Calcium Markers Research Articles

Capsaicin inhibits aortic valvular interstitial cell calcification via the redox-sensitive NFκB/AKT/ERK1/2 pathway

Calcific aortic valve stenosis (CAVS), the third most prevalent cardiovascular disorder is known to impose a huge social and economic burden on patients. However, no pharmacotherapy has yet been established. Aortic valve replacement is the only treatment option, although its lifelong efficacy is not guaranteed and involves inevitable complications. So, there is a crucial need to find novel pharmacological targets to delay or prevent CAVS progression. Capsaicin is well known for its anti-inflammatory and antioxidant properties and has recently been revealed to inhibit arterial calcification. We thus investigated the effect of capsaicin in attenuating aortic valve interstitial cells (VICs) calcification induced by pro-calcifying medium (PCM). Capsaicin reduced the level of calcium deposition in calcified VICs, along with reductions in gene and protein expression of the calcification markers Runx2, osteopontin, and BMP2. Based on Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway analysis oxidative stress, AKT and AGE–RAGE signaling pathways were selected. The AGE–RAGE signaling pathway activates oxidative stress and inflammation-mediated pathways including ERK and NFκB signaling pathways. Capsaicin successfully inhibited oxidative stress- and reactive oxygen species-related markers NOX2 and p22phox. The markers of the AKT, ERK1/2, and NFκB signaling pathways, namely, phosphorylated AKT, ERK1/2, NFκB, and IκBα were upregulated in calcified cells, while being significantly downregulated upon capsaicin treatment. Capsaicin attenuates VICs calcification in vitro by inhibition of redox-sensitive NFκB/AKT/ERK1/2 signaling pathway, indicating its potential as a candidate to alleviate CAVS.

Calcium isotopes as a biomarker for vascular calcification in chronic kidney disease.

Calcium balance is abnormal in adults with chronic kidney disease (CKD) and is associated with the development of vascular calcification. It is currently not routine to screen for vascular calcification in CKD patients. In this cross-sectional study, we investigate whether the ratio of naturally occurring calcium (Ca) isotopes, 44Ca and 42Ca, in serum could be used as a noninvasive marker of vascular calcification in CKD. We recruited 78 participants from a tertiary hospital renal center: 28 controls, 9 subjects with mild-moderate CKD, 22 undertaking dialysis and 19 who received a kidney transplant. For each participant, systolic blood pressure, ankle brachial index, pulse wave velocity, and estimated glomerular filtration rate were measured, along with serum markers. Calcium concentrations and isotope ratios were measured in urine and serum. While we found no significant association between urine Ca isotope composition (noted δ44/42Ca) between the different groups, δ44/42Ca values in serum were significantly different between healthy controls, subjects with mild-moderate CKD and those undertaking dialysis (P<0.01). Receiver operative characteristic curve analysis shows that the diagnostic utility of serum δ44/42Ca for detecting medial artery calcification is very good (AUC=0.818, sensitivity 81.8% and specificity 77.3%, P<0.01), and performs better than existing biomarkers. Although our results will need to be verified in prospective studies across different institutions, serum δ44/42Ca has the potential to be used as an early screening test for vascular calcification.

Different vitamin K forms in hemodialysis patients: a simple dietary supplement to battle vascular calcification—randomized controlled trial

Background and aimVascular calcification is a significant risk factor for cardiovascular diseases in patients with end-stage renal disease, particularly those on hemodialysis. Previous research on vitamin K found that it had a positive on calcification markers. However, clinical data is still limited. This study aimed to compare the efficacy and safety of vitamin K2 versus vitamin k1 on a calcification regulator in hemodialysis patients.MethodsA prospective randomized placebo-controlled trial was conducted on 120 patients, who were divided into three groups; group 1: administered 10 mg of vitamin K1 (phytomenadione thrice weekly); group 2: administered 90 μg of vitamin k2 (MK-7); group 3: administered placebo for 3 months. Matrix Gla protein (MGP), calcium, phosphorous, and intact parathyroid hormone levels were measured.ResultsMK-7 significantly increased active MGP levels compared to phytomenadione and placebo groups (p<0.0001). No correlations were found between calcium, phosphorous, PTH, and MGP levels at baseline or after treatment.ConclusionVitamin k supplementation was effective and tolerable in modulating MGP in hemodialysis patients, with MK-7 outperforming phytomenadione.

