Rosacea is a common chronic inflammation of sebaceous gland–rich facial skin characterized by severe skin dryness, elevated pH, transepidermal water loss, and decreased hydration levels. Until now, ...there has been no thorough molecular analysis of permeability barrier alterations in the skin of patients with rosacea. Thus, we aimed to investigate the barrier alterations in papulopustular rosacea samples compared with healthy sebaceous gland–rich skin, using RNA sequencing analysis (n = 8). Pathway analyses by Cytoscape ClueGO revealed 15 significantly enriched pathways related to skin barrier formation. RT-PCR and immunohistochemistry were used to validate the pathway analyses. The results showed significant alterations in barrier components in papulopustular rosacea samples compared with sebaceous gland–rich skin, including the cornified envelope and intercellular lipid lamellae formation, desmosome and tight junction organizations, barrier alarmins, and antimicrobial peptides. Moreover, the barrier damage in papulopustular rosacea was unexpectedly similar to atopic dermatitis; this similarity was confirmed by immunofluorescent staining. In summary, besides the well-known dysregulation of immunological, vascular, and neurological functions, we demonstrated prominent permeability barrier alterations in papulopustular rosacea at the molecular level, which highlight the importance of barrier repair therapies for rosacea.
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-1β and nuclear translocation of the NF-κβ 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.
Background and Purpose
Calcification of heart valves is a frequent pathological finding in chronic kidney disease and in elderly patients. Hydrogen sulfide (H2S) may exert anti‐calcific actions. Here ...we investigated H2S as an inhibitor of valvular calcification and to identify its targets in the pathogenesis.
Experimental Approach
Effects of H2S on osteoblastic transdifferentiation of valvular interstitial cells (VIC) isolated from samples of human aortic valves were studied using immunohistochemistry and western blots. We also assessed H2S on valvular calcification in apolipoprotein E‐deficient (ApoE−/−) mice.
Key Results
In human VIC, H2S from donor compounds (NaSH, Na2S, GYY4137, AP67, and AP72) inhibited mineralization/osteoblastic transdifferentiation, dose‐dependently in response to phosphate. Accumulation of calcium in the extracellular matrix and expression of osteocalcin and alkaline phosphatase was also inhibited. RUNX2 was not translocated to the nucleus and phosphate uptake was decreased. Pyrophosphate generation was increased via up‐regulating ENPP2 and ANK1. Lowering endogenous production of H2S by concomitant silencing of cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS) favoured VIC calcification. analysis of human specimens revealed higher Expression of CSE in aorta stenosis valves with calcification (AS) was higher than in valves of aortic insufficiency (AI). In contrast, tissue H2S generation was lower in AS valves compared to AI valves. Valvular calcification in ApoE−/− mice on a high‐fat diet was inhibited by H2S.
Conclusions and Implications
The endogenous CSE‐CBS/H2S system exerts anti‐calcification effects in heart valves providing a novel therapeutic approach to prevent hardening of valves.
Linked Articles
This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc
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.
The microbial community exhibits remarkable diversity on topographically distinct skin regions, which may be accompanied by differences in skin immune characteristics. Our aim was to compare the ...immune milieu of healthy sebaceous gland-rich (SGR) and sebaceous gland-poor skin areas, and to analyze its changes in an inflammatory disease of SGR skin. For this purpose, immunohistochemical, immunocytochemical, and quantitative real-time PCR analyses of thymic stromal lymphopoietin (TSLP) and other cytokines, phenotypic immune cell markers and transcription factors were carried out in samples from sebaceous gland-poor, SGR skin and from papulopustular rosacea. TSLP mRNA and protein production was also studied in cultured keratinocytes. In SGR skin, higher TSLP expression, dendritic cell appearance without prominent activation, and T cell presence with IL-17/IL-10 cytokine milieu were detected compared with sebaceous gland-poor skin. Linoleic acid, a major sebum component, was found to induce TSLP expression dose-dependently in keratinocytes. In papulopustular rosacea, significantly decreased TSLP level and influx of inflammatory dendritic cells and T cells with IL-17/interferon-γ cytokine milieu were observed. According to our results, SGR skin is characterized by a distinct, noninflammatory immune surveillance, which may explain the preferred localization of inflammatory skin diseases, and can influence future barrier repair therapeutic concepts.
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•H2S derived from CSE/CBS inhibits inflammation and calcification of valvular interstitial cells.•H2S releasing molecules inhibits inflammation and calcification of valvular ...interstitial cells.•H2S inhibits inflammation and calcification in aortic valve of apolipoprotein E deficient mice.•H2S suppresses nuclear translocation of NF-κB and subsequent expression of IL-1β and TNF-α.•Activation of Runx2, its nuclear translocation is mediated by NF-κB in calcifying conditions.•Inflammation and calcification are connected via master transcription factors, NF-κB and Runx2.
Hydrogen sulfide (H2S) was revealed to inhibit aortic valve calcification and inflammation was implicated in the pathogenesis of calcific aortic valve disease (CAVD).
We investigate whether H2S inhibits mineralization via abolishing inflammation.
Expression of pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased in patients with CAVD and in calcified aortic valve of ApoE-/- mice. Administration of H22S 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-1β 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-1β 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.
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.
