Wnt2 and WISP-1/CCN4 Induce Intimal Thickening via Promotion of Smooth Muscle Cell Migration

Abstract

Objective-Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. Approach and Results-Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs.Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via β-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. was not translated into protein, and recombinant Wnt2 (rWnt2) protein did not increase VSMC proliferation in vitro. 5 The Wnt pathway has also been shown to have a role in cell migration, with Wnt3a involvement in both migration and adhesion of VSMCs through integrin linked kinase regulation of β1-integrin.10 However, on initiation of this study, it was unclear which Wnt proteins modulated VSMC growth factor-induced migration and whether Wntinduced VSMC migration promoted intimal thickening in vivo. Many genes are upregulated by the Wnt pathway, some of which are known to modulate VSMC migration, including Wnt-1-inducible signaling pathway protein-1 (WISP-1/CCN4 Materials and MethodsMaterials and Methods are available in the online-only Data supplement. Nonstandard Abbreviations and Acronyms Results Wnt2 Was Upregulated in Migrating VSMCs In VitroWnt mRNA levels during VSMC migration were assessed using an in vitro scratch wound assay with multiple wounds to stimulate migration of the VSMCs. mRNA was extracted from VSMCs and applied to a focussed Wnt pathway microarray to assess whether the level of expression changed during migration. A significant increase was only observed in Wnt2 mRNA ( Figure 1A), and this change was confirmed using quantitative polymerase chain reaction ( Figure 1A). No significant change was seen in the mRNA levels of any other Wnts (see Table III in the online-only Data Supplement). It was observed by Western blotting ( Figure 1B) and immunocytochemistry ( Figure I in the online-only Data Supplement) that the increase in Wnt2 mRNA was translated into augmented Wnt2 protein levels in migrating VSMCs. Migrating VSMCs on the wound edge could be seen to express higher levels of Wnt2 protein than nonmigratory VSMCs further away from the wound edge ( Figure I in the online-only Data Supplement). Wnt2 Promoted VSMC Migration In VitroAddition of rWnt2 protein significantly increased VSMC migration in vitro, whereas knockdown of Wnt2 using silencing RNA (siRNA) inhibited migration ( Figure 1C). When rWnt2 was ad...

Reactive Oxygen Species Generation and Atherosclerosis

“…79 Wnt2 also induced WISP-1 expression in SMC in β-catenin-dependent manner. 80 Levels of Wnt2 and WISP-1 were elevated in ligated carotid arteries. Furthermore, WISP-1 overexpression promoted intimal thickening, which was reduced in Wnt2 +/− animals.…”

“…78 Wnt/β-catenin/WISP-1 (WNT1-induciblesignaling pathway protein 1) represents example of the pathway that can protect SMCs from apoptosis 79 and oxidative stress but, on the other hand, enhance SMC migration and contribute to intimal thickening. 80 Mill et al 79 showed recently that macrophage-derived Wnt5a could protect SMC from H 2 O 2 -induced apoptosis. Exogenous Wnt5a increased amounts of active and nuclear β-catenin in SMC and induced TCF (transcription factor) signaling.…”

“…Furthermore, WISP-1 overexpression promoted intimal thickening, which was reduced in Wnt2 +/− animals. 80 Finally, although high glutathione levels and, that is, high ratio of reduced to oxidized glutathione are considered protective, too much of it can also lead to the enhanced e46…”

Atherosclerotic Calcification: Wnt Is the Hint

“…Interestingly, Wnt signaling in ECs has been shown to activate proinflammatory gene expression via inhibition of canonical Wnt signaling and activation of the noncanonical Ca 2+ ‐dependent pathway via ligand Wnt5a 88, 89. Several studies have demonstrated a direct link between canonical Wnt signaling via ligands Wnt1, Wnt2, and Wnt3a and arterial smooth muscle cell proliferation, which underlies the intimal thickening stage of atherosclerosis 20, 21, 90, 91. Canonical Wnt signaling and LRP5 have also been directly implicated in the promotion of foam cell formation 6.…”

Wnt signaling in cardiovascular disease: opportunities and challenges

“…In addition to inhibiting Wnt3a-induced pancreatic beta-cell insulin secretion[68], Wnt4 stimulates VSM proliferation and promote arterial intimal thickening[69] as relevant to the cardiovascular disease of diabesity. Importantly, in very recent data, George and colleagues have identified matricellular proteins of the CCN family ( C ysteine-rich protein 61/ C onnective tissue growth factor/ N ephroblastoma overexpressed gene) as down-stream mediators of Wnt-initiated VSM migration during neointima formation[70, 71]. …”