Wnt16 attenuates TGFß-induced chondrogenic transformation in vascular smooth muscle.

OBJECTIVE: Phenotypic plasticity of vascular smooth muscle cells (VSMCs) contributes to cardiovascular disease. Chondrocyte-like transformation of VSMCs associates with vascular calcification and underlies the formation of aortic cartilaginous metaplasia induced in mice by genetic loss of matrix Gla protein (MGP). Previous microarray analysis identified a dramatic downregulation of Wnt16 in calcified MGP-null aortae, suggesting an antagonistic role for Wnt16 in the chondrogenic transformation of VSMCs. APPROACH AND RESULTS: Wnt16 is significantly downregulated in MGP-null aortae, before the histological appearance of cartilaginous metaplasia, and in primary MGP-null VSMCs. In contrast, intrinsic TGFß is activated in MGP-null VSMCs and is necessary for spontaneous chondrogenesis of these cells in high-density micromass cultures. TGFß3-induced chondrogenic transformation in wild-type VSMCs associates with Smad2/3-dependent Wnt16 downregulation, but Wnt16 does not suppress TGFß3-induced Smad activation. In addition, TGFß3 inhibits Notch signaling in wild-type VSMCs, and this pathway is downregulated in MGP-null aortae. Exogenous Wnt16 stimulates Notch activity and attenuates TGFß3-induced downregulation of Notch in wild-type VSMCs, prevents chondrogenesis in MGP-null and TGFß3-treated wild-type VSMCs, and stabilizes expression of contractile markers of differentiated VSMCs. CONCLUSIONS: We describe a novel TGFß-Wnt16-Notch signaling conduit in the chondrocyte-like transformation of VSMCs and identify endogenous TGFß activity in MGP-null VSMCs as a critical mediator of chondrogenesis. Our proposed model suggests that the activated TGFß pathway inhibits expression of Wnt16, which is a positive regulator of Notch signaling and a stabilizer of VSMC phenotype. These data advance the comprehensive mechanistic understanding of VSMC transformation and may identify a novel potential therapeutic target in vascular calcification.

Quercetin attenuates warfarin-induced vascular calcification in vitro independently from matrix Gla protein.

Warfarin can stimulate vascular calcification in vitro via activation of ß-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, ß-catenin activity is important because inhibition of ß-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of ß-catenin using the glycogen synthase kinase-3ß (GSK-3ß) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the ß-catenin pathway. Calcification in VSMCs induced by 10 µm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new ß-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

Transglutaminase inhibitors attenuate vascular calcification in a preclinical model.

OBJECTIVE: In vitro, transglutaminase-2 (TG2)-mediated activation of the ß-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo. METHODS AND RESULTS: The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of ß-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit ß-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells. CONCLUSIONS: Inhibition of the TG2/ß-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.

Transglutaminase 2-mediated activation of ß-catenin signaling has a critical role in warfarin-induced vascular calcification.

OBJECTIVE: Accumulating experimental evidence implicates ß-catenin signaling and enzyme transglutaminase 2 (TG2) in the progression of vascular calcification, and our previous studies have shown that TG2 can activate ß-catenin signaling in vascular smooth muscle cells (VSMCs). Here we investigated the role of the TG2/ß-catenin signaling axis in vascular calcification induced by warfarin. METHODS AND RESULTS: Warfarin-induced calcification in rat A10 VSMCs is associated with the activation of ß-catenin signaling and is independent of oxidative stress. The canonical ß-catenin inhibitor Dkk1, but not the Wnt antagonist Wif-1, prevents warfarin-induced activation of ß-catenin, calcification, and osteogenic transdifferentiation in VSMCs. TG2 expression and activity are increased in warfarin-treated cells, in contrast to canonical Wnt ligands. Vascular cells with genetically or pharmacologically reduced TG2 activity fail to activate ß-catenin in response to warfarin. Moreover, warfarin-induced calcification is significantly reduced on the background of attenuated TG2 both in vitro and in vivo. CONCLUSIONS: TG2 is a critical mediator of warfarin-induced vascular calcification that acts through the activation of ß-catenin signaling in VSMCs. Inhibition of canonical ß-catenin pathway or TG2 activity prevents warfarin-regulated calcification, identifying the TG2/ß-catenin axis as a novel therapeutic target in vascular calcification.

Binding of pro-migratory serum factors to electrospun PLLA nano-fibers.

