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This study aimed to determine if CD31 is a novel marker of a circulating angio-vasculogenic cell population and to establish their therapeutic effects on experimental ischemia.
Emerging evidence suggested that therapeutic mechanisms underlying various bone marrow (BM)-derived cells are due to paracrine effects. Furthermore, the vasculogenic potential of these cells is under debate. CD31 is a well known marker for endothelial cells (ECs) but is also expressed in a fraction of peripheral blood (PB) mononuclear cells.
CD31+ cells were isolated from human PB by magnetic-activated cell sorting (MACS). The gene expression profile was examined by DNA microarray and real-time RT-PCR (qRT-PCR). Various in vitro endothelial differentiation or vasculogenic assays were conducted. Finally, cells were directly implanted into a mouse hindlimb ischemia (HLI) model to test angiogenic-vasculogenic and therapeutic effects.
Fluorescent-activated cell sorter (FACS) analysis revealed that PB-CD31+ cells exhibited endothelial and hematopoietic stem/progenitor markers. CD31+ cells had higher levels of expression of pro-angiogenic genes on microarray and qRT-PCR and generated higher numbers of endothelial progenitor cells (EPCs) compared to CD31− cells. CD31+ cells spontaneously formed vascular tube-like structures and exhibited an endothelial cell phenotype in vitro. In a HLI model, CD31+ cell transplantation augmented blood perfusion and prevented limb loss. Both angiogenic cytokines and capillary density were increased, suggesting CD31+ cells augmented neovascularization.
CD31 is a novel marker that designates circulating angiogenic and vasculogenic cells. These cells are easily isolated from human PB and thus are a novel candidate for treatment of ischemic cardiovascular disease.
Ischemic cardiovascular disease is the most prevalent disease in Westernized society. Despite progress in therapies, nearly 80 million are affected yearly from heart attack, stroke and ischemic cardiomyopathy (1). As our knowledge of stem cell biology grows, cell-based therapies have become a promising strategy for tissue regeneration. Transplantation of various bone marrow (BM)-derived cells, including mononuclear cells (MNCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), were reported to induce therapeutic neovascularization in adult ischemic tissues (2–5). While cell-based therapy has great therapeutic potential, several issues remain. The use of unselected BM cells or BM-MNCs was associated with adverse events including calcification, vascular plaque growth, and worsening of tissue ischemia (6–8). EPCs and MSCs require ex vivo culture to be used clinically. For all BM-derived cells, their ability to (trans)differentiate into ECs, and the magnitude of their vasculogenic potential, has been debated (9–11). Paracrine mechanisms and/or other non-differentiation effects are the primary mediators of BM-derived cell benefit in ischemic cardiovascular disease (3,12–14) and mainly work via augmentation of neovascularization/angiogenesis or improvement of microcirculation.
Based on these new discoveries on the mechanisms and adverse effects of various BM cells over the past decade, in this study, we sought to explore whether there is a specific native population of BM cells which possesses higher paracrine or angiogenic activity, and further, to determine the therapeutic potential of this population for treating ischemic cardiovascular diseases. Additionally, given the controversy regarding the (trans)differentiation, we sought to definitively determine whether BM cells are able to give rise to ECs via vasculogenesis. We used rigorous criteria and new experimental techniques, to address the main points of criticism from the previous studies that refuted the concept of endothelial differentiation from BM cells (9–11,15).
We have found that CD31, a ubiquitous EC marker, is also expressed on the surface of various mouse hematopoietic cell lineages (16) including HSCs (17), and undifferentiated mouse embryonic stem cells (18). CD31, also known as platelet endothelial cell adhesion molecule (PECAM)-1, is a 130-kd cell surface molecule and a member of the immunoglobulin superfamily (19). CD31 plays important role for transendothelial leukocyte migration, cell-cell adhesion and anti-apoptotic signaling (20). However, the therapeutic role of the cells expressing this marker in tissue regeneration is unknown. Based on the conserved expression of CD31 on cells of the hemangioblast lineage, we hypothesized that CD31+ cells in PB have higher angiogenic and/or vasculogenic capabilities.
Through a series of in vitro and in vivo experiments, we found that CD31+ cells isolated from PB showed higher angiogenic activity and vasculogenic potential, effectively improving ischemia in mouse HLI by augmentation of neovascularization.
An expanded Methods section is available in the Online Appendix.
