Original article
Circulating human CD34+ progenitor cells modulate neovascularization and inflammation in a nude mouse model

https://doi.org/10.1016/j.yjmcc.2007.03.907Get rights and content

Abstract

CD34+ progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34+ cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34+ cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34+ implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34+ cells did not incorporate in neovessels, but formed vWF+/CD31+/VEGF+ cell clusters that were present up to day 14. However, CD34+ cells induced host neovascularization, as demonstrated by increased presence of murine CD31+ and vWF+ vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34+ implants at all time points. Gene expression of chemotactic cytokines MCP-1 and IL-8 was detected on CD34+ cells in vitro and confirmed immunohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34+ cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages.

Introduction

Cardiovascular disease, among which is coronary artery disease, is the main cause of morbidity and mortality in the Western world [1]. After an ischemic insult of the heart, regeneration and repair of the damaged tissue will occur, in which both neovascularization and inflammation (removal of damaged tissues) play an important role [2]. Currently, the therapies for coronary artery disease are limited and the search for new therapeutics continues.

Endothelial progenitor cells (EPC) are bone marrow-derived cells that circulate in peripheral blood and can contribute to neovascularization. Therefore, EPC hold potential for post-ischemic neovascularization. Currently, EPC are tested in clinical trials in patients after myocardial infarction [3]. Although these trials appear to be successful, it proves difficult to retrieve the injected EPC in or around the infarction site [4], [5]. This hampers the understanding of the therapeutic success of EPC transplantation. Therefore, the implementation of a scaffold (for example biomaterials) to immobilize EPC at the ischemic site is a good alternative to the straightforward injection of EPC.

Several surface markers are used to identify EPC, of which CD34, CD133, and KDR are generally accepted [6]. However, a definitive marker for EPC has not been discovered yet and may even not exist. In previous work, using our isolated in vivo model for subcutaneous neovascularization of Matrigel implants, we demonstrated that CD34+ cells were superior to CD133+ and KDR+ cells in neovascularization and recruitment of inflammatory cells [7].

CD34+ cells are currently applied for the induction of neovascularization, both in vitro [8], in vivo [4], [9], [10], and in clinical trials [3], [11]. The mechanism by which EPC contribute to neovascularization is unknown. It has been demonstrated that EPC can incorporate into newly formed blood vessel structures, albeit at low numbers [5], [12]. Additionally, these EPC may enhance sprouting of existing vasculature in a paracrine manner [13], [14]. The inflammatory micro-environment, the niche, into which EPC migrate probably plays a determinative role in the process of neovascularization.

Tissue repair is a strictly spatiotemporally regulated process in which the interplay between resident tissue cells and infiltrating cells determines the outcome [15]. We have previously shown that in subcutaneous Matrigel implants seeded with human CD34+ cells cellular infiltration and vascularization was present 14 days after implantation [7]. However, for potential therapeutic purposes, as well as for understanding the mechanisms governing these phenomena, a kinetic study is imperative.

Therefore, in our isolated hypoxic system, which is devoid of confounding factors that may exist after ischemic injury [7], we immobilized the implanted human CD34+ cells in a Matrigel matrix and studied their survival and differentiation longitudinally for 14 days. In addition, we dissected the influence of the human CD34+ cells on host neovascularization and inflammation in the Matrigel implants using immunohistochemistry and gene expression profiling.

Section snippets

Animals

Male, athymic nude mice (Harlan, Zeist, The Netherlands), 8–10 weeks of age, were held under specified pathogen-free conditions in IVC racks (in groups of 5 animals per cage) and received sterilized water and food ad libitum. Animal experimentation was approved by the local Ethical Committee on Animal Experiments and was performed according to governmental and international guidelines on animal experimentation.

Isolation of human CD34+ cells

Human CD34+ cells were isolated from buffy coats of 0.5 L peripheral blood healthy

Human CD34+ cells induce vascularization of Matrigel implants

We have previously shown that in subcutaneous Matrigel implants seeded with human CD34+ cells cellular infiltration and vascularization was present 14 days after implantation [7]. However, for potential therapeutic purposes, as well as for understanding the mechanisms governing these phenomena, a kinetic study is required.

The macroscopic aspect of a cell-loaded Matrigel, showing the pattern of neovascularization at 14 days after implantation, is depicted in Fig. 1A. In the current time course

Discussion

Implantation of cell-loaded scaffolds holds promise for therapeutic application after ischemic injury. Therefore, the role of immobilized, i.e. Matrigel embedded, human CD34+ cells in neovascularization and recruitment of inflammatory cells was investigated longitudinally in our model.

In this study, we show that the effects of human CD34+ cell-implantation were already visible early after implantation. Already after 2 days, an increased cellular and capillary density was observed in the

Acknowledgment

This research was financially supported by an institutional grant.

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