Abdominal aortic calcification score can predict all-cause and cardiovascular mortality in maintenance hemodialysis patients

Purpose Abdominal aortic calcification (AAC) assessed by using standard lateral lumbar radiographs can be graded, and composite summary scores (range, 0–24) have been shown to be highly predictive of subsequent cardiovascular morbidity and mortality in hemodialysis (HD) patients. However, few studies have sought to determine the optimal AAC score cutoff values for the prediction of mortality among HD patients. Methods This retrospective cohort study included 408 hemodialysis patients. AAC severity was quantified by the AAC score, which was measured by lateral lumbar radiography with complete follow-up data from January 2015 to December 2021. We used receiver operating characteristic (ROC) analysis to find the cutoff AAC value for the prediction of mortality. The Kaplan–Meier method was used to analyze all-cause and cardiovascular mortality. Results The cutoff calcification score for the prediction of mortality was 4.5 (sensitivity, 67.3%; specificity, 70.4%). The patients with AAC scores above 4.5 had significantly higher all-cause (log-rank p < 0.001) and cardiovascular (log-rank p < 0.001) mortality rates than those with AAC scores below 4.5. In the multivariate regression analyses, an AAC score above 4.5 was a significant factor associated with all-cause mortality (HR: 2.079, p = 0.002) and cardiovascular mortality (HR: 2.610, p < 0.001). Conclusions AAC is a reliable aortic calcification marker. HD patients with an AAC score > 4.5 have significantly elevated all-cause and cardiovascular mortality compared with those with an AAC score ≤ 4.5. AAC was a better predictor than cardiac valve calcification for mortality in HD patients.

Natural history of Echinococcus granulosus microcyst development in long term in vitro culture and molecular and morphological changes induced by insulin and BMP-4.

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus is a disease of worldwide public health and economic importance. The determinants and underlying cellular mechanisms of CE development and fate in intermediate hosts are largely unknown. Hormones and cytokines such as insulin and BMP-4 are the key players in the development, differentiation, and apoptosis. In this study, we evaluated the long term natural history of E. granulosus microcysts in an vitro setting and the molecular and morphological changes induced by the growth factors, insulin and BMP4 during the development of metacestode stage of E. granulosus. E. granulosus protoscoleces were cultivated and the parasite development was followed in the long term mono-phasic culture for 105 days and the morphometric, molecular and immunohistochemical changes were evaluated, including the microcysts number and size, microcysts development and deformation rates as well as the markers of calcification (Alizarin Red staining) and apoptosis (BAX, BCL2, Caspase-3, Caspase-8 and TNF-α expression) in the microcysts. Also the biological, histological and molecular consequences of insulin and BMP-4 treatment on the parasite development were evaluated. Insulin and BMP-4 treatment of microcysts resulted in significant increase in microcyst formation, increased size, reduced apoptosis and deformation of the microcysts. Alizarin red staining of the microcysts treated with the insulin and BMP-4 confirmed that calcium deposition is significantly lower than the untreated microcysts. Also Alizarin Red staining and Immunohistochemistry of the microcysts indicates that calcium accumulation in deformed microcysts is higher than the normal ones on day 105. The microcysts began to wrinkle and the germinal layer was partially detached from the laminated layer on day 84. Results of the present study suggest that the degenerative changes in hydatid cysts can be slowed down by insulin and BMP-4, indicating that cellular factors and host hormones could contribute to the longevity of hydatid cysts. Significant evidences are provided suggesting that the microcysts cultivated in vitro can undergo calcification and apoptotic processes similar to what have been observed in the natural hydatid infection in the intermediate hosts.

Can Bone-Specific Alkaline Phosphatase be a Marker of Vascular Calcification in Type 2 Diabetes Mellitus?

