NEWS
July 2021, paper published in Cells
miRNA-132/212 Gene-Deletion Aggravates the Effect of Oxygen-Glucose Deprivation on Synaptic Functions in the Female Mouse Hippocampus
https://doi.org/10.3390/cells10071709
Bormann D1,2,3,†, Stojanovic T1,†, Cicvaric A 4 , Schuld GA1, Cabatic M1, Ankersmit HA2,3,5 Monje FJ1
1 Center for Physiology and Pharmacology, Department of Neurophysiology and Neuropharmacology,
Medical University of Vienna, Schwarzspanierstraße 17, 1090 Vienna, Austria;
2 Laboratory for Cardiac and Thoracic Diagnosis, Department of Surgery, Regeneration and Applied
Immunology, Medical University of Vienna, Research Laboratories Vienna General Hospital,
Waehringer Guertel 18-20, 1090 Vienna, Austria;
3 Division of Thoracic Surgery, Medical University of Vienna,Waehringer Guertel 18-20, 1090 Vienna, Austria
4 Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine,
New York, NY 10461, USA;
5 Aposcience AG, Dresdner Straße 87/A 21, 1200 Vienna, Austria
Abstract:
Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved shortlength/ noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA- 132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia.
June 2021 - Paper accepted in Nature Communications
Single cell sequencing identifies the serine proteases dipeptidyl-peptidase 4 and urokinase as key molecules in scar formation
Vorstandlechner V1,2,3, Laggner M1,2, Copic D1,2, Klas K1,2, Direder M1,2, Chen Y4,5, Golabi B4, Haslik W3, Radtke C3, Tschachler E4, Hötzenecker K6, Ankersmit HJ1,2, Mildner M4
1 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology,
2 Department of Thoracic Surgery, Medical University of Vienna
3 Aposcience AG (FN 308089y), Dresdner Straße 87/A21, Vienna, Austria.
3 Department of Plastic and Reconstructive Surgery, Medical University of Vienna, Vienna, Austria
4 Department of Dermatology, Medical University of Vienna, Vienna, Austria
5 University of Applied Sciences FH Campus Wien, Vienna, Austria
6 Department of Thoracic Surgery, Medical University of Vienna, Vienna, Austria
* contributed equally to this work
Abstract
Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigated mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we found significant differences in gene expression in most cell types present in scar tissue. Fibroblasts (FBs) showed the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine FBs during scar development with genes highly expressed in mature human hypertrophic scars, we identified a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), were further analyzed in functional assays, revealing a role in TGFβ1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix (ECM) in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo showed anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present new insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.
Figure 9: In vivo application of BC-11 or Sitagliptin improves collagen alignment and fiber orientation in mouse scars
A) Picrosirius red staining and immunofluorescent stainings of
B) Col3a1 and
C) fibronectin in mouse skin and scars is shown. Four mice per group were analyzed. Arrows indicate areas of increased matrix density.
D) H&E images of mouse skin and scars. Squares indicate areas analyzed for collagen alignment. Histograms illustrate measurement of fiber orientation.
E) Calculation of alignment coefficient by CurveAlign in mouse skin and scar. Four to five mice were analyzed per group, and three to four Regions of interest were calculated per image. Whiskers represent range maximum and minimum values with < 1.5 interquartile range, boxes represent 25th -75th quartiles, line represents mean. Statistical significance was tested using one-way ANOVA with Tukey post1072 test. NS p>0.05, *p<0.05, **p<0.01, ***p<0.001.
June 2021 Paper published in JTCVS (The Journal of Thoracic and Cardiovascular Surgery) Open
Inflammatory immune response in Transcatheter Aortic Valve Implantation recipients
Journal Pre-Proof: JTCVS Open (2021), doi: doi.org/10.1016/j.xjon.2021.02.012.
Veraar C 1,2, Koschutnik M 3, Nitsche C 3, Laggner M 2,4, Polak D 5, Bohle B 5 , Mangold A 3, Moser B 4, Mascherbauer J 3, Ankersmit HJ 2,4
1) Department of Anaesthesiology, General Intensive Care and Pain Medicine, Division of Cardiothoracic and Vascular Anaesthesia and Intensive Care Medicine, Medical University of Vienna, Austria.
2) Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Medical University of Vienna, Austria
3) Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, Austria
4) Department of Thoracic Surgery, Medical University of Vienna, Austria
5) Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria
Abstract:
Objective: Transcatheter aortic valve implantation (TAVI) is rapidly replacing cardiac surgery due to its minimal invasiveness and practicability. Midterm immunological studies on the biocompatibility of galactose-alpha-1,3-galactose (α-Gal)-carrying bioprosthetic heart valves (BHVs) for TAVI are not available. In this study we investigated whether BHVs employed for TAVI augment an α-Gal-specific antibody-dependent and antibody-independent immune response 3 months post TAVI implantation.
Methods: This prospective observational study included 27 patients with severe aortic valve stenosis undergoing TAVI and 10 patients with severe mitral valve regurgitation treated with a transcatheter MitralClip procedure. Blood samples were drawn prior to and 90 days after treatment at a routine checkup. Serum samples were analysed using ELISA. Serum concentrations of α-Gal-specific IgG, IgG subclasses and IgE, complement C3a, NETosis specific CitH3, and the systemic inflammation markers sST2 and IL-33 were evaluated.
Results: Three months after TAVI we found significantly increased serum concentrations of α65 Gal-specific IgG3, complement factor C3a, citH3 levels, and sST2 (P = 0.002, P = 0.001, P = 0.025, and P = 0.039, respectively). Sensitization of α-Gal-specific IgE antibodies occurred in 55% of all patients after TAVI.
Conclusion: Our results indicate that TAVI elicits a mid-term, specific humoral immune response against α-Gal and causes an unspecific humoral inflammation compared with patients undergoing MitralClip implantation. This observation will lead to a better understanding of post intervention morbidity and the long-term durability of bioprostheses and indicates that caution is appropriate when designing implantation strategies for younger patients (Figure 1).
