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Haploinsufficiency of Transferrin Receptor 1 Impairs Angiogenesis with Reduced Mitochondrial Complex I in Mice with Limb Ischemia.

ABSTRACT: Limb ischemia (LI) is a major consequence of peripheral artery disease (PAD) with a high mortality rate. Iron is an essential mineral to maintain physiological function of multiple organs. Intracellular iron transport is regulated by transferrin receptor 1 (TfR1). Although increase in serum ferritin levels has been reported in PAD patients, the mechanism of iron metabolism in LI is still unclear. The aim of this study is to investigate whether TfR1 deletion attenuates LI formation. To generate LI, the left femoral artery of 8-10 week-old C57BL6/J male mice was ligated. Adductor muscles were harvested at 28 days after surgery to investigate iron metabolism. The level of ferritin, intracellular iron storage protein, was higher in ischemic adductor muscles compared to non-ischemic adductor muscles. Level of intracellular iron transport protein, TfR1, was decreased in ischemic adductor muscles. LI was then generated in TfR1 heterozygous deleted mice (TfR1+/-) to examine whether TfR1 contributes to the pathophysiology of LI. Laser Doppler blood flowmetry revealed that blood flow recovery was attenuated in TfR1+/- mice compared to wild type (WT) littermates, along with decreased expression of ferritin and CD31 in ischemic adductor muscles. Since iron is used for energy production in mitochondria, we then assessed mitochondrial complexes in the ischemic adductor muscle. Of interest, expression of mitochondrial complex I, but not complexes II, III, and V in ischemic adductor muscles was significantly reduced in TfR1+/- mice compared to WT mice. Haploinsufficiency of TfR1 attenuated angiogenesis via reduction of mitochondrial complex I in LI in mice.

SUBMITTER: Okuno K

PROVIDER: S-EPMC6754437 | biostudies-other | 2019 Sep

REPOSITORIES: biostudies-other

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Haploinsufficiency of Transferrin Receptor 1 Impairs Angiogenesis with Reduced Mitochondrial Complex I in Mice with Limb Ischemia.

Okuno Keisuke K Naito Yoshiro Y Yasumura Seiki S Sawada Hisashi H Asakura Masanori M Masuyama Tohru T Ishihara Masaharu M

Scientific reports 20190920 1

Limb ischemia (LI) is a major consequence of peripheral artery disease (PAD) with a high mortality rate. Iron is an essential mineral to maintain physiological function of multiple organs. Intracellular iron transport is regulated by transferrin receptor 1 (TfR1). Although increase in serum ferritin levels has been reported in PAD patients, the mechanism of iron metabolism in LI is still unclear. The aim of this study is to investigate whether TfR1 deletion attenuates LI formation. To generate L ...[more]

PMID: 31541184

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Project description:Older patients are thought to tolerate acute ischemia more poorly than younger patients. Since aging may depress both angiogenesis and arteriogenesis, we determined the effects of age on both angiogenesis and arteriogenesis in a model of severe acute limb ischemia.Young adult (3-months-old) and aged (18-months-old) C57BL/6 mice underwent right common iliac artery and vein ligation and transection. Data were collected on days 0, 7, and 14. Perfusion was measured with a laser Doppler scan and compared to the contralateral limb. Functional deficits were evaluated with the Tarlov scale. Capillary density and endothelial progenitor cell (EPC) number were determined by direct counting lectin-positive/alpha-actin-negative cells and VEGFR2/CXCR4 dually-positive cells, respectively; angiography was performed to directly assess arteriogenesis.Young adult and aged mice had a similar degree of decreased perfusion after iliac ligation (young, n = 15: 20.4 +/- 1.9%, vs aged, n = 20: 19.6 +/- 1.3%; P = .72, analysis of variance [ANOVA]); however, young mice recovered faster and to a greater degree than aged mice (day 7, 35 +/- 6% vs 17 +/- 4%, P = .046; day 14, 60 +/- 5% vs 27 +/- 7%, P = .0014). Aged mice had worse functional recovery by day 14 compared to young mice (2.3 +/- 0.3 vs 4.3 +/- 0.4; P = .0021). Aged mice had increased capillary density (day 7, 12.9 +/- 4.4 vs 2.8 +/- 0.3 capillaries/hpf; P = .02) and increased number of EPC incorporated into the ischemic muscle (day 7, 8.1 +/- 0.9 vs 2.5 +/- 1.9 cells; P = .007) compared to young mice, but diminished numbers of collateral vessels to the ischemic limb (1 vs 9; P = .01), as seen on angiography.After severe hind limb ischemia, aged animals become ischemic to a similar degree as young animals, but aged animals have significantly impaired arteriogenesis and functional recovery compared to younger animals. These results suggest that strategies to stimulate arteriogenesis may complement those that increase angiogenesis, and may result in improved relief of ischemia.

Project description:(1) This study tests hypothesis whether extracorporeal shock wave (ECSW) therapy effectively salvages mouse critical limb ischemia (CLI). In vitro result demonstrated that the angiogenesis parameters (i.e., tubular length/cluster/network formation) and protein expressions of EGFR/VEGFR2/RAS/c-Raf/MEK/ERK/VEGF/p-PI3K/p-Akt/p-m-TOR were significantly and progressively increased with stepwise augmentation of ECSW energy (0.1/0.14/0.20 mJ/mm2/140 impulses). On the other hand, they were suppressed by administration of Avastin (20 μM). Adult male B6 mice (n = 24) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 [CLI + ECSW (0.12 mJ/mm2/120 impulses/at days 1/3/7 after CLI induction)] and group 4 [CLI + ECSW (0.12 mJ/mm2/120 impulses) + Avastin (1 mg/intramuscular-injection)] at days 1/3/7 after CLI induction] and quadriceps were harvested by day 14. The laser Doppler result showed that the ratio of left (ischemia) to right (normal) limb blood flow was highest in group 1, lowest in group 2, and significantly higher in group 3 than in group 4 by days 7/14 after the CLI procedure (p < 0.0001). The protein expressions of cell proliferation/migration/angiogenesis receptors (EGFR/VEGFR2), angiogenesis biomarkers (VEGF/CXCR4/SDF-1) and cell proliferation/growth/survival (Ras/c-Raf/MEK/ERK)/(PI3K/Akt/m-TOR) and cell motility/proliferation (p-FAK/p-Scr) signaling biomarkers were significantly higher in group 3 than in groups 1/2/4, and significantly lower in group 1 than in groups 2/4, but they did not show a difference between groups 2 and 4 (all p < 0.001). The small vessel density and cellular levels of endothelial cell surface marker (CD31+) exhibited an identical pattern of blood flow, whereas the angiogenesis (CXCR4+/VEGF+) displayed an identical pattern of VEGFR2 among the groups (all p < 0.0001). The in vitro and in vivo studies found ECSW salvaged the CLI mainly through upregulating Ras-Raf-MEK/ERK/cell motility, cell proliferation/growth pathways and angiogenesis.

Dataset Information

Haploinsufficiency of Transferrin Receptor 1 Impairs Angiogenesis with Reduced Mitochondrial Complex I in Mice with Limb Ischemia.

Publications

Haploinsufficiency of Transferrin Receptor 1 Impairs Angiogenesis with Reduced Mitochondrial Complex I in Mice with Limb Ischemia.

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