Unveiling cellular and molecular aspects of ascending thoracic aortic aneurysms and dissections.

Ascending thoracic aortic aneurysm (ATAA) remains a significant medical concern, with its asymptomatic nature posing diagnostic and monitoring challenges, thereby increasing the risk of aortic wall dissection and rupture. Current management of aortic repair relies on an aortic diameter threshold. However, this approach underestimates the complexity of aortic wall disease due to important knowledge gaps in understanding its underlying pathologic mechanisms.Since traditional risk factors cannot explain the initiation and progression of ATAA leading to dissection, local vascular factors such as extracellular matrix (ECM) and vascular smooth muscle cells (VSMCs) might harbor targets for early diagnosis and intervention. Derived from diverse embryonic lineages, VSMCs exhibit varied responses to genetic abnormalities that regulate their contractility. The transition of VSMCs into different phenotypes is an adaptive response to stress stimuli such as hemodynamic changes resulting from cardiovascular disease, aging, lifestyle, and genetic predisposition. Upon longer exposure to stress stimuli, VSMC phenotypic switching can instigate pathologic remodeling that contributes to the pathogenesis of ATAA.This review aims to illuminate the current understanding of cellular and molecular characteristics associated with ATAA and dissection, emphasizing the need for a more nuanced comprehension of the impaired ECM-VSMC network.

Mediterranean Diet Favors Vitamin K Intake: A Descriptive Study in a Mediterranean Population.

The Mediterranean diet (MD) is associated with improved longevity and the prevention and management of chronic inflammatory diseases (CIDs). Vitamin K, which is present in MD core components such as leafy green vegetables, is also known as a protective factor for CIDs. Estimates of vitamin K intake in Mediterranean settings are still scarce, and the association between MD and vitamin K intake is yet to be established. This study analyzed vitamin K intake and MD adherence in the Algarve region, in Portugal. We conducted a cross-sectional study in a nonrandom sample of adults using an online questionnaire which included a validated food-frequency questionnaire and a screener for MD adherence. A total of 238 participants were recruited (68% women and 32% men). Adherence to the MD was low (11%). Only 10% of the participants had vitamin K intake below the adequate intake. Adherence to the MD was positively correlated with vitamin K intake (r = 0.463; p < 0.001) and age (r = 0.223; p < 0.001). Our findings underscore the importance of promoting adherence to the MD for optimal vitamin K intake, and future research should focus on developing effective interventions to promote this dietary pattern, particularly among younger individuals and men.

Fundamental considerations for designing endothelialized in vitro models of thrombosis.

Endothelialized in vitro models for cardiovascular disease have contributed greatly to our current understanding of the complex molecular mechanisms underlying thrombosis. To further elucidate these mechanisms, it is important to consider which fundamental aspects to incorporate into an in vitro model. In this review, we will focus on the design of in vitro endothelialized models of thrombosis. Expanding our understanding of the relation and interplay between the different pathways involved will rely in part on complex models that incorporate endothelial cells, blood, the extracellular matrix, and flow. Importantly, the use of tissue-specific endothelial cells will help in understanding the heterogeneity in thrombotic responses between different vascular beds. The dynamic and complex responses of endothelial cells to different shear rates underlines the importance of incorporating appropriate shear in in vitro models. Alterations in vascular extracellular matrix composition, availability of bioactive molecules, and gradients in concentration and composition of these molecules can all regulate the function of both endothelial cells and perivascular cells. Factors modulating these elements in in vitro models should therefore be considered carefully depending on the research question at hand. As the complexity of in vitro models increases, so can the variability. A bottom-up approach to designing such models will remain an important tool for researchers studying thrombosis. As new techniques are continuously being developed and new pathways are brought to light, research question-dependent considerations will have to be made regarding what aspects of thrombosis to include in in vitro models.

Plasma Dephosphorylated-Uncarboxylated Matrix Gla-Protein in Systemic Sclerosis Patients: Biomarker Potential for Vascular Calcification and Inflammation.

