Cardiac Ischemia Activates Vascular Endothelial Cadherin Promoter in Both Preexisting Vascular Cells and Bone Marrow Cells Involve
http://www.100md.com
Naoko Kogata, Yuji Arai, James T. Pearso
参见附件。
the Departments of Structural Analysis (N.K., Y.N., T.K., S.S., N.M.), Bioscience (Y.A., K.H.)
Cardiac Physiology (J.T.P.)
National Cardiovascular Center Research Institute, Osaka, Japan
the Department of Cell Differentiation, Institute for Molecular Embryology and Genetics (K.H., M.O.), Kumamoto University, Kumamoto, Japan
the Vascular Development Laboratory (R.H.A.), Cancer Research UK London Research Institute, United Kingdom
the Department of Oncogene Research (M.O.), Institute for Microbial Disease, Osaka University, Japan.
Abstract
Vascular endothelial cadherin (VE-cadherin) is expressed on vascular endothelial cells, which are involved in developmental vessel formation. However, it remains elusive how VE-cadherin–expressing cells function in postnatal neovascularization. To trace VE-cadherin–expressing cells, we developed mice expressing either green fluorescent protein or LacZ driven by VE-cadherin promoter using Cre-loxP system. Although VE-cadherin promoter is less active after birth than during embryogenesis in blood vessels, it is reactivated on cardiac ischemia. Both types of reporter-positive cells are found in the vasculature and in the infarcted myocardium. Those found in the vasculature were pre-existing endothelial cells and incorporated endothelial progenitor cells derived from extracardiac tissue. In addition to the vasculature, VE-cadherin promoter-activated cells were positive for CD45 in the bone marrow cells of the infarcted mice. VE-cadherin promoter–reactivated CD45-positive leukocytes were also found in the infarcted area. In addition, VE-cadherin promoter was activated in the bone marrow vessels of the infarcted mice. Collectively, our findings reveal a new ischemia-induced neovascularization mechanism involving VE-cadherin; the re-expressed VE-cadherin–mediated cell adhesion between cells may be involved not only in homing of bone marrow–derived cells to ischemic area but also mobilization from bone marrow.
Key Words: vasculogenesis angiogenesis hemangioblast ischemia CD45
Introduction
Cell-based therapies have been aimed at neovascularization in ischemic diseases.1 Recruitment of both angiogenic factor-producing hematopoietic cells and vasculature-constituting endothelial cells to the ischemic area contributes to neovascularization.2 Endothelial progenitor cells (EPCs) and circulating bone marrow–derived EPCs (CEPCs) are incorporated into the nascent vessels.3,4 These cells have been suggested to originate from bone marrow. On the basis of this potential differentiation capability, bone marrow cell–based therapy has been attempted and proven to be effective for ischemic heart disease and peripheral artery disease.5,6 However, it is unclear how bone marrow–derived cells are recruited to the ischemic area.
Postnatal neovascularization includes angiogenesis and vasculogenesis. Both steps cooperatively work by involving the sprouting and branching of the pre-existing endothelial cells and recruiting EPCs in the vascular tree.7 Proangiogenic factors released from ischemic tissue and infiltrating cells, including vascular endothelial growth factor (VEGF), fibroblast growth factor, granulocyte macrophage colony-stimulating factor, and placental growth factor, mobilize hematopoietic stem cells (HPCs) as well as EPCs to the infarcted area.8–10 Thus, HPCs and EPCs homing to the ischemic area are involved in angiogenesis and vasculogenesis in coordination with pre-existing vascular cells. Similar to adult neovascularization, embryonic vasculogenesis and angiogenesis are coordinated by both endothelial lineage cells and hematopoietic cells originating from hemangioblasts.
Several cell surface molecules, including CD133, VEGF receptor 2, vascular endothelial cadherin (VE-cadherin), and CD34, have been used to characterize the EPCs in the bone marrow and CEPCs in the peripheral blood.6 Although the cells purified by cell surface marker have been demonstrated to be recruited to the ischemic area when transferred for cell-based therapy, it is elusive how and what endogenous EPCs, CEPCs, and HPCs are mobilized to ischemic area for neovascularization in ischemic diseases.
VE-cadherin (Cadherin5, CD144), which belongs to cadherin super family, is expressed on cultured vascular endothelial cells and is essential for endothelial cell–cell interaction.11 Whereas N-cadherin, another cadherin expressed on the endothelial cells, is thought to function in adherens junctions between endothelial cells and mural cells (pericytes and vascular smooth muscle cells [VSMCs]).12 VE-cadherin is required in vivo in the postnatal vasculature to maintain endothelial cell integrity and barrier function.13,14 Moreover, VE-cadherin associated with VEGF receptor is involved in the regulation of permeability after myocardial ischemia.15
In the present study, we investigated the role of VE-cadherin promoter-activated cells in ischemia-induced neovascularization. We demonstrate that VE-cadherin promoter is activated in the pre-existing vascular endothelial cells of bone marrow and heart. In addition, we noticed VE-cadherin promoter is activated in the bone marrow cells and peripheral blood cells presumably corresponding to EPCs/CEPCs and CD45-positive cells preferentially homing to infarcted heart. Thus, we raise the possibility of VE-cadherin–mediated cell–cell contact for effective homing of proangiogenic cells to ischemic tissues.
Materials and Methods
Generation of Transgenic Mice
Cre recombinase driven by VE-cadherin promoter (VE-cad-Cre) mice contained the 4.2-kb VE-cad-Cre transgene (Figure 1A) excised from pBluescript-VE-cad-Cre (online supplement, available at http://circres.ahajournals.org). VE-cad-Cre mice were crossed with LacZ reporter mice (cAct-XstopX-LacZ from the Jackson Laboratory, Bar Harbor, Me) or enhanced green fluorescent protein (GFP) reporter mice (CAG-CAT–enhanced GFP [EGFP] obtained from J. Miyazaki, Osaka University).16 The offspring were named VE-cadherin promoter-driven LacZ-expressing mice (VE/Z) and VE-cadherin promoter-driven EGFP-expressing mice (VE/EG), respectively. All animal experiments were approved by the animal committee of the National Cardiovascular Center and performed according to the regulation of the National Cardiovascular Center.
