Inequalities in regional excess mortality and life expectancy during the COVID-19 pandemic in Europe.

Using data for 201 regions (NUTS 2) in Europe, we examine the mortality burden of the COVID-19 pandemic and how the mortality inequalities between regions changed between 2020 and 2022. We show that over the three years of the pandemic, not only did the level of excess mortality rate change considerably, but also its geographical distribution. Focusing on life expectancy as a summary measure of mortality conditions, we find that the variance of regional life expectancy increased sharply in 2021 but returned to the pre-pandemic level in 2022. The 2021 increase was due to a much higher-than-average excess mortality in regions with lower pre-pandemic life expectancy. While the life expectancy inequality has returned to its pre-pandemic level in 2022, the observed life expectancy in almost all regions is far below that expected without the pandemic.

Contribution of cultural heritage values to steppe conservation on ancient burial mounds of Eurasia.

Civilizations, including ancient ones, have shaped global ecosystems in many ways through coevolution of landscapes and humans. However, the cultural legacies of ancient and lost civilizations are rarely considered in the conservation of the Eurasian steppe biome. We used a data set containing more than 1000 records on localities, land cover, protection status, and cultural values related to ancient steppic burial mounds (kurgans); we evaluated how these iconic and widespread landmarks can contribute to grassland conservation in the Eurasian steppes, which is one of the most endangered biomes on Earth. Using Bayesian logistic generalized regressions and proportional odds logistic regressions, we examined the potential of mounds to preserve grasslands in landscapes with different levels of land-use transformation. We also compared the conservation potential of mounds inside and outside protected areas and assessed whether local cultural values support the maintenance of grasslands on them. Kurgans were of great importance in preserving grasslands in transformed landscapes outside protected areas, where they sometimes acted as habitat islands that contributed to habitat conservation and improved habitat connectivity. In addition to steep slopes hindering ploughing, when mounds had cultural value for local communities, the probability of grassland occurrence on kurgans almost doubled. Because the estimated number of steppic mounds is about 600,000 and similar historical features exist on all continents, our results may be applicable at a global level. Our results also suggested that an integrative socioecological approach in conservation might support the positive synergistic effects of conservation, landscape, and cultural values.

Contribución de los valores culturales para la conservación esteparia en los antiguos montículos funerarios de Eurasia Resumen Las civilizaciones modernas y antiguas han moldeado de muchas maneras los ecosistemas globales mediante la coevolución del paisaje y la humanidad. Sin embargo, pocas veces se considera el legado cultural de las civilizaciones perdidas o antiguas para la conservación del bioma de la estepa euroasiática. Usamos un conjunto de datos que contiene más de 1,000 registros de las localidades, cobertura del suelo, estado de protección y valores culturales relacionados con los antiguos montículos funerarios de esta estepa (kurgans). Después analizamos cómo estos símbolos icónicos y distribuidos extensamente pueden contribuir a la conservación de los pastizales en la estepa euroasiática, uno de los biomas en mayor peligro de extinción. Analizamos el potencial de conservación de los montículos en paisajes con diferentes niveles de transformación en el uso de suelo mediante regresiones logísticas generalizadas bayesianas y regresiones logísticas de probabilidades proporcionales. También comparamos el potencial de conservación de los montículos dentro y fuera de las áreas protegidas y evaluamos si los valores culturales locales conservan los pastizales dentro de estas mismas áreas. Los kurgans fueron de gran importancia para la conservación de los pastizales en los paisajes transformados ubicados fuera de las áreas protegidas, en donde llegaron a fungir como hábitats aislados que contribuyeron a la conservación y conectividad del hábitat. Además de que las pendientes pronunciadas impiden el arado, cuando los montículos contaban con valor cultural para las comunidades locales, la probabilidad de que el pastizal se ubicara sobre un kurgan casi se duplicó. Ya que se estima que el número de montículos esteparios ronda los 6,000 y que rasgos históricos similares existen en todos los continentes, nuestros resultados pueden aplicarse a nivel global. Nuestros resultados también sugieren que una estrategia socio-ecológica integradora para la conservación podría respaldar los efectos sinérgicos positivos de la conservación, el paisaje y los valores culturales.

