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Vasculogenic potential of long term repopulating cord blood progenitors

SARA DROETTO^*,,,1, ANDREA VIALE^*,, LUCA PRIMO^,, NOELA JORDANEY^*,, STEFANIA BRUNO^*,, MARCO PAGANO, WANDA PIACIBELLO^*,, FEDERICO BUSSOLINO^, and MASSIMO AGLIETTA^*,

^* Division of Clinical Oncology and
Division of Molecular Angiogenesis Institute for Cancer Research and Treatment (IRCC), School of Medicine,
Department of Oncological Sciences, and
Department of Biomedical Sciences and Oncology, University of Turin, Torino Italy

¹ Correspondence: Division of Molecular Angiogenesis, I.R.C.C., Strada Provinciale 142, Km 3.95, Candiolo (TO), Italy. E-mail: sara.droetto{at}ircc.it

SPECIFIC AIMS

Involvement of bone marrow (BM) derived progenitors in adult experimental tissue revascularization has been shown beyond any doubt, but the importance of such cells in restoring organ vascularization in a clinical setting remains unknown. A mouse transplantation model was engrafted by human cord blood (CB) hematopoietic stem and progenitor cells to follow the behavior of donor-derived endothelial and hematopoietic cells in the presence of a localized source of an angiogenic inducer and in response to altered homeostatic conditions.

PRINCIPAL FINDINGS

1. Human hematopoietic and endothelial cell engraftment in the BM of CB transplanted mice
To study the fate of HSC-derived cells in vivo and their role in new blood vessel formation, NOD/SCID mice were sublethally irradiated and i.v. injected with human CD34⁺ cells purified from CB. Six weeks after CB transplantation the engrafted mice were implanted s.c. with vascular endothelial growth factor (VEGF)-A₁₆₅-enriched Matrigel plugs. Ten days later, mice were killed and BM and Matrigel plugs recovered and analyzed for the presence of human endothelial and hematopoietic cells.

Transplantation of human CD34⁺ CB cells resulted in high levels of hematopoietic cell engraftment in NOD/SCID mice, being up to 75% of human CD45⁺ cells detected in the BM of animals. Flow cytometry analysis demonstrated the differentiation of human CD34⁺ CB cells toward lymphoid and myeloid lineages. In all BM of transplanted mice (CB mice), a well-represented human CD31⁺ cell population containing CD45⁺ hematopoietic cells and CD45^– endothelial cells was also detected. The CD31⁺/CD45^– endothelial subpopulation represented 3.5% of total BM cells. RT-PCR analysis of RNA isolated from the BM of engrafted mice confirmed the presence of cells expressing the human endothelial-specific marker VE-cadherin and CD31.

2. Human cells localize to the neovascularization sites of CB mice
In some experiments human CD34⁺ cells were transduced using a lentiviral-enhanced green fluorescent protein (EGFP) expression vector. In all the BM of mice (n=7) transplanted with EGFP-CD34⁺ CB cells, expression of the transgene was evident. FACS analysis revealed high levels of human engraftment (average value: 75%). Human lymphoid, myeloid, and endothelial EGFP⁺ subpopulations were found by flow cytometry in the BM of animals. PCR analysis of genomic DNA isolated from the Matrigel plugs of engrafted mice established the presence of cells carrying the EGFP transgene, demonstrating localization of human donor cells to these sites.

