Biomechanical Properties of the Aortic Wall: Changes during Vascular Calcification.

Medial vascular calcification (MAC) is characterized by the deposition of hydroxyapatite (HAP) in the medial layer of the vessel wall, leading to disruption of vessel integrity and vascular stiffness. Because currently no direct therapeutic interventions for MAC are available, studying the MAC pathogenesis is of high research interest. Several methods exist to measure and describe the pathophysiological processes in the vessel wall, such as histological staining and gene expression. However, no method describing the physiological properties of the arterial wall is currently available. This study aims to close that gap and validate a method to measure the biomechanical properties of the arterial wall during vascular calcification. Therefore, a stress-stretch curve is monitored using small-vessel-myography upon ex vivo calcification of rat aortic tissue. The measurement of biomechanical properties could help to gain further insights into vessel integrity during calcification progression.

Molecular Imaging and Quantification of Smooth Muscle Cell and Aortic Tissue Calcification In Vitro and Ex Vivo with a Fluorescent Hydroxyapatite-Specific Probe.

Vessel calcification is characterized by the precipitation of hydroxyapatite (HAP) in the vasculature. Currently, no causal therapy exists to reduce or prevent vessel calcification. Studying the underlying pathways within vascular smooth muscle cells and testing pharmacological intervention is a major challenge in the vascular research field. This study aims to establish a rapid and efficient working protocol for specific HAP detection in cells and tissue using the synthetic bisphosphonate fluorescence dye OsteoSense™. This protocol facilitates especially early quantification of the fluorescence signal and permits co-staining with other markers of interest, enabling smaller experimental set-ups with lesser primary cells consumption and fast workflows. The fluorescence-based detection of vascular calcification with OsteoSense™ combines a high specificity with improved sensitivity. Therefore, this methodology can improve research of the pathogenesis of vascular calcification, especially for testing the therapeutic benefit of inhibitors in the case of in vitro and ex vivo settings.

Uremic mouse model to study vascular calcification and "inflamm-aging".

Calcification and chronic inflammation of the vascular wall is a high-risk factor for cardiovascular mortality, especially in patients with chronic uremia. For the reduction or prevention of rapid disease progression, no specific treatment options are currently available. This study aimed to evaluate an adenine-based uremic mouse model for studying medial vessel calcification and senescence-associated secretory phenotype (SASP) changes of aortic tissue to unravel molecular pathogenesis and provide a model for therapy testing. The dietary adenine administration induced a stable and similar degree of chronic uremia in DBA2/N mice with an increase of uremia blood markers such as blood urea nitrogen, calcium, creatinine, alkaline phosphatase, and parathyroid hormone. Also, renal fibrosis and crystal deposits were detected upon adenine feeding. The uremic condition is related to a moderate to severe medial vessel calcification and subsequent elastin disorganization. In addition, expression of osteogenic markers as Bmp-2 and its transcription factor Sox-9 as well as p21 as senescence marker were increased in uremic mice compared to controls. Pro-inflammatory uremic proteins such as serum amyloid A, interleukin (Il)-1ß, and Il-6 increased. This novel model of chronic uremia provides a simple method for investigation of signaling pathways in vascular inflammation and calcification and therefore offers an experimental basis for the development of potential therapeutic intervention studies.

Medial Arterial Calcification: JACC State-of-the-Art Review.

Medial arterial calcification (MAC) is a chronic systemic vascular disorder distinct from atherosclerosis that is frequently but not always associated with diabetes mellitus, chronic kidney disease, and aging. MAC is also a part of more complex phenotypes in numerous less common diseases. The hallmarks of MAC include disseminated and progressive precipitation of calcium phosphate within the medial layer, a prolonged and clinically silent course, and compromise of hemodynamics associated with chronic limb-threatening ischemia. MAC increases the risk of complications during vascular interventions and mitigates their outcomes. With the exception of rare monogenetic defects affecting adenosine triphosphate metabolism, MAC pathogenesis remains unknown, and causal therapy is not available. Implementation of genetics and omics-based approaches in research recognizing the critical importance of calcium phosphate thermodynamics holds promise to unravel MAC molecular pathogenesis and to provide guidance for therapy. The current state of knowledge concerning MAC is reviewed, and future perspectives are outlined.