Alkaline phosphatase (ALP) enzyme has been linked to vascular calcification. Unexplained elevations in serum ALP levels have been reported in patients with type 2 diabetes mellitus (T2DM). We assessed bone-specific alkaline phosphatase (BAP) levels in patients with T2DM who had unexplained ALP elevations and studied the association between BAP and other markers of vascular calcification. Patients with T2DM who had high serum ALP in the absence of known causes of ALP elevation were studied. The control group was T2DM patients with normal ALP. We measured the serum levels of BAP along with the leptin, fetuin-A, and vitamin K2 levels. Ankle-brachial index (ABI) was also measured in both groups. Serum BAP levels were significantly higher in the group with high ALP when compared with the normal ALP group. A significant positive correlation was present between BAP and serum fetuin-A as well as between BAP and Vit K2 levels. There was no correlation between BAP and serum leptin. ABI was comparable between the two groups. Patients with T2DM may have unexplained elevation in ALP due to an increase in BAP. Elevation in BAP may be associated with other markers of vascular calcification suggesting an increased risk of vascular calcification.

Lipid profile peculiarities and matrix Gla protein concentration in Ukrainian pediatric patients with heterozygous familial hypercholesterolemia

Atherosclerotic changes in the vascular walls begin early in childhood, especially in association with familial hypercholesterolemia (FH). The process may be subclinical, which nevertheless requires therapeutic and preventive measures. Purpose - to evaluate baseline lipid profiles, the thickness of carotid intima-media complexes, blood pressure indices and the association with concentration changes of dephosphorylated-uncarboxylated matrix Gla protein (dp-uc MGP) as a marker of subclinical arterial lesions in different age groups of pediatric patients with FH. Materials and methods. Children with heterozygous FH (n=15), stratified by age and sex, were included in the study. The control group consisted of healthy peers (n=21). Blood samples were analyzed to determine levels of total (TC), low-density (LDL-C), very-low-density (VLDL-C), high-density (HDL-C), remnant (rC) and non-high-density (non-HDL-C) cholesterol, triglycerides (TG), apolipoproteins A1 (apoA1) and B (apoB), lipoprotein (a), and dp-uc MGP. The intima-media complex thickness of the common carotid artery and blood pressure were measured in all study subjects. The obtained data were processed using the accepted methods of medical statistics and SAS® OnDemand for Academics. Results. Lipid profile changes in pediatric patients with FH were characterized by high levels of LDL-C, non-HDL-C and lipoprotein (a) in the 5-9 years age group; in the 10-14 years age group - high levels of LDL-C, TG, rC, non-HDL-C and lipoprotein (a); in the 15-18 years age group - high levels of LDL-C, TG, non-HDL-C and lipoprotein (a). At the same time, the most marked dyslipidemia changes were evident in children aged 10-14 years in the FH group. apoA1 levels were significantly decreased in all FH children. Elevated levels of lipoprotein (a) (&gt;30 mg/dL) in FH children were found in all age groups, suggesting that elevated lipoprotein (a) levels can be used as a factor for cardiovascular risk stratification. Dp-uc MGP levels were significantly elevated in all age groups of FH children compared to healthy peers. Conclusions. A lipid profile examination is necessary to diagnose FH in children, along with family health history and cascade screening. As atherosclerotic changes at 5-18 years of age remain subclinical, and the instrumental tests available in routine medical practice are not sensitive enough to detect them, therefore, preventive or therapeutic measures cannot be initiated promptly. The evaluation of circulating matrix Gla protein in pediatric patients with FH can be used as a marker of vascular wall calcification, which may allow early preventive measures against microcalcification to be developed. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Imbalance in Bone Morphogenic Proteins 2 and 7 Is Associated with Renal and Cardiovascular Damage in Chronic Kidney Disease.