Detection of Fluorescence In Vivo
VE/EG embryos and dissected organs from VE/EG mice were examined using an Olympus SZX12 stereo-fluorescent microscope equipped with a VB-6010 charge-coupled device camera. The organs from control mice were imaged next to those from VE/EG mice.
Histochemistry, Immunostaining, Immunofluorescence, and In Situ Hybridization
The procedures of histological examination are described in the online supplement.
Neovascularization Models
To monitor VE-cadherin promoter–activated cells during neovascularization, we used a corneal angiogenesis model of 8-week-old VE/EG mouse and myocardial infarction model. Both methods are described in the online supplement.
Parabiosis Model
Pairs of a 6- to 10-week-old wild-type and VE/Z mice were subjected to parabiotic surgery. Mice were surgically joined from shoulder to femur. One week after parabiotic surgery, the coronary artery was ligated in the wild-type mouse.
Characterization of EGFP-Expressing Cells by Flow Cytometric Analysis
EGFP expression of these cells was analyzed by FACS (fluorescence-activated cell sorting) Calibur (BD Biosciences). EGFP together with cell surface antigen immunostained with phycoerythrin-conjugated anti-CD31 or anti-CD45 were investigated using FACS VantageSE or FACS Aria (BD Biosciences).
Results
VE-Cadherin Promoter Is Activated in Cells Responsible for Developmental Vessel Formation
To trace the VE-cadherin–expressing cells in fetal vascular development and postnatal neovascularization, we first generated transgenic mice expressing VE-cad-Cre (Figure 1A). By crossing three lines of VE-cad-Cre mice with either EGFP reporter mice or LacZ reporter mice, we obtained VE/EG and VE/Z (Figure 1B and 1C).
EGFP and LacZ expression by VE-cad-Cre–mediated recombination was observed in embryonic vascular development (Figure 1B and 1C). We examined LacZ expression in Aorta-Gonad-Mesonephros (AGM) region of VE/Z mice where hemangioblasts reside.17,18 LacZ-positive cells were detected in the dorsal aorta (Figure 1D, boxed). We noticed that LacZ-stained cells were in the lumen and in the lining cells of the ventral wall (Figure 1E, arrowhead) and in the cells that seemed to bud off from the inner layer. LacZ-stained cells were also found in the newborn VE/Z mouse liver, where hepatic hematopoiesis is organized (Figure 1F, asterisks). These results suggest that VE-cadherin promoter is activated in the cells probably corresponding to hemangioblasts responsible for fetal vasculogenesis.
VE-Cadherin Promoter Becomes Less Active After Birth
To assess the involvement of VE-cadherin in postnatal vascular development, we examined changes in EGFP reporter expression with age in tissues. Although EGFP expression in both neonatal heart and lung was noticeable, its expression was gradually decreased by aging and no longer observed by the fifth week (Figure 2A, 2C, 2E, and 2G). EGFP and CD31 (platelet and endothelial cell adhesion molecule-1) expression overlapped in the vascular endothelium of both heart and lung of the neonatal VE/EG mice, as revealed by immunohistochemical analyses using anti-GFP and anti-CD31 antibodies (Figure 2B and 2F). In clear contrast, GFP was not detected in the CD31-positive vascular endothelium of 5-week-old VE/EG mice (Figure 2D and 2H). Similarly, we found that EGFP expression was decreased with age in other organs, including the brain, liver, and kidney (data not shown).
Tie2 is a tyrosine kinase receptor for angiopoietin and is expressed in the endothelial cells and hematopoietic cells.19 Thus, we further compared the VE-cadherin promoter-dependent EGFP expression with that dependent on Tie2 promoter. EGFP expression persisted through the entire life (supplemental Figure II), supporting that the VE-cadherin promoter is more active during embryonic and prenatal vascularization than postnatal vessel maintenance. Similarly, VE-cadherin promoter-driven LacZ expression was decreased with aging (supplemental Figure II).
VE-Cadherin Is Expressed in Developing Vasculature
To confirm that VE-cadherin promoter-driven EGFP and LacZ reporter expression reflects endogenous VE-cadherin expression in vivo, we examined the expression of VE-cadherin and VE-cadherin mRNA in embryo. VE-cadherin, VE-cadherin mRNA was detected in developing vessels of the embryo stained with anti–VE-cadherin antibody (Figure 3A) and of the embryo probed with antisense–VE-cadherin cDNA (Figure 3B). VE-cadherin protein and VE-cadherin mRNA were detected in the basilar arteries, intersomitic vessels, and endocardium as reporters of VE/EG and VE/Z mice were expressed. These results indicate that VE-cadherin promoter-driven reporter expression of both VE/EG and VE/Z mice reflects the endogenous VE-cadherin expression.
VE-Cadherin Promoter-Activated Cells Are Involved in Neovascularization
EPCs are positive for VE-cadherin and involved in neovascularization.6 We hypothesized that VE-cadherin promoter is turned on in EPCs during adult vasculogenesis and that VE-cadherin promoter may be reactivated in vessels. Therefore, we first tested whether VEGF induces EGFP expression in using a cornea model of VE/EG mice. After implanting VEGF-containing pellets, EGFP expression was monitored every day. Not only new vessels growing toward the implanted pellets but also limbus vessels exhibited EGFP expression (Figure 4A), indicating that VE-cadherin promoter is activated in the vascular cells involved in neovascularization.