Vascular Calcification in Rodent Models-Keeping Track with an Extented Method Assortment.

Vascular calcification is a multifaceted disease and a significant contributor to cardiovascular morbidity and mortality. The calcification deposits in the vessel wall can vary in size and localization. Various pathophysiological pathways may be involved in disease progression. With respect to the calcification diversity, a great number of research models and detection methods have been established in basic research, relying mostly on rodent models. The aim of this review is to provide an overview of the currently available rodent models and quantification methods for vascular calcification, emphasizing animal burden and assessing prospects to use available methods in a way to address the 3R principles of Russel and Burch: "Replace, Reduce and Refine".

Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization.

Medial vascular calcification (mVC) is closely related to cardiovascular disease, especially in patients suffering from chronic kidney disease (CKD). Even after successful kidney transplantation, cardiovascular mortality remains increased. There is evidence that immunosuppressive drugs might influence pathophysiological mechanisms in the vessel wall. Previously, we have shown in vitro that mVC is induced in vascular smooth muscle cells (VSMCs) upon treatment with azathioprine (AZA). This effect was confirmed in the current study in an in vivo rat model treated with AZA for 24 weeks. The calcium content increased in the aortic tissue upon AZA treatment. The pathophysiologic mechanisms involve AZA catabolism to 6-thiouracil via xanthine oxidase (XO) with subsequent induction of oxidative stress. Proinflammatory cytokines, such as interleukin (IL)-1ß and IL-6, increase upon AZA treatment, both systemically and in the aortic tissue. Further, VSMCs show an increased expression of core-binding factor α-1, alkaline phosphatase and osteopontin. As the AZA effect could be decreased in NLRP3-/- aortic rings in an ex vivo experiment, the signaling pathway might be, at least in part, dependent on the NLRP3 inflammasome. Although human studies are necessary to confirm the harmful effects of AZA on vascular stiffening, these results provide further evidence of induction of VSMC calcification under AZA treatment and its effects on vessel structure.

Acid sphingomyelinase promotes SGK1-dependent vascular calcification.

In chronic kidney disease (CKD), hyperphosphatemia is a key factor promoting medial vascular calcification, a common complication associated with cardiovascular events and high mortality. Vascular calcification involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), but the complex signaling events inducing pro-calcific pathways are incompletely understood. The present study investigated the role of acid sphingomyelinase (ASM)/ceramide as regulator of VSMC calcification. In vitro, both, bacterial sphingomyelinase and phosphate increased ceramide levels in VSMCs. Bacterial sphingomyelinase as well as ceramide supplementation stimulated osteo-/chondrogenic transdifferentiation during control and high phosphate conditions and augmented phosphate-induced calcification of VSMCs. Silencing of serum- and glucocorticoid-inducible kinase 1 (SGK1) blunted the pro-calcific effects of bacterial sphingomyelinase or ceramide. Asm deficiency blunted vascular calcification in a cholecalciferol-overload mouse model and ex vivo isolated-perfused arteries. In addition, Asm deficiency suppressed phosphate-induced osteo-/chondrogenic signaling and calcification of cultured VSMCs. Treatment with the functional ASM inhibitors amitriptyline or fendiline strongly blunted pro-calcific signaling pathways in vitro and in vivo. In conclusion, ASM/ceramide is a critical upstream regulator of vascular calcification, at least partly, through SGK1-dependent signaling. Thus, ASM inhibition by repurposing functional ASM inhibitors to reduce the progression of vascular calcification during CKD warrants further study.

Stressor-Induced "Inflammaging" of Vascular Smooth Muscle Cells via Nlrp3-Mediated Pro-inflammatory Auto-Loop.

