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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.
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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.
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Fallo Renal Crónico , Insuficiencia Renal Crónica , Calcificación Vascular , Humanos , Riñón/patología , Insuficiencia Renal Crónica/complicaciones , Calcificación Fisiológica , Miocitos del Músculo Liso/patologíaRESUMEN
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.
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Micropartículas Derivadas de Células , Exosomas , Vesículas Extracelulares , Humanos , Anciano , Micropartículas Derivadas de Células/metabolismo , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Senescencia Celular , Cicatrización de HeridasRESUMEN
Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.
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Insuficiencia Renal Crónica , Calcificación Vascular , Calcio/metabolismo , Humanos , Músculo Liso Vascular , Miocitos del Músculo Liso , Calcificación Vascular/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/patologíaRESUMEN
RATIONALE: Vascular calcification is a prominent feature of late-stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context. METHODS AND RESULTS: In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α-SMA+ cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE-/- mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFß1, phosphate and ß-glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues. CONCLUSION: We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α-SMA+ regions of calcified cardiovascular tissues, induced by pro-inflammatory and pro-osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification.
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Proteínas de la Matriz Extracelular/farmacología , Músculo Liso/efectos de los fármacos , Osteogénesis/genética , Proteoglicanos/farmacología , Calcificación Vascular/etiología , Análisis de Varianza , Estudios de Cohortes , Estudios Transversales , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Modelos Lineales , Músculo Liso/fisiología , Países Bajos , Osteogénesis/fisiología , Estudios Prospectivos , Proteoglicanos/metabolismo , Estadísticas no Paramétricas , Suecia , Calcificación Vascular/genéticaRESUMEN
BACKGROUND: Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs). METHODS: We developed a new cell-based high-content assay, the BioHybrid assay, to measure in vitro calcification. The BioHybrid assay was compared with the o-Cresolphthalein assay and the T50 assay. Serum and plasma were derived from different cohort studies including chronic kidney disease (CKD) stages III, IV, V and VD (on dialysis), pseudoxanthoma elasticum (PXE) and other cardiovascular diseases including serum from participants with mild and extensive coronary artery calcification (CAC). hVSMCs were exposed to serum and plasma samples, and in vitro calcification was measured using AlexaFluor®-546 tagged fetuin-A as calcification sensor. RESULTS: The BioHybrid assay measured the kinetics of calcification in contrast to the endpoint o-Cresolphthalein assay. The BioHybrid assay was more sensitive to pick up differences in calcification propensity than the T50 assay as determined by measuring control as well as pre- and post-dialysis serum samples of CKD patients. The BioHybrid response increased with CKD severity. Further, the BioHybrid assay discriminated between calcification propensity of individuals with a high CAC index and individuals with a low CAC index. Patients with PXE had an increased calcification response in the BioHybrid assay as compared to both spouse and control plasma samples. Finally, vitamin K1 supplementation showed lower in vitro calcification, reflecting changes in delta Agatston scores. Lower progression within the BioHybrid and on Agatston scores was accompanied by lower dephosphorylated-uncarboxylated matrix Gla protein levels. CONCLUSION: The BioHybrid assay is a novel approach to determine the vascular calcification propensity of an individual and thus may add to personalised risk assessment for CVD.
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Músculo Liso Vascular/metabolismo , Calcificación Vascular/sangre , Biomarcadores/sangre , Proteínas de Unión al Calcio/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/tratamiento farmacológico , Células Cultivadas , Proteínas de la Matriz Extracelular/sangre , Colorantes Fluorescentes/química , Pruebas Hematológicas , Humanos , Cinética , Diálisis Renal , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/terapia , Calcificación Vascular/diagnóstico , Vitamina K 1/uso terapéutico , alfa-2-Glicoproteína-HS/química , alfa-2-Glicoproteína-HS/metabolismo , Proteína Gla de la MatrizRESUMEN
Patients with chronic kidney disease (CKD) suffer from vitamin K deficiency and are at high risk of vascular calcification (VC) and premature death. We investigated the association of functional vitamin K deficiency with all-cause mortality and whether this association is modified by the presence of VC in CKD stage 5 (CKD G5). Plasma dephosphorylated-uncarboxylated matrix Gla-protein (dp-ucMGP), a circulating marker of functional vitamin K deficiency, and other laboratory and clinical data were determined in 493 CKD G5 patients. VC was assessed in subgroups by Agatston scoring of coronary artery calcium (CAC) and aortic valve calcium (AVC). Backward stepwise regression did not identify dp-ucMGP as an independent determinant of VC. During a median follow-up of 42 months, 93 patients died. Each one standard deviation increment in dp-ucMGP was associated with increased risk of all-cause mortality (sub-hazard ratio (sHR) 1.17; 95% confidence interval, 1.01-1.37) adjusted for age, sex, cardiovascular disease, diabetes, body mass index, inflammation, and dialysis treatment. The association remained significant when further adjusted for CAC and AVC in sub-analyses (sHR 1.22, 1.01-1.48 and 1.27, 1.01-1.60, respectively). In conclusion, functional vitamin K deficiency associates with increased mortality risk that is independent of the presence of VC in patients with CKD G5.