We next tested whether ischemia triggers VE-cadherin expression in a myocardial infarction model. When the coronary artery of VE/Z mice was ligated, LacZ-positive cells were found 3 days after coronary ligation in the hearts of the infarcted mice but not those in the sham-operated mice (Figure 4B and 4C). Similarly, EGFP expression was examined by immunohistochemistry using anti-GFP antibody. In sham-operated control VE/EG mice, GFP-positive cells were not detected in the heart except a few capillaries (Figure 4D), whereas GFP-positive cells were observed in the infarcted area (Figure 4E, left). Notably, among GFP-positive cells, we found CD31-positive cells in the merged image (Figure 4E, right). More than 50% of GFP-positive cells were negative for CD31, suggesting that GFP-positive cells may include nonendothelial lineage cells. Intriguingly, the endothelial cells lining the vessels marked by CD31 in the adjacent viable region were positive for GFP (Figure 4F). In addition, we could find GFP-positive cells in the nonendothelial cell layer in the arterial walls, probably smooth muscle cell layer in the viable area (Figure 4F, arrows). Consistent with GFP reporter expression, VE-cadherin was detected by immunohistochemistry in the endothelium and smooth muscle cells in the vessels of the infarcted heart but not in the sham-operated heart (Figure 4G).
Ischemia Triggers VE-Cadherin Expression in Preexisting Vascular Cells and Mobilized Cells
To test whether the VE-cadherin promoter-activated cells are derived from extracardiac tissues of infarcted mice, we used a parabiotic pairing between a wild-type mouse and a VE/Z mouse (Figure 5, gray mouse and blue mouse, respectively). In a parabiotic mice model, circulating blood cells are mixed through vascular anastomoses that form between the two mice.20,21 LacZ-positive cells were found in both endothelial cell layer and smooth muscle cell layer of infarcted VE/Z mouse (Figure 5A, arrowhead and arrow, respectively), as confirmed by the same section immunostained with anti-CD31 and anti–-smooth muscle actin (SMA). In a parabiosis model in which the wild-type mouse was coronary ligated (indicated by cross), we observed that LacZ-positive cells were recruited to the infarcted area of the wild-type mouse (Figure 5B, denoted by cross) from the VE/Z mice (Figure 5B, left). These results indicate that VE-cadherin promoter-reactivated cells are derived from extracardiac tissues and subsequently home to the infarcted heart. Notably, these LacZ-stained cells were negative for CD31 or -SMA (Figure 5B, right). These results were consistent with those found in the infarcted area of VE/EG mice (Figure 4E). In addition to the infarcted area, LacZ-positive cells were incorporated in the vascular endothelial cell layer and VSMC layer of the viable area (Figure 5C and D), although the number of LacZ-positive cells were much less than that of unconnected mouse. Given that most of the endothelial cells lining the vessels of viable area of infarcted VE/EG mice were positive for GFP (Figure 4E) and that the number of LacZ-positive cells found in the vessels were much less in parabiotic model, VE-cadherin promoter is activated in the pre-existing vessels of viable area. Collectively, these data indicate that VE-cadherin–expressing cells, which do not correspond to vascular cells, are recruited to infarcted area from extracardiac tissue. Notably, when examining the heart of the donor VE/Z mouse without myocardial infarction, LacZ-stained cells were found in both endothelial cell layer and VSMC layer in spite of the absence of cardiac ischemia (Figure 5E), indicating that circulating stimuli may activate VE-cadherin promoter in either the pre-existing vascular cells or the mobilized cells from extracardiac tissue to the vasculature. Parabiosis itself did not trigger LacZ expression before the ischemia (supplemental Figure IIIA). Cardiac ischemia, not but other organ ischemia, is critical for VE-cadherin expression in the heart (supplemental Figure III).
VE-Cadherin Promoter Is Activated in Bone Marrow Cells and Circulating Blood Cells on Myocardial Ischemia
To investigate the origin of the cells homing to the ischemic tissues from extracardiac tissue, we explored EGFP-expressing cells in bone marrow and peripheral blood of the infarcted VE/EG mice by flow cytometry. Although EGFP-expressing cells were not detected in the bone marrow of the sham-operated mice, EGFP-expressing bone marrow cells increased after coronary ligation and reached 5% of total bone marrow mononuclear cells (Figure 6A and 6B). In parallel with the increase of EGFP-expressing cells in bone marrow, EGFP-expressing cells also increased in the peripheral blood (Figure 6C). No EGFP-expressing cells were detected in the blood from sham-operated mice. EGFP-expressing cells increased up to 6% of total circulating mononuclear cells 7 days after ligation (Figure 6D).
EGFP-expressing cells were observed in situ in the vasculature as well as in the marrow cells of infarcted VE/EG mice (Figure 6F) but not in sham-operated mice (Figure 6E), suggesting that VE-cadherin–mediated cell adhesion may be involved in mobilization of bone marrow cells into the bloodstream.
We further examined the expression of endogenous VE-cadherin in the EGFP-expressing cells sorted from the bone marrow of infarcted VE/EG mice. More than 90% and 40% of EGFP-expressing cells were positive for VE-cadherin and for CD31, respectively (Figure 6G and 6H). These data indicate that EGFP reporter expression reflects endogenous VE-cadherin expression in bone marrow cells. To characterize the CD31-negative cells, we performed flow cytometric analysis on mononuclear bone marrow cells obtained from infarcted VE/EG mice 2 days after coronary ligation using anti-CD31 and anti-CD45 because CD45 is expressed in the common origin of both myeloid cells and endothelial cells.5 Among EGFP-positive cells, 50% were positive for CD31, in agreement with the immunostaining of GFP-positive cells with anti-CD31 (Figure 6H and 6I). Of note, all EGFP-expressing cells were positive for the pan-leukocyte cell marker CD45. VE-cadherin mRNAs of bone marrow CD45-positive cells of infarcted mice were twice as much as those of sham-operated mice (supplemental Figure V). These results indicate that EGFP-expressing cells consist of either multilineage cells or distinct stages of differentiated cell from a common origin: CD45-positive EPCs, CD45-positive hematopoietic precursor cells, and CD45-positive hematopoietic cells.