3. Human endothelial and hematopoietic cells in Matrigel implants of CB mice
To determine whether donor-derived endothelial and hematopoietic cells incorporated into vascular structures formed in Matrigel plugs, we performed RT-PCR and immunohistochemical analysis. Screening for human and murine CD31 expression was performed by RT-PCR on Matrigel invading cells using specie-specific primers. Matrigel plugs established as positive for the expression of human CD31 by RT-PCR were subsequently investigated by immunohistochemistry to confirm the presence of human endothelial cells lining the nascent capillaries. Sections of Matrigel plugs were stained with antibodies raised against human and murine CD31, Von Willebrand factor (VWF), and HLA class I. Most capillaries were largely of murine origin, as demonstrated by costaining with antibodies to murine CD31 and VWF. The incorporation of human-derived endothelial cells (humanCD31⁺/VWF⁺) into neovascularization sites of VEGF-A₁₆₅-enriched Matrigel plugs was randomly detectable in 25% of engrafted mice (n=20). To quantify human-derived endothelial cells in Matrigel implants, human CD31⁺/VWF⁺ and human CD31^–/VWF⁺ capillaries were counted. Less than 1% of total Matrigel capillaries incorporated endothelial cells of donor origin. Indeed, among human-derived cells in Matrigel plugs, myelo-monocytic (CD11b⁺/CD31⁺/VWF^–) phenotypes were evident in 70% of engrafted mice. These results suggest that vasculogenesis occurred in the proposed model, but its contribution in capillary formation was not significant. Certainly, the transplanted HSCs seemed to contribute to Matrigel plug cellularity more to supply myelo-monocytic cells (CD11b⁺ and CD31+/VWF^–) than ECs (CD31⁺/VWF⁺).

4. Enhanced vasculogenesis in transplanted NOD/SCID mice after MNC injection
In several animal models it has been reported that microenvironment perturbation after administration of recombinant cytokines or allogenic cells ameliorates the incorporation of EPCs at sites of neovascular formation. Therefore, we looked at the role of conditions altering host microenvironment for the ability of transplanted endothelial progenitors to support vasculogenesis. Mice engrafted with CB-derived progenitors were i.v. injected with mononuclear cells (MNCs) separated from human peripheral blood 1 day after Matrigel implantation. To discriminate human long-term CB repopulating cells from human peripheral blood MNCs, the latter were transduced using a lentiviral EGFP expression vector. The incorporation of human-derived endothelial cells (CD31⁺/VWF⁺) into neovascularization sites of Matrigel plugs was detectable in 44% of "transplanted and MNC injected" (MNC/CB, n=18) mice vs. 25% in "only transplanted" (CB, n=20) mice (Fig. 1 ). The number of capillaries containing human endothelial cells in each plug of CB/MNC mice was significantly increased vs. plugs of CB mice. Matrigel plugs of controls mice not transplanted but injected with human MNCs were all negative for expression of human CD31. Moreover, PCR analysis performed on genomic DNA of Matrigel plug cells of CB/MNC mice was negative for EGFP insertion, indicating that EGFP-transduced and injected MNCs did not localize preferentially into Matrigel implants, and their influence on Matrigel vascularization could be probably mediated by soluble factors.

View larger version (35K): [in this window] [in a new window]   Figure 1. Matrigel cell characterization in MNC/CB mice. A) RT-PCR amplification of RNA from Matrigel plugs. RNA was isolated from the Matrigel plug cells of transplanted mice (CB mice: 23–25), transplanted and MNC-injected mice (MNC/CB mice: 26–29), or not transplanted but MNC-injected mice (MNC mice: 21–22) and analyzed for the presence of transcripts for human CD31 and human CD11b. Human CD31-positive samples were 44% (n=8) of analyzed MNC/CB mice (n=18), whereas control MNC mice (n=10) were all negative for human CD31 expression. B, C) Fluorescence micrographs of vascular development in Matrigel plugs of MNC/CB mice. Cryostat sections from Matrigel implants of MNC/CB mice stained by 2-color immunofluorescence with an anti-human CD31 MoAb (red) and an anti-VWF MoAb (green); nuclei of cells were stained with DAPI (blue); double-positive cells appear yellow and are endothelial cells of human origin, 40x. These pictures are representative of implants with similar results.