Compositional Analysis of Glycosaminoglycans in Different Lung Cancer Types-A Pilot Study.

Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.

Green Biomass-Based Protein for Sustainable Feed and Food Supply: An Overview of Current and Future Prospective.

It is necessary to develop and deploy novel protein production to allow the establishment of a sustainable supply for both humans and animals, given the ongoing expansion of protein demand to meet the future needs of the increased world population and high living standards. In addition to plant seeds, green biomass from dedicated crops or green agricultural waste is also available as an alternative source to fulfill the protein and nutrient needs of humans and animals. The development of extraction and precipitation methods (such as microwave coagulation) for chloroplast and cytoplasmic proteins, which constitute the bulk of leaf protein, will allow the production of leaf protein concentrates (LPC) and protein isolates (LPI). Obtained LPC serves as a sustainable alternative source of animal-based protein besides being an important source of many vital phytochemicals, including vitamins and substances with nutritional and pharmacological effects. Along with it, the production of LPC, directly or indirectly, supports sustainability and circular economy concepts. However, the quantity and quality of LPC largely depend on several factors, including plant species, extraction and precipitation techniques, harvest time, and growing season. This paper provides an overview of the history of green biomass-derived protein from the early green fodder mill concept by Károly Ereky to the state-of-art of green-based protein utilization. It highlights potential approaches for enhancing LPC production, including dedicated plant species, associated extraction methods, selection of optimal technologies, and best combination approaches for improving leaf protein isolation.

Use of the Modified Ramberg-Osgood Material Model to Predict Dynamic Modulus Master Curves of Asphalt Mixtures.

Dynamic modulus master curves are usually constructed by using sigmoid functions, but the coefficients of these functions are not independent of each other. For this reason, it is not possible to clearly identify their physical mean. Another way of describing the dynamic modulus master curves is to choose the Ramberg-Osgood (RAMBO) material model, which is also well-suited for modelling the cyclic behaviour of soils. The Ramberg-Osgood model coefficients are completely independent of each other, so the evaluation of the fitted curve is simple and straightforward. This paper deals with the application of the Ramberg-Osgood material model compared to the usual techniques for constructing a master curve, determining the accuracy in describing the material behaviour of asphalt mixtures, and seeking any surplus information that cannot be derived by traditional techniques. Because the dynamic modulus and phase angle master curves are strictly related, in the present study, the asymmetric bell-shaped frequency curve of Toranzos was used to describe the phase angle for four types of asphalt mixtures (RmB, PmB, RA, and NB). The results show that the RAMBO model is a good alternative to the sigmoid function in describing the master curve of the dynamic modulus. We successfully used the Toranzos asymmetric bell-shaped frequency curve to describe the phase angle master curve. We also found a promising relationship between the independent RAMBO model parameters and the physical properties of the investigated binders, but this requires further research.

Does sexual Intimate Partner Violence (IPV) increase risk of multiple high-risk fertility behaviours in India: evidence from National Family Health Survey 2015-16.

BACKGROUND: One in three women from lower and middle-income countries are subjected to physical and/or sexual intimate partner violence (IPV) in their life span. Prior studies have highlighted a range of adverse health impacts of sexual IPV. However, less is known about the link between multiple high-risk fertility behaviours and sexual intimate partner violence. The present study examines the statistical association between multiple high-risk fertility behaviours and sexual intimate partner violence among women in India. METHODS: The present study used a nationally representative dataset, the National Family Health Survey (NFHS-4) 2015-16. A total of 23,597 women were included in the study; a subsample of married women of reproductive age who have had at least one child 5 years prior to the survey and who had valid information about sexual IPV. Logistic regression models were employed alongside descriptive statistics. RESULTS: Approximately 7% of women who are or had been married face sexual IPV. The prevalence of sexual violence was higher among women who had short birth intervals and women who had given birth more than three times (12%). Around 11% of women who had experienced any high-risk fertility behaviours also experienced sexual violence. The unadjusted association suggested that multiple high-risk fertility behaviours were 32% (UORs = 1.32, 95% CI: 1.16-1.50) higher for those women who experienced sexual violence. After adjusting for other sociodemographic variables, except for women's education and wealth quantile, the odds of multiple high-risk fertility behaviours were 16% (AOR = 1.16; 95% CI: 1.02-1.34) higher among women who faced sexual violence. The inclusion of women's educational attainment and wealth status in the model made the association between sexual IPV and high-risk fertility behaviours insignificant. CONCLUSION: Sexual intimate partner violence is statistically associated with high-risk fertility behaviours among women in India. Programs and strategies designed to improve women's reproductive health should investigate the different dimensions of sexual IPV in India.