VE-Cadherin Promoter-Activated CD45-Positive Cells Are Actively Recruited to Ischemic Area
We found that VE-cadherin promoter-activated cells were positive for CD45 (Figure 6I) and that CD31-negative GFP-expressing cells were detected in the infarcted area (Figure 4E). Thus, we assumed that CD31-negative GFP-expressing cells might be CD45-positive cells in the infarcted heart. The heart of the infarcted VE/EG mice were immunostained with anti-GFP and anti-CD45. Double-positive staining was found in cells other than elongated cells that seemed to be endothelial cells (Figure 7A). The percentage of double-positive cells among CD45-positive cells in the infarcted area (>10% of CD45-positive cells were positive for GFP) was greater than that of CD45/EGFP-expressing cells in bone marrow cells as examined by FACS analysis (1.5% of CD45-positive cells were GFP positive; Figure 6I), indicating that double-positive cells were more selectively recruited to the ischemic area than single CD45-positive cells.
Discussion
Here we show for the first time that VE-cadherin promoter-activated cells are detected in both bone marrow cells and blood vessels in the infarcted mice. Although VE-cadherin promoter is less active with aging, reactivated VE-cadherin promoter drives VE-cadherin expression in the pre-existing vessels of ischemic hearts. Our results do not suggest that VE-cadherin is not expressed in mature vessels but rather suggests that VE-cadherin expression is enhanced during ischemia, as detected by anti–VE-cadherin antibody (Figure 4G). Although VE-cadherin is required in the postnatal vascular endothelial cell integrity,13,14 VE-cadherin is hardly detectable by immunohistochemistry.22 Ischemia may drive VE-cadherin promoter, resulting in detectable increased VE-cadherin expression in the pre-existing vessels.
What is the role of re-expressed VE-cadherin of the vascular vessels during ischemia VE-cadherin expression during ischemia may make conditions favorable for the homing of EPCs/CEPCs and the integration of these cells to create the neovessels. VE-cadherin may function not only as an endothelial cell–cell adhesion molecule, but as a leukocyte–endothelial cell adhesion molecule. Leukocytes extravasate across the endothelial cell layer via homophilic platelet and endothelial cell adhesion molecule-1 (CD31), binding between leukocytes and vascular endothelial cells because leukocytes do not express VE-cadherin.23,24 Because VE-cadherin promoter was activated in CD45-positive leukocytes on ischemia, VE-cadherin on both pre-existing vessels and CD45-positive cells may help to the extravasation of CD45-positive cells.
We noticed that ischemia induced VE-cadherin promoter activation of both marrow cells and endothelium in the bone (Figure 6F). In addition, VE-cadherin promoter-activated marrow cells were positive for VE-cadherin (Figure 6G).VE-cadherin re-expression induced by ischemia appears to promote the mobilization of VE-cadherin–expressing cells from bone marrow because VE-cadherin regulates the mobilization of bone marrow cells across bone marrow endothelium.25 Angiopoietin-1/Tie2 signaling maintains an HPC quiescence in bone marrow, probably by regulating N-cadherin.26 Thus, ischemia may trigger the cadherin switch from N-cadherin to VE-cadherin in cells mobilizing from the bone marrow niche. These CD45-positive cells may function as proangiogenic factor-producing cells at the ischemic tissue.
VE-cadherin may be expressed in both prenatal and postnatal hemangioblasts. In embryos and embryonic stem cells, VE-cadherin–expressing cells have the potential to generate hematopoietic precursors,27–29 which exist and repopulate in the AGM to differentiate into both hematopoietic cells and endothelial cells.17 Consistently, we found LacZ-positive cells in the lumen of dorsal aorta and those budding from the lining cells of the dorsal aorta (Figure 1D and 1E). Moreover, no blood cells were found within VE-cadherin–deficient embryos, in addition to impaired vascularization.30 During embryogenesis, vasculogenesis and hematopoiesis are coordinated by hemangioblasts residing in AGM region, including the dorsal aorta.31 VE-cadherin–expressing cells may function as adult hemangioblasts because VE-cadherin promoter–activated cells were positive for both CD31 and CD45. It should be tested in the future whether GFP-expressing CD45-positive cells in the infarcted area function as cytokine-secreting cells because EPCs are derived from monocytes and secrete angiogenic factors.2
It has been controversial whether bone marrow–derived cells are integrated into vasculature.32,33 By using parabiotic mice model, we demonstrated that cells from extracardiac tissues were incorporated into both pre-existing endothelial cell and VSMC layers (Figure 5C and 5D). Furthermore, we detected VE-cadherin in both vascular layers of infarcted mice (Figure 4G). VE-cadherin promoter-activated cell detected among smooth muscle cells may be either incorporated cell or pre-existing smooth muscle cells. The former cell may use VE-cadherin–dependent cell–cell interaction for incorporation into the smooth muscle layer across the VE-cadherin–expressing endothelial cells on ischemia. Both endothelial cells and VSMCs originate from the same lineage serving as vascular progenitor cells in embryonic stem cells.34 Even mature vascular endothelial cells can differentiate into smooth muscle cell.35 It will be necessary to test whether VE-cadherin promoter-activated cells can give rise to the smooth muscle cells to examine the potential function as vascular progenitor cells.
We obtained the data indicative of unidentified factors that drives VE-cadherin promoter in bone marrow and heart. VEGF-induced mobilization of EPCs depends on the expression of Flk-1 expressed on the EPCs as placental growth factor recruits HPCs that express Flt-1.8,36 It is of note that we find that circulating factors affect the activity of VE-cadherin promoter just in the vessels of the heart, without affecting activity in other organs (supplemental Figure III). By identifying VE-cadherin promoter-activating factors, we may augment neovascularization in combination with EPC-based cell therapy.
In conclusion, VE-cadherin promoter is reactivated in the ischemic tissue vessels and bone marrow–derived cells. Thus, reactivation of VE-cadherin may be involved in the integration of vessel-constituting cells and angiogenic factor–producing cells.
Acknowledgments
This work was supported by grants from the Ministry of Health, Labor, and Welfare of Japan, from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), from the Ministry of Education, Science, Sports and Culture of Japan, and from Takeda Medical Research Foundation. We thank M. Matsuda for comments; S.I. Nishikawa and D. Vestweber for antibodies; P. Huber for VE-cadherin promoter DNA; M. Yanagisawa, and J. Miyazaki for mice; and M. Miyabayashi, and Y. Matsuura for technical assistance.