Calcification of the vessel wall as one structural pathology of aged vessels is associated with high cardiovascular mortality of elderly patients. Aging is linked to chronic sterile inflammation and high burden of reactive oxygen species (ROS), leading to activation of pattern recognition receptors (PRRs) such as Nlrp3 in vascular cells. The current study investigates the role of PRR activation in the calcification of vascular smooth muscle cells (VSMCs). Therefore, in vitro cell culture of primary rat VSMCs and ex vivo aortic stimulations were used to analyze osteogenic, senescence and inflammatory markers via real-time PCR, in situ RNA hybridization, Western Blot, photometric assays and histological staining. Induction of ROS and DNA-damage by doxorubicin induces a shift of VSMC phenotype toward the expression of osteogenic, senescence and inflammatory proteins. Induction of calcification is dependent on Nlrp3 activity. Il-1ß as a downstream target of Nlrp3 induces the synthetic, pro-calcifying VSMC phenotype. Inhibition of PRR with subsequent reduction of chronic inflammation might be an interesting target for reduction of calcification of VSMCs, with subsequent reduction of cardiovascular mortality of patients suffering from vessel stiffness.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Efficacy of Repeat Immunoadsorption.

(1) Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmunological disease. There is evidence for an autoimmune mechanism for ME/CFS with an infection-triggered onset and dysfunction of ß2-adrenoreceptor antibodies (ß2AR-AB). In a first proof-of-concept study, we could show that IA was effective to reduce ß2AR-AB and led to improvement of various symptoms. (2) Five of the ME/CFS patients who had clinical improvement following treatment with a five-day IA were retreated in the current study about two years later with a modified IA protocol. The severity of symptoms was assessed by disease specific scores during a follow-up period of 12 months. The antibodies were determined by ELISA. (3) The modified IA treatment protocol resulted in a remarkable similar clinical response. The treatment was well tolerated and 80-90% decline of total IgG and ß2AR-AB was achieved. Four patients showed a rapid improvement in several clinical symptoms during IA therapy, lasting for six to 12 months. One patient had no improvement. (4) We could provide further evidence that IA has clinical efficacy in patients with ME/CFS. Data from our pilot trial warrant further controlled studies in ME/CFS.

A Novel Protocol for Detection of Senescence and Calcification Markers by Fluorescence Microscopy.

Vascular calcification and stiffening of the arterial wall is a systemic phenomenon that is associated with aging and it can be increased by several risk factors. The underlying mechanisms, especially the pathways of cellular senescence, are under current investigation. Easily manageable in vitro settings help to study the signaling pathways. The experimental setting presented here is based on an in vitro model using rat vascular smooth muscle cells and the detection of senescence and osteoblastic markers via immunofluorescence and RNAscope™. Co-staining of the senescence marker p21, the osteoblastic marker osteopontin, detection of senescence-associated heterochromatin foci, and senescence-associated ß-galactosidase is possible within one test approach requiring fewer cells. The protocol is a fast and reliable evaluation method for multiplexing of calcifying and senescence markers with fluorescence microscopy detection. The experimental setting enables analysis on single cell basis and allows detection of intra-individual variances of cultured cells.

Research Models for Studying Vascular Calcification.

Calcification of the vessel wall contributes to high cardiovascular morbidity and mortality. Vascular calcification (VC) is a systemic disease with multifaceted contributing and inhibiting factors in an actively regulated process. The exact underlying mechanisms are not fully elucidated and reliable treatment options are lacking. Due to the complex pathophysiology, various research models exist evaluating different aspects of VC. This review aims to give an overview of the cell and animal models used so far to study the molecular processes of VC. Here, in vitro cell culture models of different origins, ex vivo settings using aortic tissue and various in vivo disease-induced animal models are summarized. They reflect different aspects and depict the (patho)physiologic mechanisms within the VC process.

A Novel Long-Term ex vivo Model for Studying Vascular Calcification Pathogenesis: The Rat Isolated-Perfused Aorta.

The investigation of vascular calcification and its underlying cellular and molecular pathways is of great interest in current research efforts. Therefore, suitable assays are needed to allow examination of the complex calcification process under controlled conditions. The current study describes a new ex vivo model of isolated-perfused rat aortic tissue with subsequent quantification and vessel staining to analyze the calcium content of the aortic wall. A rat aorta was perfused ex vivo with control and calcification media for 14 days, respectively. The calcification medium was luminally perfused and induced a significant increase in calcium deposition within the media of the vessel wall detected alongside the elastic laminae. Perfusion with control medium induced no calcification. In addition, the mRNA expression of the osteogenic marker bone morphogenetic protein 2 (BMP-2) increased in aortic tissue after perfusion, while SM22α as smooth muscle marker decreased. This newly developed ex vivo model of isolated-perfused rat aorta is suitable for vascular calcification studies testing inducers and inhibitors of vessel calcification and studying signaling pathways within calcification progression.

