Specific heterozygous variants in MGP lead to endoplasmic reticulum stress and cause spondyloepiphyseal dysplasia.

Matrix Gla protein (MGP) is a vitamin K-dependent post-translationally modified protein, highly expressed in vascular and cartilaginous tissues. It is a potent inhibitor of extracellular matrix mineralization. Biallelic loss-of-function variants in the MGP gene cause Keutel syndrome, an autosomal recessive disorder characterized by widespread calcification of various cartilaginous tissues and skeletal and vascular anomalies. In this study, we report four individuals from two unrelated families with two heterozygous variants in MGP, both altering the cysteine 19 residue to phenylalanine or tyrosine. These individuals present with a spondyloepiphyseal skeletal dysplasia characterized by short stature with a short trunk, diffuse platyspondyly, midface retrusion, progressive epiphyseal anomalies and brachytelephalangism. We investigated the cellular and molecular effects of one of the heterozygous deleterious variants (C19F) using both cell and genetically modified mouse models. Heterozygous 'knock-in' mice expressing C19F MGP recapitulate most of the skeletal anomalies observed in the affected individuals. Our results suggest that the main underlying mechanism leading to the observed skeletal dysplasia is endoplasmic reticulum stress-induced apoptosis of the growth plate chondrocytes. Overall, our findings support that heterozygous variants in MGP altering the Cys19 residue cause autosomal dominant spondyloepiphyseal dysplasia, a condition distinct from Keutel syndrome both clinically and molecularly.

Urotensin II, urotensin-related peptide, and their receptor in aortic valve stenosis.

OBJECTIVES: Aortic valve stenosis (AVS) is the most common cause of surgical valve replacement worldwide. The vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis and several other cardiovascular diseases; however, its role in the pathogenesis of AVS remains to be determined. Here, we investigated the expression of UII, urotensin-related peptide (URP), and the urotensin receptor (UT) and the role this system plays in AVS. METHODS: Immunohistochemistry and reverse-transcriptase polymerase chain reaction were used to examine the cellular localization and mRNA expression, of UII, URP, and UT in calcified and noncalcified aortic valves. Human aortic valve interstitial cells were isolated from normal valves and treated with UII or URP, and changes in cell proliferation, cholesterol efflux, calcium deposition, and ß-catenin translocation were assessed. RESULTS: The mRNA expression of UII, URP, and UT was significantly greater in patients with AVS. There was abundant presence of UII, URP, and UT immunostaining in diseased compared with nondiseased valves and correlated significantly with presence of calcification (P < .0001) and fibrosis (P < .0001). Treating human aortic valve interstitial cells with UII or URP significantly increased cell proliferation (P < .0001) and decreased cholesterol efflux (P = .0011 and P = .0002, respectively). UII also significantly reduced ABCA1 protein expression (P = .0457) and increased ß-catenin nuclear translocation (P < .0001) and mineral deposition (P < .0001). CONCLUSIONS: Together, these data suggest that the urotensin system plays a role in the pathogenesis of AVS and warrants further investigation.

Elastin calcification in in vitro models and its prevention by MGP's N-terminal peptide.

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp-/-;Eln+/- mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N'-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP3) scaffold, where the peptide prevented mineral maturation. Overall, this work describes a novel cell culture model for elastocalcinosis and examines the inhibition of mineral deposition by the m3pS peptide in this and a cell-free elastin-based scaffold. Our study provides strong evidence suggesting the critical functional roles of MGP's phosphorylated serine residues in the prevention of elastin calcification and proposes a possible mechanism of their action.

Differences in mineral composition and morphology between men and women in aortic valve calcification.

Aortic valve calcification leads to the deposition of calcium phosphate minerals in the extracellular matrix of the aortic valve leaflets. The mineral deposits can severely narrow the opening of the aortic valve, leading to aortic stenosis. There are no therapies to halt or slow down disease progression and the mechanisms governing aortic valve calcification are still poorly understood. Recently, several studies have shown that for the same aortic stenosis severity, women present significantly lower calcification loads than men. The cause of this sex-related difference is unknown. To understand this difference, we analyzed mineral deposits from surgically excised calcified human aortic valves with different material characterization techniques. We find profound differences in mineral composition and morphology between sexes, which strongly suggest that minerals form slower in women than in men and follow a different mineralization pathway. This finding paves the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification. STATEMENT OF SIGNIFICANCE: Aortic valve calcification is a health disorder with increasing prevalence and high morbidity and mortality. Currently there is no approved effective treatment; the only available therapeutic option is invasive valve replacement, to which not all patients are suited. The main reason for such lack of treatment options is our lack of understanding of the calcification mechanism. In this study, we show profound differences in mineral composition and morphology between sexes, suggesting that aortic valve calcification follows different mineralization pathways in men and women. These findings pave the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification.

Effect of the Ionic Concentration of Simulated Body Fluid on the Minerals Formed on Cross-Linked Elastin-Like Polypeptide Membranes.