Footnotes
Original received April 20, 2005; revision received February 28, 2006; accepted March 8, 2006.
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the Departments of Structural Analysis (N.K., Y.N., T.K., S.S., N.M.), Bioscience (Y.A., K.H.)
Cardiac Physiology (J.T.P.)
National Cardiovascular Center Research Institute, Osaka, Japan
the Department of Cell Differentiation, Institute for Molecular Embryology and Genetics (K.H., M.O.), Kumamoto University, Kumamoto, Japan
the Vascular Development Laboratory (R.H.A.), Cancer Research UK London Research Institute, United Kingdom
the Department of Oncogene Research (M.O.), Institute for Microbial Disease, Osaka University, Japan.
Abstract
Vascular endothelial cadherin (VE-cadherin) is expressed on vascular endothelial cells, which are involved in developmental vessel formation. However, it remains elusive how VE-cadherin–expressing cells function in postnatal neovascularization. To trace VE-cadherin–expressing cells, we developed mice expressing either green fluorescent protein or LacZ driven by VE-cadherin promoter using Cre-loxP system. Although VE-cadherin promoter is less active after birth than during embryogenesis in blood vessels, it is reactivated on cardiac ischemia. Both types of reporter-positive cells are found in the vasculature and in the infarcted myocardium. Those found in the vasculature were pre-existing endothelial cells and incorporated endothelial progenitor cells derived from extracardiac tissue. In addition to the vasculature, VE-cadherin promoter-activated cells were positive for CD45 in the bone marrow cells of the infarcted mice. VE-cadherin promoter–reactivated CD45-positive leukocytes were also found in the infarcted area. In addition, VE-cadherin promoter was activated in the bone marrow vessels of the infarcted mice. Collectively, our findings reveal a new ischemia-induced neovascularization mechanism involving VE-cadherin; the re-expressed VE-cadherin–mediated cell adhesion between cells may be involved not only in homing of bone marrow–derived cells to ischemic area but also mobilization from bone marrow.
Key Words: vasculogenesis angiogenesis hemangioblast ischemia CD45
Introduction
Cell-based therapies have been aimed at neovascularization in ischemic diseases.1 Recruitment of both angiogenic factor-producing hematopoietic cells and vasculature-constituting endothelial cells to the ischemic area contributes to neovascularization.2 Endothelial progenitor cells (EPCs) and circulating bone marrow–derived EPCs (CEPCs) are incorporated into the nascent vessels.3,4 These cells have been suggested to originate from bone marrow. On the basis of this potential differentiation capability, bone marrow cell–based therapy has been attempted and proven to be effective for ischemic heart disease and peripheral artery disease.5,6 However, it is unclear how bone marrow–derived cells are recruited to the ischemic area.
Postnatal neovascularization includes angiogenesis and vasculogenesis. Both steps cooperatively work by involving the sprouting and branching of the pre-existing endothelial cells and recruiting EPCs in the vascular tree.7 Proangiogenic factors released from ischemic tissue and infiltrating cells, including vascular endothelial growth factor (VEGF), fibroblast growth factor, granulocyte macrophage colony-stimulating factor, and placental growth factor, mobilize hematopoietic stem cells (HPCs) as well as EPCs to the infarcted area.8–10 Thus, HPCs and EPCs homing to the ischemic area are involved in angiogenesis and vasculogenesis in coordination with pre-existing vascular cells. Similar to adult neovascularization, embryonic vasculogenesis and angiogenesis are coordinated by both endothelial lineage cells and hematopoietic cells originating from hemangioblasts.
Several cell surface molecules, including CD133, VEGF receptor 2, vascular endothelial cadherin (VE-cadherin), and CD34, have been used to characterize the EPCs in the bone marrow and CEPCs in the peripheral blood.6 Although the cells purified by cell surface marker have been demonstrated to be recruited to the ischemic area when transferred for cell-based therapy, it is elusive how and what endogenous EPCs, CEPCs, and HPCs are mobilized to ischemic area for neovascularization in ischemic diseases.
VE-cadherin (Cadherin5, CD144), which belongs to cadherin super family, is expressed on cultured vascular endothelial cells and is essential for endothelial cell–cell interaction.11 Whereas N-cadherin, another cadherin expressed on the endothelial cells, is thought to function in adherens junctions between endothelial cells and mural cells (pericytes and vascular smooth muscle cells [VSMCs]).12 VE-cadherin is required in vivo in the postnatal vasculature to maintain endothelial cell integrity and barrier function.13,14 Moreover, VE-cadherin associated with VEGF receptor is involved in the regulation of permeability after myocardial ischemia.15
In the present study, we investigated the role of VE-cadherin promoter-activated cells in ischemia-induced neovascularization. We demonstrate that VE-cadherin promoter is activated in the pre-existing vascular endothelial cells of bone marrow and heart. In addition, we noticed VE-cadherin promoter is activated in the bone marrow cells and peripheral blood cells presumably corresponding to EPCs/CEPCs and CD45-positive cells preferentially homing to infarcted heart. Thus, we raise the possibility of VE-cadherin–mediated cell–cell contact for effective homing of proangiogenic cells to ischemic tissues.
Materials and Methods
Generation of Transgenic Mice
Cre recombinase driven by VE-cadherin promoter (VE-cad-Cre) mice contained the 4.2-kb VE-cad-Cre transgene (Figure 1A) excised from pBluescript-VE-cad-Cre (online supplement, available at http://circres.ahajournals.org). VE-cad-Cre mice were crossed with LacZ reporter mice (cAct-XstopX-LacZ from the Jackson Laboratory, Bar Harbor, Me) or enhanced green fluorescent protein (GFP) reporter mice (CAG-CAT–enhanced GFP [EGFP] obtained from J. Miyazaki, Osaka University).16 The offspring were named VE-cadherin promoter-driven LacZ-expressing mice (VE/Z) and VE-cadherin promoter-driven EGFP-expressing mice (VE/EG), respectively. All animal experiments were approved by the animal committee of the National Cardiovascular Center and performed according to the regulation of the National Cardiovascular Center.
