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Abstract Background Typical bone proteins, such as sclerostin and periostin, have been associated with cardiovascular disease (CVD). Simultaneously, several risk scores have been developed to predict CVD in the general population. Therefore, we aimed to evaluate the association of these bone proteins related to CVD, with the main vascular risk scales: Framingham Risk Score (FRS), REGICOR and SCORE2-Diabetes, in patients with type 2 diabetes. We focus in particular on the SCORE2-Diabetes algorithm, which predicts 10-year CVD risk and is specific to the study population. Methods This was a cross-sectional study including 104 patients with type 2 diabetes (62 ± 6 years, 60% males). Clinical data, biochemical measurements, and serum bioactive sclerostin and periostin levels were collected, and different risk scales were calculated. The association between bioactive sclerostin or periostin with the risk scales was analyzed. Results A positive correlation was observed between circulating levels of bioactive sclerostin (p 131 pmol/L showed 51.6% sensitivity and 78.6% specificity. Similarly, serum periostin levels > 1144 pmol/L had 64.5% sensitivity and 76.2% specificity. Conclusions Sclerostin and periostin are associated with vascular risk in the SCORE2-Diabetes algorithm, opening a new line of investigation to identify novel biomarkers of cardiovascular risk in the type 2 diabetes population. Graphical Abstract

Type 2 diabetes mellitus (T2DM) is one of the most common worldwide metabolic disorders, characterized by insulin resistance (IR) and defective insulin secretion by pancreatic β-cells which leads to multiple complications such as bone fragility. This complication might be influenced by other factors such as gender and cardiovascular disease (CVD). However, it is unclear why a certain T2DM group develops bone fragility, and what the molecular mechanism is. Metabolomics is a powerful approach to the study of human metabolism, especially in complex diseases such as T2DM. Thus, this study aimed to identify significant metabolites associated with bone fragility in T2DM patients. To achieve this, 81 individuals were enrolled and classified as T2DM patients (D, n=28), T2DM with bone fragility (D-Frag, n=25), or age-matched non-diabetic subjects (ND, n=28) as the control group. Serum samples were collected and analyzed using liquid chromatography and gas chromatography, both coupled to mass spectrometry (LC-MS and GC-MS, respectively). Samples were compared within four different scenarios: 1) the classical comparison of D vs ND to corroborate previous studies; 2) D-Frag vs D to explore the metabolites mainly associated with bone fragility; 3) the same comparison using male data (MD-Frag vs MD) to study as a more homogeneous model of bone fragility as in women, bone fragility could be mainly associated with hormonal stage and pregnancy; and 4) MD-Frag-CVD vs MD-CVD to explore the influence of bone fragility in the male-based model with CVD considering that most of the T2DM patients suffer from CVD. After analysis of these scenarios, our results suggest that acylcarnitines and glycerophospholipids, among other metabolites, are involved in the development of bone fragility in T2DM.

Abstract Background Sclerostin is an inhibitor of the Wnt/b-catenin pathway, which regulates bone formation, and can be expressed in vascular smooth muscle cells (VSMCs). Type 2 diabetes (T2D) is associated with an increased risk of cardiovascular disease (CVD) and increased serum and tissue expression of sclerostin. However, whether the role of sclerostin is detrimental or protective in the development of CVD is unknown. Therefore, our aims are to determine the level of sclerostin in T2D patients with/without CVD and in controls, both at serum and vascular tissue, and to analyze the role of sclerostin in VSMCs under calcified environments. Methods Cross-sectional study including 121 controls and 139 T2D patients with/without CVD (48/91). Sclerostin levels in serum were determined by ELISA, and sclerostin expression was analyzed by RT-qPCR and immunohistochemistry in calcified and non-calcified artery of lower limb from T2D patients (n = 7) and controls (n = 3). In vitro experiments were performed in VSMCs (mock and sclerostin overexpression) under calcifying conditions analyzing the sclerostin function by determination of calcium and phosphate concentrations, and quantification of calcium deposits by Alizarin Red. Proliferation and apoptosis were analyzed by MTT assay and flow cytometry, respectively. The regulation of the expression of genes involved in bone metabolism was determined by RT-qPCR. Results A significant increase in serum sclerostin levels in T2D patients with CVD compared to T2D patients without CVD and controls (p

IntroductionHypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity. Although the main characteristic of this disease is bone involvement, it presents a great genetic and clinical variability, which makes it a systemic disease.MethodsPatients were recruited based on biochemical assessments. Diagnosis was made by measuring serum ALP and pyridoxal 5-phosphate levels and finally by Sanger sequencing of the ALPL gene from peripheral blood mononuclear cells. Characterization of the new variants was performed by transfection of the variants into HEK293T cells, where ALP activity and cellular localization were measured by flow cytometry. The dominant negative effect was analyzed by co-transfection of each variant with the wild-type gene, measuring ALP activity and analyzing cellular localization by flow cytometry.ResultsTwo previously undescribed variants were found in the ALPL gene: leucine 6 to serine missense mutation (c.17T>C, L6S) affecting the signal peptide and threonine 167 deletion (c.498_500delCAC, T167del) affecting the vicinity of the active site. These mutations lead mainly to non-pathognomonic symptoms of HPP. Structural prediction and modeling tools indicated the affected residues as critical residues with important roles in protein structure and function. In vitro results demonstrated low TNSALP activity and a dominant negative effect in both mutations. The results of the characterization of these variants suggest that the pleiotropic role of TNSALP could be involved in the systemic effects observed in these patients highlighting digestive and autoimmune disorders associated with TNSALP dysfunction.ConclusionsThe two new mutations have been classified as pathogenic. At the clinical level, this study suggests that both mutations not only lead to pathognomonic symptoms of the disease, but may also play a role at the systemic level.