BACKGROUND: Systemic sclerosis (SSc) patients face an elevated risk of cardiovascular disease (CVD), even when classic cardiovascular risk factors are considered. Plasma dephosphorylated-uncarboxylated Matrix Gla-protein (dp-ucMGP), an inactive form of MGP, is associated with increased CVD risk. Smooth muscle cells, implicated in SSc's development, are the primary dp-ucMGP producers. This study assessed dp-ucMGP levels and initial CVD events in early-diagnosed SSc patients, investigating its potential as a CVD and all-cause mortality predictor over time. METHODS: In a cohort of 87 SSc patients (excluding those with pre-existing CVD or on dialysis), baseline dp-ucMGP levels were measured, along with cardiovascular risk factors. Validation involved assessing dp-ucMGP in a subset of treatment-naive SSc patients. RESULTS: A significantly elevated median dp-ucMGP level of 634 pmol/L (IQR 301) compared with healthy controls (dp-ucMGP < 393 pmol/L; p < 0.001) was observed. Validation in a treatment-naive SSc patient subset yielded similar results (median 589 pmol/L; IQR 370). During a median 10.5-year follow-up among 78 SSc patients, 33.3% experienced their first CVD event, independent of traditional risk factors. Elevated dp-ucMGP levels (>634 pmol/L) correlated with a higher risk of CVD and/or death (log-rank test: p < 0.01). CONCLUSIONS: In summary, dp-ucMGP emerges as a novel biomarker in SSc patients, with elevated levels indicating an increased risk of CVD and/or mortality in this population.

Higher levels of circulating desphospho-uncarboxylated matrix Gla protein over time are associated with worse survival: the prospective Maastricht Intensive Care COVID cohort.

BACKGROUND: Extra-hepatic vitamin K-status, measured by dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP), maintains vascular health, with high levels reflecting poor vitamin K status. The occurrence of extra-hepatic vitamin K deficiency throughout the disease of COVID-19 and possible associations with pulmonary embolism (PE), and mortality in intensive care unit (ICU) patients has not been studied. The aim of this study was to investigated the association between dp-ucMGP, at endotracheal intubation (ETI) and both ICU and six months mortality. Furthermore, we studied the associations between serially measured dp-ucMGP and both PE and mortality. METHODS: We included 112 ICU patients with confirmed COVID-19. Over the course of 4 weeks after ETI, dp-ucMGP was measured serially. All patients underwent computed tomography pulmonary angiography (CTPA) to rule out PE. Results were adjusted for patient characteristics, disease severity scores, inflammation, renal function, history of coumarin use, and coronary artery calcification (CAC) scores. RESULTS: Per 100 pmol/L dp-ucMGP, at ETI, the odds ratio (OR) was 1.056 (95% CI: 0.977 to 1.141, p = 0.172) for ICU mortality and 1.059 (95% CI: 0.976 to 1.059, p = 0.170) for six months mortality. After adjustments for age, gender, and APACHE II score, the mean difference in plasma dp-ucMGP over time of ICU admission was 167 pmol/L (95% CI: 4 to 332, p = 0.047). After additional adjustments for c-reactive protein, creatinine, and history of coumarin use, the difference was 199 pmol/L (95% CI: 50 to 346, p = 0.010). After additional adjustment for CAC score the difference was 213 pmol/L (95% CI: 3 to 422, p = 0.051) higher in ICU non-survivors compared to the ICU survivors. The regression slope, indicating changes over time, did not differ. Moreover, dp-ucMGP was not associated with PE. CONCLUSION: ICU mortality in COVID-19 patients was associated with higher dp-ucMGP levels over 4 weeks, independent of age, gender, and APACHE II score, and not explained by inflammation, renal function, history of coumarin use, and CAC score. No association with PE was observed. At ETI, higher levels of dp-ucMGP were associated with higher OR for both ICU and six month mortality in crude and adjusted modes, although not statistically significantly.

