背景肾素-血管紧张素系统在心力衰竭的发展中起着至关重要的作用,AngII(血管紧张素II)在调节心脏纤维化中充当肾素-血管紧张素系统的关键效应物。然而,心脏纤维化的机制是复杂的,仍然没有完全了解。IGF1R(胰岛素样生长因子1受体)在维持心血管稳态方面具有多种功能,低剂量IGF1治疗可有效缓解AngII诱导的心肌纤维化。这里,我们旨在探讨IGF1R在AngⅡ诱导的心肌纤维化中的分子机制。方法和结果使用原代小鼠心脏微血管内皮细胞和成纤维细胞,进行了体外实验。使用C57BL/6J小鼠和成簇规则间隔的短回文重复序列(CRISPR)/CRISPR相关9(Cas9)介导的IGF1R杂合子敲除(Igf1r/-)小鼠,AngII诱导2周心肌纤维化小鼠模型。通过定量逆转录聚合酶链反应检测IGF1R的表达,免疫组织化学,和Westernblot。使用Masson和picrosirius红染色评估小鼠心脏组织学变化。使用定量逆转录聚合酶链反应和Westernblot检测纤维化标志物和指示Akt(蛋白激酶B)/ERK(细胞外信号调节激酶)/核因子-κB通路功能的信号分子。RNA测序用于探索小鼠心脏中IGF1R介导的靶基因,并通过免疫共沉淀鉴定了IGF1R和G蛋白偶联受体激酶5的关联。更重要的是,阻断IGF1R信号显着抑制小鼠心脏微血管内皮细胞和小鼠对转化生长因子β1或AngII的反应的内皮-间质转化,分别。IGF1R信号传导的缺乏或抑制显著减弱了在原代小鼠心脏成纤维细胞和小鼠中AngII诱导的心脏纤维化。我们进一步观察到患有心力衰竭的患者表现出比健康个体更高的IGF1和IGF1R血液水平。此外,AngII治疗在野生型小鼠中显著增加心脏IGF1R,但在Igf1r+/-小鼠中导致轻微下调。有趣的是,IGF1R缺乏可显着减轻AngII治疗小鼠的心脏纤维化。机械上,Akt和ERK的磷酸化水平在AngII处理的小鼠中上调,而阻断IGF1R信号在小鼠中抑制Akt和ERK磷酸化的这些变化。同时,在相应的小鼠组中,核因子κB的磷酸化p65表现出相似的变化。有趣的是,IGF1R直接与G蛋白偶联受体激酶5相互作用,在Igf1r/-小鼠中,这种相关性降低了约50%。此外,Grk5缺失下调Akt/ERK/核因子-κB信号通路在原代小鼠心脏成纤维细胞中的表达。结论IGF1R信号传导缺失减轻AngII诱导的心肌纤维化,至少部分通过Akt/ERK/核因子-κB途径抑制内皮-间质转化。有趣的是,G蛋白偶联受体激酶5与IGF1R信号直接相关,并且它同时充当IGF1R下游效应物。这项研究表明IGF1R作为心脏纤维化的治疗靶标具有很有希望的潜力。
Background The renin-angiotensin system plays a crucial role in the development of heart failure, and Ang II (angiotensin II) acts as the critical effector of the renin-angiotensin system in regulating cardiac fibrosis. However, the mechanisms of cardiac fibrosis are complex and still not fully understood. IGF1R (insulin-like growth factor 1 receptor) has multiple functions in maintaining cardiovascular homeostasis, and low-dose IGF1 treatment is effective in relieving Ang II-induced cardiac fibrosis. Here, we aimed to investigate the molecular mechanism of IGF1R in Ang II-induced cardiac fibrosis. Methods and Results Using primary mouse cardiac microvascular endothelial cells and fibroblasts, in vitro experiments were performed. Using C57BL/6J mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated IGF1R heterozygous knockout (Igf1r+/-) mice, cardiac fibrosis mouse models were induced by Ang II for 2 weeks. The expression of IGF1R was examined by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot. Mice heart histologic changes were evaluated using Masson and picro sirius red staining. Fibrotic markers and signal molecules indicating the function of the Akt (protein kinase B)/ERK (extracellular signal-regulated kinase)/nuclear factor-κB pathway were detected using quantitative reverse transcription polymerase chain reaction and Western blot. RNA sequencing was used to explore IGF1R-mediated target genes in the hearts of mice, and the association of IGF1R and G-protein-coupled receptor kinase 5 was identified by coimmunoprecipitation. More important, blocking IGF1R signaling significantly suppressed endothelial-mesenchymal transition in primary mouse cardiac microvascular endothelial cells and mice in response to transforming growth factor-β1 or Ang II, respectively. Deficiency or inhibition of IGF1R signaling remarkably attenuated Ang II-induced cardiac fibrosis in primary mouse cardiac fibroblasts and mice. We further observed that the patients with heart failure exhibited higher blood levels of IGF1 and IGF1R than healthy individuals. Moreover, Ang II treatment significantly increased cardiac IGF1R in wild type mice but led to a slight downregulation in Igf1r+/- mice. Interestingly, IGF1R deficiency significantly alleviated cardiac fibrosis in Ang II-treated mice. Mechanistically, the phosphorylation level of Akt and ERK was upregulated in Ang II-treated mice, whereas blocking IGF1R signaling in mice inhibited these changes of Akt and ERK phosphorylation. Concurrently, phosphorylated p65 of nuclear factor-κB exhibited similar alterations in the corresponding group of mice. Intriguingly, IGF1R directly interacted with G-protein-coupled receptor kinase 5, and this association decreased ≈50% in Igf1r+/- mice. In addition, Grk5 deletion downregulated expression of the Akt/ERK/nuclear factor-κB signaling pathway in primary mouse cardiac fibroblasts. Conclusions IGF1R signaling deficiency alleviates Ang II-induced cardiac fibrosis, at least partially through inhibiting endothelial-mesenchymal transition via the Akt/ERK/nuclear factor-κB pathway. Interestingly, G-protein-coupled receptor kinase 5 associates with IGF1R signaling directly, and it concurrently acts as an IGF1R downstream effector. This study suggests the promising potential of IGF1R as a therapeutic target for cardiac fibrosis.