a-semi-automated-reproducible-biological-based-method-to-quantify

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition <em>In Vitro</em>

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

Maastricht University

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.

Targeting Coagulation Factor Xa Promotes Regression of Advanced Atherosclerosis in Apolipoprotein-E Deficient Mice.

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.

Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease.

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.

Functional vitamin K insufficiency, vascular calcification and mortality in advanced chronic kidney disease: A cohort study.

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).

Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity.

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   and control   mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to  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% ( < 0.001) and -1.44 ± 7.05% ( = 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 ( = 0.008), this slope became insignificant after splitting ( = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density ( = 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.

Improved Quantification of Cell Density in the Arterial Wall-A Novel Nucleus Splitting Approach Applied to 3D Two-Photon Laser-Scanning Microscopy.

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.

Article	Year
Targeting Coagulation Factor Xa Promotes Regression of Advanced Atherosclerosis in Apolipoprotein-E Deficient Mice. Scientific reports\| PubMed ID: 30846818	2019
Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease. Scientific reports\| PubMed ID: 32313061	2020
Functional vitamin K insufficiency, vascular calcification and mortality in advanced chronic kidney disease: A cohort study. PloS one\| PubMed ID: 33626087	2021
Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity. Cells\| PubMed ID: 34440866	2021
Improved Quantification of Cell Density in the Arterial Wall-A Novel Nucleus Splitting Approach Applied to 3D Two-Photon Laser-Scanning Microscopy. Frontiers in physiology\| PubMed ID: 35095571	2021
Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification. Clinical and translational medicine\| PubMed ID: 35184400	2022

Armand M. G. Jaminon

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