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5.5 Wound to Blastema
One of the prominent characteristics of the blastema is its relative avascularity
compared to surrounding tissues. This is true for both the salamander limb blastema
as well as the mouse digit blastema [ 6 , 41 ]. The digit blastema is not devoid of
endothelial cells, instead they are present as individual cells dispersed randomly
throughout the blastema and not organized into functional vascular units. The endo-
thelial cells of the blastema express the stem cell marker Sca1 , suggesting that the
regeneration of vasculature in the digit tip is mediated via endothelial stem cells [ 6 ].
Associated with avascularity , the wound healing and early blastema stages is char-
acterized by the expression of the anti- angiogenic factor , pigment epithelial derived
factor ( Pedf ) (Fig. 5.5a ) and the absence of transcripts for the angiogenic factor,
vascular endothelial growth factor A ( VegfA ) [ 42 , 43 ]. The expression of the antian-
giogenic factor Pedf and the corresponding lack of expression of the angiogenic
factor Veg f , coupled with the presence of endothelial stem cells in the blastema, are
consistent with the conclusion that blastemal avascularity is causally linked to the
control of angiogenesis during blastema formation. Why is blastema avascularity
important? To test the importance of re-vascularization in regeneration, amputated
neonate digits were treated with VEGF-soaked beads after the completion of wound
closure. VEGF-treatment led to an increase in endothelial cells within the blastema
and adjacent to the VEGF source within 3 days post implantation (DPI) , an enhanced
level of vascularity by 7 DPI and the complete inhibition of the regenerative response
(Fig. 5.5b, c ) [ 42 ]. The inhibition of regeneration by VEGF was shown to be dose-
dependent, and these fi ndings provide evidence that the control of re-vascularization
during blastema formation plays a critical role in the control of regeneration.
The conclusion that enhanced re-vascularization inhibited regeneration is sug-gestive that the avascular state of the blastema is required for the regenerative
response. To address this issue we explored the role that PEDF played in the inhibi-
tion of re-vascularization. We took advantage of the observation that treatment of
the digit amputation with BMP9, a member of the TGFβ superfamily, inhibited the
regenerative response (Fig. 5.5h ). While Bmp9 is not expressed during digit tip
regeneration, BMP9 has been shown to act in a context-dependent manner acting as
either an anti-angiogenic factor or to enhance angiogenesis [ 44 – 46 ], and a microar-
ray analysis of BMP9 treated digits showed a signifi cant increase in several known
modulators of angiogenesis , including Vegfa. Further studies showed that BMP9
treatment induced an immediate upregulation of Vegfa expression, and a persistent
and expanded Vegfa expression domain at later time points (Fig. 5.5d–g ), and an
enhanced level of re- vascularization. In short, the data were consistent with the
conclusion that the BMP9 inhibition of regeneration was causally linked to the
induced expression of Vegfa , and we were able to use this model to test the role of
PEDF in blastema formation and regeneration. PEDF treatment after BMP9 inhibi-
tion markedly decreased the expression domain of Vegfa , thus restoring the avascu-
lar character of the blastema, and ultimately rescuing osteogenesis associated with
the regeneration response (Fig. 5.5h–n ). Taken together, the evidence shows PEDF
L.A. Dawson et al.