The mechanosensitive Piezo1 channels contribute to the arterial medial calcification.

Vascular calcification (VC) is associated with a number of cardiovascular diseases, as well as chronic kidney disease. The role of smooth muscle cells (SMC) has already been widely explored in VC, as has the role of intracellular Ca2+ in regulating SMC function. Increased intracellular calcium concentration ([Ca2+]i) in vascular SMC has been proposed to stimulate VC. However, the contribution of the non-selective Piezo1 mechanosensitive cation channels to the elevation of [Ca2+]i, and consequently to the process of VC has never been examined. In this work the essential contribution of Piezo1 channels to arterial medial calcification is demonstrated. The presence of Piezo1 was proved on human aortic smooth muscle samples using immunohistochemistry. Quantitative PCR and Western blot analysis confirmed the expression of the channel on the human aortic smooth muscle cell line (HAoSMC). Functional measurements were done on HAoSMC under control and calcifying condition. Calcification was induced by supplementing the growth medium with inorganic phosphate (1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L) for 7 days. Measurement of [Ca2+]i using fluorescent Fura-2 dye upon stimulation of Piezo1 channels (either by hypoosmolarity, or Yoda1) demonstrated significantly higher calcium transients in calcified as compared to control HAoSMCs. The expression of mechanosensitive Piezo1 channel is augmented in calcified arterial SMCs leading to a higher calcium influx upon stimulation. Activation of the channel by Yoda1 (10 µmol/L) enhanced calcification of HAoSMCs, while Dooku1, which antagonizes the effect of Yoda1, reduced this amplification. Application of Dooku1 alone inhibited the calcification. Knockdown of Piezo1 by siRNA suppressed the calcification evoked by Yoda1 under calcifying conditions. Our results demonstrate the pivotal role of Piezo1 channels in arterial medial calcification.

Quantification of Polyethylene Glycol 400 Excreted in the Urine by MALDI-TOF Mass Spectrometry.

Polyethylene glycol 400 (PEG 400) was used as a permeability probe to examine the gastrointestinal tract which can be involved in the pathogenesis of some inflammatory and autoimmune diseases. A novel methodology was developed and validated for the quantitation of PEG 400 excreted in human urine after oral administration using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The excretion ratios were determined for the most intense ions corresponding to nine PEG 400 oligomers. The relative error of accuracy was between -6.0% and 8.5%, and the relative standard deviation (RSD) of the precision was below 15%. Our method was successfully applied in a large-scale experimental study involving nearly two hundred volunteers. Due to the large number of measurements, detailed and reliable statistical analysis was performed. No significant difference was found between the male and female group of volunteers at 0.05 significance level, except the two largest PEG oligomers. However, the average excretion ratios of the male volunteers are greater than that of the women for all the nine PEG oligomers, suggesting a difference in the intestinal permeability between men and women.

Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis.

