Effects of STAT3 on aging-dependent neovascularization impairment following limb ischemia: from bedside to bench

Abstract

Aging is a major risk factor for ischemic hypoxia-related diseases, including peripheral artery diseases (PADs). Signal transducer and activator of transcription 3 (STAT3) is a critical transcription activator in angiogenesis. Nevertheless, the effect of aging on endothelial cells and their responses to hypoxia are not well studied. Using a hindlimb hypoxic/ischemic model of aged mice, we found that aged mice (80-100-week-old) expressed significantly lower levels of angiogenesis than young mice (10-week-old). In our in vitro study, aged endothelial cells (≥30 passage) showed a significant accumulation of β-galactosidase and a high expression of aging-associated genes, including p16, p21, and hTERT compared with young cells (<10 passage). After 24 hours of hypoxia exposure, proliferation, migration and tube formation were significantly impaired in aged cells compared with young cells. Notably, STAT3 and angiogenesis-associated proteins such as PI3K/AKT were significantly downregulated in aged mouse limb tissues and aged cells. Further, using STAT3 siRNA, we found that suppressing STAT3 expression in endothelial cells impaired proliferation, migration and tube formation under hypoxia. Correspondingly, in patients with limb ischemia we also observed a higher expression of circulating STAT3, associated with a lower rate of major adverse limb events (MALEs). Collectively, STAT3 could be a biomarker reflecting the development of MALE in patients and also a regulator of age-dependent angiogenesis post limb ischemia. Additional studies are required to elucidate the clinical applications of STAT3.

Keywords: MALE; STAT3; aging; angiogenesis; limb ischemia.

Figure 1

Impaired angiogenesis of aged mice post hindlimb ischemia surgery. (A) The study design of limb ischemia in young (10 w/o) and aged (80–100 w/o) mice. (B) Representative images of laser Doppler perfusion flow in limb ischemia in young and aged mice. (C) Representative images and weight quantifications of harvested ischemic (left) limbs compared with nonischemic (right) limbs. N = 6, ^*P < 0.05.

Figure 2

Impaired capillary density, expression of STAT3 and proliferation-associated proteins in young and aged mice post hindlimb ischemia surgery. (A) Representative images and quantification of CD31 immunostaining representing capillary density (red; left panel) and STAT3 (green) expressions in endothelial cells (red; CD 31 positive; right panel). Cell nuclei were stained with DAPI (blue); (B) The quantification of capillary density and (C) STAT3 expression; (D) Representative blots and quantification of PI3K, AKT, STAT3, VEGF, and MMP9 protein expression in the ischemic limbs of young and aged mice. The experiment was repeated in triplicate, ^*P < 0.05, ^**P < 0.01, ^**P < 0.001 and ^****P < 0.0001.

Figure 3

Aging attenuates proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). (A) Cell proliferation, evaluated by measuring BrdU incorporation into cells, (B) cell migration after 24 hours, (C) tube formation, evaluated by the branched points, and (D) expressions of P-PI3K, PI3K, P-AKT, AKT, P-STAT3, STAT3 and proliferation-associated proteins of MMP9, VEGF, VEGFR2 and the phosphorylation form of VEGFR2 in young (<10 passage) and aged HUVECs (≥30 passage) with and without hypoxia treatment for 24 hours. The experiment was conducted in triplicate, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and, ^****P < 0.0001.

Figure 4

Knocking down STAT3 suppresses proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). (A) Cell proliferation, evaluated by measuring BrdU incorporation into cells, (B) cell migration after 24 hours, (C) tube formation, evaluated by the branched points, and (D) phosphorylation protein expression of P-STAT3 and proliferation-associated proteins, including MMP9, VEGF, VEGFR2 and the phosphorylation form of VEGFR2, in HUVECs treated with scramble or STAT3 small interfering RNA (siSTAT3) under normoxia or hypoxia for 24 hours. The experiment was repeated in triplicate, ^*P < 0.05, ^***P < 0.001, and ^****P < 0.0001.

Figure 5

Lower circulating STAT3 expression in elderly individuals is associated with more major adverse limb events (MALEs). (A) The design of a clinical study focusing on patients with peripheral artery disease (PAD). (B) The expression of circulating STAT3 in young (<65 y/o) and old (≥65 y/o) patients. (C) The expression of circulating STAT3 in patients with or without MALEs. (D) A Kaplan–Meier plot of MALEs among young patients with high STAT3, young patients with low STAT3, old patients with high STAT3, and old patients with high STAT3 (N = 11–20, ^*P < 0.05), ^*The cutoff value of STAT3 is defined as 15 ng/ml.

Graphic abstract. Summary of STAT3 in regulating aging association attenuation of angiogenesis in limb ischemia.