Arterial stiffness is a major vascular complication of chronic kidney disease (CKD). The development of renal damage, hypertension, and increased pulse wave velocity (PWV) in CKD might be associated with an imbalance in bone morphogenetic proteins (BMP)-2 and BMP-7. Plasma BMP-2 and BMP-7 were determined by ELISA in CKD patients (stages I-III; n = 95) and Munich Wistar Frömter (MWF) rats. Age-matched Wistar rats were used as a control. The expression of BMP-2, BMP-7, and profibrotic and calcification factors was determined in kidney and perivascular adipose tissues (PVAT). BMP-2 was higher in stage III CKD patients compared to control subjects. BMP-7 was lower at any CKD stage compared to controls, with a significant further reduction in stage III patients. A similar imbalance was observed in MWF rats together with the increase in systolic (SBP) and diastolic blood pressure (DBP), or pulse wave velocity (PWV). MWF exhibited elevated urinary albumin excretion (UAE) and renal expression of BMP-2 or kidney damage markers, Kim-1 and Ngal, whereas renal BMP-7 was significantly lower than in Wistar rats. SBP, DBP, PWV, UAE, and plasma creatinine positively correlated with the plasma BMP-2/BMP-7 ratio. Periaortic and mesenteric PVAT from MWF rats showed an increased expression of BMP-2 and profibrotic and calcification markers compared to Wistar rats, together with a reduced BMP-7 expression. BMP-2 and BMP-7 imbalance in plasma, kidney, and PVATs is associated with vascular damage, suggesting a profibrotic/pro-calcifying propensity associated with progressive CKD. Thus, their combined analysis stratified by CKD stages might be of clinical interest to provide information about the degree of renal and vascular damage in CKD.

Serum Calcification Propensity Represents a Good Biomarker of Vascular Calcification: A Systematic Review.

Vascular calcification contributes to cardiovascular morbidity and mortality. A recently developed serum calcification propensity assay is based on the half-transformation time (T50) from primary calciprotein particles (CPPs) to secondary CPPs, reflecting the serum’s endogenous capacity to prevent calcium phosphate precipitation. We sought to identify and review the results of all published studies since the development of the T50-test by Pasch et al. in 2012 (whether performed in vitro, in animals or in the clinic) of serum calcification propensity. To this end, we searched PubMed, Elsevier EMBASE, the Cochrane Library and Google Scholar databases from 2012 onwards. At the end of the selection process, 57 studies were analyzed with regard to the study design, sample size, characteristics of the study population, the intervention and the main results concerning T50. In patients with primary aldosteronism, T50 is associated with the extent of vascular calcification in the abdominal aorta. In chronic kidney disease (CKD), T50 is associated with the severity and progression of coronary artery calcification. T50 is also associated with cardiovascular events and all-cause mortality in CKD patients, patients on dialysis and kidney transplant recipients and with cardiovascular mortality in patients on dialysis, kidney transplant recipients, patients with ischemic heart failure and reduced ejection fraction, and in the general population. Switching from acetate-acidified dialysate to citrate-acidified dialysate led to a longer T50, as did a higher dialysate magnesium concentration. Oral administration of magnesium (in CKD patients), phosphate binders, etelcalcetide and spironolactone (in hemodialysis patients) was associated with a lower serum calcification propensity. Serum calcification propensity is an overall marker of calcification associated with hard outcomes but is currently used in research projects only. This assay might be a valuable tool for screening serum calcification propensity in at-risk populations (such as CKD patients and hemodialyzed patients) and, in particular, for monitoring changes over time in T50.

Reversal Of Arterial Disease by modulating Magnesium and Phosphate (ROADMAP-study): rationale and design of a randomized controlled trial assessing the effects of magnesium citrate supplementation and phosphate-binding therapy on arterial stiffness in moderate chronic kidney disease

BackgroundArterial stiffness and calcification propensity are associated with high cardiovascular risk and increased mortality in chronic kidney disease (CKD). Both magnesium and phosphate are recognized as modulators of vascular calcification and chronic inflammation, both features of CKD that contribute to arterial stiffness. In this paper, we outline the rationale and design of a randomized controlled trial (RCT) investigating whether 24 weeks of oral magnesium supplementation with or without additional phosphate-binding therapy can improve arterial stiffness and calcification propensity in patients with stage 3–4 CKD.MethodsIn this multi-center, placebo-controlled RCT, a total of 180 participants with an estimated glomerular filtration rate of 15 to 50 ml/min/1.73 m2 without phosphate binder therapy will be recruited. During the 24 weeks intervention, participants will be randomized to one of four intervention groups to receive either magnesium citrate (350 mg elemental magnesium/day) or placebo, with or without the addition of the phosphate binder sucroferric oxyhydroxide (1000 mg/day). Primary outcome of the study is the change of arterial stiffness measured by the carotid-femoral pulse wave velocity over 24 weeks. Secondary outcomes include markers of calcification and inflammation, among others calcification propensity (T50) and high-sensitivity C-reactive protein. As explorative endpoints, repeated 18F-FDG and 18F-NaF PET-scans will be performed in a subset of participants (n = 40). Measurements of primary and secondary endpoints are performed at baseline, 12 and 24 weeks.DiscussionThe combined intervention of magnesium citrate supplementation and phosphate-lowering therapy with sucroferric oxyhydroxide, in stage 3–4 CKD patients without overt hyperphosphatemia, aims to modulate the complex and deregulated mineral metabolism leading to vascular calcification and arterial stiffness and to establish to what extent this is mediated by T50 changes. The results of this combined intervention may contribute to future early interventions for CKD patients to reduce the risk of CVD and mortality.Trial registrationNetherlands Trial Register, NL8252 (registered December 2019), EU clinical Trial Register 2019-001306-23 (registered November 2019).