Aim: The aim of our study was to explore the pathophysiologic role of oxidation of hemoglobin (Hb) to ferrylHb in human atherosclerosis. Results: We observed a severe oxidation of Hb to ferrylHb in complicated atherosclerotic lesions of carotid arteries with oxidative changes of the globin moieties, detected previously described oxidation hotspots in Hb (ß1Cys93; ß1Cys112; ß2Cys112) and identified a novel oxidation hotspot (α1Cys104). After producing a monoclonal anti-ferrylHb antibody, ferrylHb was revealed to be localized extracellularly and also internalized by macrophages in the human hemorrhagic complicated lesions. We demonstrated that ferrylHb is taken up via phagocytosis as well as CD163 receptor-mediated endocytosis and then transported to lysosomes involving actin polymerization. Internalization of ferrylHb was accompanied by upregulation of heme oxygenase-1 and H-ferritin and accumulation of iron within lysosomes as a result of heme/iron uptake. Importantly, macrophages exposed to ferrylHb in atherosclerotic plaques exhibited a proinflammatory phenotype, as reflected by elevated levels of IL-1ß and TNF-α. To find further signatures of ferrylHb in complicated lesions, we performed RNA-seq analysis on biopsies from patients who underwent endarterectomies. RNA-seq analysis demonstrated that human complicated lesions had a unique transcriptomic profile different from arteries and atheromatous plaques. Pathways affected in complicated lesions included gene changes associated with phosphoinositide 3-kinase (PI3K) signaling, lipid transport, tissue remodeling, and vascularization. Targeted analysis of gene expression associated with calcification, apoptosis, and hemolytic-specific clusters indicated an increase in the severity of complicated lesions compared with atheroma. A 39% overlap in the differential gene expression profiles of human macrophages exposed to ferrylHb and the complicated lesion profiles was uncovered. Among these 547 genes, we found inflammatory, angiogenesis, and iron metabolism gene clusters regulated in macrophages. Innovation and Conclusion: We conclude that oxidation of Hb to ferrylHb contributes to the progression of atherosclerosis via polarizing macrophages into a proatherogenic phenotype. Antioxid. Redox Signal. 35, 917-950.

Ferryl Hemoglobin and Heme Induce A1-Microglobulin in Hemorrhaged Atherosclerotic Lesions with Inhibitory Function against Hemoglobin and Lipid Oxidation.

Infiltration of red blood cells into atheromatous plaques and oxidation of hemoglobin (Hb) and lipoproteins are implicated in the pathogenesis of atherosclerosis. α1-microglobulin (A1M) is a radical-scavenging and heme-binding protein. In this work, we examined the origin and role of A1M in human atherosclerotic lesions. Using immunohistochemistry, we observed a significant A1M immunoreactivity in atheromas and hemorrhaged plaques of carotid arteries in smooth muscle cells (SMCs) and macrophages. The most prominent expression was detected in macrophages of organized hemorrhage. To reveal a possible inducer of A1M expression in ruptured lesions, we exposed aortic endothelial cells (ECs), SMCs and macrophages to heme, Oxy- and FerrylHb. Both heme and FerrylHb, but not OxyHb, upregulated A1M mRNA expression in all cell types. Importantly, only FerrylHb induced A1M protein secretion in aortic ECs, SMCs and macrophages. To assess the possible function of A1M in ruptured lesions, we analyzed Hb oxidation and heme-catalyzed lipid peroxidation in the presence of A1M. We showed that recombinant A1M markedly inhibited Hb oxidation and heme-driven oxidative modification of low-density lipoproteins as well plaque lipids derived from atheromas. These results demonstrate the presence of A1M in atherosclerotic plaques and suggest its induction by heme and FerrylHb in the resident cells.

Heme cytotoxicity is the consequence of endoplasmic reticulum stress in atherosclerotic plaque progression.

Hemorrhage and hemolysis with subsequent heme release are implicated in many pathologies. Endothelial cells (ECs) encounter large amount of free heme after hemolysis and are at risk of damage from exogenous heme. Here we show that hemorrhage aggravates endoplasmic reticulum (ER) stress in human carotid artery plaques compared to healthy controls or atheromas without hemorrhage as demonstrated by RNA sequencing and immunohistochemistry. In EC cultures, heme also induces ER stress. In contrast, if cultured ECs are pulsed with heme arginate, cells become resistant to heme-induced ER (HIER) stress that is associated with heme oxygenase-1 (HO-1) and ferritin induction. Knocking down HO-1, HO-2, biliverdin reductase, and ferritin show that HO-1 is the ultimate cytoprotectant in acute HIER stress. Carbon monoxide-releasing molecules (CORMs) but not bilirubin protects cultured ECs from HIER stress via HO-1 induction, at least in part. Knocking down HO-1 aggravates heme-induced cell death that cannot be counterbalanced with any known cell death inhibitors. We conclude that endothelium and perhaps other cell types can be protected from HIER stress by induction of HO-1, and heme-induced cell death occurs via HIER stress that is potentially involved in the pathogenesis of diverse pathologies with hemolysis and hemorrhage including atherosclerosis.

