Our findings show that administration of human CD34+ cells to immunocompromised mice 48 hours after stroke enhances neovascularization at the border of the ischemic zone followed by endogenous neurogenesis. Furthermore, suppression of the neovascularization by an antiangiogenic agent impaired neurogenesis. On the basis of these data, accelerated neovessel formation seems to be essential for enhancing endogenous neurogenesis and improving functional recovery.
The generation of new neurons in the adult is largely restricted to two regions: the SVZ lining the lateral ventricles and the subgranular zone of the dentate gyrus (25
). In the transient cerebral ischemia model, evidence has been provided that neuronal regeneration occurs (10
). However, even in a transient ischemia model in which integrity of the microcirculation is maintained, it was shown that greater than 80% of newly formed neurons died, most likely because of unfavorable environmental conditions including lack of trophic support and exposure to products of damaged tissue. These considerations may underlie the observation that only 0.2% of nonviable ischemic neurons were replaced through neurogenesis (20
). Such previous observations are consistent with our current results in animals treated with CD34–
cells or PBS, in which there was no enhancement of neovascularization, no neurogenesis, and no functional recovery.
Consistent with these data, Endostatin-mediated suppression of endothelial proliferation and direct effects on EPCs abrogated the beneficial effect CD34+
cells on neurogenesis and cortical expansion, and at the same time inhibited formation of neovasculature after stroke. In contrast, when EPO was used as a proangiogenic agent (recently, EPO has also been found to promote mobilization of EPCs) (23
), accelerated formation of neovasculature was accompanied by enhanced neurogenesis after stroke in our murine model. Our data provide the first direct link between therapeutic neovascularization after stroke and enhanced neurogenesis; formation of neovasculature after stroke supported neurogenesis. Consistent with a previous report (26
), activation of NPCs after stroke was induced in adult, as well as young, murine brain. The number of activated NPCs was less in the adult animals, but a significant increase was still observed consequent to CD34+
cells transplantation, with evidence of activated vasculature in brains of the older animals. These findings support our hypothesis that administration of CD34+
cells provides a milieu favorable for neovascularization and endogenous neurogenesis, even in the mature brain.
A relationship between angiogenesis and neurogenesis would be consistent with regeneration of parenchymal cells in other organs subject to therapeutic angiogenesis (27
). Mechanisms underlying this observation might include more optimal preparation of the ischemic tissue bed for neuronal regeneration by accelerated removal of debris and toxic products, and/or the enhanced production of chemokines and trophic agents by neovasculature. Factors involved might be FGF2 (11
), PDGF (29
), brain-derived neurotrophic factor (30
), and IL-8 (31
), which have the capacity to induce mitogenesis, differentiation, recruitment, and survival of NPCs and newly generated neurons. In addition, mediators produced by CD34+
), such as VEGF, FGF2, and IGF-1, have been shown to accelerate endogenous neurogenesis (10
). The effects of these and other factors derived from CD34+
) acting on the vasculature are also likely to have an important role in providing an environment conducive to neurogenesis. Newly formed neurons in the setting of neovascularization have been shown to integrate into neuronal networks in adult animals (34
); in fact, lack of participation of neurons in such neuronal circuits is probably associated with cell death (20
). Our results provide strong support for the hypothesis that neovascularization consequent to administration of CD34+
cells induces neurogenesis by providing the necessary supportive environment. Whereas perturbation of the neurovascular unit has been proposed to contribute to tissue damage in stroke, neoangiogenesis and accompanying neurogenesis could be considered to be rebuilding crucial elements of the neurovascular unit (35
). Previous reports have demonstrated that endothelial cells in newly formed vessels after stroke mainly originate from pre-existing endothelial cells (due to proliferation) with a contribution of circulating EPCs (36
). Consistent with these findings, we observed proliferating mouse endothelial cells around the ischemic area soon after stroke. The latter endothelial cells could be visualized, at least in part, with antibody specific for human CD31, after transplantation of human CD34+
cells. Furthermore, diminished neovascularization was observed after stroke following transplantation of CD34+
cells, compared with CD34+
cells (containing both Flk-1–
cells) despite similar activation of murine endothelial cells in the ischemic territory in each case. These results support potential contribution of CD34+
cells (a population known to be rich in EPCs) in expansion of the vascular network after stroke.
The actual pattern of the newly formed vascular network was quite different on the ipsilateral (ischemic) from the contralateral side in poststroke mice treated with CD34+ cells. This observation suggests that formation of vasculature consequent to transplantation of CD34+ cells after stroke does not simply reconstitute the original vascular network. Rather, a new vascular pattern arises (a true neovasculature) that is capable of supporting neurogenesis, followed by functional recovery, even though it displays a relatively “aberrant” pattern, at least anatomically, compared with the contralateral vasculature.
Our observations provide evidence of a crucial role for neovessel formation, achieved through the administration of CD34+
cells after stroke, in processes that underlie neurogenesis. These data strongly suggest that neovascularization is essential for neuronal regeneration after stroke and that therapeutic neovascularization is a potentially effective means of enhancing functional recovery. Our observations might explain the mild therapeutic effect achieved by neuronal cell transplantation after stroke reported in humans (38
). This leads to the hypothesis that therapeutic neovascularization may be required to achieve optimal “take” of transplanted neuronal precursors in the setting of ischemia. Although the current data bear most directly on the endogenous neuronal response to cerebrovascular ischemia, it is possible that enhanced formation of neovasculature may also be important for survival of embryonic (39
) and neural (40
) stem cell transplants in other circumstances, such as neurodegenerative disorders.