March 2021 Paper accepted in Molecular Therapy: Methods & Clinical Development
Comparing the efficacy of γ- and electron-irradiation of PBMCs to promote secretion of paracrine, regenerative factors
Laggner M1,2, Gugerell A1,2, Copic D1,2, Jeitler M3, Springer M1,2, Peterbauer A4, Kremslehner C5,6, Filzwieser-Narzt M5,6, Gruber F5,6, Madlener S7,8, Erb M9, Widder J10, Lechner W10, Mildner M5, §, Ankersmit HJ1,2,§
1 Department of Thoracic Surgery, Medical University of Vienna, 1090 Vienna, Austria
2 Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, 1090 Vienna, Austria
3 Core Facility Genomics, Medical University of Vienna, 1090 Vienna, Austria
4 Austrian Red Cross Blood Transfusion Service of Upper Austria, 4020 Linz, Austria
5 Department of Dermatology, Medical University of Vienna, 1090 Vienna, Austria
6 Christian Doppler Laboratory for Biotechnology of Skin Aging, 1090 Vienna, Austria
7 Molecular Neuro-Oncology, Department of Pediatrics and Adolescent Medicine and Institute of Neurology, Medical University of Vienna, 1090 Vienna, Austria
8 Comprehensive Cancer Center of the Medical University of Vienna, 1090 Vienna, Austria
9 SYNLAB Analytics and Services Switzerland AG, 4127 Birsfelden, Switzerland
10 Department of Radiation Oncology, Medical University of Vienna, 1090 Vienna, Austria
Abstract:
Cell-free secretomes represent a promising new therapeutic avenue in regenerative medicine and γ-irradiation of human peripheral blood mononuclear cells (PBMCs) has been shown to promote the release of paracrine factors with high regenerative potential. Recently, the use of alternative irradiation sources, such as artificially generated β- or electron-irradiation, is encouraged by authorities. Since the effect of the less hazardous electron-radiation on the production and functions of paracrine factors has not been tested so far, we compared the effects of γ- and electron-irradiation on PBMCs and determined the efficacy of both radiation sources for producing regenerative secretomes. Exposure to 60 Gy γ-rays from a radioactive nuclide and 60 Gy electron-irradiation provided by a linear accelerator comparably induced cell death and DNA damage. The transcriptional landscapes of PBMCs exposed to either radiation source shared a high degree of similarity. Secretion patterns of proteins, lipids, and extracellular vesicles displayed similar profiles after γ- and electron-irradiation. Lastly, we detected comparable biological activities in functional assays reflecting the regenerative potential of the secretomes. Taken together, we were able to demonstrate that electron-irradiation is an effective, alternative radiation source for producing therapeutic, cell-free secretomes. Our study paves the way for future clinical trials employing secretomes generated with electron-irradiation in tissue-regenerative medicine.
November 2020 Billroth Prize for Elisabeth Simader
We are happy to anounce that Dr Elisabeth Simader was awarded the Billroth Prize 2020 of the Vienna Medical Association (Wiener Ärztekammer) for her publication
Simader E, Beer L, Laggner M, Vorstandlechner V, Gugerell A, Erb M, Kalinina P, Copic D, Moser D, Spittler A, Tschachler E, Jan Ankersmit H, Mildner M. Cell Death Dis. 2019 Sep 30;10(10):729.
Our congratulations to Elisabeth on this great success!
November 2020 Paper published in International Journal of Molecular Sciences
TGF-β in the Secretome of Irradiated Peripheral Blood Mononuclear Cells Supports In Vitro Osteoclastogenesis
Int. J. Mol. Sci. 2020, 21(22), 8569; doi.org/10.3390/ijms21228569
Panahipour L1, Kargarpour Z1, Laggner M2,3,Mildner M4, Ankersmit HJ2,3, Gruber R1,5,6
1 Department of Oral Biology, University Clinics for Dentistry, Medical University of Vienna, Sensengasse 2a, 1090 Vienna, Austria
2 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Währingergürtel 18-20, 1090 Vienna, Austria
3 Division of Thoracic Surgery, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria
4 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria
5 Department of Periodontology, School of Dental Medicine, University of Bern, Freiburgstrasse 7, 3010 Bern, Switzerland
6 Austrian Cluster for Tissue Regeneration, Donaueschingenstraße 13, 1200 Vienna, Austria
Abstract
Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-β enhanced osteoclastogenesis, we confirmed the TGF-β activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-β by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-β released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.
Figure 1. The secretome increased the formation of TRAP+ multinucleated cells in bone marrow cultures. Murine bone marrow-derived macrophages were exposed to the secretome corresponding to 1 × 106 irradiated PBMCs/mL in the presence of 30 ng/mL RANKL and 20 ng/mL M-CSF for five days. The histochemical staining identified the cells staining positive for the tartrate-resistant acid phosphatase (TRAP+). The multinucleated cells with more than three nuclei and red stain were considered “osteoclast-like cells,” even though mononuclear cells also showed positive TRAP staining in the presence of RANKL and particularly when combined with 10 ng/mL TGF-β or the secretome.
August 2020 Paper published in Bioenineering & Translational Medicine
The secretome of stressed peripheral blood mononuclear cells increases tissue survival in a rodent epigastric flap model
Hacker S1,2, Mittermayr R3, Traxler D2, Keibl C3, Resch A1, Salminger S, Leiss H4, Hacker P5, Gabriel C3,6, Golabi B7, Pauzenberger R1, Slezak P3, Laggner M2, Mildner M7, Michlits W8, Ankersmit HJ2,9
1 Division of Plastic and Reconstructive Surgery, Medical University of Vienna, Vienna, Austria
2 Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, Austria
3 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
4 Division of Rheumatology, Medical University of Vienna, Vienna, Austria
5 Department of Oral- and Maxillofacial Surgery, University Clinic Sankt Poelten, Sankt Poelten, Austria
6 Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Austria
7 Department of Dermatology, Medical University of Vienna, Vienna, Austria
8 Department of Plastic and Reconstructive Surgery, Hospital Wiener Neustadt, Wiener Neustadt, Austria
9 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria
Abstract
Reconstructive surgery transfers viable tissue to cover defects and to restore aesthetic and functional properties. Failure rates after free flap surgery range from 3 to 7%. Co‐morbidities such as diabetes mellitus or peripheral vascular disease increase the risk of flap failure up to 4.5‐fold. Experimental therapeutic concepts commonly use a monocausal approach by applying single growth factors. The secretome of γ‐irradiated, stressed peripheral blood mononuclear cells (PBMCsec) resembles the physiological environment necessary for tissue regeneration. Its application led to improved wound healing rates and a two‐fold increase in blood vessel counts in previous animal models. We hypothesized that PBMCsec has beneficial effects on the survival of compromised flap tissue by reducing the necrosis rate and increasing angiogenesis. Surgery was performed on 39 male Sprague–Dawley rats (control, N = 13; fibrin sealant, N = 14; PBMCsec, N = 12). PBMCsec was produced according to good manufacturing practices (GMP) guidelines and 2 ml were administered intraoperatively at a concentration of 2.5 × 107 cells/ml using fibrin sealant as carrier substance. Flap perfusion and necrosis (as percentage of the total flap area) were analyzed using Laser Doppler Imaging and digital image planimetry on postoperative days 3 and 7. Immunohistochemical stainings for von Willebrand factor (vWF) and Vascular Endothelial Growth Factor‐receptor‐3 (Flt‐4) were performed on postoperative day 7 to evaluate formation of blood vessels and lymphatic vessels. Seroma formation was quantified using a syringe and flap adhesion and tissue edema were evaluated clinically through a cranial incision by a blinded observer according to previously described criteria on postoperative day 7. We found a significantly reduced tissue necrosis rate (control: 27.8% ± 8.6; fibrin: 22.0% ± 6.2; 20.9% reduction, p = .053 vs. control; PBMCsec: 19.1% ± 7.2; 31.1% reduction, p = .012 vs. control; 12.9% reduction, 0.293 vs. fibrin) together with increased vWF+ vessel counts (control: 70.3 ± 16.3 vessels/4 fields at 200× magnification; fibrin: 67.8 ± 12.1; 3.6% reduction, p = .651, vs. control; PBMCsec: 85.9 ± 20.4; 22.2% increase, p = .045 vs. control; 26.7% increase, p = .010 vs. fibrin) on postoperative day 7 after treatment with PBMCsec. Seroma formation was decreased after treatment with fibrin sealant with or without the addition of PBMCsec. (control: 11.9 ± 9.7 ml; fibrin: 1.7 ± 5.3, 86.0% reduction, 0.004 vs. control; PBMCsec: 0.6 ± 2.0; 94.8% reduction, p = .001 vs. control; 62.8% reduction, p = .523 vs. fibrin). We describe the beneficial effects of a secretome derived from γ‐irradiated PBMCs on tissue survival, angiogenesis, and clinical parameters after flap surgery in a rodent epigastric flap model.