Is the prediction of one or two ipsilateral positive lymph nodes by computerized tomography and ultrasound reliable enough to restrict therapeutic neck dissection in oral squamous cell carcinoma (OSCC) patients?

INTRODUCTION: Proper management of the clinically involved neck in OSCC patients continues to be a matter of debate. Our aim was to analyze the accuracy of computerized tomography (CT) and ultrasound (US) in anticipating the exact location of lymph node (LN) metastases of OSCC patients across the AAO-HNS (American Academy of Otolaryngology-Head and Neck Surgery) levels ipsi- and contralaterally. Furthermore, we wanted to assess the suitability of therapeutic selective neck dissection (SND) in patients with one or two ipsilateral positive nodes upon clinical staging (cN1/cN2a and cN2b(2/x) patients). METHODS: We prospectively analyzed the LN status of patients with primary OSCC using CT and US from 2007 to 2013. LNs were individually assigned to a map containing the AAO-HNS levels; patients bearing a single or just two ipsilateral positive nodes (designated cN1/cN2a or cN2b(2/x) patients either by CT (CT group) or US alone (US group) or in a group combining findings of CT and US (CTUS group)) received an ipsi-ND (I-V) and a contra-ND (I-IV). 78% of the LNs were sent individually for routine histopathological examination; the remaining were dissected and analyzed per neck level. RESULTS: Upon the analysis of 1.670 LNs of 57 patients, the exact location of pathology proven LN metastases in cN1 patients was more precisely predicted by US compared to CT with confirmed findings only in levels IA, IB und IIA. Clearly decreasing the number of missed lesions, the findings in the CTUS group nearly kept the spatial reliability of the US group. The same analysis for patients with exactly two supposed ipsilateral lesions (cN2b(2/x)) yielded confirmed metastases from levels I to V for both methods individually and in combination and, therefore, render SND insufficient for these cases. CONCLUSION: Our findings stress the importance of conducting both, CT and US, in patients with primary OSCC. Only the combination of their findings warrants the application of therapeutic SND in patients with a single ipsilateral LN metastasis (cN1/cN2a patients) but not in patients with more than one lesion upon clinical staging (≥ cN2b).

Expression of apoptosis repressor with caspase recruitment domain (ARC) in familial adenomatous polyposis (FAP) adenomas and its correlation with DNA mismatch repair proteins, p53, Bcl-2, COX-2 and beta-catenin.

BACKGROUND: Colorectal familial adenomatous polyposis (FAP) adenomas exhibit a uniform pathogenetic basis caused by a germline mutation in the adenomatous polyposis gene (APC), but the molecular changes leading to their development are incompletely understood. However, dysregulated apoptosis is known to substantially affect the development of colonic adenomas. One of the key regulatory proteins involved in apoptosis is apoptosis repressor with caspase recruitment domain (ARC). METHODS: The expression of nuclear and cytoplasmic ARC in 212 adenomas from 80 patients was analyzed by immunohistochemistry. We also compared expression levels of ARC with the expression levels of p53, Bcl-2, COX-2, and MMR proteins. Statistical analyses were performed by Spearman's rank correlation and linear regression test. RESULTS: ARC was overexpressed in the nuclei and cytoplasm of most FAP adenomas investigated. Cytoplasmic ARC staining was moderately stronger (score 2) in 49.1% (n = 104/212) and substantially stronger (score 3) in 32.5% (n = 69/212) of adenomas compared to non-tumorous colorectal mucosa. In 18.4% (n = 39/212) of adenomas, cytoplasmic ARC staining was equivalent to that in non-tumorous mucosa. Nuclear expression of ARC in over 75% of cells was present in 30.7% (n = 65/212) of investigated adenomas, and nuclear expression in 10-75% of cells was detected in 62.7% (n = 133/212). ARC expression in under 10% of nuclei was found in 6.6% (n = 14/212) of adenomas. The correlation between nuclear ARC expression and cytoplasmic ARC expression was highly significant (p = 0.001). Moreover, nuclear ARC expression correlated positively with overexpression of Bcl-2, COX-2 p53 and ß-catenin. Cytoplasmic ARC also correlated with overexpression of Bcl-2. Sporadic MMR deficiency was detected in very few FAP adenomas and showed no correlation with nuclear or cytoplasmic ARC. CONCLUSIONS: Our results demonstrated that both cytoplasmic and nuclear ARC are overexpressed in FAP adenomas, thus in a homogenous collective. The highly significant correlation between nuclear ARC and nuclear ß-catenin suggested that ARC might be regulated by ß-catenin in FAP adenomas. Because of its further correlations with p53, Bcl-2, and COX-2, nuclear ARC might play a substantial role not only in carcinomas but also in precursor lesions. Video Abstract.