Detection of Fluorescence In Vivo
VE/EG embryos and dissected organs from VE/EG mice were examined using an Olympus SZX12 stereo-fluorescent microscope equipped with a VB-6010 charge-coupled device camera. The organs from control mice were imaged next to those from VE/EG mice.
Histochemistry, Immunostaining, Immunofluorescence, and In Situ Hybridization
The procedures of histological examination are described in the online supplement.
Neovascularization Models
To monitor VE-cadherin promoter–activated cells during neovascularization, we used a corneal angiogenesis model of 8-week-old VE/EG mouse and myocardial infarction model. Both methods are described in the online supplement.
Parabiosis Model
Pairs of a 6- to 10-week-old wild-type and VE/Z mice were subjected to parabiotic surgery. Mice were surgically joined from shoulder to femur. One week after parabiotic surgery, the coronary artery was ligated in the wild-type mouse.
Characterization of EGFP-Expressing Cells by Flow Cytometric Analysis
EGFP expression of these cells was analyzed by FACS (fluorescence-activated cell sorting) Calibur (BD Biosciences). EGFP together with cell surface antigen immunostained with phycoerythrin-conjugated anti-CD31 or anti-CD45 were investigated using FACS VantageSE or FACS Aria (BD Biosciences).
Results
VE-Cadherin Promoter Is Activated in Cells Responsible for Developmental Vessel Formation
To trace the VE-cadherin–expressing cells in fetal vascular development and postnatal neovascularization, we first generated transgenic mice expressing VE-cad-Cre (Figure 1A). By crossing three lines of VE-cad-Cre mice with either EGFP reporter mice or LacZ reporter mice, we obtained VE/EG and VE/Z (Figure 1B and 1C).
EGFP and LacZ expression by VE-cad-Cre–mediated recombination was observed in embryonic vascular development (Figure 1B and 1C). We examined LacZ expression in Aorta-Gonad-Mesonephros (AGM) region of VE/Z mice where hemangioblasts reside.17,18 LacZ-positive cells were detected in the dorsal aorta (Figure 1D, boxed). We noticed that LacZ-stained cells were in the lumen and in the lining cells of the ventral wall (Figure 1E, arrowhead) and in the cells that seemed to bud off from the inner layer. LacZ-stained cells were also found in the newborn VE/Z mouse liver, where hepatic hematopoiesis is organized (Figure 1F, asterisks). These results suggest that VE-cadherin promoter is activated in the cells probably corresponding to hemangioblasts responsible for fetal vasculogenesis.
VE-Cadherin Promoter Becomes Less Active After Birth
To assess the involvement of VE-cadherin in postnatal vascular development, we examined changes in EGFP reporter expression with age in tissues. Although EGFP expression in both neonatal heart and lung was noticeable, its expression was gradually decreased by aging and no longer observed by the fifth week (Figure 2A, 2C, 2E, and 2G). EGFP and CD31 (platelet and endothelial cell adhesion molecule-1) expression overlapped in the vascular endothelium of both heart and lung of the neonatal VE/EG mice, as revealed by immunohistochemical analyses using anti-GFP and anti-CD31 antibodies (Figure 2B and 2F). In clear contrast, GFP was not detected in the CD31-positive vascular endothelium of 5-week-old VE/EG mice (Figure 2D and 2H). Similarly, we found that EGFP expression was decreased with age in other organs, including the brain, liver, and kidney (data not shown).
Tie2 is a tyrosine kinase receptor for angiopoietin and is expressed in the endothelial cells and hematopoietic cells.19 Thus, we further compared the VE-cadherin promoter-dependent EGFP expression with that dependent on Tie2 promoter. EGFP expression persisted through the entire life (supplemental Figure II), supporting that the VE-cadherin promoter is more active during embryonic and prenatal vascularization than postnatal vessel maintenance. Similarly, VE-cadherin promoter-driven LacZ expression was decreased with aging (supplemental Figure II).
VE-Cadherin Is Expressed in Developing Vasculature
To confirm that VE-cadherin promoter-driven EGFP and LacZ reporter expression reflects endogenous VE-cadherin expression in vivo, we examined the expression of VE-cadherin and VE-cadherin mRNA in embryo. VE-cadherin, VE-cadherin mRNA was detected in developing vessels of the embryo stained with anti–VE-cadherin antibody (Figure 3A) and of the embryo probed with antisense–VE-cadherin cDNA (Figure 3B). VE-cadherin protein and VE-cadherin mRNA were detected in the basilar arteries, intersomitic vessels, and endocardium as reporters of VE/EG and VE/Z mice were expressed. These results indicate that VE-cadherin promoter-driven reporter expression of both VE/EG and VE/Z mice reflects the endogenous VE-cadherin expression.
VE-Cadherin Promoter-Activated Cells Are Involved in Neovascularization
EPCs are positive for VE-cadherin and involved in neovascularization.6 We hypothesized that VE-cadherin promoter is turned on in EPCs during adult vasculogenesis and that VE-cadherin promoter may be reactivated in vessels. Therefore, we first tested whether VEGF induces EGFP expression in using a cornea model of VE/EG mice. After implanting VEGF-containing pellets, EGFP expression was monitored every day. Not only new vessels growing toward the implanted pellets but also limbus vessels exhibited EGFP expression (Figure 4A), indicating that VE-cadherin promoter is activated in the vascular cells involved in neovascularization.
We next tested whether ischemia triggers VE-cadherin expression in a myocardial infarction model. When the coronary artery of VE/Z mice was ligated, LacZ-positive cells were found 3 days after coronary ligation in the hearts of the infarcted mice but not those in the sham-operated mice (Figure 4B and 4C). Similarly, EGFP expression was examined by immunohistochemistry using anti-GFP antibody. In sham-operated control VE/EG mice, GFP-positive cells were not detected in the heart except a few capillaries (Figure 4D), whereas GFP-positive cells were observed in the infarcted area (Figure 4E, left). Notably, among GFP-positive cells, we found CD31-positive cells in the merged image (Figure 4E, right). More than 50% of GFP-positive cells were negative for CD31, suggesting that GFP-positive cells may include nonendothelial lineage cells. Intriguingly, the endothelial cells lining the vessels marked by CD31 in the adjacent viable region were positive for GFP (Figure 4F). In addition, we could find GFP-positive cells in the nonendothelial cell layer in the arterial walls, probably smooth muscle cell layer in the viable area (Figure 4F, arrows). Consistent with GFP reporter expression, VE-cadherin was detected by immunohistochemistry in the endothelium and smooth muscle cells in the vessels of the infarcted heart but not in the sham-operated heart (Figure 4G).