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Insuficiencia Renal Crónica/complicaciones , Calcificación Vascular/complicaciones , Deficiencia de Vitamina K/complicaciones , Vitamina K/sangre , Adulto , Anciano , Biomarcadores/sangre , Proteínas de Unión al Calcio/sangre , Estudios de Cohortes , Proteínas de la Matriz Extracelular/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/mortalidad , Insuficiencia Renal Crónica/patología , Tasa de Supervivencia , Calcificación Vascular/sangre , Calcificación Vascular/mortalidad , Calcificación Vascular/patología , Deficiencia de Vitamina K/sangre , Deficiencia de Vitamina K/mortalidad , Deficiencia de Vitamina K/patología , Proteína Gla de la MatrizRESUMEN
Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a "nucleus splitting" procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR Apoe -/- and control Apoe -/- mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed -26.6 ± 9.90% (p < 0.001) and -1.44 ± 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.
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Aims: Vascular calcification is a hallmark of atherosclerotic burden and can predict the cardiovascular outcome. Vitamin K antagonists (VKA) are widely used anticoagulant drugs to treat patients at risk of arterial and venous thrombosis but are also associated with increase vascular calcification progression. We aim to unravel the paradox that VKA suppresses plasma coagulation but promotes vascular calcification and subsequent atherosclerosis-dependent coagulability of the vessel wall. Methods and results: Apoe -/- mice were placed on western-type diet enriched with the VKA warfarin for 18 weeks to measure atherosclerotic plaque burden, calcification, and coagulation. Patients (n = 54) displaying paroxysmal atrial fibrillation with a low cardiovascular risk, who were treated with VKA were included to measure pre-thrombotic state. Finally, primary vascular smooth muscle cells (VSMC) derived from human tissue explants were used for in vitro experiments. In Apoe -/- mice, VKA increases both atherosclerotic plaque size and calcification. Higher plaque calcification was associated with increased plasma levels of thrombin-antithrombin and factor IXa-antithrombin complexes in mice and patients treated with VKA. Mechanistically, phenotypic switching of VSMC into synthetic VSMC promotes thrombin generation, which is enhanced in a tissue-factor (TF)-dependent manner by VSMC calcification. Moreover, calcified VSMC exposed to whole blood under flow significantly enhanced platelet deposition and TF-dependent fibrin formation. Conclusions: Oral anticoagulation with VKA aggravates vascular calcification and atherosclerosis. VSMC phenotype differentiation impacts coagulation potential in a TF-dependent manner. VKA-induced vascular calcification increases hypercoagulability and could thereby potentially positively affect atherothrombosis.
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Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification (VC) and requires carboxylation by vitamin K to exert calcification inhibition. Chronic kidney disease (CKD) patients undergo early vascular aging often involving extensive VC. The present cross-sectional study investigated the association between circulating dp-ucMGP levels, MGP expression in vascular tissue and MGP polymorphisms. In 141 CKD stage 5 patients, CAC score was significantly increased in the highest tertile of dp-ucMGP (p = 0.002), and a high medial VC score was associated with elevated dp-ucMGP levels. MGP vascular expression was associated with increased circulating dp-ucMGP and CAC scores. MGP SNP analysis revealed that patients homozygous for the C allele of the rs1800801 variant had a higher CAC score (median 15 [range 0-1312]) compared to patients carrying a T allele (median 0 [range 0-966] AU). These results indicate that plasma levels of dp-ucMGP are an independent predictor of increased VC in CKD5 patients and correlate with both higher CAC scores and degree of medial calcification. Additionally, high vascular expression of MGP was associated with higher CAC scores and plasma dp-ucMGP levels. Taken together, our results support that MGP is involved in the pathogenesis of VC.
