Attenuation of chondrogenic transformation in vascular smooth muscle by dietary quercetin in the MGP-deficient mouse model.

Cartilaginous metaplasia of vascular smooth muscle (VSM) is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP). A better understanding of the molecular details of this process is critical for the development of novel therapeutic approaches to VSM transformation and arterial calcification.This study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-β3 stimulation in vitro, and to characterize the associated alterations in cell signaling.Molecular analysis revealed activation of β-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of β-catenin both in vivo and in vitro. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae.Quercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin

Transglutaminase 2 regulates early chondrogenesis and glycosaminoglycan synthesis

The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-β activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering

Quercetin attenuates chondrogenic transformation and β-catenin activation in primary VSMCs.

<p><b>A</b>, Quercetin reduces deposition of GAG-positive matrix in VSMCs induced to undergo chondrogensis in high-density micromass culture in chondrogenic medium (ChoM). Quantitation of GAG deposition (<i>left graph</i>) and cell death, detected by LDH release into the culture medium (<i>right graph</i>), in the presence or absence of 50 µmol/L quercetin. N=4. <b>B</b>-<b>C</b>, Real-time PCR analysis of markers of chondrogenesis (<b>B</b>) and β-catenin target genes (<b>C</b>) in VSMC micromass cultures treated with ChoM and quercetin as indicated. N=4. <b>D</b>, Quantitation of GAG deposition by VSMC micromass cultures treated with ChoM in the presence or absence of 0.5 µg/mL Dkk1. N=4.</p

Quercetin reduces calcium accrual in <i>Mgp-/-</i> mice.

<p><b>A</b>-<b>B</b>, Total calcium in aortic tissue from 5 week old untreated <i>Mgp-/</i>- mice (N=8) or <i>Mgp-/</i>- mice treated with 0.02% w/w dietary quercetin for 2 weeks after weaning (<b>A</b>; N=16) or for 5 weeks from birth through weaning (<b>B</b>; N=9). Heterzygous (<i>Mgp+/-</i>) mice served as control (N=6). <b>C</b>, Von Kossa stain for calcified matrix deposition in aortic tissue from <i>Mgp+/-</i>, untreated <i>Mgp-/</i>-, and quercetin-treated <i>Mgp-/</i>- animals. Sections from 2 representative animals are shown. Arrows denote chondrogenic metaplasia. Scale = 50 µm.</p

Quercetin prevents chondroplasia and improves vessel morphology in <i>Mgp-/-</i> mice.

<p><b>A</b>, Serial sections spaced 100 µm apart through a 1 mm segment of descending aorta from each animal were analyzed. <b>B</b>, Representative images of serial sections from <i>Mgp+/-</i> (N=3), untreated <i>Mgp-/</i>- (N=5), and quercetin-treated <i>Mgp-/</i>- (N=6) animals stained with Alcian blue. Dashed lines denote the proportional presence of chondroplastic lesions in the vessel wall. Scale = 50 µm. <b>C</b>-<b>D</b>, Quantitative analysis of the serial sections of aortae show that quercetin diet reduces the percentage of vessel circumference occupied by chondrogenic lesions (<b>C</b>) and the cross-sectional thickness of the medial aorta (<b>D</b>).</p