Assessment of the Physical Activity of Children with Asthma Bronchiale.

Physical activity is an especially important part of everyday life for children with chronic diseases. The aim of the study was to show whether asthma is a barrier to physical activity in our society. The correlations between the severity of the disease, body mass index, and physical activity were analyzed, and parents' opinions on whether children should participate in active sports were assessed. Physical activity of children with asthma was analyzed by questionnaires; 93 parents and their 93 children were involved in the survey. The age of children was 12.6 ± 3.5 years (mean ± SD), 69.9% were boys, 30.1% were girls. A total of 93.4% of the respondents participated in a physical education program and 56.5% also attended sporting activities on a regular basis. In terms of disease severity, 61.2% of the children had mild asthma, 37.6% moderate, and 1.2% severe, and 6.5% of the respondents also stated that their children's illness had been consistently or frequently limiting their performance concerning their school or home duties over the past four weeks. Of the parents surveyed, 12% felt that physical activity was not appropriate in the context of this disease. We concluded that fear of the consequences of physical activity depends largely on education, which should involve parents, teachers, and coaches.

Distinct subsets of anti-pulmonary autoantibodies correlate with disease severity and survival in severe COVID-19 patients.

Autoantibodies targeting the lung tissue were identified in severe COVID-19 patients in this retrospective study. Fifty-three percent of 104 patients developed anti-pulmonary antibodies, the majority of which were IgM class, suggesting that they developed upon infection with SARS-CoV-2. Anti-pulmonary antibodies correlated with worse pulmonary function and a higher risk of multiorgan failure that was further aggravated if 3 or more autoantibody clones were simultaneously present (multi-producers). Multi-producer patients were older than the patients with less or no autoantibodies. One of the identified autoantibodies (targeting a pulmonary protein of ~ 50 kDa) associated with worse clinical outcomes, including mortality. In summary, severe COVID-19 is associated with the development of lung-specific autoantibodies, which may worsen the clinical outcome. Tissue proteome-wide tests, such as the ones applied here, can be used to detect autoimmunity in the post-COVID state to identify the cause of symptoms and to reveal a new target for treatment.

Prevention of Chronic Morbidities in Extremely Premature Newborns with LISA-nCPAP Respiratory Therapy and Adjuvant Perinatal Strategies.

Less invasive surfactant administration techniques, together with nasal continuous airway pressure (LISA-nCPAP) ventilation, an emerging noninvasive ventilation (NIV) technique in neonatology, are gaining more significance, even in extremely premature newborns (ELBW), under 27 weeks of gestational age. In this review, studies on LISA-nCPAP are compiled with an emphasis on short- and long-term morbidities associated with prematurity. Several perinatal preventative and therapeutic investigations are also discussed in order to start integrated therapies as numerous organ-saving techniques in addition to lung-protective ventilations. Two thirds of immature newborns can start their lives on NIV, and one third of them never need mechanical ventilation. With adjuvant intervention, these ratios are expected to be increased, resulting in better outcomes. Optimized cardiopulmonary transition, especially physiologic cord clamping, could have an additively beneficial effect on patient outcomes gained from NIV. Organ development and angiogenesis are strictly linked not only in the immature lung and retina, but also possibly in the kidney, and optimized interventions using angiogenic growth factors could lead to better morbidity-free survival. Corticosteroids, caffeine, insulin, thyroid hormones, antioxidants, N-acetylcysteine, and, moreover, the immunomodulatory components of mother's milk are also discussed as adjuvant treatments, since immature newborns deserve more complex neonatal interventions.

Comparison of flexible nasogastric tube and semi-rigid catheter during less invasive surfactant administration.

