One of the contributors to atherogenesis is enzymatically modified LDL (eLDL). eLDL was detected in all stages of aortic valve sclerosis and was demonstrated to trigger the activation of p38 mitogen-activated protein kinase (p38 MAPK), which has been identified as a pro-inflammatory protein in atherosclerosis. In this study, we investigated the influence of eLDL on IL-6 and IL-33 induction, and also the impact of eLDL on calcification in aortic valve stenosis (AS). eLDL upregulated phosphate-induced calcification in valvular interstitial cells (VICs)/myofibroblasts isolated from diseased aortic valves, as demonstrated by alizarin red staining. Functional studies demonstrated activation of p38 MAPK as well as an altered gene expression of osteogenic genes known to be involved in vascular calcification. In parallel with the activation of p38 MAPK, eLDL also induced upregulation of the cytokines IL-6 and IL-33. The results suggest a pro-calcifying role of eLDL in AS via induction of IL-6 and IL-33.

Animal C-reactive protein (CRP) has a widespread existence throughout phylogeny implying that these proteins have essential functions mandatory to be preserved. About 500 million years of evolution teach us that there is a continuous interplay between emerging antigens and components of innate immunity. The most archaic physiological roles of CRP seem to be detoxication of heavy metals and other chemicals followed or accompanied by an acute phase response and host defense against bacterial, viral as well as parasitic infection. On the other hand, unusual antigens have emerged questioning the black-and-white perception of CRP as being invariably beneficial. Such antigens came along either as autoantigens like excessive tissue-stranded modified lipoprotein due to misdirected food intake linking CRP with atherosclerosis with an as yet open net effect, or as foreign antigens like SARS-CoV-2 inducing an uncontrolled CRP-mediated autoimmune response. The latter two examples impressingly demonstrate that a component of ancient immunity like CRP should not be considered under identical “beneficial” auspices throughout phylogeny but might effect quite the reverse as well.

Acute aortic dissection (AAD) is a rare condition, but together with acute myocardial infarction (AMI) and pulmonary embolism (PE) it belongs to the most relevant and life-threatening causes of acute chest pain. Until now, there has been no specific blood test in the diagnostic workup of AAD. To identify clinically relevant biomarkers for AAD, we applied Proseek® Multiplex assays to plasma samples from patients with AAD, AMI, PE, thoracic aortic aneurysm (TAA), and non-cardiovascular chest pain (nonCVD). Subsequently, we validated top hits using conventional immunoassays and examined their expression in the aortic tissue. Interleukin 10 (IL-10) alone showed the best performance with a sensitivity of 55% and a specificity of 98% for AAD diagnosis. The combination of D-dimers, high-sensitive troponin T (hs-TnT), interleukin 6 (IL-6), and plasminogen activator inhibitor 1 (PAI1) correctly classified 75% of AAD cases, delivering a sensitivity of 83% and specificity of 95% for its diagnosis. Moreover, this model provided the correct classification of 77% of all analyzed cases. Our data suggest that IL-10 shows potential to be a rule-in biomarker for AAD. Moreover, the addition of PAI1 and IL-6 to hs-TnT and D-dimers may improve the discrimination of suspected AAD, AMI, and PE in patients presenting with acute chest pain.

Atherosclerosis research typically focuses on the evolution of intermediate or advanced atherosclerotic lesions rather than on prelesional stages of atherogenesis. Yet these early events may provide decisive leads on the triggers of the pathologic process, before lesions become clinically overt. Thereby, it is mandatory to consider extracellular lipoprotein deposition at this stage as the prerequisite of foam cell formation leading to a remarkable accumulation of LDL (Low Density Lipoproteins). As progression of atherosclerosis displays the characteristic features of a chronic inflammatory process on the one hand and native LDL lacks inflammatory properties on the other hand, the lipoprotein must undergo biochemical modification to become atherogenic. During the last 25 years, evidence was accumulated in support of a different concept on atherogenesis proposing that modification of native LDL occurs through the action of ubiquitous hydrolytic enzymes (enzymatically modified LDL or eLDL) rather than oxidation and contending that the physiological events leading to macrophage uptake and reverse transport of eLDL first occur without inflammation (initiation with reversion). Preventing or reversing initial atherosclerotic lesions would avoid the later stages and therefore prevent clinical manifestations. This concept is in accordance with the response to retention hypothesis directly supporting the strategy of lowering plasma levels of atherogenic lipoproteins as the most successful therapy for atherosclerosis and its sequelae. Apart from but unquestionable closely related to this concept, there are several other hypotheses on atherosclerotic lesion initiation favoring an initiating role of the immune system ('vascular-associated lymphoid tissue' (VALT)), defining foam cell formation as a variant of lysosomal storage disease, relating to the concept of the inflammasome with crystalline cholesterol and/or mitochondrial DAMPs (damage-associated molecular patterns) being mandatory in driving arterial inflammation and, last but not least, pointing to miRNAs (micro RNAs) as pivotal players. However, direct anti-inflammatory therapies may prove successful as adjuvant components but will likely never be used in the absence of strategies to lower plasma levels of atherogenic lipoproteins, the key point of the perception that atherosclerosis is not simply an inevitable result of senescence. In particular, given the importance of chemical modifications for lipoprotein atherogenicity, regulation of the enzymes involved might be a tempting target for pharmacological research.

