Hemoadsorption: effective in reducing circulating fragments of the endothelial glycocalyx during cardiopulmonary bypass in patients undergoing on-pump cardiac surgery?

BACKGROUND: The vascular endothelial glycocalyx is susceptible to ischemia and hypoxia. Released soluble components of the endothelial glycocalyx (EG) have been identified as potential damage associated molecular patterns (DAMPs) able to enhance an ongoing inflammatory response. Shedding of the EG has been associated with released atrial-natriuretic peptide (ANP) during cardiac surgery procedures. A novel hemoadsorption technique (CytoSorb (R)) has been shown to effectively remove molecules up to 55 kDa unspecifically from circulation. It is not known whether ANP or glycocalyx components can be removed successfully by this technique. METHODS: In 15 patients undergoing on-pump cardiac surgery, the hemoadsorption device was integrated in the cardiopulmonary bypass (CPB) circuit. Pre- and post-adsorber concentrations ofANP, heparan sulphate (HEP), syndecan-1 (SYN) and hyaluronan (HYA) were measured at 10 (T1), 30 (T2), and 60 (T3) minutes after aortic cross-clamping and complete CPB. RESULTS: Hemoadsorption significantly reduced mean HEP concentrations (-157.5 [333.4] ng/mL; P<0.001) post adsorber. For ANP and SYN no statistically significant changes were detected whereas mean [SD] HYA concentrations even increased significantly (+21.6 [43.0] ng/mL; P<0.001) post adsorber. CONCLUSIONS: In this study representing a real-life scenario, we could demonstrate that the novel hemoadsorption device (CytoSorbe (R)) was able to effectively adsorb HEP from the circulation if integrated in a CPB circuit. However, blood concentrations of HYA, SYN, and ANP could not be reduced during CPB in our investigation

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis

Background: Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined. Objective: We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment. Methods: Ex vivo and in vivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR -2)-and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining. Results: Mas-related G protein-coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1a. Indeed, IL-1a acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL 8, and stem cell factor. Conclusion: MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis

Eosinophils are a Major Source of Interleukin-31 in Bullous Pemphigoid

Bullous pemphigoid (BP) is characterized by substantial skin and blood eosinophilia as well as intensive pruritus. Since the pruritogenic cytokine interleukin (IL)-31 is increased in inflammatory skin diseases the aim of this study was to determine whether IL-31 plays a role in BP. Using immunofluorescence, IL-31 expression was analysed in eosinophils derived from blister fluids and skin from patients with BP and IL-31 levels in blister fluids, serum and culture supernatants were determined by enzyme-linked immunoassay (ELISA). High levels of IL-31 expression were observed in BP blister fluids, but they were only marginally elevated in BP serum compared with healthy controls. Eosinophils from either BP blister fluids or skin biopsies showed strong expression of IL-31. Furthermore, peripheral blood eosinophils from patients with BP, but not healthy controls, released high levels of IL-31, reflecting those in blister fluids. In conclusion, eosinophils are a major source of IL-31 in BP and this cytokine may contribute to itch in patients with BP

Microvascular perfusion, perfused boundary region and glycocalyx shedding in patients with autosomal dominant polycystic kidney disease: results from the GlycoScore III study

Lay Summary Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to kidney failure. However, ADPKD not only affects the kidney, but also involves other organ systems, including the vasculature. The pathogenesis of vascular disease is not well understood. Here we examined changes in the inner-most layer of blood vessels in ADPKD patients and found significant alterations of the endothelial surface layer (ESL). Considering the importance of the ESL in the integrity of vascular function, this finding is of great interest for ADPKD research. Background Vascular abnormalities and endothelial dysfunction are part of the spectrum of autosomal dominant polycystic kidney disease (ADPKD). The mechanisms behind these manifestations, including potential effects on the endothelial surface layer (ESL) and glycocalyx integrity, remain unknown. Methods Forty-five ambulatory adult patients with ADPKD were enrolled in this prospective, observational, cross-sectional, single-centre study. Fifty-one healthy volunteers served as a control group. All participants underwent real-time microvascular perfusion measurements of the sublingual microcirculation using sidestream dark field imaging. After image acquisition, the perfused boundary region (PBR), an inverse parameter for red blood cell (RBC) penetration into the ESL, was automatically calculated. Microvascular perfusion was assessed by RBC filling and capillary density. Concentrations of circulating glycocalyx components were determined by enzyme-linked immunosorbent assay. Results ADPKD patients showed a significantly larger PBR compared with healthy controls (2.09 +/- 0.23 mu m versus 1.79 +/- 0.25 mu m; P < .001). This was accompanied by significantly lower RBC filling (70.4 +/- 5.0% versus 77.9 +/- 5.4%; P < .001) as well as a higher valid capillary density {318/mm(2) [interquartile range (IQR) 269-380] versus 273/mm(2) [230-327]; P = .007}. Significantly higher plasma concentrations of heparan sulphate (1625 +/- 807 ng/ml versus 1329 +/- 316 ng/ml; P = .034), hyaluronan (111 ng/ml [IQR 79-132] versus 92 ng/ml [82-98]; P = .042) and syndecan-1 were noted in ADPKD patients compared with healthy controls (35 ng/ml [IQR 27-57] versus 29 ng/ml [23-42]; P = .035). Conclusions Dimensions and integrity of the ESL are impaired in ADPKD patients. Increased capillary density may be a compensatory mechanism for vascular dysfunction to ensure sufficient tissue perfusion and oxygenation

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide)

Objective: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-Cre(TG) line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. Conclusions: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease

Clinical Features and Survival of Patients with Indolent Systemic Mastocytosis defined by the Updated WHO Classification

In indolent systemic mastocytosis (ISM) several risk factors of disease progression have been identified. Previous studies, performed with limited patient numbers, have also shown that the clinical course in ISM is stable and comparable to that of cutaneous mastocytosis (CM). The aim of this project was to compare the prognosis of patients with ISM with that of patients with CM.; We employed a data set of 1993 patients from the registry of the European Competence Network on Mastocytosis (ECNM) to compare outcomes of ISM and CM.; We found that overall survival (OS) is worse in ISM compared to CM. Moreover, in patients with typical ISM, bone marrow mastocytosis (BMM) and smouldering SM (SSM), 4.1% of disease progressions have been observed (4.9% of progressions in typical ISM group, 1.7% in BMM and 9.4% in SSM). Progressions to advanced SM were observed in 2.9% of these patients. In contrast, six patients with CM (1.7%) converted to ISM and no definitive progression to advanced SM was found. No significant differences in OS and event-free survival (EFS) were found when comparing ISM, BMM and SSM. Higher risk of both progression and death was significantly associated with male gender, worse performance status and organomegaly.; Our data confirm the clinical impact of the WHO classification that separates ISM from CM and from other SM variants