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First published August 14, 2020; doi:10.1152/ajpheart.00064.2020.--Insulin-like growth factor-1 (IGF-1) decreases atherosclerosis in apolipoprotein E (Apoe)-deficient mice when administered systemically. However, mechanisms for its atheroprotective effect are not fully understood. We generated endothelium-specific IGF-1 receptor (IGF1R)-deficient mice on an Apoe-deficient background to assess effects of IGF-1 on the endothelium in the context of hyperlipidemia-induced atherosclerosis. Endothelial deficiency of IGF1R promoted atherosclerotic burden, when animals were fed on a high-fat diet for 12 wk or normal chow for 12 mo. Under the normal chow feeding condition, the vascular relaxation response to acetylcholine was increased in the endothelial IGF1R-deficient aorta; however, feeding of a high-fat diet substantially attenuated the relaxation response, and there was no difference between endothelial IGF1R-deficient and control mice. The endothelium and its intercellular junctions provide a barrier function to the vasculature. In human aortic endothelial cells, IGF-1 upregulated occludin, claudin 5, VE-cadherin, JAM-A, and CD31 expression levels, and vice versa, specific IGF1R inhibitor, picropodophyllin, an IGF1R-neutralizing antibody (IR3), or siRNA to IGF1R abolished the IGF-1 effects on junction and adherens proteins, suggesting that IGF-1 promoted endothelial barrier function. Moreover, endothelial transwell permeability assays indicated that inhibition of IGF-1 signaling elevated solute permeability through the monolayer of human aortic endothelial cells. In summary, endothelial IGF1R deficiency increases atherosclerosis, and IGF-1 positively regulates tight junction protein and adherens junction protein levels and endothelial barrier function. Our findings suggest that the elevation of the endothelial junction protein level is, at least in part, the mechanism for antiatherogenic effects of IGF-1. [ABSTRACT FROM AUTHOR]

Estrogen has been shown to affect vascular reactivity. Here, we assessed the estrogen receptor-α (ERα) dependency of estrogenic effects on vasorelaxation via a rapid nongenomic pathway in both male and ovary-intact female mice. We compared the effect of a primary estrogen, 17β-estradiol (E2) or 4,4',4''-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT; selective ERα agonist). We found that E2 and PPT induced greater aortic relaxation in female mice than in male mice, indicating ERα mediation, which was further validated by using ERα antagonism. Treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) phenol]-1H-pyrazole dihydrochloride (MPP dihydrochloride; ERα antagonist) attenuated PPT-mediated vessel relaxation in both sexes. ERα-mediated vessel relaxation was further validated by the absence of significant PPT-mediated relaxation in aortas isolated from ERα knockout mice. Treatment with a specific ERK inhibitor, PD- 98059, reduced E2-induced vessel relaxation in both sexes but to a lesser extent in female mice. Furthermore, PD-98059 prevented PPTinduced vessel relaxation in both sexes. Both E2 and PPT treatment activated ERK as early as 5-10 min, which was attenuated by PD-98059 in aortic tissue, cultured primary vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). Aortic rings denuded of endothelium showed no differences in vessel relaxation after E2 or PPT treatment, implicating a role of ECs in the observed sex differences. Here, our results are unique to show estrogen-stimulated rapid ERα signaling mediated by ERK activation in aortic tissue, as well as VSMCs and ECs in vitro, in regulating vascular function by using side-by-side comparisons in male and ovary-intact female mice in response to E2 or PPT. [ABSTRACT FROM AUTHOR]

The redox- sensitive transcription factors NF-κB and activator protein-1 (AP-1) are critical mediators of ANG II signaling. The promitogenic and promigratory factor interleukin (IL)-18 is an NF-κB- and AP-1- responsive gene. Therefore, we investigated whether ANG II-mediated smooth muscle cell (SMC) migration and proliferation involve IL-18. ANG II induced rat carotid artery SMC migration and proliferation and IL-18 and metalloproteinase (MMP)-9 expression via ANG II type 1 (AT1) receptor. ANG II-induced superoxide generation, NF-κB and AP-1 activation, and IL-18 and MMP-9 induction were all markedly attenuated by losartan, diphenyleneiodonium chloride (DPI), and Nox1 knockdown. Similar to ANG II, addition of IL-18 also induced superoxide generation, activated NF-κB and AP-1, and stimulated SMC migration and proliferation, in part via Nox1, and both ANG II and IL-18 induced NOX1 transcription in an AP-1- dependent manner. AT1 physically associates with Nox1 in SMC, and ANG II enhanced this binding. Interestingly, exogenous IL-18 neither induced AT1 binding to Nox1 nor enhanced the ANG II-induced increase in AT1/Nox1 binding. Importantly, IL-18 knockdown, or pretreatment with IL-18 neutralizing antibodies, or IL-18 binding protein, all attenuated the migratory and mitogenic effects of ANG II. Continuous infusion of ANG II for 7 days induced carotid artery hyperplasia in rats via AT1 and was associated with increased AT1/ Nox1 binding (despite lower AT1 levels); increased DPI-inhibitable superoxide production; increased phospho-IKKβ, JNK, p65, and c- Jun; and induction of IL-18 and MMP-9 in endothelium-denuded carotid arteries. These results indicate that IL-18 amplifies the ANG II-induced, redox-dependent inflammatory cascades by activating similar promitogenic and promigratory signal transduction pathways. The ANG II/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis. [ABSTRACT FROM AUTHOR]