BACKGROUND: Various flexible and semi-rigid catheter techniques have been reported for surfactant delivery during less invasive surfactant administration (LISA) in preterm infants. Data on the effect of catheter selection on procedural success rates and adverse events are limited. Our objective was to compare the rates of success and adverse events of LISA performed with nasogastric tube and semi-rigid catheter. METHODS: This was a post-hoc analysis of data from a quality improvement project. LISA was performed according to the standardized local protocol. Baseline characteristics, data on performance of LISA, degree of difficulty in laryngoscopy and vital parameters after the initiation of LISA were collected and outcomes were compared between groups. RESULTS: Fifty-six infants were included (21 with nasogastric tube, and 35 with semi-rigid catheter). Procedure success rate (defined as a single LISA attempt resulting in intratracheal administration of the planned dose of surfactant), incidence of adverse events, heart rate and oxygen saturation values and outcomes did not differ significantly between the two groups. When using a nasogastric tube for LISA, a significantly higher fraction of inspired oxygen was needed in the 3rd (0.62 vs. 0.48, P=0.024), 4th (0.61 vs. 0.37, P<0.001) and 5th minute (0.48 vs. 0.37, P=0.001) to maintain normal oxygen saturations. CONCLUSIONS: Use of the semi-rigid catheter was associated with better oxygenation during and shortly after the procedure. Our results may help neonatal units to develop local guidelines.

Interactions of reactive sulfur species with metalloproteins.

Reactive sulfur species (RSS) entail a diverse family of sulfur derivatives that have emerged as important effector molecules in H2S-mediated biological events. RSS (including H2S) can exert their biological roles via widespread interactions with metalloproteins. Metalloproteins are essential components along the metabolic route of oxygen in the body, from the transport and storage of O2, through cellular respiration, to the maintenance of redox homeostasis by elimination of reactive oxygen species (ROS). Moreover, heme peroxidases contribute to immune defense by killing pathogens using oxygen-derived H2O2 as a precursor for stronger oxidants. Coordination and redox reactions with metal centers are primary means of RSS to alter fundamental cellular functions. In addition to RSS-mediated metalloprotein functions, the reduction of high-valent metal centers by RSS results in radical formation and opens the way for subsequent per- and polysulfide formation, which may have implications in cellular protection against oxidative stress and in redox signaling. Furthermore, recent findings pointed out the potential role of RSS as substrates for mitochondrial energy production and their cytoprotective capacity, with the involvement of metalloproteins. The current review summarizes the interactions of RSS with protein metal centers and their biological implications with special emphasis on mechanistic aspects, sulfide-mediated signaling, and pathophysiological consequences. A deeper understanding of the biological actions of reactive sulfur species on a molecular level is primordial in H2S-related drug development and the advancement of redox medicine.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease.

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves. Lower levels of bioavailable H2S and higher levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine ß-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1ß and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1ß and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation. We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin/heme-mediated vascular damage in atherosclerosis.

Vulnerable atherosclerotic plaques with hemorrhage considerably contribute to cardiovascular morbidity and mortality. Calcification is the main characteristic of advanced atherosclerotic lesions and calcified aortic valve disease (CAVD). Lyses of red blood cells and hemoglobin (Hb) release occur in human hemorrhagic complicated lesions. During the interaction of cell-free Hb with plaque constituents, Hb is oxidized to ferric and ferryl states accompanied by oxidative changes of the globin moieties and heme release. Accumulation of both ferryl-Hb and metHb has been observed in atherosclerotic plaques. The oxidation hotspots in the globin chain are the cysteine and tyrosine amino acids associated with the generation of Hb dimers, tetramers and polymers. Moreover, fragmentation of Hb occurs leading to the formation of globin-derived peptides. A series of these pro-atherogenic cellular responses can be suppressed by hydrogen sulfide (H2S). Since H2S has been explored to exhibit a wide range of physiologic functions to maintain vascular homeostasis, it is not surprising that H2S may play beneficial effects in the progression of atherosclerosis. In the present review, we summarize the findings about the effects of H2S on atherosclerosis and CAVD with a special emphasis on the oxidation of Hb/heme in atherosclerotic plaque development and vascular calcification.