FIGURE 1 (a) The tissue necrosis rate was significantly reduced after a single, intraoperative application of the secretome derived from γ-irradiated PBMCs (PBMCsec). Only the combinatory use of fibrin and PBMCsec showed significantly improved results. (b) Examples for the development of tissue necrosis over the postoperative period are shown for each group. (d) Flap adherence to the underlying tissue was evaluated clinically as parameter for tissue integration. Both groups, fibrin sealant alone and in combination with PBMC secretomes, showed markedly improved rates of flap adherence on postoperative day 7.
FIGURE 4 (b) The irradiation of PBMC causes a switch toward a regenerative phenotype and induces the secretion of proteins, lipids, and EVs, which form the secretome. (c) PBMCsec resembles the physiologic environment of wound healing and regeneration and therefore has pleiotropic effects
July 2020 Paper published in Journal of Allergy and Clinical Immunology
Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic Dermatitis
Rojahn TB1, Vorstandlechner V2, Krausgruber T3, Bauer WM1, Alkon N1, Bangert C1, Thaler FM1, Sadeghyar F1, Fortelny N3, Gernedl V3, Rindler K1, Elbe-Bürger A1, Bock C4, Mildner M1, Brunner PM5
1 Department of Dermatology, Medical University of Vienna, Vienna, Austria.
2 Department of Dermatology, Medical University of Vienna, Vienna, Austria; Department of Surgery, Research Laboratory for Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
3 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
4 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
5 Department of Dermatology, Medical University of Vienna, Vienna, Austria.
Abstract
Background: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, but its complex pathogenesis is only insufficiently understood, resulting in still limited treatment options.
Objective: We sought to characterize AD on both transcriptomic and proteomic levels in humans.
Methods: We used skin suction blistering, a painless and nonscarring procedure that can simultaneously sample skin cells and interstitial fluid. We then compared results with conventional biopsies.
Results: Suction blistering captured epidermal and most immune cells equally well as biopsies, except for mast cells and nonmigratory CD163+ macrophages that were only present in biopsy isolates. Using single-cell RNA sequencing, we found comparable transcriptional profiles of key inflammatory pathways between blister and biopsy AD, but suction blistering was superior in cell-specific resolution for high-abundance transcripts (KRT1/KRT10, KRT16/KRT6A, S100A8/S100A9), which showed some background signals in biopsy isolates. Compared with healthy controls, we found characteristic upregulation of AD-typical cytokines such as IL13 and IL22 in Th2 and Th22 cells, respectively, but we also discovered these mediators in proliferating T cells and natural killer T cells, that also expressed the antimicrobial cytokine IL26. Overall, not T cells, but myeloid cells were most strongly enriched in AD, and we found dendritic cell (CLEC7A, amphiregulin/AREG, EREG) and macrophage products (CCL13) among the top upregulated proteins in AD blister fluid proteomic analyses.
Conclusion: These data show that by using cutting-edge technology, suction blistering offers several advantages over conventional biopsies, including better transcriptomic resolution of skin cells, combined with proteomic information from interstitial fluid, unraveling novel inflammatory players that shape the cellular and proteomic microenvironment of AD.
June 2020 Paper published in the International Journal of Molecular Sciences
Role for Lipids Secreted by Irradiated Peripheral Blood Mononuclear Cells in Inflammatory Resolution in Vitro.
Int. J. Mol. Sci. 2020, 21, 4694; doi:10.3390/ijms21134694.
Panahipour L, Kochergina E, Laggner M, Zimmermann M, Mildner M, Ankersmit HJ, Gruber R.
1 Department of Oral Biology, Medical University of Vienna, Sensengasse 2a, 1090 Vienna, Austria;
2 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Währingergürtel 18-20, 1090 Vienna, Austria;
3 Division of Thoracic Surgery, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria
4 Department of Oral and Maxillofacial Surgery, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria;
5 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical Universityof Vienna, Währingergürtel 18-20, 1090 Vienna, Austria;
6 Department of Periodontology, School of Dental Medicine, University of Bern, Freiburgstrasse 7, 3010 Bern, Switzerland;
7 Austrian Cluster for Tissue Regeneration, Donaueschingenstraße 13, 1200 Vienna, Austria;
Abstract:
Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1β and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.
June 2020 Paper published in Molecular Therapy
Quantitative Hybrid Cardiac [18F]FDG-PET-MRI Images for Assessment of Cardiac Repair by Preconditioned Cardiosphere-Derived Cells.
Molecular Therapy. Methods & Clinical Development. Vol. 18, 11 Sept 2020, p 354-366. doi: 10.1016/j.omtm.2020.06.008.
Winkler J, Lukovic D, Mester-Tonczar J, Zlabinger K, Gugerell A, Pavo N, Jakab A. Szanka Z, Traxler1 D, Müller C, Spannbauer A, Riesenhuber M, Hasimbegovic E, Dawkins J, Zimmermann M, Ankersmit HJ, Marban E, Gyöngyösi M.
