We show that hVEGF secretion by transplanted hCNS-SCns is directly involved in the functional recovery they elicit in vivo, as Avastin, which neutralizes human but not rodent VEGF, inhibited the hCNS-SCns-induced improvements we observed. Others have reported that overexpression of VEGF in transplanted cells further enhances recovery [8
]. However, in these studies it is unclear if VEGF acts directly on the host brain or indirectly by enhancing graft survival. Thus, such studies do not distinguish whether VEGF is necessary for recovery or alternatively amplifies recovery induced by some other mechanism. This study therefore provides the first direct evidence of a stem cell-secreted factor that is critical for cell-induced functional recovery in the post-stroke brain.
Having established a critical role for secreted hVEGF in functional recovery, we then investigated what changes hCNS-SCns elicit in the brain to enhance recovery and, in particular, which of these changes are modulated by hVEGF. We and others [12
] have postulated a role for stem cell-induced vascularization in cell-induced recovery; this fits with the essential role of hVEGF in recovery as we show the first in vivo evidence that the induced vascularization is hVEGF-dependent. We also demonstrate for the first time that hCNS-SCns-induced neovascularization is regulated in a spatio-temporal manner. The most significant increase was observed in the peri-infarct region at 2 weeks post-transplantation, with very little effect in areas closer to the graft. This implies an effect that is not simply a uniform response to a gradient of graft-secreted factors, but one that requires host tissue responsiveness to the graft. Therapeutically this is important as it shows hCNS-SCns do not readily affect vascularization in healthy tissue but primarily influence tissue already undergoing repair and re-vascularization. Furthermore, hCNS-SCns-treated animals demonstrate a profile of vessel induction followed by regression suggesting a highly regulated process rather than a continuous, aberrant, and potentially detrimental increase in blood vessel formation as is observed in certain retinopathies.
The peri-infarct tissue is presumably the most responsive region to hCNS-SCns as this area upregulates angiogenic signaling after ischemia through the VEGFR2 and the angiopoietin receptors Tie 1 and 2 [44
]. We found that hCNS-SCns treatment increases endothelial cell signaling through VEGFR2 and increases Tie2 expression in vivo prior to the observed changes in vascularization. Furthermore, we demonstrated for the first time in vivo that secretion of hVEGF by hCNS-SCns is both necessary and sufficient for the increased angiogenic signaling as it was abolished by Avastin. In addition to changes in the blood vessel number, we found that hCNS-SCns also enhances expression of β–dystroglycan in a hVEGF-dependent manner. β–dystroglycan is an extracellular matrix adhesion protein abundant in astrocytic endfeet surrounding blood vessels that may also be expressed by pericytes and endothelial cells [40
]. Thus, this data implies that the hCNS-SCns affect not only endothelial cells but also other critical components of the vasculature, which may be indicative of enhanced communication and functioning of the neurovascular unit [46
The exact contribution of enhanced neovascularization to stem cell–induced functional recovery is unclear as VEGF is known to have pleiotropic effects, including influencing neurite outgrowth and neurogenesis (reviewed in [47
]), all of which could contribute to hVEGF-induced functional recovery. What is apparent from our study is that neovascularization is unlikely to be involved in the initial phase of recovery, as hCNS-SCns significantly enhanced behavior recovery at 1 week post-transplantation, which is prior to their effects on vessel formation. Omori et al. [48
] also concluded that angiogenesis was not the only contributing factor for human mesenchymal stem cell (hMSC)-induced functional recovery, as different hMSC treatments resulted in different levels of functional recovery but had similar effects on angiogenesis.
The early cell-induced recovery at 1 week post-transplantation instead coincided with hCNS-SCns effects on inflammation and BBB repair. Previous studies have reported on the unexpected anti-inflammatory effects of neural stem cells or MSCs and their ability to improve BBB integrity [49
] when administered either before or acutely (6 – 72h) after stroke [21
]. Our study expands this therapeutic window and demonstrates that sub-acute delivery of hCNS-SCns remains immunosuppressive and can restore impaired BBB function. While the T cell deficiency of our Nude rat model could potentially bias the inflammatory data, this situation is not unlike that of the patient as they will be given immunosuppressive drugs to inhibit T cells to prevent neural graft rejection.
It is not understood how xenografts of human stem cells into rodents are immunosuppressive; our data demonstrates for the first time that secretion of hVEGF plays an important role in this immunomodulatory effect of hCNS-SCns. This was unexpected as VEGF is conventionally considered to be pro-inflammatory. However, a growing body of literature corroborates the anti-inflammatory properties of VEGF. Manoonkitiwingsa [51
] found that treatment of ischemic brains with low doses of VEGF reduced macrophage numbers, while higher doses increased macrophage density. Furthermore, hematopoietic progenitor cells express VEGF receptors and VEGF has been shown to directly inhibit the growth of myeloid progenitor cells (the monocyte and granulocyte precursors) [52
]. Additionally, VEGF reportedly suppresses the development and activation of dendritic cells (antigen presenting cells important in initiating immune responses) and T cells [53
]. Pre-clinical and clinical studies indicate that the immunosuppressive effect of VEGF may be involved in establishing immune privilege of VEGF-secreting tumors [55
]. Thus, precedence exists for an immunosuppressive role for VEGF, which may be dependent on the dose, timing, and route of VEGF delivery, suggesting the importance of further investigation of VEGF in the context of cerebral ischemia and cell transplantation.
The immunosuppressive effects of hCNS-SCns may contribute to hCNS-SCns-induced BBB repair as inflammation contributes to BBB breakdown [18
], and there is significant cross-talk between inflammatory cells and components of the neurovascular unit [20
]. Additionally, hCNS-SCns enhanced other effectors of the BBB including increased expression of Tie2 and TJ proteins. A similar result was reported by Zacharek [50
] after acute delivery of MSCs, implying this effect is independent of the cell type transplanted. Avastin blocked both hCNS-SCns-induced immunomodulation and TJ expression; these may be interrelated as inflammation is known to affect TJ proteins expression [38
]. However, although hVEGF secretion was important for these aforementioned parameters, cell-enhanced BBB integrity appeared to be hVEGF-independent as it was not blocked by Avastin. A possible explanation for this disparity is that occludin expression, although reduced in the presence of Avastin, still remained significantly elevated in cell-treated animals compared to the buffer control group, and perhaps retaining a certain threshold level of occludin may be enough for sustained BBB integrity. It remains to be determined whether transcellular influx (i.e., through the endothelial cells) of biotin through its known transporter [56
] or by pinocytosis is also affected in our experimental paradigm, as such mechanisms are less influenced by TJs than paracellular leakage (i.e., between endothelial cells).
Understanding how cell transplantation affects the host brain will lead to further improvements in cell efficacy and, perhaps more importantly, may highlight potential side effects of cell therapy. Furthermore, cell-induced changes in the brain could serve as useful surrogate clinical indicators of transplanted cell activity. For example, changes in vasculature and blood flow can be measured non-invasively in patients by perfusion studies. Moving from the bench to the clinic also raises cell manufacturing issues, in particular designing bioassays to predict clinical efficacy of the cell product [57
]. Understanding the mechanism of action of the transplanted cells will be critical for this and our data implies that secreted VEGF could be an important predictive marker of efficacy, especially as cell viability may affect the capacity of stem cells to secrete VEGF and other potential biofactors. However, future studies are required to determine the relationship between the amount of hVEGF secreted and the extent of cell-induced recovery.