Angiogenesis is inhibitory for mammalian digit regeneration

Abstract

The regenerating mouse digit tip is a unique model for investigating blastema formation and epimorphic regeneration in mammals. The blastema is characteristically avascular and we previously reported that blastema expression of a known anti-angiogenic factor gene, Pedf, correlated with a successful regenerative response (Yu, L., Han, M., Yan, M., Lee, E. C., Lee, J. & Muneoka, K. (2010). BMP signaling induces digit regeneration in neonatal mice. Development, 137, 551-559). Here we show that during regeneration Vegfa transcripts are not detected in the blastema but are expressed at the onset of differentiation. Treating the amputation wound with vascular endothelial growth factor enhances angiogenesis but inhibits regeneration. We next tested bone morphogenetic protein 9 (BMP9), another known mediator of angiogenesis, and found that BMP9 is also a potent inhibitor of digit tip regeneration. BMP9 induces Vegfa expression in the digit stump suggesting that regenerative failure is mediated by enhanced angiogenesis. Finally, we show that BMP9 inhibition of regeneration is completely rescued by treatment with pigment epithelium-derived factor. These studies show that precocious angiogenesis is inhibitory for regeneration, and provide compelling evidence that the regulation of angiogenesis is a critical factor in designing therapies aimed at stimulating mammalian regeneration.

Keywords: Angiogenesis; BMP9; PEDF; VEGF; blastema; digit; mouse; regeneration.

Figure 1

VEGF treatment inhibits digit tip regeneration (top is dorsal and proximal is to the left). (A) At 10 DPA when redifferentiation has initiated, transcripts for the anti‐angiogenic factor Pedf are abundant in both the digit stump (arrow) and blastema (arrowhead). (B) During wound healing Vegfa expression is not detected in either the stump or wound bed at 5 DPA. (C) At 9 DPA, transcripts for Vegfa can be detected in the digit stump (arrow) but not the blastema. (D) Immunohistochemical staining for von Willebrand factor (VWF, red) at 7 DPA showing endothelial cells (arrow) in the blastema. (E) VWF staining of a control sample treated with a BSA bead (*) showing endothelial cells (arrow) in the blastema. (F) VWF staining of an experimental digit treated with a VEGF bead (*) showing an enhanced population of endothelial cells (arrow) associated with the bead. Sections shown in (D)−(F) were counterstained with DAPI. (G) Alizarin Red whole mount staining of 14 DPI digits showing that regeneration is not altered by control BSA treated bead implantation. (H) Based on whole mount staining the majority of digits fail to regenerate following treatment with a VEGF microcarrier bead (*). (I) Some VEGF bead treated digits form partial regenerates that include bony spikes that extend distally from the digit stump (arrow). (J) MicroCT rendering of the P3 skeletal element shown in (G). (K) MicroCT rendering of the P3 element shown in (H). (L) Graph showing normalized volume measurements from microCT analyses of control BSA treated (blue), VEGF treated (red) and BMP9 treated (green) digits at 14 DPI.

Figure 2

BMP9 treatment inhibits digit tip regeneration (top is dorsal and proximal is to the left). (A), (B) Alizarin Red whole mount stained digit at 14 DPI treated with a BMP9 microcarrier bead (*). Truncated digit tips formed in approximately half of the BMP9 treated digits (A), whereas the remaining digits formed a shortened but anatomically distinct digit tip suggestive of a remodeling response (B). (C) The majority of BMP9 treated digit tips analyzed at 42 DPI formed shortened digit tips. (D)−(G) Section in situ hybridization studies to localize Vegfa transcripts after treatment with a control BSA or a BMP9 microcarrier bead. (D) Vegfa expression was not detected at 1 DPI in BSA treated control digits. (E) Following BMP9 treatment Vegfa transcripts were abundant in the digit stump (arrowhead) but not directly associated with the microcarrier bead (*). (F) At 7 DPI Vegfa expression was detected in BSA treated control digits at a level similar to that of untreated regenerates at a similar stage (see Fig. 1C). (G) Vegfa expression remained upregulated (arrowhead) in the digit stump and proximal blastema 7 days after treatment with BMP9.