Objective This study assessed whether apolipoprotein CIII-lipoprotein(a) complexes (ApoCIII-Lp(a)) associate with progression of calcific aortic valve stenosis (AS). Methods Immunostaining for ApoC-III was performed in explanted aortic valve leaflets in 68 patients with leaflet pathological grades of 1–4. Assays measuring circulating levels of ApoCIII-Lp(a) complexes were measured in 218 patients with mild–moderate AS from the AS Progression Observation: Measuring Effects of Rosuvastatin (ASTRONOMER) trial. The progression rate of AS, measured as annualised changes in peak aortic jet velocity (Vpeak), and combined rates of aortic valve replacement (AVR) and cardiac death were determined. For further confirmation of the assay data, a proteomic analysis of purified Lp(a) was performed to confirm the presence of apoC-III on Lp(a). Results Immunohistochemically detected ApoC-III was prominent in all grades of leaflet lesion severity. Significant interactions were present between ApoCIII-Lp(a) and Lp(a), oxidised phospholipids on apolipoprotein B-100 (OxPL-apoB) or on apolipoprotein (a) (OxPL-apo(a)) with annualised Vpeak (all p<0.05). After multivariable adjustment, patients in the top tertile of both apoCIII-Lp(a) and Lp(a) had significantly higher annualised Vpeak (p<0.001) and risk of AVR/cardiac death (p=0.03). Similar results were noted with OxPL-apoB and OxPL-apo(a). There was no association between autotaxin (ATX) on ApoB and ATX on Lp(a) with faster progression of AS. Proteomic analysis of purified Lp(a) showed that apoC-III was prominently present on Lp(a). Conclusion ApoC-III is present on Lp(a) and in aortic valve leaflets. Elevated levels of ApoCIII-Lp(a) complexes in conjunction with Lp(a), OxPL-apoB or OxPL-apo(a) identify patients with pre-existing mild–moderate AS who display rapid progression of AS and higher rates of AVR/cardiac death. Trial registration NCT00800800.

Cardiovascular diseases, such as atherosclerosis and aortic valve sclerosis (AVS) are driven by inflammation induced by a variety of stimuli, including low-density lipoproteins (LDL), reactive oxygen species (ROS), infections, mechanical stress, and chemical insults. Fibrosis is the process of compensating for tissue injury caused by chronic inflammation. Fibrosis is initially beneficial and maintains extracellular homeostasis. However, in the case of AVS and atherosclerosis, persistently active resident fibroblasts, myofibroblasts, and smooth muscle cells (SMCs) perpetually remodel the extracellular matrix under the control of autocrine and paracrine signaling from the immune cells. Myofibroblasts also produce pro-fibrotic factors, such as transforming growth factor-β1 (TGF-β1), angiotensin II (Ang II), and interleukin-1 (IL-1), which allow them to assist in the activation and migration of resident immune cells. Post wound repair, these cells undergo apoptosis or become senescent; however, in the presence of unresolved inflammation and persistence signaling for myofibroblast activation, the tissue homeostasis is disturbed, leading to excessive extracellular matrix (ECM) secretion, disorganized ECM, and thickening of the affected tissue. Accumulating evidence suggests that diverse mechanisms drive fibrosis in cardiovascular pathologies, and it is crucial to understand the impact and contribution of the various mechanisms for the control of fibrosis before the onset of a severe pathological consequence.

