integrin in the endothelium remain unclear, despite our current data establishing an absolute requirement for this integrin in the endothelium. We show here both a developmental embryonic phenotype in response to homozygous endothelial β1 integrin deletion and an abnormal adaptive response to altered flow in adult mice heterozygous for this deletion. Whether these embryonic and adult phenotypes are mechanistically linked or are the result of separate β1 integrin functions in the vasculature also remains unclear. Our analysis of the yolk sac vasculature demonstrates abnormal vascular patterning in ECβ1–/– versus control littermates. Although vascular plexi do form in ECβ1–/– yolk sacs and the vascular abnormalities noted appear to be due to defects in yolk sac angiogenesis or remodeling, our analysis does not wholly rule out that a defect in vasculogenesis contributes to the phenotype. In the embryo proper the vascular phenotype also prominently features abnormal morphology and patterning. This suggests that endothelial β1 integrin may be involved in the recognition of specific extracellular and/or cellular cues that help guide vascular patterning. Heterodimers containing β1 integrin bind a variety of extracellular matrix components. The integrin heterodimers 7β1 and 1β1, for example, bind galectin-1, a carbohydrate-binding lectin that has been shown to play a crucial role in angiogenesis and vascular patterning (7, 34). Several β1-containing integrin heterodimers bind laminin, and antibodies against the laminin receptor 6β1 block angiogenesis (2, 8). β1 heterodimers such as 5β1 also bind fibronectin, and blocking this interaction inhibits tumor angiogenesis in vivo (18, 35). Thus, the interactions of β1-containing integrin complexes with extracellular matrix components appear to indeed play an essential role in defining vascular patterning. Whether this involves downstream signaling events triggered by β1 integrin binding remains unclear. Interestingly, abnormal vascular patterning has also been documented in the absence of the integrin-linked kinase (ILK), a serine/threonine protein kinase that binds several integrins and regulates the activation state of β1 integrin (10). ILK has also been linked to endothelial progenitor cell recruitment and vasculogenesis, although it is not clear that these functions are mediated via ILK effects on β1 integrin (21).
The abnormal remodeling we see in the CCAs of ECβ1+/– adult mice in response to external carotid ligation features increased vessel wall thickness in response to reduced flow and a loss of the differences in right versus left carotid diameter that normally occur in this model (16, 29). These changes appear to be distinct from the abnormalities seen in developmental vascular patterning in the ECβ1–/– embryos and may involve a different vascular function or functions of β1 integrin in this context, perhaps as a flow sensor. Although we show via in situ zymography and gene expression analysis that external carotid ligation does indeed induce gelatinase expression and activity in the ipsilateral common carotid at the 24-hour time point we examined, we did not see a significant difference in these parameters between ECβ1+/– and control vessels. Similarly, the marked induction of MCP-1 expression observed in the ipsilateral common carotid was also not significantly different between ECβ1+/– vessels and controls. These data do not exclude the possibility that these parameters are different at other time points and do contribute to the phenotype; however, the precise mechanism whereby heterozygous deletion of β1 integrin in the endothelium is sufficient to induce altered flow-mediated remodeling remains unclear. Regarding flow, it is interesting to note that in our study expression of MCP-1 is markedly increased in both ECβ1+/– and control CCAs exposed to decreased flow (i.e., ipsilateral to external carotid ligation). Expression of MCP-1 is known to be inducible by shear stress, and although the physical determinants of shear stress are complex, decreased flow is generally associated with a reduction of shear stress in this model (16). Nonetheless, we observed MCP-1 expression as markedly induced in this setting of decreased flow. This is illustrative, in our view, of the complexity relating physical parameters such as flow mechanistically to downstream molecular events associated with vascular remodeling.
Irrespective of the precise underlying mechanisms, the fact that heterozygous deletion of β1 integrin in the endothelium is sufficient to reduce β1 integrin protein levels in EC and alter flow-mediated vascular remodeling is both interesting and supportive of the clinical relevance of partial loss or dysregulation of this integrin. Other integrin genes have been shown to be susceptible to silencing, for example, via DNA methylation, and it is intriguing to consider that epigenetic phenomena could alter the transcriptional expression of β1 integrin sufficiently to contribute to the pathogenesis of specific disease entities. Moreover, alterations of β1 integrin activation, contextually modulated by ILK or other regulatory partners, could also alter β1 function sufficiently to have significant vascular effects. Of particular interest in the studies we present here are the findings that flow-mediated vascular remodeling is altered in mice heterozygous for endothelial β1 integrin. This suggests that altered expression levels of β1 integrin could have clinically relevant effects on vascular remodeling. The mechanisms whereby this effect is mediated are not clear, but it has previously been suggested that the β1 integrin can act as a shear stress or flow sensor (3). This raises the question of whether altered β1 integrin expression may play a role in the genesis of pathological vascular remodeling, such as coronary atherosclerosis, that is associated with altered flow patterns that occur at vascular branch points. The fact that ECβ1+/– mice are viable provides a powerful opportunity to answer this question in relevant in vivo models of vascular remodeling.
Cutaneous wound healing is abnormal in mice null for β1 integrin in keratinocytes (13). Conversely, global deletion of the β5 integrin does not effect wound healing (14), suggesting a uniquely important role of the β1 integrin in this process. Our data demonstrate that heterozygous endothelial loss of β1 integrin does not significantly delay cutaneous wound healing. This may be attributable to a keratinocyte-specific function of β1 integrin that is essential for wound healing, or it may reflect a dosage effect since our wound healing studies were conducted with mice with only heterozygous deletion of endothelial β1 integrin. There was a trend toward reduced vessel counts in the wound margins in the ECβ1+/– mice, which suggests that loss of a single endothelial β1 integrin allele is not sufficient to prevent angiogenesis but that a sufficient reduction in EC β1 integrin levels is capable of delaying or attenuating angiogenesis in the adult. Further investigation of wound healing and angiogenic responses in these haploid EC β1 integrin mice will be required to more fully understand the dose-dependent role of EC β1 integrin in these processes.
In summary, we have shown here that endothelial expression of the β1 integrin is absolutely required for normal embryonic vascular development and survival. We also present data that support the hypothesis that cavernous malformation syndrome, caused by defects in the KRIT-1 gene, is a consequence of abnormal β1 integrin regulation. Further, we show that a reduction of endothelial expression of β1 integrin to haploid levels is sufficient to mediate altered vascular remodeling in vivo. Cumulatively these data establish definitively that endothelial expression of the β1 integrin is essential for normal vascular development and remodeling and that this is a gene dose-dependent phenomenon. The clinical implications of these findings in human vascular disease, and in the CCM syndrome, are significant, and these data add further mechanistic complexity to the processes of angiogenesis and vascular remodeling.
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ACKNOWLEDGMENTS |
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We thank William C. Sessa for his expertise and assistance in performing the carotid ligation studies and review of the manuscript.
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FOOTNOTES |
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Published ahead of print on 5 November 2007.
L.L. and D.L. contributed equally to this study.
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E9.5 and that embryonic development was delayed prior to lethality. Documentation that the homozygous deletion of β1 integrin in the endothelium resulted in embryonic lethality was obtained by PCR genotyping of embryos. The embryos shown are littermates and reflect relative sizes of β1 integrin null and wild-type littermates for each developmental stage. (C) Hematoxylin- and eosin-stained sections of wild-type littermate (left) and endothelial β1 integrin null (right) embryos at 