1 Department of Cardiology, Medical University of Vienna, Vienna, Austria
2 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
3 Center for MR-Research, University Children’s Hospital Zurich, Steinwiesstrasse 7e, 80cb Zurich, Switzerland
4 Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
5 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria
Abstract:
Cardiosphere-derived cells (CDCs) are progenitor cells derived from heart tissue and have shown promising results in preclinical models. APOSEC, the secretome of irradiated peripheral blood mononuclear cells, has decreased infarct size in acute and chronic experimental myocardial infarction (MI). We enhanced the effect of CDCs with APOSEC preconditioning (apoCDC) and investigated the reparative effect in a translational pig model of reperfused MI. Supernatants of CDCs, assessed by proteomic analysis, revealed reduced production of extracellular matrix proteins after in vitro APOSEC preconditioning. In a porcine model of catheter-based reperfused anterior acute MI (AMI), CDCs with (apoCDC, n = 8) or without APOSEC preconditioning (CDC, n = 6) were infused intracoronary, 15 min after the start of reperfusion. Untreated AMI animals (n = 7) and sham procedures (n = 5) functioned as controls. 2-deoxy-2-(18 F)-fluoro-D-glucose-positron emission tomography-magnetic resonance imaging ([18F]FDG-PET-MRI), with late enhancement after 1 month, showed reduced scar volume and lower transmurality of the infarcted area in CDC and apoCDC compared to AMI controls. Segmental quantitative PET images displayed indicated more residual viability in apoCDC. The left-ventricle (LV) ejection fraction was improved nonsignificantly to 45.8% ± 8.6% for apoCDC and 43.5% ± 7.1% for CDCs compared to 38.5% ± 4.4% for untreated AMI. Quantitative hybrid [18F]FDG-PET-MRI demonstrated improved metabolic and functional recovery after CDC administration, whereas apoCDCs induced preservation of viability of the infarcted area.
May 2020 Paper published in EBioMedicine
Therapeutic potential of lipids obtained from γ-irradiated PBMCs in dendritic cell-mediated skin inflammation.
EBioMedicine. 2020 May;55:102774. doi: 10.1016/j.ebiom.2020.102774. Epub 2020 May 8.
Maria Laggner 1, Dragan Copic 1, Lucas Nemec 2, Vera Vorstandlechner 1, Alfred Gugerell 1, Florian Gruber 2, Anja Peterbauer 3, Hendrik J Ankersmit 4, Michael Mildner 5
1 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Vienna, Austria; Division of Thoracic Surgery, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
2 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Lazarettgasse 14, 1090 Vienna, Austria.
3 Austrian Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria.
4 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Vienna, Austria; Division of Thoracic Surgery, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
5 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Lazarettgasse 14, 1090 Vienna, Austria.
Abstract
Background: Since numerous pathological conditions are evoked by unwanted dendritic cell (DC) activity, therapeutic agents modulating DC functions are of great medical interest. In regenerative medicine, cellular secretomes have gained increasing attention and valuable immunomodulatory properties have been attributed to the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs). Potential effects of the PBMC secretome (PBMCsec) on key DC functions have not been elucidated so far.
Methods: We used a hapten-mediated murine model of contact hypersensitivity (CH) to study the effects of PBMCsec on DCs in vivo. Effects of PBMCsec on human DCs were investigated in monocyte-derived DCs (MoDC) and ex vivo skin cultures. DCs were phenotypically characterised by transcriptomics analyses and flow cytometry. DC function was evaluated by cytokine secretion, antigen uptake, PBMC proliferation and T-cell priming.
Findings: PBMCsec significantly alleviated tissue inflammation and cellular infiltration in hapten-sensitized mice. We found that PBMCsec abrogated differentiation of MoDCs, indicated by lower expression of classical DC markers CD1a, CD11c and MHC class II molecules. Furthermore, PBMCsec reduced DC maturation, antigen uptake, lipopolysaccharides-induced cytokine secretion, and DC-mediated immune cell proliferation. Moreover, MoDCs differentiated with PBMCsec displayed diminished ability to prime naïve CD4+T-cells into TH1 and TH2 cells. Furthermore, PBMCsec modulated the phenotype of DCs present in the skin in situ. Mechanistically, we identified lipids as the main biomolecule accountable for the observed immunomodulatory effects.
Interpretation: Together, our data describe DC-modulatory actions of lipids secreted by stressed PBMCs and suggest PBMCsec as a therapeutic option for treatment of DC-mediated inflammatory skin conditions.
Fig. 1:
Fig. 1 PBMC secretome alleviates ear swelling in DNFB-induced CH and abolishes immune cell recruitment to challenged skin.
(a) Hematoxylin/eosin-stained ears 24 h after DNFB re-challenge with vehicle or PBMCsec treatment. Naïve ears served as untreated controls. Representative micrographs of n = 5 mice per treatment condition are shown. ac, auricular cartilage; c, cornified epidermis; d, dermis; epi, epidermis. Scale bar, 200 µm.
(b) Increase in ear thickness assessed by micrometer-assisted measurements 24 h post DNFB elicitation. * indicates p<.05 PBMCsec versus vehicle. N = 5 mice per group. Ordinary one-way ANOVA was performed. Dunnett's multiple comparison test was carried out to compare groups versus vehicle control.
(c) Number of cells per field. * denotes p<.05 PBMCsec versus vehicle. N = 5 mice per group. Ordinary one-way ANOVA was performed. Dunnett's multiple comparison test was carried out to compare groups versus vehicle control.
(d) qPCR analysis of genes encoding cytokines and chemokines in ears of DNFB-sensitized mice. Red and green dots indicate up- and down-regulated genes, respectively, in PBMCsec- versus medium-treated ears. Blue dots represent genes considered not differentially regulated. Dotted lines denote log2-transformed 1.3-fold regulations when comparing PBMCsec and medium. Solid gray line represents log2 transformation of PBMCsec / medium = 1. Solid black line indicates -log10 of p = .05. N = 5 mice per condition. p-values were calculated by one-tailed student's t-test with equal variances between the two samples.
March 2020 Paper published in FASEB J
Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing.
FASEB J. 2020 Mar;34(3):3677-3692. doi: 10.1096/fj.201902001RR. Epub 2020 Jan 12.
Vera Vorstandlechner 1, Maria Laggner 1, Polina Kalinina 2, Werner Haslik 3, Christine Radtke 3, Lisa Shaw 4, Beate Maria Lichtenberger 5, Erwin Tschachler 2, Hendrik Jan Ankersmit 1, Michael Mildner 2
1 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
2 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
3 Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria.