AimPatients with type 2 diabetes (T2DM) have more risk of bone fractures. However, areal bone mineral density (aBMD) by conventional dual-energy x-ray absorptiometry (DXA) is not useful for identifying this risk. This study aims to evaluate 3D-DXA parameters determining the cortical and trabecular compartments in patients with T2DM compared to non-diabetic subjects and to identify their determinants.Materials and methodsCase-control study in 111 T2DM patients (65.4 ± 7.6 years old) and 134 non-diabetic controls (64.7 ± 8.6-year-old). DXA, 3D-DXA modelling via 3D-Shaper software and trabecular bone score (TBS) were used to obtain aBMD, cortical and trabecular parameters, and lumbar spine microarchitecture, respectively. In addition, biochemical markers as 25-hydroxyvitamin d, type I procollagen N-terminal propeptide (P1NP), C-terminal telopeptide of type I collagen (CTX), and glycated haemoglobin (HbA1c) were analysed.ResultsMean-adjusted values showed higher aBMD (5.4%-7.7%, ES: 0.33-0.53) and 3D-DXA parameters (4.1%-10.3%, ES: 0.42-0.68) in the T2DM group compared with the control group. However, TBS was lower in the T2DM group compared to the control group (-14.7%, ES: 1.18). In addition, sex (β = 0.272 to 0.316) and body mass index (BMI) (β = 0.236 to 0.455) were the most consistent and positive predictors of aBMD (p ≤ 0.01). BMI and P1NP were negative predictors of TBS (β = -0.530 and -0.254, respectively, p ≤ 0.01), while CTX was a positive one (β = 0.226, p=0.02). Finally, BMI was consistently the strongest positive predictor of 3D-DXA parameters (β = 0.240 to 0.442, p

Hypophosphatasia (HPP) a rare disease caused by mutations in the ALPL gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ALPL results in high clinical heterogeneity, which makes it difficult to establish a specific HPP symptomatology. In the present study, three variants of ALPL gene with uncertain significance and no previously described (p.Del Glu23_Lys24, p.Pro292Leu and p.His379Asn) were identified in heterozygosis in patients diagnosed with HPP. These variants were characterized at phenotypic, functional and structural levels. All genetic variants showed significantly lower in vitro ALP activity than the wild-type (WT) genotype (p-value

The identification of common targets in Alzheimer’s disease (AD) and cardiovascular disease (CVD) in recent years makes the study of the CVD/AD axis a research topic of great interest. Besides aging, other links between CVD and AD have been described, suggesting the existence of common molecular mechanisms. Our study aimed to identify common targets in the CVD/AD axis. For this purpose, genomic data from calcified and healthy femoral artery samples were used to identify differentially expressed genes (DEGs), which were used to generate a protein–protein interaction network, where a module related to AD was identified. This module was enriched with the functionally closest proteins and analyzed using different centrality algorithms to determine the main targets in the CVD/AD axis. Validation was performed by proteomic and data mining analyses. The proteins identified with an important role in both pathologies were apolipoprotein E and haptoglobin as DEGs, with a fold change about +2 and −2, in calcified femoral artery vs healthy artery, respectively, and clusterin and alpha-2-macroglobulin as close interactors that matched in our proteomic analysis. However, further studies are needed to elucidate the specific role of these proteins, and to evaluate its function as biomarkers or therapeutic targets.

Cardiovascular diseases are a health problem throughout the world, especially in people with diabetes. The identification of cardiovascular disease biomarkers can improve risk stratification. Sclerostin is a modulator of the Wnt/β-catenin signalling pathway in different tissues, and it has recently been linked to vascular biology. The current study aimed to evaluate the relationship between circulating sclerostin levels and cardiovascular and non-cardiovascular mortality in individuals with and without type 2 diabetes. We followed up a cohort of 130 participants (mean age 56.8 years; 48.5% females; 75 with type 2 diabetes; 46 with prevalent cardiovascular disease) in which serum sclerostin levels were measured at the baseline. Time to death (both of cardiovascular and non-cardiovascular causes) was assessed to establish the relationship between sclerostin and mortality. We found that serum sclerostin concentrations were significantly higher in patients with prevalent cardiovascular disease (p

ObjectivesDickkopf-1 (DKK1) is a potent inhibitor of Wnt signalling, which exerts anabolic effects on bone and also takes part in the regulation of vascular cells. Our aims were to evaluate serum DKK1 in type 2 diabetes (T2DM) patients and to analyze its relationships with cardiovascular disease (CVD). We also evaluated the relationship between DKK1 and bone metabolism.DesignWe conducted a cross-sectional study in which we measured serum DKK1 (ELISA, Biomedica) in 126 subjects: 72 patients with T2DM and 54 non-diabetic subjects. We analysed its relationship with clinical CVD, preclinical CVD expressed as carotid intima media thickness (IMT), and bone metabolism.ResultsT2DM patients with CVD (P = 0,026) and abnormal carotid IMT (P = 0,038) had higher DKK1 concentrations. DKK1 was related to the presence of CVD in T2DM, independently of the presence of risk factors for atherosclerosis. Therefore, for each increase of 28 pg/ml of serum DKK1 there was a 6,2% increase in the risk of CVD in T2DM patients. The ROC curve analysis to evaluate the usefulness of DKK1 as a marker for high risk of CVD showed an area under the curve of 0,667 (95% CI: 0,538-0,795; P = 0,016). In addition, there was a positive correlation between serum DKK1 and spine bone mineral density in the total sample (r = 0,183; P = 0,048).ConclusionIn summary, circulating DKK1 levels are higher in T2DM with CVD and are associated with an abnormal carotid IMT in this cross-sectional study. DKK1 may be involved in vascular disease of T2DM patients.