The majority of severe COVID-19 patients develop anti-cardiac autoantibodies.

Severe cases of COVID-19 are characterized by an inflammatory burst, which is accompanied by multiorgan failure. The elderly population has higher risk for severe or fatal outcome for COVID-19. Inflammatory mediators facilitate the immune system to combat viral infection by producing antibodies against viral antigens. Several studies reported that the pro-inflammatory state and tissue damage in COVID-19 also promotes autoimmunity by autoantibody generation. We hypothesized that a subset of these autoantibodies targets cardiac antigens. Here we aimed to detect anti-cardiac autoantibodies in severe COVID-19 patients during hospitalization. For this purpose, 104 COVID-19 patients were recruited, while 40 heart failure patients with dilated cardiomyopathy and 20 patients with severe aortic stenosis served as controls. Patients were tested for anti-cardiac autoantibodies, using human heart homogenate as a bait. Follow-up samples were available in 29 COVID-19 patients. Anti-cardiac autoantibodies were detected in 68% (71 out of 104) of severe COVID-19 patients. Overall, 39% of COVID-19 patients had anti-cardiac IgG autoantibodies, while 51% had anti-cardiac autoantibodies of IgM isotype. Both IgG and IgM anti-cardiac autoantibodies were observed in 22% of cases, and multiple cardiac antigens were targeted in 38% of COVID-19 patients. These anti-cardiac autoantibodies targeted a diverse set of myocardial proteins, without apparent selectivity. As controls, heart failure patients (with dilated cardiomyopathy) had similar occurrence of IgG (45%, p = 0.57) autoantibodies, while significantly lower occurrence of IgM autoantibodies (30%, p = 0.03). Patients with advanced aortic stenosis had significantly lower number of both IgG (11%, p = 0.03) and IgM (10%, p < 0.01) type anti-cardiac autoantibodies than that in COVID-19 patients. Furthermore, we detected changes in the anti-cardiac autoantibody profile in 7 COVID-19 patients during hospital treatment. Surprisingly, the presence of these anti-cardiac autoantibodies did not affect the clinical outcome and the prevalence of the autoantibodies did not differ between the elderly (over 65 years) and the patients younger than 65 years of age. Our results demonstrate that the majority of hospitalized COVID-19 patients produce novel anti-cardiac IgM autoantibodies. COVID-19 also reactivates resident IgG autoantibodies. These autoantibodies may promote autoimmune reactions, which can complicate post-COVID recuperation, contributing to post-acute sequelae of COVID-19 (long COVID).

A Randomized, Controlled Trial to Investigate the Efficacy of Nebulized Poractant Alfa in Premature Babies with Respiratory Distress Syndrome.

OBJECTIVE: To investigate the efficacy and safety of nebulized poractant alfa (at 200 and 400 mg/kg doses) delivered in combination with nasal continuous positive airway pressure compared with nasal continuous positive airway pressure alone in premature infants with diagnosed respiratory distress syndrome. STUDY DESIGN: This randomized, controlled, multinational study was conducted in infants at 280/7 to 326/7 weeks of gestation. The primary outcome was the incidence of respiratory failure in the first 72 hours of life, defined as needing endotracheal surfactant and/or mechanical ventilation owing to prespecified criteria. Secondary outcomes included the time to respiratory failure in the first 72 hours, duration of ventilation, mortality, incidence of bronchopulmonary dysplasia, and major associated neonatal comorbidities. In addition, the safety and tolerability of the treatments were assessed reporting the number and percentage of infants with treatment-emergent adverse events and adverse drug reactions during nebulization. RESULTS: In total, 129 infants were randomized. No significant differences were observed for the primary outcome: 24 (57%), 20 (49%), and 25 (58%) infants received endotracheal surfactant and/or mechanical ventilation within 72 hours in the poractant alfa 200 mg/kg, poractant alfa 400 mg/kg, and nasal continuous positive airway pressure groups, respectively. Similarly, secondary respiratory outcomes did not differ among groups. Enrollment was halted early owing to a change in the benefit-risk balance of the intervention. Nebulized poractant alfa was well-tolerated and safe, and no serious adverse events were related to the study treatment. CONCLUSIONS: The intervention did not decrease the likelihood of respiratory failure within the first 72 hours of life. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03235986.