The eLDL (enzymatically modified LDL) hypothesis proposes that modification of LDL during atherogenesis occurs through the action of ubiquitous hydrolytic enzymes. eLDL is recognized by multiple macrophage receptors. Following cellular uptake, eLDL triggers atherosclerotic lesion initiation with reversion or progression depending on the balance between cholesterol insudation and depletion. The effects of eLDL on cellular constituents of the atherosclerotic lesion comprise both pro- and anti-inflammatory mechanisms. eLDL triggered complement activation is centrally involved in atherosclerosis with the first CRP (C-reactive protein)-dependent activation step to prevail at the early stages of atherogenesis (lesion initiation with reversion), and the second situation to gain dominance as local concentrations of eLDL surpass a critical threshold (lesion initiation with progression). Thus, CRP-mediated lipoprotein removal likely underlies the regression of early lesions, which occurs continuously through life. Perhaps CRP should be considered as an antiatherogenic agent and the question whether it is an innocent bystander or proatherogenic culprit is not really to the point. The observed association between CRP and atherosclerosis might simply be reverse causation: atherosclerotic disease progression induces CRP.

Atherosclerosis and aortic valve sclerosis are cardiovascular diseases with an increasing prevalence in western societies. Statins are widely applied in atherosclerosis therapy, whereas no pharmacological interventions are available for the treatment of aortic valve sclerosis. Therefore, valve replacement surgery to prevent acute heart failure is the only option for patients with severe aortic stenosis. Both atherosclerosis and aortic valve sclerosis are not simply the consequence of degenerative processes, but rather diseases driven by inflammatory processes in response to lipid-deposition in the blood vessel wall and the aortic valve, respectively. The p38 mitogen-activated protein kinase (MAPK) is involved in inflammatory signaling and activated in response to various intracellular and extracellular stimuli, including oxidative stress, cytokines, and growth factors, all of which are abundantly present in atherosclerotic and aortic valve sclerotic lesions. The responses generated by p38 MAPK signaling in different cell types present in the lesions are diverse and might support the progression of the diseases. This review summarizes experimental findings relating to p38 MAPK in atherosclerosis and aortic valve sclerosis and discusses potential functions of p38 MAPK in the diseases with the aim of clarifying its eligibility as a pharmacological target.

The first ATP‐competitive p38a MAPK/MAPK14 inhibitor with excellent in vivo efficacy and selectivity, skepinone‐L, is now available. We investigated the impact of selective p38a MAPK/MAPK14 inhibition on enzymatically modified LDL (eLDL) stimulated human monocytes with its implications for atherosclerosis. Among the different p38 MAPK isoforms, p38a/MAPK14 was the predominantly expressed and activated isoform in isolated human peripheral blood monocytes. Moreover, eLDL colocalized with macrophages positive for p38a MAPK/ MAPK14 in human carotid endarterectomy specimens. Using the human leukemia cell line THP‐1 and/or primary monocyte‐derived macrophages, skepinone‐L inhibited eLDL‐induced activation of the p38 MAPK pathway, inhibited eLDL induced expression of both cluster of differentiation 36 (CD36) and ATP‐binding cassette, subfamily A, member 1 (ABCA1), without a net effect on foam cell formation, had a cell‐and time‐dependent effect on eLDLtriggered apoptosis, and inhibited eLDL‐stimulated secretion of IL‐8 and MIP‐1b/CCL4 (macrophage inflammatory protein‐1b/chemokine, CC motif, ligand 4). Inhibition of a key signaling molecule of the p38 MAPK pathway, p38a MAPK/MAPK14, by selective inhibitors like skepinone‐L, conclusively facilitates elucidation of the impact of the complex network of p38 MAPK signaling on atherogenesis and might provide a promising therapeutic tool to prevent inflammatory cascades in atherosclerosis.—Cheng, F., Twardowski, L., Fehr, S., Aner, C., Schaeffeler, E., Joos, T., Knorpp, T., Dorweiler, B., Laufer, S., Schwab, M., Torzewski, M. Selective p38a MAP kinase/MAPK14 inhibition in enzymatically modified LDL‐stimulated human monocytes: implications for atherosclerosis. FASEB J. 31, 674–686 (2017). www.fasebj.org

The LPA gene is the only monogenetic risk factor for calcific aortic valve stenosis (CAVS). Oxidized phospholipids (OxPL) and lysophosphatidic acid generated by autotaxin (ATX) from OxPL are pro-inflammatory. Aortic valve leaflets were categorized pathologically from Both ATX-apoB and ATX-apo(a) were measureable in plasma. Lp(a), autotaxin, OxPL and MDA epitopes progressively increased in immunostaining (p<0.001 for all). Six species of OxPL and LysoPA were identified following extraction from valve leaflets. The presence of a constellation of pathologically-linked, Lp(a)-associated molecules in plasma and in aortic valve leaflets of patients with CAVS suggest that Lp(a) is a key etiological factor in CAVS.