FACS analysis on PB-CD31+ cells showed that > 95% of MACS-isolated CD31+ cells express CD31 (Fig. 1A). Approximately 40% of CD31+ cells expressed CD14, a monocyte/macrophage marker and more than 99% of CD31+ cells expressed CD45, a pan-hematopoietic marker; this suggests that CD31+ cells are not circulating ECs (Fig. 1A and 1C). CD31+ cells preferentially expressed endothelial markers (CD105, CD141, CD144 and von Willebrand Factor [vWF]; p < 0.05) and stem cell or progenitor markers (CD34, CD133, CD117 and KDR [VEGFR-2]; p < 0.05) (Fig. 1B and Online Fig. 1A). To investigate the hematopoietic progenitor cell (HPC) properties, a clonogenic assay was performed. CD31+ cells generated a significantly higher number of hematopoietic colonies compared to CD31− cells such as colony forming unit-erythroid (CFU-E), burst forming unit-erythroid (BFU-E), colony forming unit granulocyte/macrophage (CFU-GM), and colony forming unit-granulocyte/erythroid/macrophage-/megakaryocyte (CFU-GEMM) (p < 0.05) (Fig. 1C and Online Fig. 1B). These data show that CD31+ cells have characteristics of HSC/HPCs as well as ECs.
To compare global gene expression patterns between CD31+ and CD31− cells, we carried out microarray analysis. Hierarchical cluster analysis showed that gene expression in CD31+ cells are distinct from CD31− cells in that 749 genes were upregulated and 26 genes were downregulated by more than two-fold in the CD31+ cells compared to CD31− cells (Fig. 2A). Further characterization with gene ontology data base (GO, http://www.geneontolgy.org) demonstrated that genes involved in angiogenesis, cell adhesion, transmembrane structure, chemokine production and reception, and extracellular matrix were highly and preferentially expressed in CD31+ cells (Fig. 2B, Table 1 and Online Table S1 to S3) (21).
To confirm the results of microarray data, we performed qRT-PCR analysis. Major angiogenic factors such as vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF) and angiopoietin (Ang)-1, and an adhesion molecule, VE-cadherin, were more highly expressed in CD31+ cells compared to CD31− cells or MNCs (Fig. 2C). Chemokines such as monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8, which play important role for neovascularization, were also significantly upregulated (22,23). Collectively, these findings show that CD31+ cells define a population enriched with angiogenic, chemoattractant and cell adhesion genes.
We next investigated the in vitro endothelial differentiation potential of CD31+ cells. First, we performed EPC culture assay. The number of EPCs and EPC colonies were markedly higher in the CD31+ cell group compared to the CD31-cell group (all p < 0.01) (Fig. 3A and 3B). Second, we carried out a cell adhesion assay. The number of adhered cells to various extracellular matrix proteins, fibronectin, vitronectin, collagen I and laminin, was significantly higher in the CD31+ cells than in the CD31− cells (all p < 0.001) (Fig. 3C). In ECs, adhesion capacity is an indicator of cell engraftment and cell survival.
To induce EC differentiation, CD31+ cells were cultured in endothelial growth media (EGM)-2, which is endothelia cell basal media (EBM)-2 with 15% FBS and cytokine cocktail (SingleQuots), for 28 days. CD31+ cells exhibited EC markers such as vWF, KDR, VE-cadherin and CD31 by immunocytochemistry (Fig. 3D). CD31− cells were not culturable under these conditions. During this culture, we made an intriguing observation that some CD31+ cells spontaneously formed tubular structures. In the first week of culture, CD31+ cells aggregated, around day 10 the cells started to form tubular structures within the cell clusters, and between day 12 to 16 the cell clusters underwent morphologic changes to form linear tubular structures, mimicking in vivo vasculogenesis (Fig. 3E to 3I). These tubular structures stained positive for lectin and took up Dil-acLDL, characteristics of ECs. We further measured the expression of endothelial and angiogenic genes during the culture of CD31+ cells over 28 days by qRT-PCR. Over 28 days the expression of KDR, VE-cadherin, HGF and insulin-like growth factor (IGF)-1 gradually increased suggesting differentiation into a more vasculogenic and angiogenic phenotype (Fig. 3J). Together, these data demonstrate the vasculogenic and angiogenic potential of CD31+ cells in vitro.