4 Department of Dermatology, Medical University of Vienna, Vienna, Austria.
5 Skin and Endothelium Research Division, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
Abstract
Though skin fibroblasts (FB) are the main cell population within the dermis, the different skin FB subsets are not well characterized and the traditional classification into reticular and papillary FBs has little functional relevance. To fill the gap of knowledge on FB diversity in human skin, we performed single-cell RNA sequencing. Investigation of marker genes for the different skin cell subtypes revealed a heterogeneous picture of FBs. When mapping reticular and papillary FB markers, we could not detect cluster specificity, suggesting that these two populations show a higher transcriptional heterogeneity than expected. This finding was further confirmed by in situ hybridization, showing that DPP4 was expressed in both dermal layers. Our analysis identified six FB clusters with distinct transcriptional signatures. Importantly, we could demonstrate that in human skin DPP4+ FBs are the main producers of factors involved in extracellular matrix (ECM) assembly. In conclusion, we provide evidence that hitherto considered FB markers are not ideal to characterize skin FB subpopulations in single-cell sequencing analyses. The identification of DPP4+ FBs as the main ECM-producing cells in human skin will foster the development of anti-fibrotic treatments for the skin and other organs.
January 2020 Paper published in Stem Cell Research & Therapy
Reproducibility of GMP-compliant production of therapeutic stressed peripheral blood mononuclear cell-derived secretomes, a novel class of biological medicinal products.
Stem Cell Res Ther. 2020 Jan 3;11(1):9. doi: 10.1186/s13287-019-1524-2.
Maria Laggner 1 2, Alfred Gugerell 1 2, Christiane Bachmann 3, Helmut Hofbauer 1 2, Vera Vorstandlechner 1 2, Marcus Seibold 2, Ghazaleh Gouya Lechner 2, Anja Peterbauer 4, Sibylle Madlener 5 6, Svitlana Demyanets 7, Dirk Sorgenfrey 8, Tobias Ostler 9, Michael Erb 9, Michael Mildner 10, Hendrik Jan Ankersmit 11 12
1 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
2 Aposcience AG, Vienna, Austria.
3 Independent Consultant, Mainaschaff, Germany.
4 Austrian Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria.
5 Molecular Neuro-Oncology, Department of Pediatrics and Adolescent Medicine and Institute of Neurology, Medical University of Vienna, Vienna, Austria.
6 Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria.
7 Department for Laboratory Medicine at the Medical University of Vienna, Vienna, Austria.
8 Dr. Regenold GmbH, Badenweiler, Germany.
9 SYNLAB Analytics and Services Switzerland AG, Birsfelden, Switzerland.
10 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
11 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
12 Aposcience AG, Vienna, Austria.
Abstract
Background: The recent concept of secretome-based tissue regeneration has profoundly altered the field of regenerative medicine and offers promising novel therapeutic options. In contrast to medicinal products with a single active substance, cell-derived secretomes comprise pleiotropic bioactive ingredients, representing a major obstacle for reproducible drug product efficacy and warranting patient safety. Good manufacturing practice (GMP)-compliant production guarantees high batch-to-batch consistency and reproducible efficacy of biological medicinal products, but different batches of cellular secretomes produced under GMP have not been compared yet, and suitable quality control parameters have not been established. To this end, we analyzed diverse biological and functional parameters of different batches produced under GMP of the secretome obtained from γ-irradiated peripheral blood mononuclear cells with proven tissue regenerative properties in infarcted myocardium, stroke, spinal cord injury, and skin wounds.
Methods: We quantified key secretome ingredients, including cytokines, lipids, and extracellular vesicles, and functionally assessed potency in tube formation assay, ex vivo aortic ring sprouting assay, and cell-based protein and reporter gene assays. Furthermore, we determined secretome stability in different batches after 6 months of storage at various ambient temperatures.
Results: We observed that inter-batch differences in the bioactive components and secretome properties were small despite considerable differences in protein concentrations and potencies between individual donor secretomes. Stability tests showed that the analytical and functional properties of the secretomes remained stable when lyophilisates were stored at temperatures up to + 5 °C for 6 months.
Conclusions: We are the first to demonstrate the consistent production of cell-derived, yet cell-free secretome as a biological medicinal product. The results from this study provide the basis for selecting appropriate quality control parameters for GMP-compliant production of therapeutic cell secretomes and pave the way for future clinical trials employing secretomes in tissue regenerative medicine.
December 2019 Paper published in Critical Care
Non-pulsatile blood flow is associated with enhanced cerebrovascular carbon dioxide reactivity and an attenuated relationship between cerebral blood flow and regional brain oxygenation.
Crit Care. 2019 Dec 30;23(1):426. doi: 10.1186/s13054-019-2671-7.
Cecilia Maria Veraar 1, Harald Rinösl 2, Karina Kühn 3, Keso Skhirtladze-Dworschak 1, Alessia Felli 1, Mohamed Mouhieddine 1, Johannes Menger 1, Ekaterina Pataraia 4, Hendrik Jan Ankersmit 5, Martin Dworschak 6
1 Division of Cardiothoracic and Vascular Anaesthesia and Intensive Care Medicine, Department of Anaesthesia, Intensive Care Medicine, and Pain Medicine, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, 1090, Vienna, Austria.
2 Department of Anaesthesia and Intensive Care Medicine, LKH Feldkirch, Feldkirch, Austria.
3 Department of Anaesthesia, Intensive Care Medicine and Pain Medicine, Klinikum Traunstein, Traunstein, Germany.
4 Department of Neurology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
5 Division of Thoracic Surgery, Department of Surgery, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
6 Division of Cardiothoracic and Vascular Anaesthesia and Intensive Care Medicine, Department of Anaesthesia, Intensive Care Medicine, and Pain Medicine, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, 1090, Vienna, Austria.
Abstract
Background: Systemic blood flow in patients on extracorporeal assist devices is frequently not or only minimally pulsatile. Loss of pulsatile brain perfusion, however, has been implicated in neurological complications. Furthermore, the adverse effects of absent pulsatility on the cerebral microcirculation are modulated similarly as CO2 vasoreactivity in resistance vessels. During support with an extracorporeal assist device swings in arterial carbon dioxide partial pressures (PaCO2) that determine cerebral oxygen delivery are not uncommon-especially when CO2 is eliminated by the respirator as well as via the gas exchanger of an extracorporeal membrane oxygenation machine. We, therefore, investigated whether non-pulsatile flow affects cerebrovascular CO2 reactivity (CVR) and regional brain oxygenation (rSO2).
