Metformin reduced the level of Flk1 in OIR mouse retinas.

<p>Flk1 expression in mouse eyes was quantified using RT-PCR and in retinal section with immunostaining. (A) RT-PCR data showed that Flk1 mRNA levels were not significantly changed in retinas of saline-treated OIR (OIR) or metformin-treated OIR (+Met) mice (n = 6). (B) Immunostaining showed a decrease in Flk1 expression in metformin-treated OIR mice (+Met) compared to saline-treated OIR (OIR) mice at P17 and P21. GCL; ganglion cell layer, INL; inner nuclear layer. Original magnification, ×400; scale bar, 100 μm.</p

Analysis of VEGF expression level in OIR mouse eyes.

<p>ELISAs revealed higher levels of VEGF expression in the eyes of mice in the saline-treated OIR group (OIR; n = 9) and metformin-treated OIR mice (+Met; n = 10) than in those of the control, reared in normoxic condition, (CTL; n = 5) and metformin-treated control (Met; n = 4) group at post natal day 17 (P17). Elevated VEGF levels in both OIR groups decreased at post natal day 21 (P21). But metformin treatment did not affect VEGF level. VEGF levels in metformin-treated OIR mice (+Met; n = 12) were not different from those in saline-treated mice (OIR; n = 12) at P17 or P21. *P < 0.05 and **P < 0.01 compared to metformin-treated CTL (Met; n = 4). (Mann-Whitney U test).</p

Metformin inhibited VEGF-induced proliferation of HUVECs and reduced Flk1 expression and pFlk1 levels.

<p>(A) VEGF-treatment increased the proliferation of HUVECs compared to non-treated control cells (CTL), and metformin inhibited VEGF-induced proliferation. (n = 10, CTL; control HUVECs, VEGF; VEGF-treated HUVECs, +Met; VEGF plus metformin treated HUVECs, Met; metformin-treated HUVECs) (B) RT-PCR data showed that Flk1 mRNA level was not significantly changed in HUVECs treated with VEGF or VEGF plus metformin (n = 3). (C) Western blotting for pFlk1 and Flk1 in HUVECs after exposed to VEGF or VEGF plus metformin (n = 4). (D, E and F) Densitometric analysis of Western blots showed that VEGF increased the levels of pFlk1 and Flk1 in HUVECs, and demonstrated that metformin inhibited these increases. *P < 0.05, **P < 0.01, ^#P < 0.005 and ^##P < 0.001.</p

Fundus and fluorescein angiographic images of OIR mice.

<p>Control (CTL) mice housed under normal room air oxygen saturation showed fully grown retinal vessels with no avascular areas, tortuous vessels, vessel tufts, or fluorescein leakage at both P17 and P21. Retinas of mice in both saline-treated OIR (OIR) and metformin-treated OIR (+Met) groups exhibited central avascular areas, tortuous vessels, vessel tufts, and fluorescein leakage. At P17, the retinopathy features were similar between the two OIR groups, but at P21, metformin-treated OIR showed poorer vessel growth and diminished retinopathy regression.</p