Cardioprotective Role of BGP-15 in Ageing Zucker Diabetic Fatty Rat (ZDF) Model: Extended Mitochondrial Longevity.

Impaired mitochondrial function is associated with several metabolic diseases and health conditions, including insulin resistance and type 2 diabetes (T2DM), as well as ageing. The close relationship between the above-mentioned diseases and cardiovascular disease (CVD) (diabetic cardiomyopathy and age-related cardiovascular diseases) has long been known. Mitochondria have a crucial role: they are a primary source of energy produced in the form of ATP via fatty acid oxidation, tricarboxylic acid (TCA) cycle, and electron transport chain (ETC), and ATP synthase acts as a key regulator of cardiomyocyte survival. Mitochondrial medicine has been increasingly discussed as a promising therapeutic approach in the treatment of CVD. It is well known that vitamin B3 as an NAD+ precursor exists in several forms, e.g., nicotinic acid (niacin) and nicotinamide (NAM). These cofactors are central to cellular homeostasis, mitochondrial respiration, ATP production, and reactive oxygen species generation and inhibition. Increasing evidence suggests that the nicotinic acid derivative BGP-15 ((3-piperidine-2-hydroxy-1-propyl)-nicotinic amidoxime) improves cardiac function by reducing the incidence of arrhythmias and improves diastolic function in different animal models. Our team has valid reasons to assume that these cardioprotective effects of BGP-15 are based on its NAD+ precursor property. Our hypothesis was supported by an animal experiment where ageing ZDF rats were treated with BGP-15 for one year. Haemodynamic variables were measured with echocardiography to detect diabetic cardiomyopathy (DbCM) and age-related CVD as well. In the ZDF group, advanced HF was diagnosed, whereas the BGP-15-treated ZDF group showed diastolic dysfunction only. The significant difference between the two groups was supported by post-mortem Haematoxylin and eosin (HE) and Masson's trichrome staining of cardiac tissues. Moreover, our hypothesis was further confirmed by the significantly elevated Cytochrome c oxidase (MTCO) and ATP synthase activity and expression detected with ELISA and Western blot analysis. To the best of our knowledge, this is the first study to demonstrate the protective effect of BGP-15 on cardiac mitochondrial respiration in an ageing ZDF model.

Incidence, predictors of success and outcome of LISA in very preterm infants.

OBJECTIVES: The aim of this study was to examine the success rate of less invasive surfactant administration (LISA), to identify early predictive factors for the outcome of LISA, and to compare neonatal outcomes between the LISA failure group and the group of infants who were successfully treated with LISA. DESIGN: A retrospective cohort study. PATIENTS: Infants born at less than 33 weeks of gestation (n = 158) and treated with LISA for respiratory distress syndrome. RESULTS: LISA was successful in 86 cases (54.4%); 72 preterm infants (45.6%) needed additional surfactant therapy and/or mechanical ventilation in the first 72 h (LISA failure). In a multivariate logistic regression analysis, six independent predictors of LISA success were identified: core temperature at the time of admission (adjusted odds ratio (OR): 3.56), dose of poractant alfa (<200 mg/kg; adjusted OR: 0.254), elevated C-reactive protein (>10 mg/L) at 24 h of life (adjusted OR: 0.28), highest respiratory severity score (RSS) during the first hour of life or at the time of LISA (adjusted OR: 0.463), maternal age (adjusted OR: 0.923), and birth weight (adjusted OR: 1.003). The receiver operating curve created by using the identified factors indicates good predictive power with an area under the curve of 0.85. LISA failure was associated with a substantially higher risk of complications. CONCLUSION: LISA success can be predicted by variables available before the intervention. Failure of LISA is relatively frequent event in very preterm infants and is associated with adverse outcomes. Prevention of hypothermia during early stabilization and appropriate dosing of surfactant may increase LISA success rates and improve patient outcome.

