M1巨噬细胞和VLA4/VCAM-1通路在风湿性心脏病所致瓣膜损害中的潜在作用

Xian Shenglin, Li Yuan, Bai Ling, Tang Senhu, Meng Zhongyuan, Wen Hong, Huang Feng, Zeng Zhiyu

Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, 530021 Nanning, Guangxi, China.

Guangxi Key Laboratory of Precision Medicine in Cardio-cerebrovascular Diseases Control and Prevention, Guangxi Medical University, 530021 Nanning, Guangxi, China.

Front Biosci (Landmark Ed). 2024 Jun 18;29(6):219. doi: 10.31083/j.fbl2906219.

BACKGROUND

Rheumatic heart disease (RHD) is caused by inflammatory cells mistakenly attacking the heart valve due to Group A Streptococcus (GAS) infection, but it is still unclear which cells or genes are involved in the process of inflammatory cells infiltrating the valve. Inflammatory infiltration into the target tissue requires an increase in the expression of phosphorylated vascular endothelial-cadherin (p-VE-cad), p-VE-cad can increase the endothelial permeability and promote the migration of inflammatory cells across the endothelium. P-VE-cad is potentially regulated by RAS-related C3 botulinum substrate 1 (RAC1), together with phosphorylated proline-rich tyrosine kinase 2 (p-PYK2). While RAC1/p-PYK2/p-VE-cad is triggered by the activation of vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is related to M1 macrophages adhering to the endothelium via very late antigen 4 (VLA4). Inflammatory infiltration into the valve is extremely important in the early pathogenesis of RHD. However, there is no relevant research on whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved in RHD; therefore, what we explored in this study was whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved.

METHODS

We established a rat model of RHD and a cell model of M1 macrophage and endothelial cell cocultivation. Subsequently, we measured the degree of inflammatory cell infiltration, the levels of IL-6/IL-17, the degree of fibrosis (COL3/1), and the expression levels of fibrosis markers (FSP1, COL1A1 and COL3A1) in the heart valves of RHD rats. Additionally, we detected the expression of M1/M2 macrophage biomarkers in rat model and cell model, as well as the expression of M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad. We also tested the changes in endothelial permeability after coculturing M1 macrophages and endothelial cells.

RESULTS

Compared to those in the control group, the levels of inflammatory cell infiltration and fibrotic factors in the heart valves of RHD rats were significantly higher; the expression of M1 macrophage biomarkers (iNOS, CD86 and TNF-α) in RHD rats was significantly higher; and significantly higher than the expression of M2 macrophage biomarkers (Arg1 and TGF-β). And the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad in the hearts of RHD rats were significantly higher. At the cellular level, after coculturing M1 macrophages with endothelial cells, the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad were significantly higher, and the permeability of the endothelium was significantly greater due to cocultivation with M1 macrophages.

CONCLUSIONS

All the results suggested that M1 macrophages and the VLA4/VCAM-1 pathway are potentially involved in the process of inflammatory infiltration in RHD.

背景

风湿性心脏病（RHD）是由A组链球菌（GAS）感染导致炎症细胞错误攻击心脏瓣膜引起的，但目前仍不清楚炎症细胞浸润瓣膜的过程中涉及哪些细胞或基因。炎症细胞浸润到靶组织需要磷酸化血管内皮钙黏蛋白（p-VE-cad）的表达增加，p-VE-cad可增加内皮通透性并促进炎症细胞跨内皮迁移。p-VE-cad可能受RAS相关C3肉毒杆菌底物1（RAC1）以及磷酸化富含脯氨酸的酪氨酸激酶2（p-PYK2）的调控。而RAC1/p-PYK2/p-VE-cad是由血管细胞黏附分子-1（VCAM-1）的激活所触发。VCAM-1与M1巨噬细胞通过极晚期抗原4（VLA4）黏附到内皮细胞有关。炎症细胞浸润到瓣膜在RHD早期发病机制中极为重要。然而，关于M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad是否参与RHD尚无相关研究；因此，本研究探讨的是M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad是否参与其中。

方法

我们建立了RHD大鼠模型以及M1巨噬细胞与内皮细胞共培养的细胞模型。随后，我们测量了RHD大鼠心脏瓣膜中炎症细胞浸润程度、IL-6/IL-17水平、纤维化程度（COL3/1）以及纤维化标志物（FSP1、COL1A1和COL3A1）的表达水平。此外，我们检测了大鼠模型和细胞模型中M1/M2巨噬细胞生物标志物的表达，以及M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad的表达。我们还测试了M1巨噬细胞与内皮细胞共培养后内皮通透性的变化。

结果

与对照组相比，RHD大鼠心脏瓣膜中炎症细胞浸润水平和纤维化因子水平显著更高；RHD大鼠中M1巨噬细胞生物标志物（诱导型一氧化氮合酶、CD86和肿瘤坏死因子-α）的表达显著更高；且显著高于M2巨噬细胞生物标志物（精氨酸酶1和转化生长因子-β）的表达。并且RHD大鼠心脏中VLA4/VCAM-1以及RAC1/p-PYK2/p-VE-cad的表达水平显著更高。在细胞水平上，M1巨噬细胞与内皮细胞共培养后，VLA4/VCAM-1以及RAC1/p-PYK2/p-VE-cad的表达水平显著更高，并且与M1巨噬细胞共培养导致内皮通透性显著增加。