Metallothionein Enhances Angiogenesis and Arteriogenesis by Modulating Smooth Muscle Cell and Macrophage Function

Abstract

Objective-In a previous study we identified metallothionein (MT) as a candidate gene potentially influencing collaterogenesis. In this investigation, we determined the effect of MT on collaterogenesis and examined the mechanisms contributing to the effects we found. Methods and Results-Collateral blood flow recovery was assessed using laser Doppler perfusion imaging, and angiogenesis was measured using a Matrigel plug assay. Smooth muscle cells were isolated from MT knockout (KO) mice for functional assays. Gene expression of matrix metalloproteinase-9, platelet-derived growth factor, vascular endothelial growth factor, and Fat cadherin in smooth muscle cells was measured by real-time polymerase chain reaction, and protein levels of vascular endothelial growth factor and matrix metalloproteinase-9 were determined using enzyme-linked immunosorbent assay and Western blot. CD11b ϩ macrophages were tested for invasiveness using a real-time impedance assay. Both flow recovery and angiogenesis were impaired in MT KO mice. Proliferation, migration, and invasion were decreased in MT KO smooth muscle cells, and matrix metalloproteinase-9, plateletderived growth factor, and vascular endothelial growth factor expression were also decreased, whereas FAT-1 cadherin expression was elevated. MT KO CD11b ϩ cells were more invasive than wild-type cells. Conclusion-MT plays an important role in collateral flow recovery and angiogenesis, an activity that appears to be mediated, in part, by the effects of MT on the functionality of 3 cell types essential for these processes: endothelial cells, smooth muscle cells, and macrophages. Key Words: angiogenesis Ⅲ endothelial cell Ⅲ macrophage Ⅲ neovascularization Ⅲ smooth muscle cell I n a study in which we examined the temporal patterns of gene expression after acute hind limb ischemia in mice, we identified metallothionein (MT) as a candidate gene whose role in collaterogenesis and angiogenesis was worthy of further investigation. 1 MT are low-molecular-weight, cysteine-rich metal-binding proteins that play a role in the regulation of zinc and copper homeostasis, constituting a cellular defense against heavy metal toxicity and free radical activity. 2 MT is also a stress-response protein, induced by oxidative stressors and acute phase cytokines such as IL-1, 3 IL-6, and tumor necrosis factor-␣. 4 Mice overexpressing MT are protected from focal cerebral ischemia, 5-7 ischemia-reperfusion injury in the heart, 8,9 and apoptosis in cardiomyocytes. 10 -13 In MT knockout (KO) mice, impaired angiogenesis and wound healing have been reported in a model of cortical freeze injury. 14,15 In addition, there are numerous reports of MT having immunomodulatory effects. 16 -20 The objective of this study was to examine the role of MT in collateral vessel formation. To this end, both arteriogenesis and angiogenesis were assessed in MT KO mice. Additional experiments to explore possible mechanisms by which MT contributes to flow recovery were performed using aortic smooth muscle cells (SMC) and C...

Expression, Regulation and Function of Human Metallothioneins in Endothelial Cells

“…As a result, effective angiogenesis in vivo following MT-I knockdown is hampered. This was confirmed by Zbinden et al [9], who found decreased vessel density and less EC migration in matrigel plugs implanted in MTI/II knockout mice as compared to wild-type animals. …”

“…At the same time, MTs have been shown to be involved in neovessel formation, i.e. angiogenesis, both in vitro and in vivo [8,9]. By inducing angiogenesis, MTs could provide a possible feedback mechanism to counteract oxidative stress.…”

Bmi1 enhances skeletal muscle regeneration through MT1-mediated oxidative stress protection in a mouse model of dystrophinopathy

“…Genome-wide transcriptome analysis of satellite cells overexpressing Bmi1 revealed significantly increased expression of MT1, which has been shown to be necessary to maintain proliferation and normal cellular functions under stress conditions, including when cells reenter the cell cycle from a quiescent state (Zbinden et al, 2010). In keeping with this notion, improvement of muscle strength is seen only in those mdx mice in which up-regulation of MT1 occurred concomitantly with overexpression of Bmi1 in satellite cells.…”

Hypoxia and Adipose Tissue Function and Dysfunction in Obesity

“…Indeed, there was a Ͼ600-fold increase in MT3 mRNA level, although such an apparently large response reflects, to some extent, the fact that expression is minimal in adipocytes incubated under normoxic conditions (208). The metallothioneins are low molecular weight (ϳ6,000 M r ) cysteine-rich, metal-binding proteins, with several presumed functions which include angiogenesis and antioxidant defense (129,150,230). A proangiogenic role in adipose tissue would be consistent with the major induction of MT3 expression by hypoxia.…”