Vascular Smooth Muscle LRP6 Limits Arteriosclerotic Calcification in Diabetic LDLR ^−/− Mice by Restraining Noncanonical Wnt Signals

Abstract

Rationale Wnt signaling regulates key aspects of diabetic vascular disease. Objective We generated SM22-Cre;LRP6(fl/fl);LDLR-/- mice to determine contributions of Wnt co-receptor LRP6 in the vascular smooth muscle lineage (VSM) of male LDLR-null mice, a background susceptible to diet (HFD) - induced diabetic arteriosclerosis. Methods and Results As compared to LRP6(fl/fl);LDLR-/- controls, SM22-Cre;LRP6(fl/fl);LDLR-/- (LRP6-VKO) siblings exhibited increased aortic calcification on HFD without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an USF-activated cognate inhibited by LRP6. RNAi revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO VSM, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and VSM calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT) - 1, and nuclear asymmetric dimethylarginine modification was increased with LRP6-VKO. RNAi demonstrated that PRMT1 inhibits OPN and TNAP while PRMT4 supports expression. USF1 complexes containing the H3R17Me2a signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1:H3R17Me2a complex formation and transactivation. Conclusions LRP6 restrains VSM noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation – dependent relays.

Fibrotic Aortic Valve Stenosis in Hypercholesterolemic/Hypertensive Mice

“…The balance of these changes may contribute to the finding that the mice do not develop the calcific type of aortic stenosis (CAVS). Expression of some other genes (LDLr-related protein 6 and TNFRSFIIB [which encodes osteoprotegerin]) 18, 19 that inhibit calcification in blood vessels is not increased in the aortic valve of HC/HT mice.…”

“…Because this model of FAVS is characterized by only minimal calcification, one of our goals was to look with RNA-Seq for genes that promote or limit calcification and fibrosis in blood vessels. 17–19 …”

Wnt signaling in cardiovascular disease: opportunities and challenges

“…While sources of circulating Wnts have yet to be robustly established in vivo, adipose and skeletal muscle[68] and VSM[27] can all produce Wnt4. 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.…”

“…LRP6 is also highly expressed in VSM, and recent studies have focused upon Wnt signaling in this cell type in cardiovascular disease[27, 72]. In addition to providing structure, ductility, and contractility, VSMs participate in local signal relays via endocrine/paracrine cues.…”

Harnessing low-density lipoprotein receptor protein 6 (LRP6) genetic variation and Wnt signaling for innovative diagnostics in complex diseases

“…was associated with carotid artery atherosclerosis and speculated that LRP6 rs2302685 was likely to be an independent risk factor of atherosclerotic plaque formation through retrospective analysis of 334 hypertensive patients. In addition, Cheng et al 44 studied the role of LRP6 in atherosclerosis in the SM22-Cre;LRP6(fl/fl); LDLR − / − animal model, conditionally depleting LRP6 in the VSM lineage in male LDLR-null mice; the authors discovered that the conditional knockout of LRP6 enhanced aortic calcification and vessel stiffening. One explanation may be that non-canonical Wnt signals promoted mature tissue calcification, whereas LRP6 restrained this pathway in part by sequestering certain Fz receptors.…”