Cytologic examination of cerebrospinal fluid (CSF) is a technically simple, yet productive diagnostic procedure. The cytocentrifuge technique is the most commonly utilized method to concentrate the generally scant cellular components of CSF. There are several preanalyti-cal and analytical pitfalls causing artefacts and making proper assessment of the CSF cell preparation more difficult or even impossible. The common cell types of CSF are lymphocytes and monocytes including their activated forms. Cytologic examination of inflammatory conditions puts emphasis on the cellular composition of CSF caused by bacterial infections compared to viral infections and noninfectious inflammatory diseases of the brain. Concerning non-neoplastic disorders, diagnosis of subarach-noidal hemorrhage is of special interest and a main field of application of CSF cytology. The cytology of neoplastic disorders encounters three typical constellations the investigator is usually confronted with: Either a primary malignancy is already known and dissemination to the meninges shall be evaluated or clinical and neuroradio-logical findings are suggestive of neoplastic meningitis though without sufficient evidence of the primary tumor. And third, a spinal tap is performed for other reasons and malignant cells are an incidental finding.

Zusammenfassung: Die Liquorzytologie ist eine technisch vergleichsweise einfache, dabei aber diagnostisch aussagefähige, schnell durchzuführende und kostengünstige Untersuchung, die auch im Rahmen des Notfall- und Basisprogramms der Liquordiagnostik nicht fehlen sollte. Die korrekte Durchführung und Interpretation setzt allerdings einige Erfahrung voraus. Anhand ausgewählter Beispiele zu den Themen Blutung, Meningitis und Meningeosis wird die Wertigkeit der Zytologie demonstriert, die oftmals nicht nur eine Ergänzung zu den üblichen quantitativen Bestimmungen ist, sondern nicht selten auch erst den diagnostisch entscheidenden Hinweis auf die zugrundliegende Erkrankung liefert: Bei der Subarachnoidalblutung ist sie hinsichtlich der Sensitivität der kranialen Computertomographie oftmals überlegen. Bei einer parasitären Meningitis liefert sie aufgrund des Nachweises eosinophiler Granulozyten erst die wesentliche Differentialdiagnose. Auch hinter einer normalen Zellzahl verbirgt sich gelegentlich eine Meningeois neoplastica. Tumorzellen lassen sich dabei mit Hilfe der Immunzytologie näher zuordnen. Entscheidend für eine aussagekräftige Zytologie ist allerdings die strikte Einhaltung präanalytischer Anforderungen.

This study focused on the unique properties of both the Ldlr knockout defect (closely mimicking the human situation) and the BALB/c (C) inbred mouse strain (Th-2 slanted immune response). We generated two immunodeficient strains with severe combined B- and T-cell immunodeficiency with or without a complete lack of natural killer cells to revisit the role of adaptive immune responses on atherogenesis. C-Ldlr-/- Rag1-/- mice, which show severe combined B- and T-cell immunodeficiency and C-Ldlr-/- Rag1-/- Il2rg-/- mice, which combine the T- and B-cell defect with a complete lack of natural killer cells and inactivation of multiple cytokine signalling pathways were fed an atherogenic Western type diet (WTD). Both B6-Ldlr-/- and C-Ldlr-/- immunocompetent mice were used as controls. Body weights and serum cholesterol levels of both immunodeficient strains were significantly increased compared to C-Ldlr-/- controls, except for cholesterol levels of C-Ldlr-/- Rag1-/- double mutants after 12 weeks on the WTD. Quantification of the aortic sinus plaque area revealed that both strains of immunodeficient mice developed significantly more atherosclerosis compared to C-Ldlr-/- controls after 24 weeks on the WTD. Increased atherosclerotic lesion development in C-Ldlr-/- Rag1-/- Il2rg-/- triple mutants was associated with significantly increased numbers of macrophages and significantly decreased numbers of smooth muscle cells compared to both C-Ldlr-/- wild type and C-Ldlr-/- Rag1-/- double mutants pointing to a plaque destabilizing effect of NK cell loss. Collectively, the present study reveals a previously unappreciated complexity with regard to the impact of lymphocytes on lipoprotein metabolism and the role of lymphocyte subsets in plaque composition.