Comparability of Plasma Iohexol Clearance Across Population-Based Cohorts.

RATIONALE & OBJECTIVE: Glomerular filtration rate (GFR) estimation based on creatinine or cystatin C level is currently the standard method for assessing GFR in epidemiologic research and clinical trials despite several important and well-known limitations. Plasma iohexol clearance has been proposed as an inexpensive method for measuring GFR that could replace estimated GFR in many research projects. However, lack of standardization for iohexol assays and the use of different protocols such as single- and multiple-sample methods could potentially hamper comparisons across studies. We compared iohexol assays and GFR measurement protocols in 3 population-based European cohorts. STUDY DESIGN: Cross-sectional investigation. SETTING & PARTICIPANTS: Participants in the Age, Gene/Environment Susceptibility-Kidney Study (AGES-Kidney; n=805), the Berlin Initiative Study (BIS, n=570), and the Renal Iohexol Clearance Survey Follow-up Study (RENIS-FU; n=1,324). TESTS COMPARED: High-performance liquid chromatography analyses of iohexol. Plasma iohexol clearance calculated using single- versus multiple-sample protocols. OUTCOMES: Measures of agreement between methods. RESULTS: Frozen samples from the 3 studies were obtained and iohexol concentrations were remeasured in the laboratory at the University Hospital of North Norway. Lin's concordance correlation coefficient ρ was>0.96 and Cb (accuracy) was>0.99 for remeasured versus original serum iohexol concentrations in all 3 cohorts, and Passing-Bablok regression did not find differences between measurements, except for a slope of 1.025 (95% CI, 1.006-1.046) for the log-transformed AGES-Kidney measurements. The multiple-sample iohexol clearance measurements in AGES-Kidney and BIS were compared with single-sample GFRs derived from the same iohexol measurements. Mean bias for multiple-sample relative to single-sample GFRs in AGES-Kidney and BIS were-0.25 and-0.15mL/min, and 99% and 97% of absolute differences were within 10% of the multiple-sample result, respectively. LIMITATIONS: Lack of comparison with an independent gold-standard method. CONCLUSIONS: Agreement between the iohexol assays and clearance protocols in the 3 investigated cohorts was substantial. Our findings indicate that plasma iohexol clearance measurements can be compared across these studies.

The cardiovascular phenotype of adult patients with phenylketonuria.

BACKGROUND: Patients with Phenylketonuria (PKU) are exposed to multiple cardiovascular risk factors, but the clinical significance of these abnormalities is yet unknown. The purpose of this study was to characterize the cardiovascular phenotype in adult patients with PKU by clinical and dietary data, measurements of biochemical markers, and non-invasive examination of vascular functions. RESULTS: Twenty-three adult patients with PKU (age: 18-47 y; 30.8 ± 8.4 y) and 28 healthy controls (age: 18-47 y; 30.1 ± 9.1 y) were included in this study. PKU patients had significantly higher systolic and diastolic blood pressure, increased resting heart rate and a higher body mass index. Total cholesterol and non-HDL cholesterol levels were significantly increased in PKU patients, whereas plasma levels of HDL cholesterol and its subfraction HDL2 (but not HDL3) were significantly decreased. The inflammatory markers C-reactive protein and serum amyloid A protein and the serum oxidative stress marker malondialdehyde were significantly higher in patients with PKU. Venous occlusion plethysmography showed marked reduction in post-ischemic blood flow and the carotid to femoral pulse wave velocity was significantly increased demonstrating endothelial dysfunction and increased vascular stiffness. CONCLUSIONS: This study shows that the cardiovascular phenotype of adult PKU patients is characterized by an accumulation of traditional cardiovascular risk factors, high levels of inflammatory and oxidative stress markers, endothelial dysfunction and vascular stiffness. These data indicate the need for early cardiovascular risk reduction in patients with PKU.