Randomized Controlled Clinical Trial of the Effect of Treatment with Vitamin K2 on Vascular Calcification in Hemodialysis Patients (Trevasc-HDK).

Introduction: Vitamin K deficiency among patients on hemodialysis (HD) affects the function of matrix GLA protein (MGP), a potent vitamin K-dependent inhibitor of vascular calcification (VC). Methods: We conducted a single-center randomized controlled trial (RCT) on maintenance HD patients to examine if vitamin K2 supplementation can reduce progression of coronary artery calcification (CAC) over an 18-month study period. Patients were randomized to vitamin K2 group receiving menaquinone-7360 µg 3 times/wk or control group. The primary outcome was CAC scores at the end of the study period. The secondary outcomes were aortic valve calcification (AVC), carotid-femoral pulse wave velocity (cfPWV), aortic augmentation index (AIx), dephosphorylated undercarboxylated MGP (dp-ucMGP) levels, major adverse cardiac events (MACE), and vascular access events. Results: Of the 178 patients randomized, follow-up was completed for 138 patients. The CAC scores between the 2 groups were not statistically different at the end of 18 months (relative mean difference [RMD] 0.85, 95% CI 0.55-1.31). The secondary outcomes did not differ significantly in AVC (RMD 0.82, 95% CI 0.34-1.98), cfPWV (absolute mean difference [AMD] 0.55, 95% CI -0.50 to 1.60), and AIx (AMD 0.13, 95% CI -3.55 to 3.80). Supplementation with vitamin K2 did reduce dp-ucMGP levels (AMD -86, 95% CI -854 to -117). The composite outcome of MACE and mortality was not statistically different between the 2 groups (Hazard ratio = 0.98, 95% CI 0.50-1.94). Conclusion: Our study did not demonstrate a beneficial effect of vitamin K2 in reducing progression of VC in this population at the studied dose and duration.

The Maastricht Acquisition Platform for Studying Mechanisms of Cell-Matrix Crosstalk (MAPEX): An Interdisciplinary and Systems Approach towards Understanding Thoracic Aortic Disease.

Current management guidelines for ascending thoracic aortic aneurysms (aTAA) recommend intervention once ascending or sinus diameter reaches 5-5.5 cm or shows a growth rate of >0.5 cm/year estimated from echo/CT/MRI. However, many aTAA dissections (aTAAD) occur in vessels with diameters below the surgical intervention threshold of <55 mm. Moreover, during aTAA repair surgeons observe and experience considerable variations in tissue strength, thickness, and stiffness that appear not fully explained by patient risk factors. To improve the understanding of aTAA pathophysiology, we established a multi-disciplinary research infrastructure: The Maastricht acquisition platform for studying mechanisms of tissue-cell crosstalk (MAPEX). The explicit scientific focus of the platform is on the dynamic interactions between vascular smooth muscle cells and extracellular matrix (i.e., cell-matrix crosstalk), which play an essential role in aortic wall mechanical homeostasis. Accordingly, we consider pathophysiological influences of wall shear stress, wall stress, and smooth muscle cell phenotypic diversity and modulation. Co-registrations of hemodynamics and deep phenotyping at the histological and cell biology level are key innovations of our platform and are critical for understanding aneurysm formation and dissection at a fundamental level. The MAPEX platform enables the interpretation of the data in a well-defined clinical context and therefore has real potential for narrowing existing knowledge gaps. A better understanding of aortic mechanical homeostasis and its derangement may ultimately improve diagnostic and prognostic possibilities to identify and treat symptomatic and asymptomatic patients with existing and developing aneurysms.

The BioHybrid Assay: A Novel Method for Determining Calcification Propensity.

