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

Figure 1.

Figure 1

SHP-2 Inactivation Inhibits Hypoxia-Induced Angiogenesis and HIF-1α Accumulation and Activity In Vitro

(A) Overexpression of SHP-2 WT, SHP-2 CS (dominant negative mutant), or SHP-2 E76A (constitutively active mutant) was achieved by lentiviral transduction (see Figure S1). Proliferation of endothelial cells expressing the different SHP-2 constructs upon 24 hr hypoxia treatment was assessed by MTT reduction 96 hr post transduction (*p < 0.05, n = 5 in triplicates). The enhanced proliferation in SHP-2 E76A cells was inhibited by treatment with 10 ng/mL echinomycin (Ecm) (p < 0.05, n = 5 in triplicates). (B) Formation of capillary like structures in Matrigel during hypoxia (24 hr) in SHP-2 WT, CS, and E76A expressing endothelial cells (*p < 0.05, n = 5 each, 4 fields of view). The Matrigel assay was performed 96 hr after lentiviral transduction. (C) Vessel sprouting from aortic rings ex vivo expressing SHP-2 WT, CS, or E76A and exposed to hypoxia (24 hr) (*p < 0.05, n = 4–5). The isolated vessels were immediately transduced using magnetic targeting of MNP-LV complexes to the vessel wall. At 48 hr later, aortas were cut into rings, embedded in Matrigel, and exposed to hypoxia. (D) Hypoxia-induced (4 hr) VEGF production from SHP-2 WT, CS, or E76A expressing endothelial cells, as assessed by ELISA 96 hr after transduction (*p < 0.05, n = 3–6). (E) Analysis of hypoxia-induced (4 hr) HIF-1α accumulation in endothelial cells lentivirally transduced with SHP-2 WT, CS, E76A, or Y2F. The graph shows protein band densities of HIF-1α normalized to actin (*p < 0.05, n = 6). (F) HIF-1 DNA binding activity in nuclear extracts of cells expressing SHP-2 WT or CS upon hypoxia (4 hr) was measured using a HIF-1 transcription factor assay kit 96 hr post transduction (*p < 0.05, n = 3 in duplicates). (G) HIF-1α activity upon hypoxia (4 hr) in endothelial cells expressing SHP-2 WT or CS was detected 72 hr upon co-transduction of SHP-2 WT or CS in combination with the HIF1-TRE-Luc or Control-Luc reporter construct (*p < 0.05, n = 5). All of the quantitative data are represented as mean ± SEM.