Changes in the SARS-CoV-2 cellular receptor ACE2 levels in cardiovascular patients: a potential biomarker for the stratification of COVID-19 patients.

Angiotensin-converting enzyme 2 (ACE2) is essential for SARS-CoV-2 cellular entry. Here we studied the effects of common comorbidities in severe COVID-19 on ACE2 expression. ACE2 levels (by enzyme activity and ELISA measurements) were determined in human serum, heart and lung samples from patients with hypertension (n = 540), heart transplantation (289) and thoracic surgery (n = 49). Healthy individuals (n = 46) represented the controls. Serum ACE2 activity was increased in hypertensive subjects (132%) and substantially elevated in end-stage heart failure patients (689%) and showed a strong negative correlation with the left ventricular ejection fraction. Serum ACE2 activity was higher in male (147%), overweight (122%), obese (126%) and elderly (115%) hypertensive patients. Primary lung cancer resulted in higher circulating ACE2 activity, without affecting ACE2 levels in the surrounding lung tissue. Male sex resulted in elevated serum ACE2 activities in patients with heart transplantation or thoracic surgery (146% and 150%, respectively). Left ventricular (tissular) ACE2 activity was unaffected by sex and was lower in overweight (67%), obese (62%) and older (73%) patients with end-stage heart failure. There was no correlation between serum and tissular (left ventricular or lung) ACE2 activities. Neither serum nor tissue (left ventricle or lung) ACE2 levels were affected by RAS inhibitory medications. Abandoning of ACEi treatment (non-compliance) resulted in elevated blood pressure without effects on circulating ACE2 activities. ACE2 levels associate with the severity of cardiovascular diseases, suggestive for a role of ACE2 in the pathomechanisms of cardiovascular diseases and providing a potential explanation for the higher mortality of COVID-19 among cardiovascular patients. Abandoning RAS inhibitory medication worsens the cardiovascular status without affecting circulating or tissue ACE2 levels.

BGP-15 Inhibits Hyperglycemia-Aggravated VSMC Calcification Induced by High Phosphate.

Vascular calcification associated with high plasma phosphate (Pi) level is a frequent complication of hyperglycemia, diabetes mellitus, and chronic kidney disease. BGP-15 is an emerging anti-diabetic drug candidate. This study was aimed to explore whether BGP-15 inhibits high Pi-induced calcification of human vascular smooth muscle cells (VSMCs) under normal glucose (NG) and high glucose (HG) conditions. Exposure of VSMCs to Pi resulted in accumulation of extracellular calcium, elevated cellular Pi uptake and intracellular pyruvate dehydrogenase kinase-4 (PDK-4) level, loss of smooth muscle cell markers (ACTA, TAGLN), and enhanced osteochondrogenic gene expression (KLF-5, Msx-2, Sp7, BMP-2). Increased Annexin A2 and decreased matrix Gla protein (MGP) content were found in extracellular vesicles (EVs). The HG condition markedly aggravated Pi-induced VSMC calcification. BGP-15 inhibited Pi uptake and PDK-4 expression that was accompanied by the decreased nuclear translocation of KLF-5, Msx-2, Sp7, retained VSMC markers (ACTA, TAGLN), and decreased BMP-2 in both NG and HG conditions. EVs exhibited increased MGP content and decreased Annexin A2. Importantly, BGP-15 prevented the deposition of calcium in the extracellular matrix. In conclusion, BGP-15 inhibits Pi-induced osteochondrogenic phenotypic switch and mineralization of VSMCs in vitro that make BGP-15 an ideal candidate to attenuate both diabetic and non-diabetic vascular calcification.