Integrin-Linked Kinase Expression in Human Valve Endothelial Cells Plays a Protective Role in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is highly prevalent during aging. CAVD initiates with endothelial dysfunction, leading to lipid accumulation, inflammation, and osteogenic transformation. Integrin-linked kinase (ILK) participates in the progression of cardiovascular diseases, such as endothelial dysfunction and atherosclerosis. However, ILK role in CAVD is unknown. First, we determined that ILK expression is downregulated in aortic valves from patients with CAVD compared to non-CAVD, especially at the valve endothelium, and negatively correlated with calcification markers. Silencing ILK expression in human valve endothelial cells (siILK-hVECs) induced endothelial-to-mesenchymal transition (EndMT) and promoted a switch to an osteoblastic phenotype; SiILK-hVECs expressed increased RUNX2 and developed calcified nodules. siILK-hVECs exhibited decreased NO production and increased nitrosative stress, suggesting valvular endothelial dysfunction. NO treatment of siILK-hVECs prevented VEC transdifferentiation, while treatment with an eNOS inhibitor mimicked ILK-silencing induction of EndMT. Accordingly, NO treatment inhibited VEC calcification. Mechanistically, siILK-hVECs showed increased Smad2 phosphorylation, suggesting a TGF-β-dependent mechanism, and NO treatment decreased Smad2 activation and RUNX2. Experiments performed in eNOS KO mice confirmed the involvement of the ILK-eNOS signaling pathway in valve calcification, since aortic valves from these animals showed decreased ILK expression, increased RUNX2, and calcification. Our study demonstrated that ILK endothelial expression participates in human CAVD development by preventing endothelial osteogenic transformation.

Abstract P3059: Hydrogen Sulfide Inhibits Aortic Valve Fibrosis, Inflammation And Calcification By Inducing Pro-autophagy Effect In Human Valvular Interstitial Cells

Background: As of now, there is no pharmacological treatment for aortic valve stenosis (AS) except for valve replacement. In the previously studied proteomics analysis, we demonstrated that the expression of sulfide:quinone oxidoreductase (SQOR) was significantly lower in aortic valve interstitial cells (VICs) isolated from AS patients than those of normal patients. However, the role of SQOR in AS pathophysiology has not been clarified yet. Methods: To elucidate the cellular mechanism of SQOR and hydrogen sulfide (H2S) as therapeutic aspects in AS, we studied the effects of NaHS (H2S-releasing compound) on diseased aortic VICs using real-time PCR and western blots. Results: NaHS treatment led to increased SQOR expression in diseased aortic VICs and induced an increase of the Nrf2-target genes, especially NQO1. It showed that the effects of NaHS decreased not only fibrosis and inflammation markers (MMP9, TNF-α, IL6) but also calcification markers (ALP, osteocalcin, RUNX2, COL1A1). In addition, AMPK and Beclin-1 significantly increased by NaHS while Akt and mTOR decreased which is expected to have pro-autophagy effect. Conclusion: H2S releasing compound enhances the expression of SQOR and activates AMPK/mTOR mediated pro-autophagy function resulting in inhibition of fibrosis, inflammation and calcification in human VICs. Therefore, H2S can be a potential therapeutic target in AS.

