58 research outputs found
Coronavirus Disease 2019-Related Alterations of Total and Anti-Spike IgG Glycosylation in Relation to Age and Anti-Spike IgG Titer
The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been affecting the world since January 2020 and has caused millions of deaths. To gain a better insight into molecular changes underlying the COVID-19 disease, we investigated here the N-glycosylation of three immunoglobulin G (IgG) fractions isolated from plasma of 35 severe COVID-19 patients, namely total IgG(1), total IgG(2), and anti-Spike IgG, by means of MALDI-TOF-MS. All analyses were performed at the glycopeptide level to assure subclass- and site-specific information. For each COVID-19 patient, the analyses included three blood withdrawals at different time-points of hospitalization, which allowed profiling longitudinal alterations in IgG glycosylation. The COVID-19 patients presented altered IgG N-glycosylation profiles in all investigated IgG fractions. The most pronounced COVID-19-related changes were observed in the glycosylation profiles of antigen-specific anti-Spike IgG(1). Anti-Spike IgG(1) fucosylation and galactosylation showed the strongest variation during the disease course, with the difference in anti-Spike IgG(1) fucosylation being significantly correlated with patients' age. Decreases in anti-Spike IgG(1) galactosylation and sialylation in the course of the disease were found to be significantly correlated with the difference in anti-Spike IgG plasma concentration. The present findings suggest that patients' age and anti-S IgG abundance might influence IgG N-glycosylation alterations occurring in COVID-19
Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells
.Here we monitored profilin-1 expression in human atherosclerotic plaques by immunofluorescent staining. The effects of recombinant profilin-1 on atherogenic signaling pathways and cellular responses such as DNA synthesis (BrdU-incorporation) and chemotaxis (modified Boyden-chamber) were evaluated in cultured rat aortic and human coronary vascular smooth muscle cells (VSMCs). Furthermore, the correlation between profilin-1 serum levels and the degree of atherosclerosis was assessed in humans.<0.001 vs. no atherosclerosis or control group).Profilin-1 expression is significantly enhanced in human atherosclerotic plaques compared to the normal vessel wall, and the serum levels of profilin-1 correlate with the degree of atherosclerosis in humans. The atherogenic effects exerted by profilin-1 on VSMCs suggest an auto-/paracrine role within the plaque. These data indicate that profilin-1 might critically contribute to atherogenesis and may represent a novel therapeutic target
Interference with platelet-derived growth factor-induced activation of vascular smooth muscle cells and stenosing vascular remodeling
Gefäß- und ihre Folgeerkrankungen stellen die häufigste Todesursache in der
westlichen Welt dar. Daher sind Strategien zur Reduzierung stenosierender,
vaskulärer Umbauprozesse therapeutisch wünschenswert. Dies schließt die genaue
Kenntnis der zugrunde liegenden zellulären und molekularen Mechanismen von
Gefäßerkrankungen, wie der Atherosklerose und der Restenose, mit ein.
Experimentelle Daten stützen die Annahme, dass die Platelet-derived Growth
Factor (PDGF)-Signaltransduktion von zentraler Bedeutung im Rahmen dieser
Prozesse ist. Verschiedene Aspekte stenosierender, vaskulärer
Gewebeumbauprozesse, insbesondere Komponenten der PDGF-Signaltransduktion,
wurden in der vorliegenden Habilitationsschrift untersucht. So konnte in der
Originalarbeit 1 gezeigt werden, dass 17-beta Östradiol die PDGF-BB-induzierte
Signaltransduktion und Zellfunktionen in glatten Gefäßmuskelzellen in vitro
signifikant beeinflusst. Während ein Einfluss auf die PDGF β-Rezeptor
Expression, Liganden-induzierte Rezeptor-Tyrosinphosphorylierung sowie
Rekrutierung zytosolischer Bindungsproteine ausgeschlossen wurde, bestand eine
konzentrationsabhängige Reduktion der PDGF-BB-induzierten DNA-Synthese und
Zellmigration. Dieser inhibitorische Effekt durch 17-beta Östradiol wurde
downstream des PDGF β-Rezeptors durch Verminderung der Aktivierung des kleinen
GTP-bindenden Proteins rac-1 vermittelt. So führte auch die transiente
Überexprimierung von dominant-negativem rac-1 sowie ein rac-1 Inhibitor zur
Hemmung der PDGF-BB-induzierten Migration glatter Gefäßmuskelzellen. Diese
Daten bieten eine mögliche molekulare Erklärung vaskuloprotektiver Effekte von
Östrogenen. Die Bedeutung antiretroviraler Therapeutika für Zellfunktionen
glatter Gefäßmuskelzellen in vitro sowie im Rahmen experimentell induzierter
vaskulärer Gewebeumbauprozesse in vivo wurde in den Originalarbeiten 2 und 3
untersucht. Während die Analyse der direkten Effekte von Ritonavir auf glatte
Gefäßmuskelzellen eine Interferenz mit PDGF-BB-induzierter
Tyrosinphosphorylierung des PDGF β-Rezeptors sowie der DNA-Synthese sowie
Zellmigration und damit einen Wachstum-inhibierenden Einfluss von Ritonavir
außerhalb von zytotoxischen und pro-apoptotischen Effekten zeigte
(Originalarbeit 2), bestand eine Progression der vaskulären Umbauprozesse und
eine Hemmung endothelialer Regenerationskapazität durch HAART in einem
Tiermodell vaskulärer Schädigung (Originalarbeit 3). Somit stehen potentiell
direkte, protektive Effekte in vitro unerwünschten Effekten in vivo gegenüber.