Author Correction: Beta Trace Protein does not outperform Creatinine and Cystatin C in estimating Glomerular Filtration Rate in Older Adults.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Dysfunctional high-density lipoprotein activates toll-like receptors via serum amyloid A in vascular smooth muscle cells.

Serum amyloid A (SAA) is an uremic toxin and acute phase protein. It accumulates under inflammatory conditions associated with high cardiovascular morbidity and mortality in patients with sepsis or end-stage renal disease (ESRD). SAA is an apolipoprotein of the high-density lipoprotein (HDL). SAA accumulation turns HDL from an anti-inflammatory to a pro-inflammatory particle. SAA activates monocyte chemoattractant protein-1 (MCP-1) in vascular smooth muscle cells. However, the SAA receptor-mediated signaling pathway in vascular cells is poorly understood. Therefore, the SAA-mediated signaling pathway for MCP-1 production was investigated in this study. The SAA-induced MCP-1 production is dependent on the activation of TLR2 and TLR4 as determined by studies with specific receptor antagonists and agonists or siRNA approach. Experiments were confirmed in tissues from TLR2 knockout, TLR4 deficient and TLR2 knock-out/TLR4 deficient mice. The intracellular signaling pathway is IκBα and subsequently NFκB dependent. The MCP-1 production induced by SAA-enriched HDL and HDL isolated from septic patients with high SAA content is also TLR2 and TLR4 dependent. Taken together, the TLR2 and TLR4 receptors are functional SAA receptors mediating MCP-1 release. Furthermore, the TLR2 and TLR4 are receptors for dysfunctional HDL. These results give a further inside in SAA as uremic toxin involved in uremia-related pro-inflammatory response in the vascular wall.

In times of tobacco-free nicotine consumption: The influence of nicotine on vascular calcification.

BACKGROUND: Smoking remains the most important avoidable cause of global mortality. Even though the number of cigarette smokers declines in first world countries, the uses of alternative nicotine delivery products increase and may even surpass the sells of cigarettes. In this light, the explicit role of nicotine in the development of cardiovascular diseases should be elucidated. OBJECTIVES: This narrative review attempts to connect current literature about possible effects of nicotine on the environment of the vasculature to the pathogenesis of vascular calcification, focusing on the tunica media of the vessel wall. METHODS: For this review, papers found on Pubmed and Medline until December 2018 by searching for the keywords nicotine, vascular calcification, oxidative stress, osteoblastic transdifferentiation and matrix degradation were considered. RESULTS: Nicotine creates an environment that probably facilitates and maybe even induces osteogenic transdifferentiation of VSMC by inflammation, endothelial dysfunction and reactive oxygen species. This process is believed to be a key event in calcification of the tunica media of the vessel wall. Furthermore, nicotine could lead to the formation of nucleation sites for hydroxyapatite by facilitating matrix vesicles and extracellular matrix degradation. CONCLUSIONS: There is a growing body of evidence implicating that nicotine alone could impair vascular function and lead to vascular calcification. Further research is necessary to elucidate the explicit influence of nicotine on arteriosclerosis.

Control of blood pressure and risk of mortality in a cohort of older adults: the Berlin Initiative Study.

AIMS: To assess whether blood pressure (BP) values below 140/90 mmHg during antihypertensive treatment are associated with a decreased risk of all-cause mortality in community-dwelling older adults. METHODS AND RESULTS: Within the Berlin Initiative Study, we assembled a cohort of patients ≥70 years treated with antihypertensive drugs at baseline (November 2009-June 2011). End of prospective follow-up was December 2016. Cox proportional hazards models yielded adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause mortality associated with normalized BP [systolic BP (SBP) <140 mmHg and diastolic BP (DBP) <90 mmHg] compared with non-normalized BP (SBP ≥140 mmHg or DBP ≥90 mmHg) overall and after stratification by age or previous cardiovascular events. Among 1628 patients (mean age 81 years) on antihypertensive drugs, 636 exhibited normalized BP. During 8853 person-years of follow-up, 469 patients died. Compared with non-normalized BP, normalized BP was associated with an increased risk of all-cause mortality (incidence rates: 60.3 vs. 48.5 per 1000/year; HR 1.26; 95% CI 1.04-1.54). Increased risks were observed in patients ≥80 years (102.2 vs. 77.5 per 1000/year; HR 1.40; 95% CI 1.12-1.74) and with previous cardiovascular events (98.3 vs. 63.6 per 1000/year; HR 1.61; 95% CI 1.14-2.27) but not in patients aged 70-79 years (22.6 vs. 22.7 per 1000/year; HR 0.83; 95% CI 0.54-1.27) or without previous cardiovascular events (45.2 vs. 44.4 per 1000/year; HR 1.16, 95% CI 0.90-1.48). CONCLUSION: Blood pressure values below 140/90 mmHg during antihypertensive treatment may be associated with an increased risk of mortality in octogenarians or elderly patients with previous cardiovascular events.