Assesment of relationship between sarcopenia and vascular calcification in patients with severe aortic stenosis

Background and Aims : Sarcopenia is a progressive disorder involving the accelerated loss of muscle mass and function. Sarcopenia becomes more frequent by ageing. Sarcopenia is associated with inflammation and increased risk for coronary artery disease. Sarcopenia may be diagnosed by several techniques including measurement of spinal muscle area in computed tomography (CT) images. We aimed to evaluate relationship between sarcopenia and vascular calcification in patients scheduled for TAVR.Methods: All of the patients who underwent CT for TAVR planning between 2013 and 2020 were screened and skeletal muscle area at L3 vertebrae level was measured. Parameters regarding vascular calcification were also measured from CT images.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Conclusions: Sarcopenia is an important marker of vascular calcification and it may be measured easily from CT images performed for any indication. MAC and all-cause mortality rate during follow-uo are more frequent in patients with sarcopenia. Background and Aims : Sarcopenia is a progressive disorder involving the accelerated loss of muscle mass and function. Sarcopenia becomes more frequent by ageing. Sarcopenia is associated with inflammation and increased risk for coronary artery disease. Sarcopenia may be diagnosed by several techniques including measurement of spinal muscle area in computed tomography (CT) images. We aimed to evaluate relationship between sarcopenia and vascular calcification in patients scheduled for TAVR. Methods: All of the patients who underwent CT for TAVR planning between 2013 and 2020 were screened and skeletal muscle area at L3 vertebrae level was measured. Parameters regarding vascular calcification were also measured from CT images. Conclusions: Sarcopenia is an important marker of vascular calcification and it may be measured easily from CT images performed for any indication. MAC and all-cause mortality rate during follow-uo are more frequent in patients with sarcopenia.

Sex-Related Signaling of Aldosterone/Mineralocorticoid Receptor Pathway in Calcific Aortic Stenosis

There are sex differences in the pathophysiology of aortic valve (AV) calcification in patients with aortic stenosis, although the molecular and cellular mechanisms have not been elucidated. Aldosterone (Aldo) promotes proteoglycan synthesis in valve interstitial cells (VICs) from mitral valves via the mineralocorticoid receptor (MR). We investigated the influence of sex in the role of Aldo/MR pathway in AV alterations in patients with aortic stenosis. MR was expressed by primary aortic VICs and in AVs from patients with aortic stenosis. MR expression positively correlated with VIC activation markers in AVs from both sexes. However, MR expression was positively associated with molecules involved in AV calcification only in AV from men. Aldo enhanced VIC activation markers in cells from men and women. Interestingly, Aldo increased the expression of calcification markers only in VICs isolated from men. In female VICs, Aldo enhanced fibrotic molecules. MR antagonism (spironolactone) blocked all the above effects. Cytokine arrays showed ICAM (intercellular adhesion molecule)-1 and osteopontin to be specifically increased by Aldo in male VICs. In AVs from men, MR expression positively associated with both ICAM-1 (intercellular adhesion molecule-1) and osteopontin. Only in female VICs, estradiol treatment blocked Aldo-induced VICs activation, inflammation, and fibrosis. These findings demonstrate that the Aldo/MR pathway could play a role in early stages of aortic stenosis by promoting VICs activation, fibrosis, and ulterior calcification. Importantly, Aldo/MR pathway is involved in fibrosis in women and in early AV calcification only in men. Accordingly, MR antagonism emerges as a new sex-specific pharmacological treatment to prevent AV alterations.

Targeting Fatty Acid-Binding Protein 4 Improves Pathologic Features of Aortic Stenosis.

Aortic stenosis (AS) is a fibrocalcific disease of the aortic valves (AVs). Sex-differences in AS pathophysiology have recently been described. High levels of fatty acid-binding protein 4 (FAPB4) in atherosclerotic plaques have been associated with increased local inflammation, endothelial dysfunction, and plaque vulnerability. FABP4 pharmacological blockade has been shown to be effective for the treatment of atherosclerosis by modulating metabolic and inflammatory pathways. We aimed to analyze the sex-specific expression of FABP4 in AS and its potential role as a therapeutic target. A total of 226 patients (61.5% men) with severe AS undergoing surgical AV replacement were recruited. The FABP4 levels were increased in the AVs of AS patients compared to the control subjects, showing greater expression in the fibrocalcific regions. Male AVs exhibited higher levels of FABP4 compared to females, correlating with markers of inflammation (IL-6, Rantes), apoptosis (Bax, caspase-3, Bcl-2), and calcification (IL-8, BMP-2 and BMP-4). VICs derived from AS patients showed the basal expression of FABP4 in vitro. Osteogenic media induced upregulation of intracellular and secreted FABP4 levels in male VICs after 7 days, along with increased levels of inflammatory, pro-apoptotic, and osteogenic markers. Treatment with BMS309403, a specific inhibitor of FABP4, prevented from all of these changes. Thus, we propose FABP4 as a new sex-specific pharmacological therapeutic target in AS.