Diese Daten zeigen, dass zum einen anti-restenotische Eigenschaften von
Ritonavir auf einer zellulären und funktionellen Ebene bestehen, zum anderen,
dass HAART möglicherweise negativen Einfluss auf den endothelialen
Heilungsprozess bzw. den vaskulären Umbau nach Gefäßverletzung besitzt. Neben
der Liganden-Exposition wird die Aktivität von Rezeptortyrosinkinasen wie dem
PDGF β-Rezeptor maßgeblich durch Protein-Tyrosin-Phosphatasen (PTPs)
reguliert. Dies beinhaltet auch die Beeinflussung der nachgeschalteten
Signaltransduktion sowie zellulärer Funktionen. Die Bedeutung von PTPs in
stenosierenden, vaskulären Umbauprozessen wurde in Modellen der vaskulären
Schädigung in vivo analysiert. Zum einen konnte gezeigt werden, dass PTPs
zeit- und schicht-spezifisch während der Neointimaentwicklung reguliert wurden
und dass dies die Aktivität des PDGF β-Rezeptors beeinflusste. Hierbei scheint
insbesondere die transiente Herunterregulierung der PTP DEP-1 von Bedeutung zu
sein (Originalarbeit 4). Zum anderen wurde die Hypothese getestet und
bestätigt, dass die Applikation von Antioxidantien durch Hemmung der Oxidation
des active-site Cysteins in der katalytischen Domäne PTPs (re-)aktiviert, was
zur Antagonisierung des PDGF β-Rezeptors in vivo führte und konsekutiv die
Neotintimaentwicklung nach Gefäßverletzung reduzierte (Originalarbeit 5).
Hierin liegt möglicherweise ein therapeutischer Ansatz zur Modifizierung von
dysregulierten Gewebeumbauprozessen, die mit gesteigerter Aktivität von
Rezeptortyrosinkinasen vergesellschaftet sind.Vascular diseases and their consequences represent the most frequent cause of
death in the Western world. Thus, strategies to reduce stenosing, vascular
remodeling processes are of high therapeutic value. A prerequisite for
therapeutic strategies is the in-depth knowledge of underlying cellular and
molecular mechanisms in vascular disease, such as atherosclerosis and
restenosis. Experimental data underline the assumption that platelet-derived
growth factor (PDGF)-singal transduction is of crucial importance for these
processes. A variety of aspects of stenosing, vascular remodeling processes,
in particular components of PDGF-signal transduction, have been analyzed in
this work. Data of article no. 1 demonstrate that 17-beta estradiol (E2)
significantly impacts on PDGF-BB-induced signal transduction and cellular
responses in vascular smooth muscle cells in vitro. While an impact of E2 on
PDGF β-receptor expression, ligand-induced receptor tyrosine-phosphorylation,
and recruitment of cytosolic binding proteins was excluded, a concentration-
dependent reduction of PDGF-BB-induced DNA-synthesis and cell migration was
demonstrated. This inhibitory effect of E2 was induced by reduced activation
of the small GTP-binding protein rac-1. In addition, transient overexpression
of dominant-negative rac-1, and a rac-1 inhibitor led to impairment of PDGF-
BB-induced migration of vascular smooth muscle cells. Taken together, these
observations offer a molecular explanation for the vasoprotective effects of
estrogens. The impact of anti-retroviral drugs on cellular functions in
vascular smooth muscle cells in vitro, and during experimentally induced
vascular remodeling processes in vivo, were evaluated in articles no. 2 and 3.
While analysis on direct effects of Ritonavir on vascular smooth muscle cells
demonstrated an interference with both PDGF-BB-induced tyrosine-
phosphorylation of the PDGF β-receptor and DNA-synthesis, and with cell
migration, and thus demonstrating a growth-inhibiting impact of Ritonavir
without exhibiting cytotoxic and apoptotic effects (article no. 2), a
progression of vascular remodeling processes and an inhibition of endothelial
regenerative capacity was observed due to treatment with highly-active anti-
retroviral therapy (HAART) in an animal model of vascular injury (article no.
3). Thus, potentially direct, protective effects in vitro are facing unwanted
effects in vivo. These data show that on one hand anti-restenotic properties
of Ritonavir are evident on a cellular and functional level, while on the
other hand HAART possibly possesses negative impact on the endothelial healing
process and on vascular remodeling following vascular injury. Besides ligand
binding the activity of receptor tyrosine kinases, such as the PDGF
β-receptor, is critically controlled by protein tyrosine phosphatases (PTPs).