Methods: In this prospective, single-centre case-control trial, we studied 32 patients undergoing elective cardiac surgery. Blood flow velocity in the middle cerebral artery (MCAv) as well as rSO2 was determined during step changes of PaCO2 between 30, 40, and 50 mmHg. Measurements were conducted on cardiopulmonary bypass during non-pulsatile and postoperatively under pulsatile blood flow at comparable test conditions. Corresponding changes of CVR and concomitant rSO2 alterations were determined for each flow mode. Each patient served as her own control.
Results: MCAv was generally lower during hypocapnia than during normocapnia and hypercapnia (p < 0.0001). However, the MCAv/PaCO2 slope during non-pulsatile flow was 14.4 cm/s/mmHg [CI 11.8-16.9] and 10.4 cm/s/mmHg [CI 7.9-13.0] after return of pulsatility (p = 0.03). During hypocapnia, non-pulsatile CVR (4.3 ± 1.7%/mmHg) was higher than pulsatile CVR (3.1 ± 1.3%/mmHg, p = 0.01). Independent of the flow mode, we observed a decline in rSO2 during hypocapnia and a corresponding rise during hypercapnia (p < 0.0001). However, the relationship between ΔrSO2 and ΔMCAv was less pronounced during non-pulsatile flow.
Conclusions: Non-pulsatile perfusion is associated with enhanced cerebrovascular CVR resulting in greater relative decreases of cerebral blood flow during hypocapnia. Heterogenic microvascular perfusion may account for the attenuated ΔrSO2/ΔMCAv slope. Potential hazards related to this altered regulation of cerebral perfusion still need to be assessed.
Trial registration: The study was retrospectively registered on October 30, 2018, with Clinical Trial.gov (NCT03732651).
November 2019 Paper accepted in Cell Stress & Chaperones
Heat shock protein 27 as a predictor of prognosis in patients admitted to hospital with acute COPD exacerbation
Zimmermann Matthias1,2, Traxler Denise1,3, Bekos Christine1, Simader Elisabeth1,4, Mueller Thomas5, Graf Alexandra6, Lainscak Mitja7,8, Marčun Robert9, Korošec Peter9, Košnik Mitja9, Fležar Matjaž9, Rozman Aleš9, Klepetko Walter10, Moser Bernhard10, Ankersmit Hendrik J1,10
1 Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Medical University of Vienna, Austria
2 Department of Maxillofacial and Oral Surgery, Medical University of Vienna, Austria
3 Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Austria
4 Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Austria.
5 Department of Clinical Pathology, Hospital of Bolzano, Bolzano, Italy
6 Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Vienna, Austria
7 Division of Cardiology, General Hospital Murska Sobota, Murska Sobota, Slovenia
8 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
9 University Clinic of Pulmonary and Allergic Diseases Golnik, Golnik, Slovenia
10 Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Austria
Abstract
Background: Episodes of acute exacerbations are major drivers of hospitalization and death from COPD. To date there are no objective biomarkers of disease activity or biomarkers to predict patient outcome.
Methods
In this prospective study 217 patients hospitalized for an acute exacerbation of COPD have been included. At the time of admission routine blood tests have been performed including complete blood count, C-reactive protein, cardiac troponin T and NT-proBNP. Heat Shock Protein 27 (HSP27) serum concentrations were determined at time of admission, discharge and 180 days after discharge post hoc by ELISA.
Results: We were able to demonstrate significantly increased serum HSP27 concentrations in COPD patients at time of admission to hospital due to an episode of acute exacerbation as compared to the corresponding concentrations after decline of acute symptoms. However we did not see any significant correlation with disease severity. Furthermore we identified a HSP27 serum cut off value serving as potential predictor for mortality. Kaplan-Meier survival curves showed significantly lower survival rates in patients with increased HSP27 concentrations (p=0.02). Further potential risk factors for 3-year mortality were selected by univariable analyses. After adjusting the model for the univariable significant markers in a multivariable Cox regression analysis, HSP27 retained its prognostic ability for the short-term up to 1 year follow-up. A significantly larger risk of 180 days-mortality after discharge was found for patients with lower HSP27 concentrations at admission (Hazard Ratio: 4.4; 95% confidence interval, 2.0-9.4).
Conclusions: Elevated serum HSP27 concentrations significantly predicted short-term mortality in patients admitted to hospital with acute exacerbation of COPD and could help to improve outcomes by identifying high-risk patients.
Figure 2: Hazard ratios (HR) and 95% Confidence Interval (CI) in univariable (grey bar) and multivariable (red bar) Cox regression analysis after 90 days (A), 180 days (B), 1 year (C) and 3 years (D). Variables remaining in the model after multivariable analyses are presented in red.
November 2019 Paper accepted in Scientific Reports
Follistatin impacts Tumor Angiogenesis and Outcome in Thymic Epithelial Tumors
Janik S1,2, Bekos C1,3, Hacker P1,4, Raunegger T1,4, Schiefer A-I5, Müllauer L5, Veraar C6, Dome B7, Klepetko W4, Ankersmit HJ1,4,8, Moser B1,4.
1 Christian Doppler Laboratory for Diagnosis and Regeneration of Cardiac and Thoracic Diseases, Medical University Vienna, Vienna, Austria
2 Department of Otorhinolaryngology, Head and Neck Surgery, Medical University Vienna, Vienna, Austria
3 Department of Obstetrics and Gynecology, Division of General Gynecology and Gynecologic Oncology, Medical University Vienna, Vienna, Austria.
4 Division of Thoracic Surgery, Department of Surgery, Medical University Vienna, Vienna, Austria
5 Clinical Institute of Pathology, Medical University Vienna, Vienna, Austria
6 Department of Anaesthesiology, General Intensive Care and Pain Medicine, Medical University of Vienna, Vienna, Austria
7 Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Centre Vienna, Medical University Vienna, Vienna, Austria
8 Head FFG Project “APOSEC“, FOLAB Surgery, Medical University Vienna, Vienna, Austria
Abstract
Tumor angiogenesis is a key factor in the progression of thymic epithelial tumors (TETs). Activin A, a member of the TGFβ family, and its antagonist Follistatin are involved in several human malignancies and angiogenesis. We investigated Activin A and Follistatin in serum and tumor tissue of patients with TETs in relation to microvessel density (MVD), WHO histology classification, tumor stage and outcome. Membranous Activin A expression was detected in all tumor tissues of TETs, while Follistatin staining was found in tumor nuclei and cytoplasm. Patients with TETs presented with significantly higher Activin A and Follistatin serum concentrations compared to healthy volunteers, respectively. Follistatin serum concentrations correlated significantly with tumor stage and decreased to physiologic values after complete tumor resection. Follistatin serum concentrations correlated further with MVD and were associated with significantly worse freedom from recurrence (FFR). Low numbers of immature tumor vessels repre ented even an independent worse prognostic factor for FFR at multivariable analysis. To conclude, the Activin A - Follistatin axis is involved in the pathogenesis of TETs. Further study of Follistatin and Activin A in TETs is warranted as the molecules may serve as targets to inhibit tumor angiogenesis and tumor progression.
