Introduction
Vascular access is the Achilles’ heel of patients undergoing hemodialysis. A functioning vascular access is necessary for the patients to receive successful life-sustaining hemodialysis. Arteriovenous fistula (AVF) is a surgical connection of an artery (such as radial artery) directly to a vein (cephalic vein), the venous limb remodels adequately and successfully to support repeated cannulations, acting as a bridge between blood and hemodialysis machine. KDOQI (Kidney Disease Outcomes Quality Initiative) vascular access guideline sets Fistula First as the goal for hemodialysis because the thrombosis rates, infection rates, access-related expenditures, and total healthcare expenditures all are lower for patients with fistulas than for those with either synthetic AV grafts or central venous catheters [
1]. Unfortunately, about 40–60% of primary vascular accesses fail in two years and require medical interventions that cost of > $1 billion annually. Multicenter, randomized clinical trials targeting thrombosis have produced only a limited improvement in the patency of AVFs and AV grafts [
2‐
8]. Therefore, uncovering cellular mechanisms and molecular pathways that regulate vascular remodeling could help develop clinical strategies to benefit maturation and patency of AVFs.
In AVFs, the vein anastomosis is subjected to increased blood flow/pressure. This causes mechanical stretching, which leads to the development of a dilated, thickened vein wall to withstand high hemodynamic forces and repeated cannulations. The venous outflow limb must dilate while remaining sufficiently pliable to maintain blood flow/pressure after moderate extracellular matrix deposition and cellular migration/proliferation. These components must be integrated to form a functional AVF [
9‐
11]. AVF can fail if the venous arm does not remodel adequately (arterialization) to support hemodialysis or if the venous arm develops neointimal hyperplasia [
12]. Excessive neointimal formation will narrow the lumen and block blood flow, causing AVF dysfunction and thrombosis. Hemodialysis Fistula Maturation Study Group reported the prevalence of stenosis detected on ultrasound was 14% at 1 day and increased to 30% from 2 to 6 weeks after creation of AVF. Notably, these postoperative fistulae venous stenosis causing by intimal hyperplasia significantly associates with fistula maturation failure [
13]. Therefore, facilitating AVF adaptive remodeling while controlling neointima formation is a current challenge.
Understanding the cellular and molecular events during AVF remodeling will lead to solutions to a targeted therapeutic strategy to improve AVF patency. We have shown that VSMCs from anastomosed arteries are dedifferentiated and migrate into the venous anastomosis to form neointima in AVFs [
14,
15]. Although multiple signaling pathways have been involved in regulating vascular remodeling [
16], Notch signaling determines VSMC differentiation and artery formation during development [
17]. Whether manipulating Notch signals can balance the lack of remodeling and neointima hyperplasia to improve AVF maturation has not been studied.
Notch and its ligands transduce signals between neighboring cells from signal-giving cells expressing Notch ligands to signal-receiving cells expressing Notch receptors. When a Notch ligand binds to Notch, the extracellular domain of the Notch receptor is cleaved, resulting in release of an intracellular Notch domain (abbreviated NICD). NICD can interact with RBP-Jκ, a primary transcription factor of Notch signaling [
18]. NICD displaces co-repressors from RBP-Jκ, and replaces them with co-activators [e.g., master mind 1 (MAML1)]. The binding of MAML1 to RBP-Jκ results in transcription of target genes, Hes1/5, Hey2, and VSMC markers, Myh11 and α-SMA [
19‐
23]. A dominant negative MAML1 (dnMAML1) can block Notch activation. Therefore in this study we studied the roles of Notch transcription factor (RBP-Jκ and MAML1) on VSMC differentiation and neointima formation during AVF remodeling. We used temporal controlled expression of RBP-Jκ or dnMAML1 to manipulate Notch activation in VSMCs to improve AVF adaptive remodeling and maturation.
Discussion
The vascular access is the lifeline for hemodialysis patients. Successful maturation following surgery requires functional and structural adaptations to arterial blood flows. Insufficient thickening of the AVF wall (maturation/remodeling) or excessive VSMC accumulation (neointima formation) leads to AVF dysfunction. In this study, we uncovered evidence showing that the Notch signaling pathway can be fine-tuned to improve AVF adaptive remodeling with limited neointima formation. Firstly, we found that the VSMCs at the anastomosis of AVFs became dedifferentiated. Secondly, the dedifferentiated, activated VSMCs migrated into the venous side and regained VSMC phenotype after expressing the contractile markers. Thirdly, inhibition of Notch signaling (KO of RBP-Jκ) blocks VSMC differentiation from dedifferentiated VSMCs in neointima and thus ablates AVF remodeling. We found that Notch signaling is required to initiate differentiation of VSMC progenitors into VSMCs, but it is not fully required to maintain VSMC phenotype. Fourthly, we showed that AVFs with insufficient venous wall thickening have endothelial barrier dysfunction and increased inflammation. Finally, we demonstrated that temporal control of Notch activation by KO of RBP-Jκ or OE of dnMAML1 improves AVF maturation.
