Zinc deficiency impairs ischemia-induced angiogenesis

Abstract

Objective: Zinc is an important essential trace metal involved in many physiologic functions, and its deficiency can affect the development of multiple organs, including the vasculature. However, clarity is lacking regarding the effects of zinc deficiency in the regulation of angiogenesis. We investigated the effects of zinc deficiency on the revascularization process through animal experiments and examined the relationship between the circulating zinc levels and tissue blood perfusion in patients with chronic limb-threatening ischemia (CLTI).

Methods: Zinc-deficient mice and control wild-type mice had undergone surgery to create unilateral hindlimb ischemia. Next, we examined the relationship between the serum zinc levels and skin perfusion pressure (SPP) as an index of tissue blood perfusion in patients with CLTI. A total of 51 patients with CLTI who had been referred for de novo revascularization for CLTI due to arteriosclerosis obliterans at our hospital from May 2012 to March 2016 were enrolled.

Results: The zinc-deficient mice showed a significant reduction in blood flow recovery rates in the ischemic limb and capillary density in the ischemic adductor muscle fibers compared with the control wild-type mice. The zinc-deficient mice also showed increased reactive oxygen species production after hindlimb ischemia. Nicotinamide adenine dinucleotide phosphate oxidase inhibitors ameliorated the zinc deficient-induced impairment of revascularization. The serum zinc levels were positively associated with the SPP in the CLTI patients. Multivariate regression analysis also revealed that the serum zinc levels were significantly correlated with the SPP in patients with CLTI.

Conclusions: Zinc deficiency impaired the rate of ischemia-induced revascularization through enhanced oxidative stress rates, suggesting that nutritional management for zinc sufficiency could be useful in CLTI prevention and treatment.

Keywords: Angiogenesis; Ischemia; Zinc.

Fig 1

Zinc-deficient (ZD) mice showing reductions in the rates of perfusion recovery and capillary vessel formation in ischemic limbs. A, Representative laser Doppler blood flow (LDBF) images of the ischemic limb of ZD mice or control wild-type (WT) mice. A low perfusion signal (dark blue) was observed in the ischemic hindlimb of ZD mice. In contrast, a high perfusion signal (red) was detected in the control WT mice at postoperative days 14, 21, and 28. B, Quantitative analysis of the ischemic/nonischemic LDBF ratio in the ZD mice or control WT mice before surgery and at different points after surgery. Results are presented as the mean ± standard deviation (n = 8 in each group). ∗P < .05 vs ZD mice. C, Fluorescence staining of ischemic tissues with anti-CD31 monoclonal antibody (red) on postoperative day 28. D and E, Quantitative analysis of capillary density in ZD mice or control WT mice on postoperative day 28. Capillary density expressed as the number of capillaries per high power field (×400) (Left) and capillaries per muscle fiber (Right). F, Messenger RNA (mRNA) levels of vascular endothelial growth factor (VEGF) in the ischemic muscle in the ZD mice or control WT mice on postoperative day 28. mRNA levels were measured using the real-time polymerase chain reaction (PCR) method (n = 8 in each group). All results were normalized to glyceraldehyde 3-phosphate dehydrogenase. ∗P < .01 vs control. Results presented as mean ± standard deviation (n = 8 in each group).

Fig 2

Zinc deficiency (ZD) increased the rate of oxidative damage in ischemic muscles. A, Production of reactive oxygen species (ROS) was evaluated by immunostaining with dihydroethidium (DHE) on postoperative day 28 (×400 for each field; red). Serum levels of the derivatives of reactive oxygen metabolites (d-ROMs; B) and nitrotyrosine (C) in the ZD mice or control wild-type (WT) mice after hindlimb surgery (n = 8 in each group). D, Messenger RNA (mRNA) levels of Nox2, p22^phox, p47^phox, and p67^phox in the ischemic muscle in the ZD mice or control WT mice on postoperative day 28. mRNA levels were measured using the real-time polymerase chain reaction (PCR) method (n = 8 in each group). All results were normalized to glyceraldehyde 3-phosphate dehydrogenase. ∗P < .01 vs control.

Fig 3

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin, restored ischemia-induced angiogenesis in zinc-deficient (ZD) mice. A, Schematic illustration of the experimental protocol. ZD and control mice were treated with both a ZD diet and an NADPH oxidase inhibitor, apocynin (300 mg/kg/d), in drinking water from 3 weeks of age. Next, the mice had undergone unilateral hindlimb surgery at 10 weeks of age. B, Quantitative analysis of the ischemic/nonischemic laser Doppler blood flow (LDBF) ratio in the ZD mice treated with apocynin at different points after surgery. C, Quantitative analysis of capillary density of the ischemic muscle in the ZD mice treated with apocynin at 14 days after surgery. Results presented as the mean ± standard deviation (n = 8 in each group). ∗P < .05. NS, Nonsignificant.

Fig 4

Association of serum zinc level with skin perfusion pressure (SPP) as an index of tissue blood perfusion in patients with chronic limb-threatening ischemia (CLTI). A total of 51 patients with CLTI who had been referred for de novo revascularization were enrolled in the present study. The results are presented as the mean ± standard error.

Supplementary Fig 1

Bar graph showing serum zinc levels in control and zinc-deficient (ZD) mice.

Supplementary Fig 2

CD31 immunostaining results showing alkaline phosphatase-positive cells. ZD, Zinc deficiency.