VAMP3 and SNAP23 mediate the disturbed flow-induced endothelial microRNA secretion and smooth muscle hyperplasia

Abstract

Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm 2 ) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm 2 ), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm 2 ), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicleassociated membrane protein 3 (VAMP3) and synaptosomalassociated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNARE activation and its contribution to the miRNA-mediated EC-SMC communication. T he interaction between hemodynamics and vascular endothelium is an important determinant of vascular homeostasis (1). In the straight part of the arteries, endothelial cells (ECs) are exposed to laminar blood flow with high shear stress that protects these parts of the vessels from atherogenesis, whereas in the branches and curvatures, the ECs experiencing disturbed flow with reciprocating and low shear stress stimulate the smooth muscle cells (SMCs) within the tunica media to become proliferative and migratory. The activated SMCs migrate into the intima, where they come into close contact with ECs and undergo phenotypic changes to lead to atherosclerosis (2). We recently demonstrated that vascular ECs repress expressions of forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 in the adjacent SMCs by producing microRNA-126-3p (miR-126-3p), and hence induce SMC turnover, and that the application of atheroprotective laminar shear (LS) at 12 dynes/cm 2 to ECs inhibits this paracrine effect (3). Although extracellular microRNAs (miRNAs) have been shown to convey messages from donor cells to recipient cells, how fluid shear stress regulates endothelial miRNA secretion remains to be elucidated.Emerging evidence has suggested that extracellular miRNAs can serve as not only pu...

Exogenous H2S Induces Hrd1 S-sulfhydration and Prevents CD36 Translocation via VAMP3 Ubiquitylation in Diabetic Hearts

“…Previous studies have identified proteins that mediate regulated exocytosis in cardiomyocytes, such as VAMP3 [20], VAMP7/8 [21], and syntaxin [22]. Most of them belong to a superfamily of transmembrane proteins named SNAREs, which mediate the trafficking of cellular materials between intracellular compartments [23]. The SNARE protein VAMP3 is known to be involved in vesicle fusion and trafficking [24].…”

Endosomal and Phagosomal SNAREs

“…The essential (161) Qbc-SNARE SNAP23 is best known for its role in plasma membrane trafficking. SNAP23 mediates exocytosis in many cell types (109,110,174,178,219,234,256,312,346), for instance in neurons where it mediates the post-synaptic surface delivery of the glutamate receptor at dendritic spines (296). The Qb-and Qc-SNARE-motifs of SNAP23 are connected by a linker region containing five palmitoylated cysteines (272) that anchor it at the plasma membrane, recycling and late endosomes, and the trans-Golgi network (88,94,104,109,166,223,275,309,312,346).…”

“…SNAP23 mediates exocytosis in many cell types (109,110,174,178,219,234,256,312,346), for instance in neurons where it mediates the post-synaptic surface delivery of the glutamate receptor at dendritic spines (296). The Qb-and Qc-SNARE-motifs of SNAP23 are connected by a linker region containing five palmitoylated cysteines (272) that anchor it at the plasma membrane, recycling and late endosomes, and the trans-Golgi network (88,94,104,109,166,223,275,309,312,346). SNAP23 is also found at phagosomes (38,51,79,215,221,271,286), and, although it might be recruited at several stages during phagosomal maturation (267), most quantitative proteomics show an overall decrease of phagosomal SNAP23 following uptake (FIGURE 5).…”

“…VAMP3 mediates exocytosis mainly by complexing with Stx4 (Qa) and SNAP23 (Qbc) (87,91,112,153,219,243,312,313,346). In addition, trafficking from early/recycling endosomes to the Golgi apparatus is mediated by complexing of VAMP3 with Stx16 (Qa), Vti1a (Qb), and Stx6 or Stx10 (Qc) (105,193,265), whereas trafficking in the reverse direction is mediated by complexing with Stx7 (Qa), Vti1b (Qb), and Stx6 (196,219,220).…”

MicroRNA Exocytosis by Vesicle Fusion in Neuroendocrine Cells

“…SNARE proteins are considered to constitute the fusion machinery that draws two opposing membranes close together ( 66 ). Neuronal SNAREs including VAMP-2, SNAP-25A, and syntaxin-1A mediate miRNA exocytosis in neuroendocrine cells (Figure 1 A) ( 24 ), whereas VAMP-3 and SNAP-23 mediate secretion of vesicle-free miRNA in vascular endothelial cells ( 67 ). miRNA exocytosis is completely inhibited when neuronal SNAREs are absent in the in vitro reconstitution system, suggesting that neuronal SNAREs mediate the release of miRNAs in chromaffin cells ( 24 ).…”