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.

Hydrogen sulfide inhibits aortic valve calcification in heart via regulating RUNX2 by NF-κB, a link between inflammation and mineralization.

INTRODUCTION: Hydrogen sulfide (H2S) was revealed to inhibit aortic valve calcification and inflammation was implicated in the pathogenesis of calcific aortic valve disease (CAVD). OBJECTIVES: We investigate whether H2S inhibits mineralization via abolishing inflammation. METHODS AND RESULTS: Expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were increased in patients with CAVD and in calcified aortic valve of ApoE-/- mice. Administration of H2 2S releasing donor (4-methoxyphenyl piperidinylphosphinodithioc acid (AP72)) exhibited inhibition on both calcification and inflammation in aortic valve of apolipoprotein E knockout mice (ApoE-/-) mice is reflected by lowering IL-1ß and TNF-α levels. Accordingly, AP72 prevented the accumulation of extracellular calcium deposition and decreased nuclear translocation of nuclear factor-κB (NF-κB) in human valvular interstitial cells (VIC). This was also accompanied by reduced cytokine response. Double-silencing of endogenous H2S producing enzymes, Cystathionine gamma-lyase (CSE) and Cystathionine beta-synthase (CBS) in VIC exerted enhanced mineralization and higher levels of IL-1ß and TNF-α. Importantly, silencing NF-κB gene or its pharmacological inhibition prevented nuclear translocation of runt-related transcription factor 2 (Runx2) and subsequently the calcification of human VIC. Increased levels of NF-κB and Runx2 and their nuclear accumulation occurred in ApoE-/- mice with a high-fat diet. Administration of AP72 decreased the expression of NF-κB and prevented its nuclear translocation in VIC of ApoE-/- mice on a high-fat diet, and that was accompanied by a lowered pro-inflammatory cytokine level. Similarly, activation of Runx2 did not occur in VIC of ApoE-/- mice treated with H2S donor. Employing Stimulated Emission Depletion (STED) nanoscopy, a strong colocalization of NF-κB and Runx2 was detected during the progression of valvular calcification. CONCLUSIONS: Hydrogen sulfide inhibits inflammation and calcification of aortic valve. Our study suggests that the regulation of Runx2 by hydrogen sulfide (CSE/CBS) occurs via NF-κB establishing a link between inflammation and mineralization in vascular calcification.

Therapeutic Potential of Carbon Monoxide (CO) and Hydrogen Sulfide (H2S) in Hemolytic and Hemorrhagic Vascular Disorders-Interaction between the Heme Oxygenase and H2S-Producing Systems.

Over the past decades, substantial work has established that hemoglobin oxidation and heme release play a pivotal role in hemolytic/hemorrhagic disorders. Recent reports have shown that oxidized hemoglobins, globin-derived peptides, and heme trigger diverse biological responses, such as toll-like receptor 4 activation with inflammatory response, reprogramming of cellular metabolism, differentiation, stress, and even death. Here, we discuss these cellular responses with particular focus on their mechanisms that are linked to the pathological consequences of hemorrhage and hemolysis. In recent years, endogenous gasotransmitters, such as carbon monoxide (CO) and hydrogen sulfide (H2S), have gained a lot of interest in connection with various human pathologies. Thus, many CO and H2S-releasing molecules have been developed and applied in various human disorders, including hemolytic and hemorrhagic diseases. Here, we discuss our current understanding of oxidized hemoglobin and heme-induced cell and tissue damage with particular focus on inflammation, cellular metabolism and differentiation, and endoplasmic reticulum stress in hemolytic/hemorrhagic human diseases, and the potential beneficial role of CO and H2S in these pathologies. More detailed mechanistic insights into the complex pathology of hemolytic/hemorrhagic diseases through heme oxygenase-1/CO as well as H2S pathways would reveal new therapeutic approaches that can be exploited for clinical benefit.

Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres.

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20-300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE-/- mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE-/- mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.