The Wnt/β-catenin signaling pathway is involved in the response to oxidative stress stimuli and leads to white matter lesions and central nervous system inflammation and injury [20–22], and inhibition of the abnormal activation of the Wnt/β-catenin signaling pathway was shown to delay coronary artery disease (CAD) in the myocardial tissue of rats [23] and the process of RI/R injury involving oxidative stress in the kidney [24]. Inhibition of the Wnt/β-catenin signaling pathway inhibited the progression of neuroblastoma and attenuated axonal degeneration in Parkinson's disease models [15, 16, 25]. These studies suggest that activation of Wnt/β-catenin signaling promotes disease, and inhibition of Wnt/β-catenin signaling may have a protective effect. However, to date, studies on how the Wnt/β-catenin signaling pathway is involved in CVSD are lacking, and our study showed that miR-320e could inhibit oxidative stress-induced Wnt2-mediated enhancement of the Wnt/β-catenin signaling pathway in endothelial cells. Thus, we speculate that miR-320e could protect vascular endothelial cells through this mechanism and is a protective factor.
Small vessel lesions are the result of a combination of factors. Because of the scarcity or absence of smooth muscle content in small arteries, small vessel diastolic function and substance screening are dependent on endothelial cells to perform normal physiological functions [26–29]. Recent studies have shown that exosomes transport complex substances freely to and from the BBB, and the miRNAs they carry are protected by the exosome membrane from degradation by RNA enzymes so that they can effectively act on target cells; thus, the microenvironment of intracranial vascular endothelial cells is affected by exosome inclusion bodies [30, 31]. Previous studies have found that neuronal-secreted exosomal miR-132 could regulate cerebrovascular integrity [32]; exosomal miR-451a may play a protective role in ischemia‒reperfusion injury [9]; and exosomal miR-124a from mesenchymal stem cells can target and inhibit FOX A2 to suppress glioblastoma [10]. Our study showed that exosomes could carry miR-320e and release it into target cells, allowing miR-320e to exert targeted inhibition of Wnt2, thereby inhibiting Wnt2-mediated enhancement of the Wnt/β-catenin signaling pathway during oxidative stress.
To verify the inhibitory effect of miR-320e on the Wnt2-mediated Wnt/β-catenin signaling pathway, we selected the target genes of miR-320e, Wnt2, and the downstream mRNAs FZD2, Axin2, GSK3β and total cellular β-catenin. Our experiment showed that after oxidative stress stimulation, the expression of the above genes was significantly inhibited by miR-320e, and the enhancement of the Wnt/β-catenin signaling pathway was suppressed. In addition, we found that miR-320e could be transported by exosomes to reach target cells and continue to exert its inhibitory effect on Wnt2/β-catenin signaling. Furthermore, we found that GSK3β was inhibited by miR-320e to a much lesser extent than the other genes we selected. GSK3β is involved in multiple signaling pathways in addition to its role in the Wnt/β-catenin signaling pathway, representing a class of molecules that exert pathway interactions during cell signaling [33], which may suggest that other exosomal miRNAs expressed in our exosome differential expression profile from CVSD plasma may contribute to CVSD development through other mechanisms.
The Wnt signaling pathway has been suggested to exhibit different responses when subjected to different time frames of oxidative stress, with transiently activated Wnt signaling pathways generally inducing neovascularization and protecting vascular structure and cellular function [34, 35], whereas continuously activated Wnt signaling pathways lead to senescence and basement membrane disruption [36]. In CVSD, when the small vessels that develop lesions are not in critical areas of the white matter or cortex or when CVSD is masked by more severe ischemia and hemorrhage, small vessel lesions could be detected at a more advanced stage, while various aggressive factors may allow sustained activation of Wnt/β-catenin, thus promoting CVSD progression. Because of the ability of exosomal miR-320e to target Wnt2 and thereby inhibit abnormal activation of Wnt/β-catenin signaling, the significantly lower expression of miR-320e in plasma exosomes of CVSD patients may lead to a lack of protection by this effect. Moreover, other miRNAs in the differential expression profile may exert protective effects in combination with miR-320e, which may guide further investigation and validation.
The normal function of the brain depends on the integrity of the cerebrovascular system, and several underlying pathophysiological mechanisms have been shown to be involved in the process of CSVD, such as blood‒brain barrier damage, small vessel stiffness, venous collagen proliferation, inflammation, and myelin damage [37, 38]. Endothelial cells are the cornerstone of small vessel function, and with the natural onset of aging and the onslaught of various risk factors, intracranial vascular function gradually declines and structural integrity is gradually lost, which has long been seen as a vital process in SVD [30, 39, 40]. Currently, there are few studies on the mechanisms of exosomal miRNAs in CVSD. In summary, by using bioinformatics analysis and endothelial cell experiments, this study predicted and confirmed that Wnt2 is an important target of miR-320e. MiR-320e could target and inhibit the Wnt2/β-catenin signaling pathway and suppress the enhancement of this pathway in response to oxidative stress stimulation, which may play a protective role in the progression of CVSD.