Background

Moyamoya disease is a cerebrovascular disease with an unclear etiology that mainly occurs in East Asia. Its characteristic lesion is progressive stenosis at the end of the bilateral internal carotid arteries, the anterior cerebral arteries, and the beginning of the middle cerebral arteries, accompanied by abnormal vascular network formation at the skull base [1, 2]. There is still a lack of effective drug treatments for moyamoya disease, and vascular reconstruction surgery is the main treatment method. The main methods of surgery include direct bypass surgery and indirect bypass surgery [3]. Currently, most centers perform both direct and indirect bypass surgery in one surgery and combine the advantages of both, known as combined vascular reconstruction surgery [4, 5].

Encephalo-myo-synangiosis (EMS) is a widely used indirect bypass surgery for Moyamoya disease and is also a part of combined vascular reconstruction surgery [6,7,8]. The temporal muscle of the patient was dissected and covered on the cerebral hemispheric surface with insufficient perfusion in this surgery. The principle of the surgery is to induce neovascularization of the temporal muscle and supply blood to the brain to alleviate the degree of cerebral ischemia [9]. However, some of the cohort studies showed that a certain proportion of the patients who underwent EMS surgery failed to achieve the expected results. Failure to achieve the expected results of EMS surgery is potentially due to effective collateral circulation between the temporal muscle and the cerebral hemisphere not being well established [10, 11]. Therefore, promoting the collateral circulation of EMS will largely improve the outcome for these patients.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types, including endothelial cells, chondrocytes, myocytes, adipocytes, and even glial cells [12, 13]. Furthermore, an increasing number of studies have suggested that MSCs exhibit an active treatment effect against various disorders, including inflammation, neuron degeneration diseases, cancers, ischemia diseases, etc. [14,15,16,17]. Studying the underlying mechanisms of this omnipotent therapeutic effect has become a hot topic. Some research has shown that MSCs may release many cytokine factors and noncoding RNAs, such as miRNAs and lncRNAs, which can effectively promote angiogenesis [18, 38]. These studies suggest that MSC treatment alone results in significant treatment efficacy in animal models. Moreover, several clinical trials around the world have demonstrated the safety of MSC transplantation in brain ischemia [39]. In recent decades, EMS surgery has become popular and has resulted in superior curative effects in treating chronic brain ischemia, such as Moyamoya disease and middle cerebral artery stenosis [40, 41]. However, there is a lack of studies evaluating the effect of MSC transplantation in combination with surgery. Our present study provides evidence that local MSC transplantation remarkably improved post-EMS neovascularization.

Transplantation of stem cells can be done using either freshly prepared cells or cryopreserved cells, but cryopreservation can in some cases adversely affect adult stem cell (and stem cell-containing) populations, which may lead to therapeutic failure [42], therefore, freshly prepared stem cells were used in our study for the experiments.

The mechanisms by which MSCs promote neovascularization comprise multiple signals [43,44,45]. Due to their differentiation potential, MSCs are able to directly differentiate into cells participating in neovascularization, including pericytes, endothelial cells, and smooth muscle cells [46], which have been verified in the treatment of a variety of diseases. Numerous circumstances, such as hypoxia or inflammation, initiate MSC differentiation [47]. In our current study, MSCs were unlikely to differentiate into these vascular cells but were likely to secrete functional factors that promote angiogenesis. According to the our array result, multiple pro-angiogenesis such as MMP-3, MMP-9, IGFBP-2 or IGFBP-3 were expressed at much higher level in the MSC transplantation group. Indeed, Matrix metalloproteinases (MMPs) are proteases that exerts pro-angiogenesis effect by degradation of the vascular basement membrane, participating in extracellular matrix remodeling and releasing of angiogenic mediators [48]. Also, numerous studies revealed that Insulin-like growth factor binding proteins (IGFBPs), such as IGFBP-2, exert angiogenic function in both IGF and IGFR1 dependent pathways [49]. Our findings suggested the potential angiogenic mechanisms of MSC transplantation, while more detail investigations are needed to identify the major effectors. Moreover, previous studies revealed that MSCs release numerous proangiogenic growth factors, such as VEGF and PDGF [50]. In addition, MSC exosomal noncoding RNAs, such as miRNAs or circRNAs, were also shown to regulate endothelial cell gene expression, which facilitates vascular generation [51, 52]. Therefore, we also evaluated the gene expression of known pro-angiogenesis factors. Indeed, MSC or MSC supernatant transplantation notably increases VEGF, and TGF expression. Despite the well-known VEGF and TGFβ, our observation revealed TGFα as the most predominant upregulated factor after BMSC-CM treatment, indicating its crucial effect on neovascularization. Transforming growth factor alpha (TGFα) belongs to the TGF superfamily, and its structure is more similar to that of the EGF family [53, 54].There are no overwhelming investigations on the pro-angiogenesis impact of TGFα, while several studies have demonstrated its important role in stem cell self-renewal and differentiation.

Current strategies for MSC treatment are predominantly cell transplantation, which comes from the umbilical cord, bone marrow, or adipose tissue [55, 56]. Ethical problems or unpredicted tumorigenesis of MSC transplantation hinder the clinical application of MSC treatment [57]. Our study provides evidence that the supernatant of MSCs exhibits a pro-angiogenesis effect equivalent to that of cell transplantation in EMS, which further avoids the malpractice of cell transplantation. Furthermore, implementation of the supernatant application is easy to accomplish during EMS by injecting it onto temporal muscle [58]. Further studies are needed to examine whether transdermal injection to the temporalis muscle after EMS is applicable.

In summary, our present study revealed improvement of neovascularization and cognitive recovery in chronic brain ischemia post-EMS by using MSC as well as supernatant treatment, which provides a novel combination of therapeutic methods for the clinical treatment of chronic brain ischemia.

In addition, a limitation in the study was the randomization of the experimental animals into sham-operated and "model" groups during experimental grou**, which did not make sense because it automatically prevented allocation concealment. Moreover, the model used in the study only mimics vascular cognitive impairment to a certain extent, and we will use a combined model of vascular cognitive impairment for more in-depth exploration in future studies [59, 60].

Conclusions

In conclusion, transplantation of MSCs during EMS can promote angiogenesis, and concentrated BMSC-CM can also achieve similar effects. Our study illustrated that MSC locally transplantation can be potential therapeutical options for improving EMS treatment efficiency which might be translated into clinical application.