Ischemia Triggers VE-Cadherin Expression in Preexisting Vascular Cells and Mobilized Cells
To test whether the VE-cadherin promoter-activated cells are derived from extracardiac tissues of infarcted mice, we used a parabiotic pairing between a wild-type mouse and a VE/Z mouse (Figure 5, gray mouse and blue mouse, respectively). In a parabiotic mice model, circulating blood cells are mixed through vascular anastomoses that form between the two mice.20,21 LacZ-positive cells were found in both endothelial cell layer and smooth muscle cell layer of infarcted VE/Z mouse (Figure 5A, arrowhead and arrow, respectively), as confirmed by the same section immunostained with anti-CD31 and anti–-smooth muscle actin (SMA). In a parabiosis model in which the wild-type mouse was coronary ligated (indicated by cross), we observed that LacZ-positive cells were recruited to the infarcted area of the wild-type mouse (Figure 5B, denoted by cross) from the VE/Z mice (Figure 5B, left). These results indicate that VE-cadherin promoter-reactivated cells are derived from extracardiac tissues and subsequently home to the infarcted heart. Notably, these LacZ-stained cells were negative for CD31 or -SMA (Figure 5B, right). These results were consistent with those found in the infarcted area of VE/EG mice (Figure 4E). In addition to the infarcted area, LacZ-positive cells were incorporated in the vascular endothelial cell layer and VSMC layer of the viable area (Figure 5C and D), although the number of LacZ-positive cells were much less than that of unconnected mouse. Given that most of the endothelial cells lining the vessels of viable area of infarcted VE/EG mice were positive for GFP (Figure 4E) and that the number of LacZ-positive cells found in the vessels were much less in parabiotic model, VE-cadherin promoter is activated in the pre-existing vessels of viable area. Collectively, these data indicate that VE-cadherin–expressing cells, which do not correspond to vascular cells, are recruited to infarcted area from extracardiac tissue. Notably, when examining the heart of the donor VE/Z mouse without myocardial infarction, LacZ-stained cells were found in both endothelial cell layer and VSMC layer in spite of the absence of cardiac ischemia (Figure 5E), indicating that circulating stimuli may activate VE-cadherin promoter in either the pre-existing vascular cells or the mobilized cells from extracardiac tissue to the vasculature. Parabiosis itself did not trigger LacZ expression before the ischemia (supplemental Figure IIIA). Cardiac ischemia, not but other organ ischemia, is critical for VE-cadherin expression in the heart (supplemental Figure III).
VE-Cadherin Promoter Is Activated in Bone Marrow Cells and Circulating Blood Cells on Myocardial Ischemia
To investigate the origin of the cells homing to the ischemic tissues from extracardiac tissue, we explored EGFP-expressing cells in bone marrow and peripheral blood of the infarcted VE/EG mice by flow cytometry. Although EGFP-expressing cells were not detected in the bone marrow of the sham-operated mice, EGFP-expressing bone marrow cells increased after coronary ligation and reached 5% of total bone marrow mononuclear cells (Figure 6A and 6B). In parallel with the increase of EGFP-expressing cells in bone marrow, EGFP-expressing cells also increased in the peripheral blood (Figure 6C). No EGFP-expressing cells were detected in the blood from sham-operated mice. EGFP-expressing cells increased up to 6% of total circulating mononuclear cells 7 days after ligation (Figure 6D).
EGFP-expressing cells were observed in situ in the vasculature as well as in the marrow cells of infarcted VE/EG mice (Figure 6F) but not in sham-operated mice (Figure 6E), suggesting that VE-cadherin–mediated cell adhesion may be involved in mobilization of bone marrow cells into the bloodstream.
We further examined the expression of endogenous VE-cadherin in the EGFP-expressing cells sorted from the bone marrow of infarcted VE/EG mice. More than 90% and 40% of EGFP-expressing cells were positive for VE-cadherin and for CD31, respectively (Figure 6G and 6H). These data indicate that EGFP reporter expression reflects endogenous VE-cadherin expression in bone marrow cells. To characterize the CD31-negative cells, we performed flow cytometric analysis on mononuclear bone marrow cells obtained from infarcted VE/EG mice 2 days after coronary ligation using anti-CD31 and anti-CD45 because CD45 is expressed in the common origin of both myeloid cells and endothelial cells.5 Among EGFP-positive cells, 50% were positive for CD31, in agreement with the immunostaining of GFP-positive cells with anti-CD31 (Figure 6H and 6I). Of note, all EGFP-expressing cells were positive for the pan-leukocyte cell marker CD45. VE-cadherin mRNAs of bone marrow CD45-positive cells of infarcted mice were twice as much as those of sham-operated mice (supplemental Figure V). These results indicate that EGFP-expressing cells consist of either multilineage cells or distinct stages of differentiated cell from a common origin: CD45-positive EPCs, CD45-positive hematopoietic precursor cells, and CD45-positive hematopoietic cells.
VE-Cadherin Promoter-Activated CD45-Positive Cells Are Actively Recruited to Ischemic Area
We found that VE-cadherin promoter-activated cells were positive for CD45 (Figure 6I) and that CD31-negative GFP-expressing cells were detected in the infarcted area (Figure 4E). Thus, we assumed that CD31-negative GFP-expressing cells might be CD45-positive cells in the infarcted heart. The heart of the infarcted VE/EG mice were immunostained with anti-GFP and anti-CD45. Double-positive staining was found in cells other than elongated cells that seemed to be endothelial cells (Figure 7A). The percentage of double-positive cells among CD45-positive cells in the infarcted area (>10% of CD45-positive cells were positive for GFP) was greater than that of CD45/EGFP-expressing cells in bone marrow cells as examined by FACS analysis (1.5% of CD45-positive cells were GFP positive; Figure 6I), indicating that double-positive cells were more selectively recruited to the ischemic area than single CD45-positive cells.