Vascular calcification is an active pathological process, characterised by cellular dysregulation and subsequent changes to the extracellular environment. In vivo detection of vascular calcification is only possible late stage via computed tomography, and there is no single biomarker for detecting progression of vascular calcification. There is an unmet clinical need to determine progression of vascular calcification in vulnerable patients. This is especially needed in chronic kidney disease (CKD) patients where there is a correlation of cardiovascular disease with declining renal status. We hypothesised that the entirety of circulating components should be taken into consideration with vessel wall cells to determine real-time vascular calcification development. In this protocol we describe the isolation and characterisation of human primary vascular smooth muscle cells (hpVSMCs), and the addition of human serum or plasma to hpVSMCs in a calcification assay and analysis. The BioHybrid analysis of biological changes to in vitro hpVSMC calcification is reflective of in vivo vascular calcification status. We suggest this analysis can discriminate between CKD patient cohorts and has the potential for wider application for risk factor determination in CKD and the general population.

Microvesicle-Mediated Tissue Regeneration Mitigates the Effects of Cellular Ageing.

Extracellular vesicles (EVs), comprising microvesicles (MVs) and exosomes (Exos), are membranous vesicles secreted by cells which mediate the repair of cellular and tissue damage via paracrine mechanisms. The action of EVs under normative and morbid conditions in the context of ageing remains largely unexplored. We demonstrate that MVs, but not Exos, from Pathfinder cells (PCs), a putative stem cell regulatory cell type, enhance the repair of human dermal fibroblast (HDF) and mesenchymal stem cell (MSC) co-cultures, following both mechanical and genotoxic stress. Critically, this effect was found to be both cellular age and stress specific. Notably, MV treatment was unable to repair mechanical injury in older co-cultures but remained therapeutic following genotoxic stress. These observations were further confirmed in human dermal fibroblast (HDF) and vascular smooth muscle cell (VSMC) co-cultures of increasing cellular age. In a model of comorbidity comprising co-cultures of HDFs and highly senescent abdominal aortic aneurysm (AAA) VSMCs, MV administration appeared to be senotherapeutic, following both mechanical and genotoxic stress. Our data provide insights into EVs and the specific roles they play during tissue repair and ageing. These data will potentiate the development of novel cell-free therapeutic interventions capable of attenuating age-associated morbidities and avoiding undesired effects.

New Food Frequency Questionnaire to Estimate Vitamin K Intake in a Mediterranean Population.

Vitamin K is a multifunctional micronutrient essential for human health, and deficiency has been linked to multiple pathological conditions. In this study, we aimed to develop and validate a new food frequency questionnaire (FFQ) to estimate total vitamin K intake, over the course of a 30-day interval, in a Portuguese, Mediterranean-based, population. We conducted a prospective study in a non-random sample of 38 healthy adult volunteers. The FFQ was designed based on a validated Portuguese FFQ used in nationally representative studies and on literature reviews, to include foods containing ≥5 µg of vitamin K/100 g and foods with a lower vitamin K content, yet commonly included in a Mediterranean diet. Vitamin K intake was estimated from 24 h recalls and six days of food records. The final FFQ included 54 food items which, according to regression analyses, explains 90% of vitamin K intake. Mean differences in vitamin K intake based on food records (80 ± 47.7 µg/day) and on FFQ (96.5 ± 64.3 µg/day) were statistically non-significant. Further, we found a strong correlation between both methods (r = 0.7; p = 0.003). Our results suggest that our new FFQ is a valid instrument to assess the last 30 days of vitamin K intake in the Portuguese Mediterranean population.

Common Histological Features Suggesting Enchondral Ossification Pathways in Calciphylaxis of Various Origins: A Study of Human Subcutaneous Tissue Biopsies.