Architecture of the poly(l-lactic acid) (PLLA) scaffolds is known to affect protein affinity and binding strength. Here, we demonstrate that nanofibrous electrospun PLLA scaffolds reversibly absorb the pro-migratory serum factors that stimulate migration of vascular smooth muscle via an NFkB-dependent mechanism. Further, we demonstrate that mesenchymal stem cells seeded on the PLLA scaffolds do not enhance muscle migration but may maintain the ability of induced cells to migrate in an NFkB-independent manner. These findings further support the promising application of PLLA scaffolds for therapeutic angiogenesis and vascular graft engineering.

Cellular functions of tissue transglutaminase.

Transglutaminase 2 (TG2 or tissue transglutaminase) is a highly complex multifunctional protein that acts as transglutaminase, GTPase/ATPase, protein disulfide isomerase, and protein kinase. Moreover, TG2 has many well-documented nonenzymatic functions that are based on its noncovalent interactions with multiple cellular proteins. A vast array of biochemical activities of TG2 accounts for its involvement in a variety of cellular processes, including adhesion, migration, growth, survival, apoptosis, differentiation, and extracellular matrix organization. In turn, the impact of TG2 on these processes implicates this protein in various physiological responses and pathological states, contributing to wound healing, inflammation, autoimmunity, neurodegeneration, vascular remodeling, tumor growth and metastasis, and tissue fibrosis. TG2 is ubiquitously expressed and is particularly abundant in endothelial cells, fibroblasts, osteoblasts, monocytes/macrophages, and smooth muscle cells. The protein is localized in multiple cellular compartments, including the nucleus, cytosol, mitochondria, endolysosomes, plasma membrane, and cell surface and extracellular matrix, where Ca(2+), nucleotides, nitric oxide, reactive oxygen species, membrane lipids, and distinct protein-protein interactions in the local microenvironment jointly regulate its activities. In this review, we discuss the complex biochemical activities and molecular interactions of TG2 in the context of diverse subcellular compartments and evaluate its wide ranging and cell type-specific biological functions and their regulation.

Nuclear ferritin: a ferritoid-ferritin complex in corneal epithelial cells.

PURPOSE: Ferritin is an iron storage protein that is generally cytoplasmic. However, in embryonic avian corneal epithelial (CE) cells, the authors previously observed that the ferritin was predominantly nuclear. They also obtained evidence that this ferritin protects DNA from oxidative damage by UV light and hydrogen peroxide and that the nuclear localization involves a tissue-specific nuclear transporter, termed ferritoid. In the present investigation, the authors have determined additional properties of the nuclear ferritoid-ferritin complexes. METHODS: For biochemical characterization, a combination of molecular sieve chromatography, immunoblotting, and nuclear-cytoplasmic fractionation was used; DNA binding was analyzed by electrophoretic mobility shift assay. RESULTS: The CE nuclear ferritin complex has characteristics that differentiate it from a "typical" cytoplasmic ferritin, including the presence of ferritin and ferritoid subunits; a molecular weight of approximately 260 kDa, which is approximately half that of cytoplasmic ferritin; its iron content, which is below our limits of detection; and its ability to bind to DNA. CONCLUSIONS: Within CE cell nuclei, ferritin and ferritoid are coassembled into stable complex(es) present in embryonic and adult corneas. Thus, ferritoid not only serves transiently as a nuclear transporter for ferritin, it remains as a component of a unique ferritoid-ferritin nuclear complex.

Effective expression of recombinant cytotoxic protein via its attachment to a polyglutamine domain.

Inadvertent cytotoxicity may hinder the expression of many recombinant proteins that are of industrial or medicinal importance. Here, we show that covalent binding of the influenza A cytotoxic protein M2 to a polyglutamine domain (polyQ-M2; QM2) results in significant delay of its cytotoxic effects when compared to wild-type protein (M2wt). We also show that while expression of recombinant M2wt from A/WSN/1933 strain could not be attained in vaccinia virus (VV), polyQ-M2 was successfully expressed in this system. Moreover, we demonstrate that in cell culture, the polyQ domain is cleaved off following 48 h of expression, thus releasing free and active M2. Similarly, we show the spontaneous cleavage and polyQ release from fusion with another distinct polypeptide, green fluorescent protein (GFP). Expression of M2 from QM2 construct was more prolonged than one based on M2wt-expressing construct, markedly exceeding it at the later time points. Therefore, cell death caused by a toxic polypeptide may be suppressed via genetic fusion with polyQ, resulting in its enhanced expression, followed by slow release of the free polypeptide from the fusion. Collectively, covalent fusion with polyQ or other aggregate-forming domains presents a novel approach for industrial production of cytotoxic proteins and also holds promise for gene therapy applications.