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. 2017 Apr 20;25(7):1616–1627. doi: 10.1016/j.ymthe.2017.04.007

Figure 5.

Figure 5

SHP-2 Activity Is Necessary for HIF-1α Dependent Angiogenesis In Vivo

(A) HIF-1α expression and activity in wounds were detected by local expression of a lentiviral HIF-1 transcriptional luciferase reporter construct (HIF1-TRE-Luc) in the wound and compared to transduction of a control luciferase reporter construct missing the HIF1-TRE (Control-Luc) (*p < 0.05, n = 8 animals). The representative bioluminescence image of luciferase activity in wounds of the dorsal skinfold chamber after magnetic assisted transduction with HIF1-TRE-Luc and Control-Luc, respectively, is shown (right). 1: HIF1-TRE-Luc transduction without wounding; 2: Control-Luc transduction of wound; and 3: HIF1-TRE-Luc transduction of wound. (B) Influence of SHP-2 WT, CS, and E76A expression in the wound area on vessel growth and wound closure (*p < 0.05, n = 5 animals). The expression of SHP-2 constructs was achieved using magnetic targeting of MNP-LV complexes. The wound sizes were normalized to respective wounds from day 3. The representative intravital microscopy images taken after intravenous injection of FITC-Dextran at day 3 and 6 after wounding showing the vessel growth into the wounds of one animal simultaneously expressing different SHP-2 constructs in separate wounds are shown (right). The white dotted lines mark the initial wound area of the respective wound as measured on day 3. The bar in photos represents 500 μm. (C) Luciferase expression and thus HIF-1α transcriptional activity 6 days post co-transduction of HIF1-TRE-Luc and different SHP-2 mutant constructs in wounds in the mouse dorsal skinfold chamber by magnetic targeting of MNP-LV complexes (*p < 0.05, n = 4 animals). The representative bioluminescence images next to the graph show wounds transduced with control-Luc (left) or HIF1-TRE-Luc (right) vectors in combination with SHP-2 WT and mutant constructs, respectively. (D) mRNA expression of VEGF-A, PDGF-B, and MMP-2 was measured in wounds and healthy tissue of the mouse dorsal skin by qRT-PCR (left graph) (*p < 0.05, n = 5), as well as in wounds expressing SHP-2 WT or SHP-2 CS (right graph) (*p < 0.05, n = 3–5). All of the quantitative data are represented as mean ± SEM.