Calciphylaxis is a rare, yet underdiagnosed condition causing high mortality in patients with severe renal and cardiovascular disease. Since knowledge of the pathophysiology of calciphylaxis is limited, a differential analysis of histological alterations in patient subgroups with various comorbidities might expose different disease phenotypes and allow deeper insights into the pathophysiology of the condition. Histological markers of osteogenesis and calcification were investigated in a group of 18 patients with clinically and histologically verified calciphylaxis, using immunohistochemical staining. Analysis of staining intensity and distribution of marker proteins in histological structures was performed to evaluate distinct patterns between subgroups with different clinical comorbidities in comparison with a control group. In all cases, immunohistochemical staining for bone matrix proteins, bone-morphogenic proteins and matrix-Gla proteins co-localized with subcutaneous vascular and interstitial calcifications. Significant expression of bone-morphogenic protein-7 and active matrix-Gla protein was observed. Mortality was associated with renal comorbidities and increased expression of bone-morphogenic protein-7. However, no distinct histological patterns were found between subgroups with renal disease, warfarin intake or coexisting micro- and macro-angiopathies. The upregulation of osteogenic markers (including bone-morphogenic protein-7) plays a major role in the development of calciphylaxis. Clinical outcome correlates with kidney function and phosphate handling, suggesting different pathophysiological mechanisms. However, biopsy  at late-stage disease shows a common histological phenotype, involving enchondral ossification.

Vitamin K and Hallmarks of Ageing: Focus on Diet and Gut Microbiome.

Vitamin K and vitamin K-dependent proteins have been reported to be associated with a large spectrum of age-related diseases. While most of these associations have been deduced from observational studies, solid evidence for the direct impact of vitamin K on cellular senescence remains to be proven. As vitamin K status reflects the complexity of interactions between dietary intake, gut microbiome activity and health, we will demonstrate the pivotal role of the diet-microbiome-health axis in human ageing and exemplify how vitamin K is implicated therein. We propose that food quality (i.e., food pattern) should be highlighted beyond the quantity of total vitamin K intake. Instead of focusing on a single nutrient, exploring a healthy diet containing vitamin K may be more strategic. As such, healthy eating patterns can be used to make dietary recommendations for the public. Emerging evidence suggests that dietary vitamin K is a modulator of the diet-microbiome-health axis, and this needs to be incorporated into the investigation of the impact of vitamin K on gut microbial composition and metabolic activities, along with host health outcomes. In addition, we highlight several critical caveats that need to be acknowledged regarding the interplay between diet, vitamin K, gut microbiome and host health that is pivotal for elucidating the role of vitamin K in ageing and responding to the urgent call of healthy eating concerning public health.

Biodegradable Poly(ester) Urethane Acrylate Resins for Digital Light Processing: From Polymer Synthesis to 3D Printed Tissue Engineering Constructs.

Digital light processing (DLP) is an accurate and fast additive manufacturing technique to produce a variety of products, from patient-customized biomedical implants to consumer goods. However, DLP's use in tissue engineering has been hampered due to a lack of biodegradable resin development. Herein, a library of biodegradable poly(esters) capped with urethane acrylate (with variations in molecular weight) is investigated as the basis for DLP printable resins for tissue engineering. The synthesized oligomers show good printability and are capable of creating complex structures with mechanical moduli close to those of medium-soft tissues (1-3 MPa). While fabricated films from different molecular weight resins show few differences in surface topology, wettability, and protein adsorption, the adhesion and metabolic activity of NCTC clone 929 (L929) cells and human dermal fibroblasts (HDFs) are significantly different. Resins from higher molecular weight oligomers provide greater cell adhesion and metabolic activity. Furthermore, these materials show compatibility in a subcutaneous in vivo pig model. These customizable, biodegradable, and biocompatible resins show the importance of molecular tuning and open up new possibilities for the creation of biocompatible constructs for tissue engineering.

Mast cells participate in smooth muscle cell reprogramming and atherosclerotic plaque calcification.