Recent data indicate that distinct skin areas show different microbial/chemical milieu. Keratinocytes (KC) respond to these stimuli by producing cytokine mediators. Therefore, we aimed to determine ...KC‐derived cytokine expression in distinct healthy skin regions (gland‐poor GP, sebaceous gland‐rich SGR and apocrine gland‐rich AGR), and their changes in skin diseases of the given regions (atopic dermatitis AD, papulopustular rosacea PPR and psoriasis). Cytokines were analysed at the mRNA and protein levels, and literature analysis was performed for functional categorization. The three regions showed characteristically different cytokine patterns. GP was featured by an IL‐25/IL‐33/IL‐36RA/IL‐38/IL‐18 cytokine milieu, SGR was characterized by IL‐23/IL‐17C/IL‐18, and AGR skin exhibited a mixed IL‐25/IL‐33/IL‐23/IL‐18 profile. Literature analyses revealed different homeostatic and proinflammatory roles of these cytokine patterns (Th2 related in GP, Th17 related in SGR and mixed Th2/Th17 in AGR). In skin diseases which are primarily epidermal cytokine‐driven (AD, PPR), the level of the regionally characteristic cytokines were further elevated, in contrast to the autoantigen‐driven psoriasis, where the cytokine pattern was independent from the localization. Healthy skin regions are equipped with different KC‐derived cytokine profiles, which may influence each region's capability of mediator production in certain types of dermatoses.
Atopic dermatitis is an inflammatory skin disease characterized by significant permeability barrier damage. Regulation and maintenance of permeability and antimicrobial skin barriers are strongly ...connected. There is a lack of comprehensive studies of the expression of all 5 major antimicrobial peptide functional groups in atopic dermatitis. The aim of this study was to investigate the major antimicrobial peptide functional groups in lesional atopic dermatitis, non-lesional atopic dermatitis, and healthy control samples, using real-time quantitative PCR and immunohistochemistry. Lesional psoriatic skin was also examined as a diseased control. No differences in mRNA levels were detected between non-lesional atopic dermatitis and healthy control skin, and, at the protein level, the only change was the significantly decreased LL-37 in non-lesional atopic dermatitis. In lesional atopic dermatitis, several antimicrobial peptides were significantly altered at the mRNA level, while, at the protein level, all antimicrobial peptides were significantly upregulated or unchanged, except for LL-37, which decreased, compared with healthy controls. Antimicrobial peptides were similarly elevated in lesional atopic dermatitis and lesional psoriatic skin, with somewhat higher expression in lesional psoriatic skin, except for LL-37. In conclusion, LL-37 was the only antimicrobial peptide that was impaired in both non-lesional and lesional atopic dermatitis, highlighting its potential pathogenetic or exacerbating role in the initial stages of the disease.
The chemical milieu, microbiota composition, and immune activity show prominent differences in distinct healthy skin areas. The objective of the current study was to compare the major permeability ...barrier components (stratum corneum and tight junction (TJ)), investigate the distribution of (corneo)desmosomes and TJs, and measure barrier function in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation, and (corneo)desmosome and TJ organization were investigated at the mRNA and protein levels using qRT-PCR and immunohistochemistry. The distribution of junction structures was visualized using confocal microscopy. Transepidermal water loss (TEWL) functional measurements were also performed. CE intracellular structural components were similarly expressed in gland-rich (SGR and AGR) and GP areas. In contrast, significantly lower extracellular protein levels of (corneo)desmosomes (DSG1 and CDSN) and TJs (OCLN and CLDN1) were detected in SGR/AGR areas compared to GP areas. In parallel, kallikrein proteases were significantly higher in gland-rich regions. Moreover, gland-rich areas were characterized by prominently disorganized junction structures ((corneo)desmosomes and TJs) and significantly higher TEWL levels compared to GP skin, which exhibited a regular distribution of junction structures. According to our findings, the permeability barrier of our skin is not uniform. Gland-rich areas are characterized by weaker permeability barrier features compared with GP regions. These findings have important clinical relevance and may explain the preferred localization of acantholytic skin diseases on gland-rich skin regions (e.g., Pemphigus foliaceus, Darier’s disease, and Hailey–Hailey disease).
Background
Acne vulgaris provides a unique disease setting in which a prominent skin inflammation is coupled with the overproduction of lipid‐rich sebum.
Objectives
Our goal was to evaluate the ...expression of barrier molecules in papular acne skin samples obtained from untreated patients and compare those to the results of healthy and of papulopustular rosacea‐involved ones at the mRNA and protein levels. In addition, we aimed to assess the effects of various sebum composing lipids on the expression of proteins involved in barrier formation in keratinocytes.
Methods
Available microarray data sets of papular acne and papulopustular rosacea‐affected skin samples were re‐analysed with a focus on epidermal barrier‐related pathways. Immunohistochemistry was performed to detect barrier molecules in the interfollicular regions of human acne and healthy skin samples. Protein levels of barrier‐related genes were measured by western blot in samples of HaCaT keratinocytes treated with selected lipids.
Results
Meta‐analysis of whole transcriptome data sets revealed that barrier‐related pathways are significantly affected in acne vulgaris skin samples. While an altered expression of key molecules in maintaining barrier functions such as filaggrin, keratin 1, involucrin, desmoglein 1, kallikrein 5 and 7, was also observed at the protein levels, our data demonstrated that sebum composing lipids may selectively modify the levels of epidermal barrier‐related molecules.
Conclusions
Our results suggest that although not as prominently as in the dry papulopustular rosacea skin, the epidermal barrier in the interfollicular region may be damaged also in the lipid‐rich skin samples of papular acne. Furthermore, our findings indicating diverse regulatory effects of various sebum lipids on the expression of barrier molecules in keratinocytes suggest, that they may influence the moisturization of the skin as well. Altogether, our findings could have implications in the development of sebum‐modulating anti‐acne therapies and even in the care of symptom‐free skin.
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