Deposition of calcium phosphate minerals on the elastin-rich medial layers of arteries can cause severe cardiovascular complications. There are no available treatments for medial calcification, and the mechanism of mineral formation on elastin layers is still unknown. We recently developed an in vitro model of medial calcification using cross-linked elastin-like polypeptide (ELP) membranes immersed in simulated body fluid (SBF). While mineral phase evolution matched that observed in a mouse model of medial calcification, the long incubation required was a practical limitation of this model. Using higher SBF ion concentrations could be a solution to speed up mineral deposition, but its effect on the mineralization process is still not well understood. Here we analyze mineral formation and phase transformation on ELP membranes immersed in high concentration SBF. We show that while mineral deposition is significantly accelerated in these conditions, the chemistry and morphology of the minerals deposited on the ELP membranes and the overall mineralization process are strongly affected. Overall, this work suggests that while the use of low concentration SBF in this in vitro model is more appropriate to study medial calcification associated with the loss of calcification inhibitors, higher SBF ion concentration may be more relevant to study medial calcification in patients with life-threatening diseases such as chronic kidney disease.

Cross-Linked Elastin-like Polypeptide Membranes as a Model for Medial Arterial Calcification.

Calcium phosphate minerals deposit on the elastin-rich medial layers of arteries in the majority of seniors, diabetic, and chronic kidney disease patients, causing severe cardiovascular complications. There is no cure for medial calcification, and the mechanism of mineral formation on elastin layers is unknown. Here we propose cross-linked elastin-like polypeptide membranes as models to study medial calcification. Calcium phosphates deposit first on fibers and filaments and then spread to globular structures present in the membranes. Mineral phase evolution analyzed by near-edge X-ray spectroscopy matches that previously observed in a mouse model of medial calcification, showing that this simple system captures some of the key in vivo findings. This work shows how minerals form and evolve upon nucleation on elastin and provides an in vitro model that can be tuned to study hypotheses related to arterial calcification mechanisms and test drugs to stop or revert mineralization.

Raman Spectroscopy for Inverted Papilloma: A Proof-of-Concept Study.

Inverted papillomas are tumors of the sinonasal tract with a propensity to recur. Raman spectroscopy can potentially identify inverted papillomas from other tissue based on biochemical signatures. A pilot study comparing Raman spectroscopy to histopathology for 3 types of sinonasal tissue was performed. Spectral data of biopsies from patients with normal sinonasal mucosa, chronic rhinosinusitis, and inverted papillomas are compared to histopathology using principal component analysis and linear discriminant analysis after data preprocessing. A total of 18 normal, 15 chronic rhinosinusitis, and 18 inverted papilloma specimens were evaluated. The model distinguished normal sinonasal mucosa, chronic rhinosinusitis, and inverted papilloma tissue with an overall accuracy of 90.2% (95% confidence interval, 0.86-0.94). In conclusion, Raman spectroscopy can distinguish inverted papilloma, normal sinonasal mucosa, and chronically rhinosinusitis tissue with acceptable accuracy.

Role of Noncanonical Wnt Signaling Pathway in Human Aortic Valve Calcification.

OBJECTIVE: The mechanisms underlying the pathogenesis of aortic valve calcification remain unclear. With accumulating evidence demonstrating that valve calcification recapitulates bone development, the crucial roles of noncanonical Wnt ligands WNT5a, WNT5b, and WNT11 in osteogenesis make them critical targets in the study of aortic valve calcification. APPROACH AND RESULTS: Using immunohistochemistry, real-time qPCR, Western blotting, and tissue culture, we examined the tissue distribution of WNT5a, WNT5b, and WNT11 in noncalcified and calcified aortic valves and their effects on human aortic valve interstitial cells (HAVICs). Only focal strong immunostaining for WNT5a was seen in and around areas of calcification. Abundant immunostaining for WNT5b and WNT11 was seen in inflammatory cells, fibrosis, and activated myofibroblasts in areas of calcified foci. There was significant correlation between WNT5b and WNT11 overall staining and presence of calcification, lipid score, fibrosis, and microvessels (P<0.05). Real-time qPCR and Western blotting revealed abundant expression of both Wnts in stenotic aortic valves, particularly in bicuspid valves. Incubation of HAVICs from noncalcified valves with the 3 noncanonical Wnts significantly increased cell apoptosis and calcification (P<0.05). Treatment of HAVICs with the mitogen-activated protein kinase-38ß and GSK3ß inhibitors significantly reduced their mineralization (P<0.01). Raman spectroscopy identified the inorganic phosphate deposits as hydroxyapatite and showed a significant increase in hydroxyapatite deposition in HAVICs in response to WNT5a and WNT11 (P<0.05). Similar crystallinity was seen in the deposits found in HAVICs treated with Wnts and in calcified human aortic valves. CONCLUSIONS: These findings suggest a potential role for noncanonical Wnt signaling in the pathogenesis of aortic valve calcification.