CONCLUSIONS

The behavior of human long-term repopulating progenitors in neovascularization processes during adult life was investigated in vivo. To simulate clinical settings as far as possible and to reduce radiation-induced tissue damage, animals were sublethally irradiated just before HSC transplantation. Moreover, the performed vasculogenic assay, which consisted in s.c. implantation of Matrigel plugs enriched of angiogenic inducers, was not as aggressive as the acute injury models. Under these experimental conditions, engraftment of human hematopoietic and endothelial cells could be visualized in CB transplanted NOD/SCID mice. Transplantation of CD34⁺ CB cells resulted in subsequent lymphomyeloid reconstitution and the majority of human cells generated in mice grafted with CB cells belonged to the B-lymphoid lineage, in agreement with published data. The CD34⁺ EPCs have been successfully cultured and in vitro differentiated from CD34⁺-enriched peripheral blood cells by Ashara and co-workers. It was likely that this cell population or its progeny developed in our long-term xenotransplantation model. Our results demonstrate that a human population showing endothelial features resides in the mouse BM after transplantation of CB-derived CD34⁺ cells, and this cell population always engrafts in NOD/SCID mice, as indicated by detection of human CD31⁺/CD45^– and human VE-cadherin⁺ populations in all BM of transplanted mice.

We show that after CB CD34⁺ transplantation, human EPCs in hematopoietic stem cell grafts can be incorporated at low levels into new blood vessels of implanted Matrigel plugs containing simply an angiogenic inducer. Treating mice with human MNCs greatly enhanced incorporation of human cells into nascent vessels in the plug, suggesting that perturbation of homeostatic conditions is necessary for the positive modulation of the angiogenic response. Enhanced vasculogenesis observed after human MNC injection could be mediated by secreted factors, as suggested by the absence of these injected cells into the Matrigel plugs. Hematopoietic cells release angiogenic factors, including VEGF-A angiopoietin-1 and matrix metalloproteinase, described to affect endothelial progenitor cell (EPC) mobilization from BM. A possible mechanism underlying the enhanced vasculogenesis seen in our model after MNC administration involves cytokine-induced mobilization of human EPCs from BM to neovascularization peripheral sites.

Our results agree with the recent observation that the contribution in vascular formation of the BM-derived cells may depend on the severity of the vascular injury. The low frequency of human EPCs incorporation into new formed capillaries observed in our model in the absence of MNC injection could reflect the fact that the angiogenic factors contained in the Matrigel matrix could be just sufficient to recruit circulating EPCs, which in physiological conditions represent a small proportion of total circulating cells. Our findings are consistent with recent reports that questioned the proposed major role and the biological significance of BM-derived EPCs in tissue revascularization. Variation in mechanical or biophysical properties inherent to a specific vascular locus, the absence or presence of inflammatory stimuli, and the unique microenvironment within different organ beds may all significantly affect the recruitment of circulating EPCs, thereby explaining the apparent differences in contribution from the BM.

Our data indicate that a human population of myelo-monocytic origin is localized into Matrigel of CB-transplanted NOD/SCID mice. This graft-derived population could cooperate to sustain neovascularization processes. BM-derived monocytes have been shown to exert a crucial role in some model of tumor angiogenesis, probably by producing angiogenic factors; in human tumors, monocyte infiltration has been correlated with invasion and angiogenesis.

These results indicate that human CB-derived EPCs, long-term engrafting a xeno-transplantation model have hematopoietic and endothelial developmental potential that can be modulated by altering the homeostatic conditions of a host microenvironment. A better knowledge of the mechanisms regulating mobilization, homing, and differentiation of HSC and EPCs to new vessels is needed to indicate new therapeutic applications in many different fields from cardiovascular diseases to tumor angiogenesis. Our long-term transplantation model allows further treatment of mice by systemic injection of cells during and after engraftment. It therefore is suitable for studying the role of different leukocyte subpopulations or mediators in modulating vessel formation.

View larger version (47K): [in this window] [in a new window]   Figure 2. Schematic representation of adult vasculogenesis sustained by transplanted CB progenitors in mice treated or not with MNC cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1444fje; doi: 10.1096/fj.03-1444fje

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