BACKGROUND: Calcification, a key feature of advanced human atherosclerosis, is positively associated with vascular disease burden and adverse events. We showed that macrocalcification can be a stabilizing factor for carotid plaque molecular biology, due to inverse association with immune processes. Mast cells (MCs) are important contributors to plaque instability, but their relationship with macrocalcification is unexplored. With a hypothesis that MC activation negatively associates with carotid plaque macrocalcification, we aimed to investigate the link between MCs and carotid plaque vulnerability, and study MC role in plaque calcification via smooth muscle cells (SMCs). METHODS: Pre-operative computed tomography angiographies of patients (n = 40) undergoing surgery for carotid stenosis were used to characterize plaque morphology. Plaque microarrays (n = 40 and n = 126) were used for bioinformatic deconvolution of immune cell populations. Tissue microarrays (n = 103) were used to histologically validate the contribution of activated and resting MCs in plaques. RESULTS: Activated MCs and their typical markers were negatively correlated with macrocalcification. The ratio of activated vs. resting MCs was increased in low-calcified plaques from symptomatic patients. There was no modulating effect of medication on MC ratios. In vitro experiments showed that SMC calcification attenuated MC activation, while both active and resting MCs stimulated SMC calcification and induced dedifferentiation towards a pro-inflammatory-, osteochondrocyte-like phenotype, without modulating their migro-proliferative function. CONCLUSIONS: Integrative analyses from human plaques showed that MC activation is inversely associated with macrocalcification and positively with parameters of plaque vulnerability. Mechanistically, MCs induce SMC osteogenic reprograming, while matrix calcification in turn attenuates MC activation, offering new therapeutic avenues for exploration.

Association of Red Blood Cell Distribution Width and Neutrophil-to-Lymphocyte Ratio with Calcification and Cardiovascular Markers in Chronic Kidney Disease.

We aimed to investigate the association between Red Blood Cell Distribution Width (RDW) and Neutrophil-to-Lymphocyte Ratio (NLR), simple, rapidly assessed markers from the complete blood count with vascular calcification (VC)/stiffness and cardiovascular disease (CVD) in chronic kidney disease (CKD). Dephosphorylated, uncarboxylated matrix Gla-protein (dp-ucMGP), and central/peripheral hemodynamics' parameters were measured in 158 CKD patients, including Hemodialysis and Peritoneal Dialysis. Spearman's rho analysis showed that RDW correlated with C-reactive protein (CRP) (r = 0.29, p < 0.001), dp-ucMGP (r = 0.43, p = < 0.0001), central diastolic blood pressure (DBP) (r = -0.19, p = 0.02), and albuminuria (r = -0.17, p = 0.03). NLR correlated with the duration of CVD (r = 0.32, p < 0.001), CRP (r = 0.27, p = 0.01), dp-ucMGP (r = 0.43, p < 0.0001), central DBP (r = -0.32, p < 0.0001) and eGFR (r = -0.25, p = 0.04). In multiple regression models, circulating dp-ucMGP was an independent predictor of RDW (ß = 0.001, p = 0.001) and NLR (ß = 0.002, p = 0.002). In CKD patients, RDW and NLR are associated with traditional and novel markers of VC and CVD.

Inhibition of Neutral Sphingomyelinase 2 by Novel Small Molecule Inhibitors Results in Decreased Release of Extracellular Vesicles by Vascular Smooth Muscle Cells and Attenuated Calcification.

Vascular calcification (VC) is an important contributor and prognostic factor in the pathogenesis of cardiovascular diseases. VC is an active process mediated by the release of extracellular vesicles by vascular smooth muscle cells (VSMCs), and the enzyme neutral sphingomyelinase 2 (nSMase2 or SMPD3) plays a key role. Upon activation, the enzyme catalyzes the hydrolysis of sphingomyelin, thereby generating ceramide and phosphocholine. This conversion mediates the release of exosomes, a type of extracellular vesicles (EVs), which ultimately forms the nidus for VC. nSMase2 therefore represents a drug target, the inhibition of which is thought to prevent or halt VC progression. In search of novel druglike small molecule inhibitors of nSMase2, we have used virtual ligand screening to identify potential ligands. From an in-silico collection of 48,6844 small druglike molecules, we selected 996 compounds after application of an in-house multi-step procedure combining different filtering and docking procedures. Selected compounds were functionally tested in vitro; from this, we identified 52 individual hit molecules that inhibited nSMase2 activity by more than 20% at a concentration of 150 µM. Further analysis showed that five compounds presented with IC50s lower than 2 µM. Of these, compounds ID 5728450 and ID 4011505 decreased human primary VSMC EV release and calcification in vitro. The hit molecules identified here represent new classes of nSMase2 inhibitors that may be developed into lead molecules for the therapeutic or prophylactic treatment of VC.