Proteomic Analysis of Serum Proteins from Patients with Severe Coronary Artery Calcification.

Proteomic studies investigating novel molecular markers of coronary artery calcification (CAC) are scarce.This study compared the protein expression in the serum of patients with severe CAC and non-CAC. The serum from 30 patients with severe CAC and 30 matched-controls were screened by data-independent acquisition(DIA)-based proteomic technology. Bioinformatics analysis tools were used to analyze the underlying molecular mechanisms of the differentially expressed proteins. Candidate proteins were further validated by an enzyme-linked immunosorbent assay (ELISA) in an independent cohort. A receiver operating characteristic (ROC) curve was used to estimate the diagnostic power of the candidate proteins. Among the 110 identified proteins, the expression of 81 was significantly upregulated, whereas 29 proteins were downregulated (fold change 1.5; p 0.05) between patients with and without CAC. Bioinformatics analysis indicated that the differential proteins are involved in complement and coagulation cascades, platelet activation, regulation of actin cytoskeleton, or glycolysis/gluconeogenesis pathways. Further verification showed that serum levels of complement C5 (C5), fibrinogen gamma (FGG), pyruvate kinase isoform M2 (PKM2), and tropomyosin 4 (TPM4) were consistent with the proteomic findings, which could allow discrimination between CAC and non-CAC patients. This study revealed that high serum levels of serum C5, FGG, PKM2, and TPM4 proteins were linked to severe CAC. These proteins may be developed as biomarkers to predict coronary calcification.

Dysregulation of the endomembrane homeostasis triggers vascular smooth muscle cell phenotype switching

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft Background Cardiovascular diseases are a leading cause of mortality globally, with cardiovascular calcification as a prominent predictor and contributor. Notably, microcalcifications might cause atherosclerotic plaque rupture, leading to myocardial infarction. Calcified plaque remodeling accompanies phenotypic modulation of vascular smooth muscle cells (SMCs). Previously, we showed that extracellular vesicles (EVs) released from calcifying SMCs are pivotal for microcalcification formation. EVs originate from the endolysosomal system. The lipid kinase FYVE-type zinc finger containing phosphoinositide kinase (PIKfyve) plays a role in the endolysosomal maturation. Here, we investigated how modulation of the endolysosomal system intersects with SMC phenotype regulation. Methods and Results In calcified arteries and SMCs, we observed increased PIKfyve expression. Inhibition of PIKfyve with the small molecule Apilimod inhibited osteogenic differentiation of SMCs in an in vitro calcification model. Apilimod reduced tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA (-79%, p&amp;lt;0.01), protein (-96%, p&amp;lt;0.01) and activity (-92%, p&amp;lt;0.01) levels. Additionally, matrix mineralization and collagen network formation are inhibited. SMC-secreted EVs exhibited a lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Transcriptome analyses revealed that the anti-calcifying effect is accompanied by a decreased expression of contractile SMC genes. Validation by RT-PCR supported that SMC-specific markers (ACTA2, SM22) and the key SMC transcription factor MYOCD were downregulated, while the expression of macrophage-like SMC markers (LGALS3, CD68) was induced. On the other hand, Apilimod increased the expression of adipogenic transcription factors (CEBPA, PPARG) and genes of the cholesterol and fatty acid metabolism pathways (SREBF2, HMGCR, FABP3, CD36) and induced the nuclear translocation of PPARγ, the main regulator of adipogenic differentiation. Fluorescence-based assays revealed increased fatty acid and disrupted low-density lipoprotein (LDL) uptake in Apilimod-treated SMCs. In Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks and treated with Apilimod for 5 weeks, we found differentially distributed plaque lipid depositions detected by Oil Red O staining. Conclusion Disrupting the endolysosomal maturation by PIKfyve inhibition ameliorates SMC calcification and induces a phenotypic adipogenic-like, pro-inflammatory adaption. Our data elucidates the importance of SMC phenotype monitoring during therapeutic interference. The exact lineage characteristics of these SMCs remain to be further investigated.