September 2019 Paper published in Cell Death and Disease
Tissue-regenerative potential of the secretome of γ-irradiated peripheral blood mononuclear cells is mediated via TNFRSF1B-induced necroptosis
Simader E1,2,3, Beer L4,5, Laggner M2,3,6, Vorstandlechner V2,3,6, Gugerell A2,3,6, Erb M7, Kalinina P8, Copic D2,3,6, Moser D9, Spittler A10, Tschachler E8, Jan Ankersmit H11,12,13, Mildner M14.
1 Department of Internal Medicine III, Division of Rheumatology, Medical University of Vienna, Vienna, Austria.
2 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
3 FFG Project 852748 "APOSEC", Medical University of Vienna, Vienna, Austria.
4 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria.
5 Department of Radiology and Cancer Research UK Cambridge Center, Cambridge, CB2 0QQ, UK.
6 Vienna Business Agency Project 2343727 "APOSEC to clinic", Medical University Vienna, Vienna, Austria
7 Synlab Analytics and Services Switzerland AG, Birsfelden, Switzerland.
8 Research Division of Biology and Pathobiology of the SkinDepartment of Dermatology, Research Division of Biology and Pathobiology of the Skin, Medical University of Vienna, Vienna, Austria.
9 Division of Oral and Maxillofacial Surgery, Medical University of Vienna, Vienna, Austria.
10 Research Laboratories, Core Facility Flow Cytometry, Medical University of Vienna, Vienna, Austria.
11 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
12 FFG Project 852748 "APOSEC", Medical University of Vienna, Vienna, Austria.
13 Vienna Business Agency Project 2343727 "APOSEC to clinic", Medical University Vienna, Vienna, Austria.
14 Research Division of Biology and Pathobiology of the SkinDepartment of Dermatology, Research Division of Biology and Pathobiology of the Skin, Medical University of Vienna, Vienna, Austria.
Abstract
Peripheral blood mononuclear cells (PBMCs) have been shown to produce and release a plethora of pro-angiogenetic factors in response to γ-irradiation, partially accounting for their tissue-regenerative capacity. Here, we investigated whether a certain cell subtype of PBMCs is responsible for this effect, and whether the type of cell death affects the pro-angiogenic potential of bioactive molecules released by γ-irradiated PBMCs. PBMCs and PBMC subpopulations, including CD4+ and CD8+ T cells, B cells, monocytes, and natural killer cells, were isolated and subjected to high-dose γ-irradiation. Transcriptome analysis revealed subpopulation-specific responses to γ-irradiation with distinct activation of pro-angiogenic pathways, cytokine production, and death receptor signalling. Analysis of the proteins released showed that interactions of the subsets are important for the generation of a pro-angiogenic secretome. This result was confirmed at the functional level by the finding that the secretome of γ-irradiated PBMCs displayed higher pro-angiogenic activity in an aortic ring assay. Scanning electron microscopy and image stream analysis of γ-irradiated PBMCs revealed distinct morphological changes, indicative for apoptotic and necroptotic cell death. While inhibition of apoptosis had no effect on the pro-angiogenic activity of the secretome, inhibiting necroptosis in stressed PBMCs abolished blood vessel sprouting. Mechanistically, we identified tumor necrosis factor (TNF) receptor superfamily member 1B as the main driver of necroptosis in response to γ-irradiation in PBMCs, which was most likely mediated via membrane-bound TNF-α. In conclusion, our study demonstrates that the pro-angiogenic activity of the secretome of γ-irradiated PBMCs requires interplay of different PBMC subpopulations. Furthermore, we show that TNF-dependent necroptosis is an indispensable molecular process for conferring tissue-regenerative activity and for the pro-angiogenic potential of the PBMC secretome. These findings contribute to a better understanding of secretome-based therapies in regenerative medicine.
Fig. 6
Fig. 6: Induction of necroptosis is necessary for the pro-angiogenic capacity of the PBMC secretome.
a Immunoblot analysis of phosphorylated MLKL and cleaved caspase (c-cas) 3 in non-irradiated and γ-irradiated PBMCs is shown. Irradiated PBMCs were cultivated with either zVAD, necrostatin-1, or both.
b Representative images of murine aortic rings after 3 days of cultivation with conditioned medium from γ-irradiated PBMC treated with zVAD, necrostatin-1, or both. Viable cells were visualized with calcein (green). Scale bar, 200 µm.
c Box plot diagrams of vessel areas are shown. Whiskers indicate minimal and maximal values. Necrostatin-1 added to irradiated PBMC significantly reduced sprout-inducing ability of the PBMC secretome, while addition of zVAD did not compromise pro-angiogenic potential.
d Endothelial cells were incubated with the secretome of γ-irradiated PBMCs alone or treated with zVAD or necrostatin for 3 h after overnight starvation. The PBMC secretome treated with necrostatin-1 lead to significantly reduced tube formation. e Bar graph depicting a significant decrease in the number of nodes and junction after treatment with necrostatin-1. Scale bar, 200 µm. *P values below 0.05 compared to PBMC. n = 3
March 2019 Paper published in Scientific Reports
Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease
Silvio Wuschko1§, Alfred Gugerell2,3§, Monika Chabicovsky4, Helmut Hofbauer2, MariaLaggner2, Michael Erb5, Tobias Ostler5, Anja Peterbauer6, Susanne Suessner6, Svitlana Demyanets7, Jost Leuschner8, Bernhard Moser2, Michael Mildner9 and Hendrik J.Ankersmit2,10*
§ share first authorship
* corresponding author
1 Drug and Chemical Safety Research & Toxicology, Consultant, Alland, Austria
2 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
3 Department of Cardiology, Department of Internal Medicine II, Medical University of
Vienna, Vienna, Austria.
4 MC Toxicology Consulting GmbH, Vienna, Austria.
5 Synlab, Birsfelden, Switzerland.
6 Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria.
7 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
8 LPT - Laboratory of Pharmacology and Toxicology GmbH & Co KG, Hamburg, Germany.
9 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology,
Medical University of Vienna, Vienna, Austria.
10 FFG Projects "APOSEC" 852748 and 862068, Medical University Vienna, Vienna, Austria.
Abstract
A cell-free approach using secretomes derived from stem cells or peripheral bloodmononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non-clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully
tested in a toxicology program, supporting clinical use of the new drug candidate. Here, toallow for topical, dermal treatment of patients with diabetic foot ulcer, several nonclinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for Skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm²/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.
