High resolution monitoring of valvular interstitial cell driven pathomechanisms in procalcific environment using label-free impedance spectroscopy

IntroductionFibro-calcific aortic valve disease has high prevalence and is associated with significant mortality. Fibrotic extracellular matrix (ECM) remodeling and calcific mineral deposition change the valvular microarchitecture and deteriorate valvular function. Valvular interstitial cells (VICs) in profibrotic or procalcifying environment are frequently used in vitro models. However, remodeling processes take several days to weeks to develop, even in vitro. Continuous monitoring by real-time impedance spectroscopy (EIS) may reveal new insights into this process.MethodsVIC-driven ECM remodeling stimulated by procalcifying (PM) or profibrotic medium (FM) was monitored by label-free EIS. Collagen secretion, matrix mineralization, viability, mitochondrial damage, myofibroblastic gene expression and cytoskeletal alterations were analyzed.Results and DiscussionEIS profiles of VICs in control medium (CM) and FM were comparable. PM reproducibly induced a specific, biphasic EIS profile. Phase 1 showed an initial impedance drop, which moderately correlated with decreasing collagen secretion (r = 0.67, p = 0.22), accompanied by mitochondrial membrane hyperpolarization and cell death. Phase 2 EIS signal increase was positively correlated with augmented ECM mineralization (r = 0.97, p = 0.008). VICs in PM decreased myofibroblastic gene expression (p < 0.001) and stress fiber assembly compared to CM. EIS revealed sex-specific differences. Male VICs showed higher proliferation and in PM EIS decrease in phase 1 was significantly pronounced compared to female VICs (male minimum: 7.4 ± 4.2%, female minimum: 26.5 ± 4.4%, p < 0.01). VICs in PM reproduced disease characteristics in vitro remarkably fast with significant impact of donor sex. PM suppressed myofibroblastogenesis and favored ECM mineralization. In summary, EIS represents an efficient, easy-to-use, high-content screening tool enabling patient-specific, subgroup- and temporal resolution

Amphetamine increases vascular permeability by modulating endothelial actin cytoskeleton and NO synthase via PAR-1 and VEGF-R

Abstract Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction–force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (−75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (−50.8±3.7%, p<0.001) and Threonine495 (−44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse

Anti-inflammatory HDL effects are impaired in atrial fibrillation

High-density lipoprotein (HDL), best known for cholesterol transport, also has anti-inflammatory effects. Previous studies suggest involvement of myeloperoxidase (MPO) in modification of HDL. HDL bound Sphingosine-1-phosphate (S1P) has been implied to be an essential protein regarding beneficial HDL effects. In this study, we analyzed anti-inflammatory HDL properties in patients with atrial fibrillation (AF), a disease involving atrial inflammation, compared to non-AF controls and whether anti-inflammatory properties improve upon catheter ablation. Additionally, association with serum concentrations of MPO and S1P were assessed. We isolated HDL from 25 AF patients, 13 non-AF individuals and 14 AF patients at follow-up (FU) after catheter ablation. S1P was measured in a cohort of 141 AF and 21 FU patients. Following preincubation with HDL from either group, bovine aortic endothelial cells were stimulated using tumor necrosis factor α and expression of pro-inflammatory genes intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), E-selectin (SELE) and P-selectin (SELP) was assessed using qPCR. Concentrations of circulating protein of these genes as well as MPO and S1P were measured in serum samples. Compared to non-AF individuals HDL from AF patients suppressed gene expression of the pro-inflammatory adhesion molecules ICAM1, VCAM1, SELE and SELP 27%, 18%, 21% and 57% less, respectively (p &amp;lt; 0.05 for all except SELE p = 0.06). In FU patients, the anti-inflammatory HDL activity was improved (suppression of ICAM1 + 22%, VCAM1 + 10%, SELE + 38% and SELP + 75%, p &amp;lt; 0.05 for all except VCAM1 p = 0.08). AF patients using angiotensin converting enzyme inhibitors or angiotensin receptor blockers had better anti-inflammatory HDL properties than non-users (gene expression suppression at least 28% more, p &amp;lt; 0.05 for all except ICAM1 p = 0.051). Circulating protein concentrations were not correlated with in vitro gene-expression, but circulating P-selectin was generally elevated in AF and FU patients compared to non-AF patients. MPO plasma concentration was positively associated with gene-expression of ICAM1, VCAM1 and SELP (