48 research outputs found

    Research Models for Studying Vascular Calcification

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    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

    Die Bedeutung des vaskulĂ€ren „Inflammaging“ fĂŒr die Pathogenese der medialen arteriellen Kalzifizierung

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    Stand der Forschung: KardiovaskulĂ€re Erkrankungen sind weltweit die fĂŒhrende Todesursache, wobei insbesondere altersbedingte vaskulĂ€re VerĂ€nderungen in den Fokus rĂŒcken. Die Arteriosklerose ist eine wichtige EntitĂ€t systemischer GefĂ€ĂŸverĂ€nderungen und gekennzeichnet durch eine Kalzifizierung der tunica media („medial arterial calcification“, MAC). Ein bedeutender Risikofaktor fĂŒr die MAC ist die chronische Nierenkrankheit. Verschiedene Mediatoren und Inhibitoren beeinflussen die MAC Pathogenese. Hierbei rĂŒcken evolutionĂ€r konservierte Mechanismen der Mustererkennung, u.a. die Aktivierung des „NOD-, LRR- and pyrin domain-containing protein“ 3 (Nlrp3) Inflammasom und das einhergehende „Inflammaging“ in den Fokus der Untersuchungen. Fragestellung: Das Ziel dieser kumulativen Arbeit ist Etablierung von geeigneten Modellen und die Charakterisierung zentraler Signalwege in glatten GefĂ€ĂŸmuskelzellen („vascular smooth muscle cell“, VSMC) innerhalb des urĂ€mischen „Inflammaging“. Ergebnisse: Um die multifaktorielle MAC Genese und insbesondere die Interaktion der Signalwege untersuchen zu können, wurde in der ersten Arbeit ein Einzelzell-basiertes Modell zum Nachweis verschiedener Seneszenz- und Kalzifizierungsmarker etabliert. Durch die Etablierung des neuen ex vivo Modells der isoliert-perfundierten Arterie im Rahmen der zweiten Arbeit kann die MAC Pathogenese unter Perfusionsbedingungen analysiert werden. Die Bedeutung reaktiver Sauerstoffspezies (ROS) im Signalweg der MAC konnte in der dritten und vierten Arbeit sowohl in vitro als auch in vivo gezeigt werden. ROS aktivieren das Nlrp3 Inflammasom als intrazellulĂ€ren Mustererkennungsrezeptor. Über die Sekretion von Interleukin 1ÎČ werden pro-inflammatorische und osteogene Signalwege in VSMC induziert, die mit einer PhĂ€notyptransformation einhergehen und so die MAC Pathogenese beeinflussen. Diskussion & Ausblick: ZellulĂ€rer Stress der VSMC resultiert im „Inflammaging“ und osteogenem PhĂ€notyp, die letztendlich in Mechanismen der Kalzifizierung mĂŒnden. In AbhĂ€ngigkeit von der Art des Stressors nehmen die VSMC zusĂ€tzlich einen seneszenten PhĂ€notyp an, der „Inflammaging“ und Kalzifizierung verstĂ€rkt. Das Nlrp3 Inflammasom nimmt eine zentrale Rolle im intrazellulĂ€ren Signalweg der Pathogenese ein. Diese Defektheilung geht mit erheblichen strukturellen und rheologischen VerĂ€nderungen im GefĂ€ĂŸ einher, stabilisieren aber die GefĂ€ĂŸintegritĂ€t. Die UrĂ€mie ist ein prĂ€destiniertes Krankheitsbild fĂŒr die Ausbildung eines chronisch-urĂ€mischen „Inflammaging“ und einer vaskulĂ€ren Progerie. Die Modulation des chronisch-urĂ€mischen „Inflammaging“ kann ein therapeutisches Ziel fĂŒr die urĂ€mische Progerie sein. Neben der Suche nach neuen Wirkstoffkandidaten können etablierte Wirkstoffe therapeutische Optionen bieten, um die hohe kardiovaskulĂ€re MorbiditĂ€t und MortalitĂ€t von niereninsuffizienten Patienten reduzieren zu können

    The cardiovascular phenotype of adult patients with phenylketonuria

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    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

    Induction of Mineralization

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    Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans- differentiation of rat vascular smooth muscle cells in vitro. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in in vitro and ex vivo models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis

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

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    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

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

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    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-1 beta 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

    Mass-spectrometric identification of a novel angiotensin peptide in human plasma

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    Objective— Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. Methods and Results— Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A / Ang II was higher in end-stage renal failure patients. Conclusion— Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II. In this study, a new angiotensin-peptide of human plasma is described, which is characterized as a strong AT2-receptor agonist

    Comparability of Plasma Iohexol Clearance Across Population-Based Cohorts.

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    To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadRationale & 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. Keywords: Renal clearance; accuracy; agreement; concordance correlation; glomerular filtration rate (GFR); iohexol; kidney function tests; measured GFR; measurement error; multiple-sample; single-sample.United States Department of Health & Human Services National Institutes of Health (NIH) - USA National Institute on Aging, United States Hjartavernd, Iceland (Icelandic Heart Association) Icelandic Parliament (Althingi) KfH-Foundation of Preventive Medicine, Germany Dr. Werner Jackstadt Foundation, Germany Northern Norway Regional Health Authority Boehringer Ingelhei

    Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization

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    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

    Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization

    Get PDF
    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 alpha-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
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