Aerial Laser Scanning Data as a Source of Terrain Modeling in a Fluvial Environment: Biasing Factors of Terrain Height Accuracy.

Airborne light detection and ranging (LiDAR) scanning is a commonly used technology for representing the topographic terrain. As LiDAR point clouds include all surface features present in the terrain, one of the key elements for generating a digital terrain model (DTM) is the separation of the ground points. In this study, we intended to reveal the efficiency of different denoising approaches and an easy-to-use ground point classification technique in a floodplain with fluvial forms. We analyzed a point cloud from the perspective of the efficiency of noise reduction, parametrizing a ground point classifier (cloth simulation filter, CSF), interpolation methods and resolutions. Noise filtering resulted a wide range of point numbers in the models, and the number of points had moderate correlation with the mean accuracies (r = -0.65, p < 0.05), indicating that greater numbers of points had larger errors. The smallest differences belonged to the neighborhood-based noise filtering and the larger cloth size (5) and the smaller threshold value (0.2). The most accurate model was generated with the natural neighbor interpolation with the cloth size of 5 and the threshold of 0.2. These results can serve as a guide for researchers using point clouds when considering the steps of data preparation, classification, or interpolation in a flat terrain.

Ferryl Hemoglobin Inhibits Osteoclastic Differentiation of Macrophages in Hemorrhaged Atherosclerotic Plaques.

Intraplaque hemorrhage frequently occurs in atherosclerotic plaques resulting in cell-free hemoglobin, which is oxidized to ferryl hemoglobin (FHb) in the highly oxidative environment. Osteoclast-like cells (OLCs) derived from macrophages signify a counterbalance mechanism for calcium deposition in atherosclerosis. Our aim was to investigate whether oxidized hemoglobin alters osteoclast formation, thereby affecting calcium removal from mineralized atherosclerotic lesions. RANKL- (receptor activator of nuclear factor kappa-Β ligand-) induced osteoclastogenic differentiation and osteoclast activity of RAW264.7 cells were studied in response to oxidized hemoglobin via assessing bone resorption activity, expression of osteoclast-specific genes, and the activation of signalization pathways. OLCs in diseased human carotid arteries were assessed by immunohistochemistry. FHb, but not ferrohemoglobin, decreased bone resorption activity and inhibited osteoclast-specific gene expression (tartrate-resistant acid phosphatase, calcitonin receptor, and dendritic cell-specific transmembrane protein) induced by RANKL. In addition, FHb inhibited osteoclastogenic signaling pathways downstream of RANK (receptor activator of nuclear factor kappa-Β). It prevented the induction of TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) and c-Fos, phosphorylation of p-38 and JNK (c-Jun N-terminal kinase), and nuclear translocation of NFκB (nuclear factor kappa-Β) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1). These effects were independent of heme oxygenase-1 demonstrated by knocking down HO-1 gene in RAW264.7 cells and in mice. Importantly, FHb competed with RANK for RANKL binding suggesting possible mechanisms by which FHb impairs osteoclastic differentiation. In diseased human carotid arteries, OLCs were abundantly present in calcified plaques and colocalized with regions of calcium deposition, while the number of these cells were lower in hemorrhagic lesions exhibiting accumulation of FHb despite calcium deposition. We conclude that FHb inhibits RANKL-induced osteoclastic differentiation of macrophages and suggest that accumulation of FHb in a calcified area of atherosclerotic lesion with hemorrhage retards the formation of OLCs potentially impairing calcium resorption.

Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction.

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1ß and nuclear translocation of the NF-κß transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.