Discussion
Here we show for the first time that VE-cadherin promoter-activated cells are detected in both bone marrow cells and blood vessels in the infarcted mice. Although VE-cadherin promoter is less active with aging, reactivated VE-cadherin promoter drives VE-cadherin expression in the pre-existing vessels of ischemic hearts. Our results do not suggest that VE-cadherin is not expressed in mature vessels but rather suggests that VE-cadherin expression is enhanced during ischemia, as detected by anti–VE-cadherin antibody (Figure 4G). Although VE-cadherin is required in the postnatal vascular endothelial cell integrity,13,14 VE-cadherin is hardly detectable by immunohistochemistry.22 Ischemia may drive VE-cadherin promoter, resulting in detectable increased VE-cadherin expression in the pre-existing vessels.
What is the role of re-expressed VE-cadherin of the vascular vessels during ischemia VE-cadherin expression during ischemia may make conditions favorable for the homing of EPCs/CEPCs and the integration of these cells to create the neovessels. VE-cadherin may function not only as an endothelial cell–cell adhesion molecule, but as a leukocyte–endothelial cell adhesion molecule. Leukocytes extravasate across the endothelial cell layer via homophilic platelet and endothelial cell adhesion molecule-1 (CD31), binding between leukocytes and vascular endothelial cells because leukocytes do not express VE-cadherin.23,24 Because VE-cadherin promoter was activated in CD45-positive leukocytes on ischemia, VE-cadherin on both pre-existing vessels and CD45-positive cells may help to the extravasation of CD45-positive cells.
We noticed that ischemia induced VE-cadherin promoter activation of both marrow cells and endothelium in the bone (Figure 6F). In addition, VE-cadherin promoter-activated marrow cells were positive for VE-cadherin (Figure 6G).VE-cadherin re-expression induced by ischemia appears to promote the mobilization of VE-cadherin–expressing cells from bone marrow because VE-cadherin regulates the mobilization of bone marrow cells across bone marrow endothelium.25 Angiopoietin-1/Tie2 signaling maintains an HPC quiescence in bone marrow, probably by regulating N-cadherin.26 Thus, ischemia may trigger the cadherin switch from N-cadherin to VE-cadherin in cells mobilizing from the bone marrow niche. These CD45-positive cells may function as proangiogenic factor-producing cells at the ischemic tissue.
VE-cadherin may be expressed in both prenatal and postnatal hemangioblasts. In embryos and embryonic stem cells, VE-cadherin–expressing cells have the potential to generate hematopoietic precursors,27–29 which exist and repopulate in the AGM to differentiate into both hematopoietic cells and endothelial cells.17 Consistently, we found LacZ-positive cells in the lumen of dorsal aorta and those budding from the lining cells of the dorsal aorta (Figure 1D and 1E). Moreover, no blood cells were found within VE-cadherin–deficient embryos, in addition to impaired vascularization.30 During embryogenesis, vasculogenesis and hematopoiesis are coordinated by hemangioblasts residing in AGM region, including the dorsal aorta.31 VE-cadherin–expressing cells may function as adult hemangioblasts because VE-cadherin promoter–activated cells were positive for both CD31 and CD45. It should be tested in the future whether GFP-expressing CD45-positive cells in the infarcted area function as cytokine-secreting cells because EPCs are derived from monocytes and secrete angiogenic factors.2
It has been controversial whether bone marrow–derived cells are integrated into vasculature.32,33 By using parabiotic mice model, we demonstrated that cells from extracardiac tissues were incorporated into both pre-existing endothelial cell and VSMC layers (Figure 5C and 5D). Furthermore, we detected VE-cadherin in both vascular layers of infarcted mice (Figure 4G). VE-cadherin promoter-activated cell detected among smooth muscle cells may be either incorporated cell or pre-existing smooth muscle cells. The former cell may use VE-cadherin–dependent cell–cell interaction for incorporation into the smooth muscle layer across the VE-cadherin–expressing endothelial cells on ischemia. Both endothelial cells and VSMCs originate from the same lineage serving as vascular progenitor cells in embryonic stem cells.34 Even mature vascular endothelial cells can differentiate into smooth muscle cell.35 It will be necessary to test whether VE-cadherin promoter-activated cells can give rise to the smooth muscle cells to examine the potential function as vascular progenitor cells.
We obtained the data indicative of unidentified factors that drives VE-cadherin promoter in bone marrow and heart. VEGF-induced mobilization of EPCs depends on the expression of Flk-1 expressed on the EPCs as placental growth factor recruits HPCs that express Flt-1.8,36 It is of note that we find that circulating factors affect the activity of VE-cadherin promoter just in the vessels of the heart, without affecting activity in other organs (supplemental Figure III). By identifying VE-cadherin promoter-activating factors, we may augment neovascularization in combination with EPC-based cell therapy.
In conclusion, VE-cadherin promoter is reactivated in the ischemic tissue vessels and bone marrow–derived cells. Thus, reactivation of VE-cadherin may be involved in the integration of vessel-constituting cells and angiogenic factor–producing cells.
Acknowledgments
This work was supported by grants from the Ministry of Health, Labor, and Welfare of Japan, from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), from the Ministry of Education, Science, Sports and Culture of Japan, and from Takeda Medical Research Foundation. We thank M. Matsuda for comments; S.I. Nishikawa and D. Vestweber for antibodies; P. Huber for VE-cadherin promoter DNA; M. Yanagisawa, and J. Miyazaki for mice; and M. Miyabayashi, and Y. Matsuura for technical assistance.
Footnotes
Original received April 20, 2005; revision received February 28, 2006; accepted March 8, 2006.
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