In the present study, the influence of mechanical loading on the paracrine stimulation of angiogenesis by MSCs was investigated. CM from untreated MSCs was not capable of stimulating angiogenesis. However, enhanced tube formation in endothelial cells cultivated with CM from unstimulated MSCs has recently been reported . This discrepancy might be due to the different experimental settings used (e.g., a 3D system in fibrin to approach physiological conditions versus a 2D cultivation of MSCs on tissue culture plastic). The data from the present study suggest that unstimulated MSCs lack the ability to promote angiogenesis. Instead, the cells seem to gain this capability in response to changes in their mechanical boundary conditions. This regulatory mechanism might be of high physiological relevance, since angiogenesis is an essential process for tissue regeneration  but on the other hand needs to be tightly controlled spatially and temporally to prevent tumor formation .
It has been shown that ECs and osteoprogenitor cells interact by gap junctions . The results presented here demonstrate that paracrine mechanisms for a cross-talk between MSCs and ECs, independent of direct cell-cell contacts, appear to exist in response to mechanical loading of MSCs. The transcription factor HIF-1α and its downstream target VEGF could represent candidate mediators for the translation of mechanical signals into a proangiogenic response, since these angiogenesis regulators were shown to be upregulated because of mechanical stress [30, 31]. However, under the conditions in this study, neither HIF-1α nor VEGF expression was enhanced after mechanical stimulation of MSCs. Since these factors are also induced by hypoxia [30, –32], it is important to note that the mechanical loading setting described was demonstrated to run under nonhypoxic conditions by direct oxygen measurement. Furthermore, the upregulation of MMP-2 indicates a nonhypoxic environment, since a report of Annabi et al.  showed that hypoxia downregulates MMP-2 expression in MSCs. The hypothesis that VEGF is not the mediating factor of the stimulatory effect of mechanical loading on angiogenesis shown in this study is further supported by the inability of a VEGF inhibitory antibody to repress the proangiogenic response. However, our data indicate that although the promotion of tube formation occurs independently of VEGF, there is still a dependence on the activity of the VEGFR pathway. This suggests that the effect might be mediated by cross-talk to another pathway. Additional candidates for soluble factors mediating the observed effect are the angiogenesis regulators MMP-2, TGF-β1, and bFGF, which were shown to be upregulated in response to mechanical loading. Since the proproliferative molecule VEGF [34, 35] was not enhanced in response to mechanical stimulation, the reported effect of angiogenesis promotion by mechanically loaded MSCs is not a likely result of this factor. However, microvascular cells have the potential to respond not only to VEGF but also to TGF-β1 and bFGF, since they were shown to express the corresponding cell surface receptors [36, 37]. Indeed, we could demonstrate that FGFR signaling, which is known to have the potential to stimulate survival, proliferation, migration, and differentiation of endothelial cells [34, 35, 38], is involved in angiogenesis stimulation by CM from mechanically loaded MSCs. MMP-2 is postulated to be essential for the initiation of angiogenesis . Our results suggest that MMP-2 is not involved in the enrichment of CM by TGF-β1 or bFGF but might contribute to angiogenesis by other mechanisms, such as the removal of mechanical barriers by extracellular matrix degradation or the generation of regulatory breakdown products from the extracellular matrix . In fact, 3D sprouting assays hint at a potential involvement of MMP-2 in mediating the observed effect. TGF-β1 seems to play a dual role in angiogenesis. Low concentrations (≤0.5 ng/ml) stimulate tube formation, whereas higher concentrations (1–5 ng/ml) are inhibitory [41, –43]. Similar effects are seen for EC proliferation . ELISA results from this study point to TGF-β1 concentrations lower than 0.2 ng/ml in CM from mechanically stimulated MSCs. Therefore, the proproliferative and tube formation-enhancing effect on ECs observed in this study could be mediated by TGF-β1. Indeed, inhibition of TGF-β1 showed a tendency to repress the enhancement of tube formation. In addition to their paracrine implications, the mechanically stimulated factors that we report here may also act directly on MSCs, since they express the appropriate cell surface receptors, such as FGFR, TGF-β1R, and TGF-β2R, and MSC function is known to be influenced by MMPs . Such paracrine and autocrine mechanisms are likely to act together to determine the consequences of mechanical loading on the signaling between MSCs and ECs.