Diabetes Mellitus in Pregnancy Leads to Growth Restriction and Epigenetic Modification of the Srebf2 Gene in Rat Fetuses.

Diabetic pregnancy is correlated with increased risk of metabolic and neurological disorders in the offspring putatively mediated epigenetically. Little is known about epigenetic changes already present in fetuses of diabetic pregnancies. We aimed at characterizing the perinatal environment after preexisting maternal diabetes mellitus and at identifying relevant epigenetic changes in the fetus. We focused on the transcription factor Srebf2 (sterol regulatory element binding transcription factor 2), a master gene in regulation of cholesterol metabolism. We tested whether diabetic pregnancy induces epigenetic changes in the Srebf2 promoter and if they become manifest in altered Srebf2 gene expression. We worked with a transgenic rat model of type 2 diabetes mellitus (Tet29) in which the insulin receptor is knocked down by doxycycline-induced RNA interference. Doxycycline was administered preconceptionally to Tet29 and wild-type control rats. Only Tet29 doxycycline dams were hyperglycemic, hyperinsulinemic, and hyperlipidemic. Gene expression was analyzed with quantitative real-time reverse transcriptase polymerase chain reaction and CpG promoter methylation with pyrosequencing. Immunohistochemistry was performed on fetal brains. Fetuses from diabetic Tet29 dams were hyperglycemic and growth restricted at the end of pregnancy. They further displayed decreased liver and brain weight with concomitant decreased microglial activation in the hippocampus in comparison to fetuses of normoglycemic mothers. Importantly, diabetic pregnancy induced CpG hypermethylation of the Srebf2 promoter in the fetal liver and brain, which was associated with decreased Srebf2 gene expression. In conclusion, diabetic and hyperlipidemic pregnancy induces neurological, metabolic, and epigenetic alterations in the rat fetus. Srebf2 is a potential candidate mediating intrauterine environment-driven epigenetic changes and later diabetic offspring health.

Beta Trace Protein does not outperform Creatinine and Cystatin C in estimating Glomerular Filtration Rate in Older Adults.

Despite intense research the optimal endogenous biomarker for glomerular filtration rate (GFR) estimation has not been identified yet. We analyzed if ß-trace protein (BTP) improved GFR estimation in elderly. 566 participants aged 70+ from the population-based Berlin Initiative Study were included in a cross-sectional validation study. BTP, standardized creatinine and cystatin C were measured in participants with iohexol clearance measurement as gold standard method for measured GFR (mGFR). In a double logarithmic linear model prediction of mGFR by BTP was assessed. Analyses with BTP only and combined with creatinine and cystatin C were performed. Additionally, performance of GFR estimating equations was compared to mGFR. We found that the combination of all three biomarkers showed the best prediction of mGFR (r2 = 0.83), whereat the combination of creatinine and cystatin C provided only minimally diverging results (r2 = 0.82). Single usage of BTP showed worst prediction (r2 = 0.67) within models with only one biomarker. Subgroup analyses (arterial hypertension, diabetes, body mass index ≤23 and >30) demonstrated a slight additional benefit of including BTP into the prediction model for diabetic, hypertensive and lean patients. Among BTP-containing GFR equations the Inker BTP-based equation showed superior performance. Especially the use of cystatin C renders the addition of BTP unnecessary.