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

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

Pulmonary arterial remodelling by deficiency of peroxisome proliferator-activated receptor- in murine vascular smooth muscle cells occurs independently of obesity-related pulmonary hypertension

BackgroundObesity is associated with cardiovascular complications, including pulmonary hypertension (PH). Reports suggest that peroxisome proliferator-activated receptor- (PPAR) has direct action in preventing vascular remodelling in PH. Here we dissected the specific role of high-fat-diet (HFD)-induced obesity and vascular smooth muscle cell (VSMC)-PPAR for remodelling of small pulmonary arteries.MethodsWild-type (WT) and VSMC-specific PPAR-knockout (SmPpar(-/-)) mice were fed a low-fat-diet (LFD, 10% kcal from fat) or HFD (60% kcal from fat) for 24weeks. Mice were metabolically phenotyped (e.g. weight development, insulin/glucose tolerance) at the beginning, and after 12 and 24weeks, respectively. At 24weeks additionally pulmonary pressure, heart structure, pulmonary vascular muscularization together with gene and protein expression in heart and lung tissues were determined.ResultsHFD increased right ventricular systolic pressure (RVSP) to a similar extent in WT and SmPpar(-/-) mice. HFD decreased glucose tolerance and insulin sensitivity in both WT and SmPpar(-/-) mice. Importantly, the increase in RVSP correlated with the degree of insulin resistance. However, VSMC-PPAR deficiency increased pulmonary vascular muscularization independently of the diet-induced rise in RVSP. This increase was associated with elevated expression of early growth response protein 1 in heart and osteopontin in lung tissue.ConclusionsHere we demonstrate a correlation of insulin resistance and pulmonary pressure. Further, deficiency of PPAR in VSMCs diet-independently leads to increased pulmonary vascular muscularization