The Pleiotropic Role of Vitamin K in Multimorbidity of Chronic Obstructive Pulmonary Disease.

Although defined by the presence of airflow obstruction and respiratory symptoms, patients with chronic obstructive pulmonary disease (COPD) are characterized by multimorbidity. Numerous co-occurring conditions and systemic manifestations contribute to the clinical presentation and progression of COPD; however, underlying mechanisms for multimorbidity are currently not fully elucidated. Vitamin A and vitamin D have been related to COPD pathogenesis. Another fat-soluble vitamin, vitamin K, has been put forward to exert protective roles in COPD. Vitamin K is an unequivocal cofactor for the carboxylation of coagulation factors, but also for extra-hepatic proteins including the soft tissue calcification inhibitor matrix Gla-protein and the bone protein osteocalcin. Additionally, vitamin K has been shown to have anti-oxidant and anti-ferroptosis properties. In this review, we discuss the potential role of vitamin K in the systemic manifestations of COPD. We will elaborate on the effect of vitamin K on prevalent co-occurring chronic conditions in COPD including cardiovascular disorders, chronic kidney disease, osteoporosis, and sarcopenia. Finally, we link these conditions to COPD with vitamin K as a connecting factor and provide recommendations for future clinical studies.

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions.

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

Effect of vitamin K supplementation on serum calcification propensity and arterial stiffness in vitamin K-deficient kidney transplant recipients: A double-blind, randomized, placebo-controlled clinical trial.

Vitamin K deficiency is common among kidney transplant recipients (KTRs) and likely contributes to progressive vascular calcification and stiffness. In this single-center, randomized, double-blind, placebo-controlled trial, we aimed to investigate the effects of vitamin K supplementation on the primary end point, serum calcification propensity (calciprotein particle maturation time, T50), and secondary end points arterial stiffness (pulse wave velocity [PWV]) and vitamin K status in 40 vitamin K-deficient KTRs (plasma dephosphorylated uncarboxylated matrix Gla protein [dp-ucMGP] ≥500 pmol/L). Participants (35% female; age, 57 ± 13 years) were randomized 1:1 to vitamin K2 (menaquinone-7, 360 µg/day) or placebo for 12 weeks. Vitamin K supplementation had no effect on calcification propensity (change in T50 vs baseline +2.3 ± 27.4 minutes) compared with placebo (+0.8 ± 34.4 minutes; Pbetween group = .88) but prevented progression of PWV (change vs baseline -0.06 ± 0.26 m/s) compared with placebo (+0.27 ± 0.43 m/s; Pbetween group = .010). Vitamin K supplementation strongly improved vitamin K status (change in dp-ucMGP vs baseline -385 [-631 to -269] pmol/L) compared with placebo (+39 [-188 to +183] pmol/L; Pbetween group < .001), although most patients remained vitamin K-deficient. In conclusion, vitamin K supplementation did not alter serum calcification propensity but prevented progression of arterial stiffness, suggesting that vitamin K has vascular effects independent of calciprotein particles. These results set the stage for longer-term intervention studies with vitamin K supplementation in KTRs. TRIAL REGISTRY: EU Clinical Trials Register (EudraCT Number: 2019-004906-88) and the Dutch Trial Register (NTR number: NL7687).

Human and murine fibroblast single-cell transcriptomics reveals fibroblast clusters are differentially affected by ageing and serum cholesterol.

AIMS: Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. METHODS AND RESULTS: Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. CONCLUSION: We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.