After identifying the angiogenic and vasculogenic characteristics of CD31+ cells in vitro, we investigated the therapeutic potential of CD31+ cells to repair experimentally induced ischemia in vivo. Laser Doppler perfusion image (LDPI) analysis demonstrated that blood perfusion was significantly higher in the CD31+ cell injected limbs versus CD31- cell- or PBS-injected limbs at day 7, 14 and 21 (Fig. 4A and 4B). The CD31+ cell-treated group showed a significantly lower limb loss score than the PBS control group (p < 0.05) (Fig. 4C and 4D). The capillary density and vascular maturation in the ischemic hindlimb adductor muscle after surgery was also significantly higher in the CD31+ group than in the CD31− group or the PBS group (p < 0.01) (Fig. 5A, 5B and Online Fig. 2). We also found that functional capillaries examined after intracardiac injection of ILB4 were significantly higher in the CD31+ group compared to the other groups (Online Fig. 3). In addition TUNEL assay, together with ILB4 staining, demonstrated that transplantation of CD31+ cells significantly reduced endothelial cell apoptosis compared to control groups suggesting protective effects on existing vessels (Online Fig. 4). Collectively, these findings suggest that CD31+ cells can augment functional neovascularization and preserve existing vessels in vivo and are effective for treating ischemic vascular disease.
To determine the effects of CD31+ cell transplantation on cytokine expression in ischemic hindlimbs, mice were sacrificed and hindlimb tissues collected. The expression levels of VEGF-A, Ang-1, FGF-2, stromal cell-derived factor (SDF)-1 and CD31, and were significantly increased in the CD31+ cell injected limbs compared to the CD31− cell- or PBS-injected limbs (Fig. 5C). These data suggest that CD31+ cell transplantation upregulates the expression of multiple biological factors associated with neovascularization and bone marrow cell mobilization.
We further sought to determine the potential and magnitude of the contribution of CD31+ cells to ECs generation or vasculogenesis. To track the transplanted cells, we intramuscularly injected 1×106 Dil-labeled CD31+ cells into the ischemic hindlimb of nude mice. Histologic analysis demonstrated that a larger part of the injected CD31+ cells were concentrated in the pericytic or perivascular areas (Fig. 6A to 6D and see Online Video 1) and a smaller population of CD31+ cells exhibited an ECs specific marker, isolectin B4 (ILB4), within the vascular structure at 2–8 weeks (Fig. 6E to 6G and see Online Video 2). This suggests that CD31+ cells generated ECs in vivo. To confirm this, we performed fluorescent in situ hybridization (FISH) using human Y chromosome probe (24,25). FISH data showed that Y chromosome signals from transplanted human CD31+ cells were detected within the nuclei of ECs (Fig. 6I). Collectively, these data indicate that CD31+ cells can give rise to ECs, suggesting their vasculogenic potential.
It has been difficult to quantify the rate of differentiation of BM-derived cells in vivo. Here, to more accurately quantify the number of functional ECs derived from transplanted CD31+ cells, we conducted FACS analysis on enzymatically digested muscle following systemic injection of FITC-labeled ILB4. FACS analysis revealed that 2.2 ± 0.8 % of the cells were double positive for DiI and ILB4 at 4 weeks, suggesting that significant portion of functional ECs were derived from transplanted CD31+ cells (Fig. 7A). Next, to further confirm whether these double positive cells were truly derived from the injected human CD31+ cells, they were subjected to FISH with human Y chromosome. FISH demonstrated that the ILB4- and Dil-positive cells from recipient mice were of human donor origin, confirming differentiation of CD31+ cells into ECs (Fig. 7B).
In this study, we show that CD31 is a unique and comprehensive marker to represent potent angiogenic and vasculogenic cells in human PB and that these cells are therapeutically effective for recovering tissue from ischemia. First, we demonstrated that human CD31+ cells highly express HSC/HPC and EC markers. Second, CD31+ cells are enriched with pro-angiogenic genes. Third CD31+ cells generated more EPCs compared to CD31− cells and demonstrated EC differentiation and vascular-like tube formation in culture. Fourth, implantation of CD31+ cells into ischemic hindlimb enhanced recovery of tissue from ischemic injury and increased the rate of limb salvage as well as the expression of angiogenic factors. Fifth, implanted CD31+ cells show clear vasculogenic potential in ischemic limb tissues.