IMBALANCE IN BONE MORPHOGENIC PROTEINS 2 AND 7 IS ASSOCIATED WITH HYPERTENSION AND ARTERIAL STIFFNESS IN CHRONIC KIDNEY DISEASE

Objective: Patients with chronic kidney disease (CKD) exhibit an elevated cardiovascular risk from the early stages (I-III) with an exponentially increasing risk of cardiovascular death as kidney function worsens. The hypothesis of this study is that there is a imbalance in bone morphogenetic proteins BMP-2 (procalcifying) and BMP-7 (renoprotective) at early CKD stages associated with the development of hypertension, vascular stiffness and progressive kidney damage. Design and method: BMP-2 and BMP-7 levels were determined by ELISA in samples of CKD patients (stages I-III; n = 95) and of Munich Wistar Frömter (MWF) rats (22 weeks old; n = 6), a genetic model with spontaneous nondiabetic CKD, compared to age-matched Wistar rats. Results: Plasma BMP-2 levels were significantly higher in stage III CKD patients compared to controls. BMP-7 was, however, significantly lower at any stage of CKD compared to the control group with a significant further reduction in stage III patients. MWF rats presented significantly higher BMP-2 plasma levels compared to Wistar rats, whereas BMP-7 level was lower. The MWF strain showed an elevation of both systolic (SBP) and diastolic blood pressure (DBP), as well as pulse wave velocity (PWV) compared to the Wistar group. A positive correlation was observed between the BMP-2/BMP-7 ratio and SBP, DBP, and PWV, respectively. Plasma 25-OH cholecalciferol concentration was lower in MWF showing a negative correlation with the BMP-2/BMP-7 plasma ratio. Urinary albumin excretion (UAE), tubular damage markers, Kim-1 and Ngal, as well as renal BMP-2 were significantly higher in the MWF group compared to Wistar, whereas renal BMP-7 expression was significantly lower. A positive correlation was detected between the BMP-2/BMP-7 ratio and both the UAE and plasma creatinine level. Periaortic and mesenteric perivascular adipose tissue (PVAT) from MWF rats showed an altered expression of BMP-2, BMP-7, profibrotic and calcification markers. Conclusions: Our data show an imbalance in plasma, kidney and PVAT-derived BMP-2 and BMP-7 levels associated with hypertension and arterial stiffness. This might reflect a profibrotic/pro-calcifying propensity related with progressive CKD.

WITHDRAWN: Wnt signaling pathway in the development of atherosclerosis: Sclerostin as a new surrogate marker of global vascular calcification?

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Utilité du score calcique coronariens et des marqueurs biologiques de calcification dans l'évaluation du risque cardiovasculaire individuel

AbstractQuantification of coronary calcification using a CT-scanner without contrast, makes it possible to calculate a calcification score (CAC). CAC improves individual cardiovascular risk (CVR) prediction compared to population models alone (Framingham, SCORE,.). A CAC of 0 excludes the risk of a CV event at ten years, whereas a CAC > 100, especially in an asymptomatic patient, allows to re-allocate the patient in a high-risk category requiring a medical follow up. The CAC is simple to implement, using low irradiation levels and intended to better predict the CVR risk at ten years of asymptomatic patients at intermediate risk. Besides CAC, several circulating biological markers such as Fetuine A, Matrix Gla Protein (MGP), Tissue-Non specific Alkaline Phosphatase (TNAP) or serum T50 which measures the propensity of the serum to calcify in-vitro, have been proposed to assess the risk of cardiovascular calcification. The values of these calcification markers are weakly associated with CAC but their clinical use for determining CVR has yet to be demonstrated. However, some of them, such as T50, are associated with cardiovascular mortality especially in patients with renal insufficiency and could therefore constitute a new independent and modifiable risk factor for cardiovascular risk.