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Proteínas de Unión al Calcio/sangre , Proteínas de la Matriz Extracelular/sangre , Fallo Renal Crónico/sangre , Insuficiencia Renal Crónica/sangre , Calcificación Vascular/sangre , Adulto , Biomarcadores/sangre , Proteínas de Unión al Calcio/genética , Grosor Intima-Media Carotídeo , Proteínas de la Matriz Extracelular/genética , Femenino , Regulación de la Expresión Génica/genética , Humanos , Fallo Renal Crónico/genética , Fallo Renal Crónico/patología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Factores de Riesgo , Calcificación Vascular/genética , Calcificación Vascular/patología , Proteína Gla de la MatrizRESUMEN
Atherosclerosis is a progressive inflammatory vascular disorder, complicated by plaque rupture and subsequently atherothrombosis. In vitro studies indicate that key clotting proteases, such as factor Xa (FXa), can promote atherosclerosis, presumably mediated through protease activated receptors (PARs). Although experimental studies showed reduced onset of atherosclerosis upon FXa inhibition, the effect on pre-existing plaques has never been studied. Therefore, we investigated effects of FXa inhibition by rivaroxaban on both newly-formed and pre-existing atherosclerotic plaques in apolipoprotein-e deficient (ApoE-/-) mice. Female ApoE-/- mice (age: 8-9 weeks, n = 10/group) received western type diet (WTD) or WTD supplemented with rivaroxaban (1.2 mg/g) for 14 weeks. In a second arm, mice received a WTD for 14 weeks, followed by continuation with either WTD or WTD supplemented with rivaroxaban (1.2 mg/g) for 6 weeks (total 20 weeks). Atherosclerotic burden in aortic arch was assessed by haematoxilin & eosin immunohistochemistry (IHC); plaque vulnerability was examined by IHC against macrophages, collagen, vascular smooth muscle cells (VSMC) and matrix metalloproteinases (MMPs). In addition, PAR1 and -2 expressions and their main activators thrombin and FXa in the plaque were determined in the plaque. Administration of rivaroxaban at human therapeutic concentrations reduced the onset of atherosclerosis (-46%, p < 0.05), and promoted a regression of pre-existing plaques in the carotids (-24%, p < 0.001). In addition, the vulnerability of pre-existing plaques was reduced by FXa inhibition as reflected by reduced macrophages (-39.03%, p < 0.05), enhanced collagen deposition (+38.47%, p < 0.05) and diminished necrotic core (-31.39%, p < 0.05). These findings were accompanied with elevated vascular smooth muscle cells and reduced MMPs. Furthermore, expression of PARs and their activators, thrombin and FXa was diminished after rivaroxaban treatment. Pharmacological inhibition of FXa promotes regression of advanced atherosclerotic plaques and enhances plaque stability. These data suggest that inhibition of FXa may be beneficial in prevention and regression of atherosclerosis, possibly mediated through reduced activation of PARs.
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Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Inhibidores del Factor Xa/uso terapéutico , Placa Aterosclerótica/tratamiento farmacológico , Rivaroxabán/uso terapéutico , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores del Factor Xa/farmacología , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Rivaroxabán/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: In end-stage renal disease (ESRD), coronary artery calcification (CAC) and inflammation contribute to cardiovascular disease (CVD). Statins do not improve survival in patients with ESRD, and their effect on vascular calcification is unclear. We explored associations between CAC, inflammatory biomarkers, statins and mortality in ESRD. MATERIALS AND METHODS: In 240 patients with ESRD (63% males; median age 56 years) from cohorts including 86 recipients of living donor kidney transplant (LD-Rtx), 96 incident dialysis patients and 58 prevalent peritoneal dialysis patients, associations of CAC score (Agatston Units, AUs), interleukin-6 (IL-6) with high-sensitivity C-reactive protein (hsCRP), tumour necrosis factor (TNF), use of statins and all-cause mortality were analysed. Cardiac CT was repeated in 35 patients after 1·5 years of renal replacement therapy. In vitro, human vascular smooth muscle cells (hVSMCs) were used to measure vitamin K metabolism. RESULTS: Among 240 patients, 129 (53%) had a CAC score > 100 AUs. Multivariate analysis revealed that independent predictors of 1-SD higher CAC score were age, male gender, diabetes and use of statins. The association between CAC score and mortality remained significant after adjustment for age, gender, diabetes, CVD, use of statins, protein-energy wasting and inflammation. Repeated CAC imaging in 35 patients showed that statin therapy was associated with greater progression of CAC. In vitro synthesis of menaquinone-4 by hVSMCs was significantly impaired by statins. CONCLUSION: Elevated CAC score is a mortality risk factor in ESRD independent of inflammation. Future studies should resolve if statins promote vascular calcification and inhibition of vitamin K synthesis in the uremic milieu.