This involves also the regulation of downstream signal transduction processes
and cellular functions. The role of PTPs in stenosing, vascular remodeling
processes was investigated in models of vascular injury in vivo. It was
demonstrated that PTPs were time- and layer-specifically regulated during the
process of neointima formation, along with changes of the PDGF β-receptor. In
particular, the transient down-regulation of the PTP DEP-1 seemed to be
crucial for the tissue remodeling process (article no. 4). Furthermore, the
hypothesis that application of antioxidants leads to inhibition of oxidation
of the active-site cysteine in the catalytic domain of PTPs, thus resulting in
(re-)activation of PTPs, was tested and eventually verified. Antioxidants led
to antagonizing of the phosphorylation of the PDGF β-receptor in vivo, and
consecutively to reduced neointima formation following vascular injury
(article no. 5). These data implicate a therapeutic approach to modify
dysregulated tissue remodeling processes that are associated with increased
activity of receptor tyrosine kinases
Platelet-derived growth factor receptor β activation and regulation in murine myelofibrosis
There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor β (PDGFRβ) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRβ signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRβ signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRβ and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRβ–PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRβ tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRβ by protein tyrosine phosphatases as endogenous PDGFRβ antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRβ-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRβ–T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRβ phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis
The effect of blood sampling and preanalytical processing on human N-glycome.
Glycome modulations have been described in the onset and progression of many diseases. Thus, many studies have proposed glycans from blood glycoproteins as disease markers. Astonishingly, little effort has been given unraveling preanalytical conditions potentially influencing glycan analysis prior to blood biomarker studies. In this work, we evaluate for the first time the effect of hemolysis, storage and blood collection, but also influence of various times and temperatures between individual processing steps on the total N-glycome and on a glycan-biomarker score. Venous blood was collected from 10 healthy donors in 11 blood collection tubes with different additives, processed variously to obtain 16 preanalytical variables and N-glycans released from serum or plasma were analyzed by MALDI-TOF-MS and capillary electrophoresis coupled with fluorescence detection (CE-LIF) for the first time. Long time storage of deep frozen samples at -20°C or -80°C exerted only a minor influence on the glycome as demonstrated by CE-LIF. The N-glycome was very stable evidenced by MALDI-TOF when stored at 4°C for at least 48 hours and blood collected in tubes devoid of additives. The glycome was stable upon storage after centrifugation and aliquoting, which is an important information considering future diagnostic applications. Hemolysis, however, negatively correlated with an established glycan score for ovarian cancer, when evaluated by MALDI-TOF-MS measurement by affecting relative intensities of certain glycans, which could lead to false negative / positive results in glycan biomarker studies
Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes
Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)-CB1R agonist, rimonabant-CB1R antagonist, John W. Huffman (JWH133)-CB2R agonist and CB2R antagonist-6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation
Quantitative protein biomarker panels: a path to improved clinical practice through proteomics
The utilisation of protein biomarker panels, rather than individual protein biomarkers, offers a more comprehensive representation of human physiology. It thus has the potential to improve diagnosis, prognosis and the differentiation of responders from nonresponders in the context of precision medicine. Although several proteomic techniques exist for measuring biomarker panels, the integration of proteomics into clinical practice has been limited. In this Commentary, we highlight the significance of quantitative protein biomarker panels in clinical medicine and outline the challenges that must be addressed in order to identify the most promising panels and implement them in clinical routines to realise their medical potential. Furthermore, we argue that the absolute quantification of protein panels through targeted mass spectrometric assays remains the most promising technology for translating proteomics into routine clinical applications due to its high flexibility, low sample costs, independence from affinity reagents and low entry barriers for its integration into existing laboratory workflows
Targeting hepatocyte growth factor in epithelial-stromal interactions in an in vitro experimental model of human periodontitis
Periodontitis is a chronic inflammatory disease leading to progressive connective tissue degradation and loss of the tooth-supporting bone. Clinical and experimental studies suggest that hepatocyte growth factor (HGF) is involved in the dysregulated fibroblast-epithelial cell interactions in periodontitis. The aim of this study was to explore effects of HGF to impact fibroblast-induced collagen degradation. A patient-derived experimental cell culture model of periodontitis was applied. Primary human epithelial cells and fibroblasts isolated from periodontitis-affected gingiva were co-cultured in a three-dimensional collagen gel. The effects of HGF neutralizing antibody on collagen gel degradation were tested and transcriptome analyses were performed. HGF neutralizing antibody attenuated collagen degradation and elicited expression changes of genes related to extracellular matrix (ECM) and cell adhesion, indicating that HGF signaling inhibition leads to extensive impact on cell-cell and cell-ECM interactions. Our study highlights a potential role of HGF in periodontitis. Antagonizing HGF signaling by a neutralizing antibody may represent a novel approach for periodontitis treatment
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