VSMCs play an important role in neointima formation in AVF. Characterization of VSMC phenotype switching can provide insight to improve AVF maturation. Based on this study, there are at least two remarkable cellular events that occur after anastomosing end of vein to the artery. At beginning, the VSMCs in the anastomosis and in the venous arm of AVF are subjected to surgery trauma. Combined with the increased mechanical and shear stress after AVF operation, they become activated and dedifferentiated. These dedifferentiated VSMCs lose the expression of contractile VSMC markers (α-SMA and MHY11). They migrate, proliferate, and produce extracellular matrix in AVF, contributing to both vascular wall thickening and neointima formation. Then, the dedifferentiated VSMCs can regain the expression of VSMC markers in the neointima in AVFs. Dysregulation of these cellular events results in un-arterialized wall of AVF. As reported by Dardik, insufficient VSMC proliferation in the venous arm of the AVF will cause unsuccessful AVF maturation, while excessive VSMC accumulation will lead to AVF failure [
12]. In addition, CKD uremic toxins promote VSMC hyperplasia and accelerate neointima formation leading to stenosis in AVFs. Thus, figuring out the molecular signaling pathways that are involved in VSMC phenotype switching in AVF will guide the development of targeted therapeutic strategy to properly regulate these cellular events and improve the AVF patency.
Notch signaling has been shown to regulate VSMC differentiation and artery formation during development. The VSMC recruitment into the arteries are severely blocked without Notch molecules [
17]. Moreover, Notch signaling is one of the most important pathway leading to VSMC phenotype changes and accumulation in AVFs. We previously showed that sustained activation of Notch stimulates VSMC accumulation and neointima formation in AVF [
33,
34]. Notch signaling promotes VSMC proliferation in response to cyclic stretch and CKD uremic toxins, which is closely associated with AVF failure [
28,
35]. Superficially, we hypothesized that KO of Notch signaling could prevent neointima hyperplasia and improve AVF maturation. To our surprise, blockade of Notch signaling suppressed VSMC accumulation and completely blocked the expressions of contractile markers (MYH11 and α-SMA) in VSMCs, resulting in failure of VSMC accumulation and AVF remodeling and maturation. Others also reported that inhibition of Notch signaling blocks VSMC terminal differentiation from these SMC progenitors [
36].
One of our interesting findings is that gaining VSMC markers (MYH11, ACTA2 and SM22) are essential for AVF function. Without expression of those contraction proteins, the dedifferentiated SMCs become more vulnerable to surrounding stimulus and lead to inflammation [
37,
38]. Indeed, failed AVF remodeling impaired EC integrity and induced infiltration by inflammatory cells. In AVF created in RBP-Jκ global or VSMC-specific KO mice, the endothelial regeneration was delayed, and the leakage was observed indicating barrier dysfunction of the AVFs. This impaired EC barrier function could be caused by the lack of VSMC coverage. In AVF and AV graft models, a newly formed endothelium without VSMC coverage is vulnerable to stresses arising from arterial blood pressure [
26,
39]. Consistent with this finding in AVF, we uncovered that KO of Jagged1 in VSMCs destabilize newly regenerated ECs in AV graft [
40].
Another important finding of the study is that fine tuning Notch activation during AVF remodeling to manipulate the VSMC activation and differentiation could be the key to maintain this balance of AVF remodeling and neointima formation. We proposed that a temporally controlling Notch activation in VSMCs would both improve adaptive remodeling of AVF while preventing neointima hyperplasia. Importantly, Notch signaling is required to initiate VSMC differentiation, but not to maintain VSMC phenotype. We found that inhibition of Notch signaling after the appearance of VSMC markers in neointimal cells improved AVF remodeling and maturation. On the other hand, Doxycycline-induced “switch” to turn “On” and “Off” the Notch signaling by overexpressing dnMAML1 is even a better choice for temporal control of Notch activation. In this case, Notch activation can be inhibited or restored by using this “switch”, while Cre-mediated cleavage of foxed RBP-Jκ causes permanent deficiency of Notch activation, which has been shown to be related with other vascular diseases [
41,
42].
VSMCs synthesize contractile markers to maintain artery homeostasis. Although Notch signaling is required to trigger progenitor cell differentiation into VSMCs, KO of RBP-Jκ did not significantly block the expression of VSMC contractile markers in cultured VSMCs or arteries of adult mice. It is possible that Notch stimulates and/or activates other signaling pathways to maintain VSMC phenotype in adult life. PDGFRβ, which has been reported to promote VSMC differentiation from stem cells, is one of the candidates that is regulated by Notch activation [
43]. Others also showed that RBP-Jκ binding sites were present in the promoter of PDGFRβ [
44]. Although other signals such as SRF/myocardin and Yes associated protein pathway also control PDGFRβ expression and VSMC differentiation [
45‐
47], Notch is the master gene to determine VSMC fate.
Conclusions
In summary, our results from this study demonstrate that Notch signaling plays a dominant role in determining VSMCs fate in AVF remodeling. Activated Notch signaling pathway promotes VSMC activation and differentiation, while deficiency of Notch impairs VSMC accumulation and induces EC barrier dysfunction and inflammation, leading to AVF maturation failure. Temporal control of Notch activation can improve AVF maturation. These findings could provide insight for developing therapeutic strategies by targeting the Notch signaling pathway to improve AVF maturation.
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