Fig. 7: QKI-7 affected blood reperfusion of the ischemic hindlimbs.

Matrigel plug assay was carried out using QKI-7 overexpressing miPS-ECs or control cells. In contrast to the control group, QKI-7 overexpression group formed much fewer capillary structures (p value: 0.0096) with irregular organization, suggesting an interrupted angiogenetic capacity of ECs (a images and quantification from 9 plugs per group based on n = 3 biological replicates). Hindlimb ischemia was induced in STZ-diabetic mice and QKI-7 overexpressing or control miPS-ECs were injected into the adductors immediately after induction of hind limb ischemia. Laser Doppler images of blood flow in the lower limbs of mice in prone position, with the ischemic leg highlighted by the yellow rectangle. After 14 days, delivery of control miPS-ECs significantly promoted the blood flow recovery of the ischemic hind limb, which was substantially compromised in the QKI-7 overexpression group (p values: 0.0018, 0.0037) (b). In vivo QKI-7 knockdown was achieved by intramuscular injection of shRNA Lentivirus construct tagged with a CD144 promoter and GFP to target ECs, and blood flow recovery in STZ-diabetic mice was investigated. The QKI-7 knockdown group showed over 60% of perfusion ratio to the opposite intact limb which was significantly higher than the scrambled Lentivirus control group (p value: 0.0081) (c). The effects of blood flow recovery in the QKI-7 knockdown group were also confirmed by hematoxylin and eosin staining where increased capillary density is shown (p value: 0.0062) (d). The downregulation of QKI-7 by shRNA lentivirus was verified by immunohistochemistry, when the adductor tissue was harvested, fixed, cryosectioned, and stained. Compared with the scrambled control, the QKI-7 knockdown group showed significantly higher expression of EC marker CD144 along with QKI-7 suppression (p values: 0.0072, 0.0056) (e). GFP staining clearly shown that the Lentiviral constructs tagged with GFP targeted ECs in vivo (e). Scale bar: a 200 μm; d 100 μm; e 200 μm. Data are from three biologically independent experiments. Error bars represent mean ± SEM (n = 3). P values are shown: **p < 0.01, ns: not significant (two-tailed t test). Source data are provided as a Source data file.