Since CD31 is a ubiquitous endothelial marker, we tested the hypothesis that the cells expressing CD31 in PB have angiogenic and/or vasculogenic capabilities. FACS showed that the CD31+ cells expressed HSC/HPC markers and well-known EC markers, suggesting dual properties of CD31+ cells. Unbiased microarray data and qRT-PCR results indicated that angiogenic and other genes beneficial for vascular regeneration are highly enriched in the CD31+ cell population. Another distinguishing feature of CD31+ cells is that they gave rise to EPCs in the culture while CD31− cells did not. CD31+ cells frequently formed vascular-like tubes in culture without the aid of biomaterials such as Matrigel, mimicking the developmental process of vascular tube formation from angioblasts or EPCs (Fig. 3E to 3I). This finding is one of the most unprecedented of this study, supporting the vasculogenic potential of CD31+ cells. These data suggest that, CD31 is an antigenic signpost for robust pro-angiogenic and vasculogenic cells in PB.
In vivo cell transplantation studies further confirmed these results. CD31+ cell transplantation induced higher expression of multiple paracrine factors in the ischemic tissues during the critical period of ischemia repair. Our data suggest that the major mechanism of CD31+ cells would be a non-differentiation mechanism because the rate of endothelial differentiation was too small to account for the magnitude of therapeutic effects seen. CD31+ cells highly expressed crucial angiogenic and arteriogenic factors including VEGF, Ang-1, FGF-2 and MCP-1. Synergistic effects of these angiogenic factors have been reported to induce therapeutic neovascularization (26). Also tissues injected with CD31+ cells expressed high levels of SDF-1 which are known to contribute to a pro-neovascularization environment. For optimal neovascularization, such an environment is required to recruit necessary cell types including monocytes and macrophages (27,28). Another characteristic that may make CD31+ cells particularly effective is their high adhesion capacity. Cell adhesion signals control cell viability and are essential for regulated angiogenesis and vasculogenesis via cell-cell and cell-matrix interactions (9,11,15).
The controversy remains regarding the (trans)differentiation potential of BM-derived cells into ECs. To clarify this, we meticulously evaluated hindlimb muscles using multiple techniques. First, we used 3D reconstruction of confocal microscopic images to examine at least 1200 tissue sections collected from 10 animals from each group. Although most CD31+ cells were localized to the interstitium or perivascular region, some cells were incorporated into vasculature and expressed functional EC markers (Fig. 7A). Second, FISH using Y chromosome probes showed positive donor cell-derived signals within the nuclei of ECs suggesting endothelial differentiation of CD31+ cells. Third, FACS analysis confirmed and quantified ECs derived from CD31+ cells injected into HLI. FACS analysis revealed 2.2% of ECs in hindlimb muscle were derived from implanted CD31+ cells. We used these techniques to avoid sampling errors inherent in histologic analysis. To the best of our knowledge, this is the first study to employ this technology to prove and quantify endothelial differentiation from injected BM cells. Finally, we further confirmed that these FACS-isolated (trans)differentiated ECs are human origin by FISH. These results indicate that a specific population of PB-MNCs can (trans)differentiate into ECs although the differentiation efficiency is low.
Cell selection using the surface antigen CD31 has several advantages over to more specific hematopoietic stem/progenitor markers CD133+ or CD34 (29,30). Approximately 80% of CD133+ cells and 85% of CD34+ cells are contained within the CD31+ cell fraction (Fig. 1 and Online Fig. 1). Thus CD31+ cells can exert most of the beneficial effects of both cell types while still providing additional paracrine or vascularizing effects through other cell types. Clinically, as CD31+ cells occupy around 30% of PB MNCs, these cells do not require mobilization to obtain enough to be used for cell therapy.
In conclusion, PB-derived CD31+ cells contain robust angiogenic and vasculogenic abilities that ameliorate hindlimb ischemia. Although we only showed ischemic limb recovery in this study, this therapy would also be effective for ischemic heart disease given their broad neovascularizing, paracrine and cell adhesion capacity. Accordingly, these cells can serve as a highly promising and novel therapeutic option for ischemic cardiovascular disease. Further investigation will be needed to determine if CD31+ cells are capable of treating human ischemic cardiovascular diseases.
This work was supported in part by National Institute of Health grants (HL079137, HL084471, HL097353), Research grant (SC4300) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea, Overseas post-doctoral fellowship grant from the Korea Research Foundation, Republic of Korea (MOEHRD, to SW Kim) and Atlanta Clinical Translational Research Institute (ACTSI)-Georgia Tech Emory Collaboration for Regenerative Medicine and Engineering (GTEC) pilot grant.
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