Background We have demonstrated previously that enzymatically degraded low‐density lipoprotein (eLDL) is an essential causative component for the initiation of atherosclerosis. Here, we investigated the different stages of human aortic valve sclerosis for the presence of eLDL and effectors of the innate immune system, as well as the interaction of eLDL with isolated valvular interstitial cells/myofibroblasts to discover possible pathways leading to aortic valve sclerosis. Methods and Results Human aortic valvular tissue was obtained from 68 patients undergoing valve replacement surgery. Patients were classified into 3 groups (mild, moderate, or severe aortic valve sclerosis), and clinical data for statistical analysis were gathered from all patients. Immunohistochemical staining demonstrated extensive extracellular deposits of eLDL throughout all grades of aortic valve sclerosis. Complementary analysis of lipid composition revealed higher concentrations of the decisive components of eLDL (ie, unesterified cholesterol and linoleic acid) compared with internal control tissues. Further, the complement component C3d and terminal complement complexes colocalized with eLDL compatible with the proposal that subendothelially deposited eLDL is enzymatically transformed into a complement activator at early stages of valvular cusp lesion development. Gene expression profiles of proteases and complement components corroborated by immunohistochemistry demonstrated an upregulation of the protease cathepsin D (a possible candidate for LDL degradation to eLDL) and the complement inhibitor CD55. Surprisingly, substantial C‐reactive protein expression was not observed before grade 2 aortic valve sclerosis as investigated with microarray analysis, reverse transcription–polymerase chain reaction analysis, and immunohistochemistry. Finally, we demonstrated cellular uptake of eLDL by valvular interstitial cells/myofibroblasts. Conclusions The present study is a startup of a hypothesis on the pathogenesis of aortic valve sclerosis declaring extracellular lipoprotein modification, subsequent complement activation, and cellular uptake by valvular interstitial cells/myofibroblasts as integral players.

As the main theme of this special issue, CRP not only is an inflammatory marker but also has diverse biological functions associated with different diseases. To investigate CRP’s physiologies and their relationship with human pathological significance, it is essential to use appropriate animal models for translational research. The most popular models for the study of CRP are transgenic mice. However, researchers should be careful when extrapolating the findings derived from these animal models. This review will discuss the current concerns on CRP transgenic mice and rabbits.

Abstract With increasing life expectancy, neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), as well as neurovascular diseases such as cerebral ischemia, are becoming a rapidly growing worldwide problem causing an increased burden on society. The development of biomarkers is aimed to enable the identification of at-risk individuals, enable early diagnosis, and reflect the progression of disease. Future challenges refer to the need for standardization of known biomarkers on the one hand and the identification of new biomarkers on the other hand. The former is impressively exemplified by a recent study addressing the impact of various types of cerebrospinal fluid collection tubes for detection of total tau, phosphorylated tau, and β-amyloid 1–42 on the diagnosis of AD. This issue was identified as a key issue by the Alzheimer’s Biomarkers Standardization Initiative (ABSI) and recommendations were listed which should be used to enable better standardization between laboratories and enable results for the AD biomarkers to be comparable between different sites. The latter challenge in particular concerns PD and is addressed by the Parkinson Progression Marker Initiative (PPMI), a comprehensive observational, international, multi-center study designed to identify PD progression biomarkers both to improve understanding of disease etiology and course and to provide crucial tools to enhance the likelihood of success of PD modifying therapeutic trials. With regard to cerebral ischemia, at present there are no clinically validated biomarkers. Nevertheless, the development of a clinically validated biomarker of acute cerebral ischemia would have the potential to facilitate the use of time-sensitive reperfusion strategies, allow for individualization of patient care by predicting relative risk of hemorrhage and volume of penumbral tissue, and add valuable prognostic information for patients presenting with acute stroke. Thereby, it is expected that a panel of biomarkers rather than one single marker will meet these criteria in the future. Unlike cerebral ischemia, cerebral hemorrhage biomarkers will probably be limited to the assessment of prognosis in the course of the disease.