Some clinical studies have suggested that lower IGF-I levels may be associated with an increased risk of ischemic heart disease. We generated atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice with 6T alleles (6T/ApoE-/- mice) with a 20% decline in circulating IGF-I and fed these mice and control ApoE-/- mice with normal chow or a Western diet for 12 wk to evaluate the effect of low serum IGF-I on atherosclerosis progression. We found that the 6T/ApoE-/- phenotype was characterized by an increased atherosclerotic burden, elevated plaque macrophages, and increased proinflammatory cytokine TNF-α levels compared with ApoE-/- controls. 6T/ApoE-/- mice had similar body weight, blood pressure, serum total cholesterol levels, total plaque and smooth muscle cell apoptosis rates, and circulating levels of endothelial progenitor cells as ApoE-/- mice. 6T/ApoE-/- mice fed with normal chow had reduced vascular endothelial nitric oxide synthase mRNA levels and a trend to increased aortic expression of chemokine (C-C motif) receptor (CCR)1, CCR2, and monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2. Western diet-fed 6T/ApoE-/- mice had a trend to increased expression of macrophage scavenger receptor-1/scavenger receptor-A, osteopontin, ATP-binding cassette (subfamily A, member 1), and angiotensin-converting enzyme and elevated circulating levels of the neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 (KC). Our data establish a link between lower circulating IGF-I and increased atherosclerosis that has important clinical implications. [ABSTRACT FROM AUTHOR]

Congestive heart failure is associated with activation of the reninangiotensin system and skeletal muscle wasting. Angiotensin II (ANG II) has been shown to increase muscle proteolysis and decrease circulating and skeletal muscle IGF-1. We have shown previously that skeletal muscle-specific overexpression of IGF-1 prevents proteolysis and apoptosis induced by ANG II. These findings indicated that downregulation of IGF-1 signaling in skeletal muscle played an important role in the wasting effect of ANG II. However, the signaling pathways and mechanisms whereby IGF-1 prevents ANG II-induced skeletal muscle atrophy are unknown. Here we show ANG II-induced transcriptional regulation of two ubiquitin ligases atroginI and muscle ring finger-I (MuRF-1) that precedes the reduction of skeletal muscle IGF-1 expression, suggesting that activation of atroginI and MuRF-1 is an initial mechanism leading to skeletal muscle atrophy in response to ANG II. IGF-1 overexpression in skeletal muscle prevented ANG II-induced skeletal muscle wasting and the expression of atroginI, but not MuRF-1. Dominant-negative Akt and constitutively active Foxo-1 blocked the ability of IGF-1 to prevent ANG IImediated upregulation of atrogin-1 and skeletal muscle wasting. Our findings demonstrate that the ability of IGF-1 to prevent ANG II-induced skeletal muscle wasting is mediated via an Aktand Foxo1-dependent signaling pathway that results in inhibition of atrogin-1 but not MuRFI expression. These data suggest strongly that atrogin-1 plays a critical role in mechanisms of ANG 11-induced wasting in vivo. [ABSTRACT FROM AUTHOR]

Oxidized low-density lipoprotein (LDL) is proatherogenic and induces smooth muscle cell apoptosis, which contributes to atherosclerotic plaque destabilization. We showed previously that oxidized LDL downregulates insulin-like growth factor-1 receptor in human smooth muscle cells and that this is critical for induction of apoptosis. To identify mechanisms, we exposed smooth muscle cells to 60 µg/ml oxidized LDL or native LDL and assessed insulin-like growth factor-1 receptor mRNA levels, protein synthesis rate, and receptor protein stability. Oxidized LDL decreased insulin-like growth factor-1 receptor mRNA levels by 30% at 8 h compared with native LDL, and this decrease was maintained for up to 20 h. However, insulin-like growth factor-1 receptor protein synthesis rate was not altered by oxidized LDL. Pulse-chase labeling experiments revealed that oxidized LDL reduced insulin-like growth factor-1 receptor protein half-life to 12.2 ± 1.7 h from 24.4 ± 4.7 h with native LDL. This destabilization of insulin-like growth factor-1 receptor protein was accompanied by enhanced receptor ubiquitination. Overexpression of dominant-negative Nedd4 prevented oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor, suggesting that Nedd4 was the ubiquitin ligase that mediated receptor downregulation. However, the proteasome inhibitors lactacystin, MG-132, and proteasome inhibitor-1 failed to block oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor. Thus oxidized LDL downregulates insulin-like growth factor-1 receptor by destabilizing the protein via Nedd4- enhanced ubiquitination, leading to degradation via a proteasome-independent pathway. This finding provides novel insights into oxidized LDL-triggered oxidant signaling and mechanisms of smooth muscle cell depletion that contribute to plaque destabilization and coronary events. [ABSTRACT FROM AUTHOR]