Figure 3

BMP9 treatment delays ossification during regeneration (top is dorsal and proximal is to the left). The microcarrier bead associated with the treatment is indicated with an asterisk. (A)−(D) BSA treated control digits. (A′)−(D′) BMP9 treated digits. (A)−(C), (A′)−(C′) Mallory's triple stained histological sections of amputated digit tips. By 3 DPI the difference between control regenerates (A) and BMP9 treated digit amputations (A′) is subtle. Both accumulate blastema cells distally but ossification of BMP9 treated digit stumps appears to be delayed. By 5 DPI ossification is prominent in the stump and proximal region of control regenerates (B) whereas ossification is clearly delayed following BMP9 treatment (B′). By 7 DPI ossification is occurring throughout the control regenerate (C) whereas ossification is restricted to the stump region of BMP9 treated digits (C′). In situ hybridization to localize transcripts for the osteogenic marker gene, Osteocalcin (Ocn), validate the histological results. Ocn is expressed throughout the control regenerate at 7 DPI (D) whereas transcripts are localized to the periphery (arrowheads) of the stump in BMP9 treated digits (D′).

Figure 4

Blastema formation is not influenced by BMP9 or VEGF treatment. The microcarrier bead associated with the treatment is indicated with an asterisk. (A)−(C) BrdU incorporation in digits at 3 DPI shows proliferation in mesenchymal cells of control BSA treated digits (A) and that proliferation is not inhibited by treatment with BMP9 (B) or VEGF (C). (D)−(F) Msx1 expression was analyzed by section in situ hybridization in digits at 3 DPI. Msx1 expression (arrowhead) is localized to cells in the dorsal mesenchyme in control BSA treated (D), BMP9 treated (E), and VEGF treated (F) digits indicating that regenerative inhibition is not associated with the suppression of Msx1 expression. (G)−(I) Pedf expression was analyzed by section in situ hybridization in digits at 3 DPI. Pedf transcripts (arrowhead) are localized to cells associated with the control BSA treated bead (G), the BMP9 treated bead (H), and the VEGF treated bead (I), indicating that Pedf expression is not inhibited by BMP9 or VEGF treatment.

Figure 5

(A)−(C) Revascularization based on immunohistochemical analysis for VWF (red) 7 days after microcarrier bead (*) implantation. Sections were counterstained with DAPI, red blood cells (RBC, yellow) are autofluorescent, distal is to the right. (A) Staining of control BSA treated digits show blood vessels in the periphery of the digit stump (arrows) and sparse staining in the blastema. (B) VWF staining of BMP9 treated digits show blood vessels throughout the digit stump (arrows) and extending into the blastema (arrowhead). (C) After VEGF treatment an extensive vascular response is evident in the digit stump (arrow) and in blastema cells (arrowhead) associated with the bead (*). (D)−(F) The inhibitor activity of BMP9 is rescued following treatment with the anti‐angiogenic factor PEDF. (D) Whole mount skeletal staining shows that the BMP9 inhibited regenerative response is not modified by a control BSA bead implanted 1 day later. (E) BMP9 treatment followed by the implantation of a PEDF bead 1 day later restores the regenerative ability as shown by whole mount skeletal staining of digits at 14 DPI. (F) Bone volume analyzed by microCT is significantly increased by PEDF treatment. Data are normalized to the BMP9 inhibited BSA control digits, P < 0.01 (*). (G)−(J) In situ hybridization of control and PEDF rescued regenerates 7 days after implantation. (G) BMP9 inhibited Ocn expression (arrowheads) is not modified by BSA control bead implantation. (H) In PEDF rescued BMP9 inhibited digits Ocn expression extends distally (arrowhead) into the blastema region. (I) Upregulation of Vegfa transcripts (arrowhead) in the stump of BMP9 inhibited digits is not modified by control BSA treatment. (J) In PEDF rescued BMP9 inhibited digits Vegfa expression is downregulated and appears similar to untreated regenerates at a similar stage (see Fig. 1C).