February 2019 Paper published in Scientific Reports
Exercise-induced bronchoconstriction, temperature regulation and the role of heat shock proteins in non-asthmatic recreational marathon and half-marathon runners
Christine Bekos1,2, Matthias Zimmermann1, Lukas Unger1, Stefan Janik1, Andreas Mitterbauer1, Michael Koller3, Robert Fritz3, Christian Gäbler3, Jessica Didcock1, Jonathan Kliman1, Walter Klepetko4, Hendrik Jan Ankersmit1,4 and Bernhard Moser1,4*
1 Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration
2 Medical University of Vienna, Comprehensive Cancer Center, Department of Obstetrics and Gynecology, Division of General Gynecology and Gynecologic Oncology
3 Sportordination, Alserstraße 28, Vienna, Austria
4 Department of Surgery, Division of Thoracic Surgery, Medical University Vienna, Austria
Abstract
Objective: Exercise is the most common trigger of bronchospasm. Heat shock protein (HSP) expression was linked to asthmatic patients. The prevalence and pathophysiology of exercise-induced bronchoconstriction (EIB) in non-professional non-asthmatic runners is unknown.
We sought to investigate the frequency of EIB and cytokine changes in non-professional non-asthmatic marathon and half marathoners with and without EIB.
Methods: Testing was performed before the marathon (baseline), immediately post-marathon at the finish area (peak), and 2-7 days after the marathon (recovery): immunosorbent assays for measurement of HSP70, blood count analysis, spirometry and temperature measurements.
Results: We experienced a decline in FEV1 of ≥10% in 35.29% of marathon and 22.22% of half marathon runners. Runners with EIB had significantly higher HSP70 serum concentrations at baseline than those without EIB (987.4±1486.7 vs. 655.6±1073.9; p=0.014). Marathoners with EIB had significantly increased WBC before participating in the competition (7.4±1.7 vs. 6.0±1.5; p=0.021). After recovery we found increased HSP70 serum concentrations in marathoners with EIB compared to those without (2539.2±1692.5 vs. 1237.2±835.2; p=0.032), WBC (7.6±1.8 vs. 6.4±1.6; p=0.048) and PLT (273.0±43.0 vs 237.2±48.3; p=0.040).
At all measured skin sites skin temperatures in runners were significantly lower immediately after participating in the competition when compared to temperature before the race (skin temperature baseline vs. peak: abdominal: 33.1±0.2 vs. 30.0±0.4; p<0.001; upper arm: 31.6±0.2 vs. 29.4±0.3; p<0.001; upper leg: 30.7±0.3 vs. 29.4±0.2; p=0.014; lower leg: 30.6±1.0 vs. 30.2±1.5; p=0.007).
Conclusions: We found a higher than expected number of non-professional athletes with EIB. HSP70 serum concentrations and elevated WBC could indicate a predisposition to EIB.
Fig 1b. Predicted FVC % in marathoners and half-marathoners at baseline, peak and recovery compared to sedentary controls. Predicted FVC % significantly decreased after participating in a M and remained stable in HM. There were no differences detectable between M and HM runners and sedentary controls. M, marathon; HM, half-marathon; baseline, 1-2 days before the run; peak, immediately after the run in the finishing area; recovery, after 2-7 days of recovery; FVC, forced vital capacity. ** p<0.01.
Fig 1c. FEV1/FVC ratio in marathoners and half-marathoners at baseline, peak and recovery compared to sedentary controls. FEV1/FVC ratio remained stable in M and HM runners. There were no differences detectable between M and HM runners and sedentary controls. M, marathon; HM, half-marathon; baseline, 1-2 days before the run; peak, immediately after the run in the finishing area; recovery, after 2-7 days of recovery; FVC, Forced vital capacity. ** p<0.01.
Fig 2b. Heat shock protein 27 in marathoners and half-marathoners at baseline, peak and recovery compared to sedentary controls. Heat shock protein 27 increased significantly after running a M or HM and returned to baseline concentrations again after 2 to 7 days of recovery. M, marathon; HM, half-marathon; baseline, 1-2 days before the run; peak, immediately after the run in the finishing area; recovery, after 2-7 days of recovery; ** p<0.01, *** p<0.001.
Fig. 3b. White blood cell counts in marathoners with and without exercise-induced bronchoconstriction before the competition. White blood cell counts in marathoners at baseline are significantly increased in runners showing signs of EIB during the competition compared to runners without EIB. EIB, exercise-induced bronchoconstriction; baseline, 1-2 days before the run; * p<0.05.
Fig. 3c. HSP70 in marathoners with and without exercise-induced bronchoconstriction after 2 to 7 days of recovery. After 2 to 7 days of recovery, HSP70 serum concentrations in marathoners are significantly increased in runners showing signs of EIB during the competition compared to runners without EIB. EIB, exercise-induced bronchoconstriction; recovery, after 2-7 days of recovery; * p<0.05.
Fig. 3d. White blood cell counts in marathoners with and without exercise-induced bronchoconstriction after 2 to 7 days of recovery. After 2 to 7 days of recovery, white blood cell counts in marathoners are significantly increased in runners showing signs of EIB during the competition compared to runners without EIB. EIB, exercise-induced bronchoconstriction; recovery, after 2-7 days of recovery; * p<0.05.
Fig. 3e. Platelets in marathoners with and without exercise-induced bronchoconstriction after 2 to 7 days of recovery. After 2 to 7 days of recovery, platelets in marathoners are significantly increased in runners showing signs of EIB during the competition compared to runners without EIB. EIB, exercise-induced bronchoconstriction; recovery, after 2-7 days of recovery; * p<0.05.
February 2019 Paper published in Blood Transfusion
Viral safety of APOSECTM: a novel peripheral blood mononuclear cell derived-biological for regenerative medicine
Gugerell A, Sorgenfrey D, Laggner M, Raimann J, Peterbauer A, Bormann D, Suessner S, Gabriel C, Moser B, Ostler T, Mildner M, Ankersmit HJ
Abstract
Background. Viral reduction and inactivation of cell-derived biologicals is paramount for patients' safety and so viral reduction needs to be demonstrated to regulatory bodies in order to obtain marketing authorisation. Allogeneic human blood-derived biological medicinal products require special attention. APOSECTM, the secretome harvested from selected human blood cells, is a new biological with promising regenerative capabilities. We evaluated the effectiveness of inactivation of model viruses by methylene blue/light treatment, lyophilisation, and gamma irradiation during the manufacturing process of APOSECTM.
Materials and methods. Samples of intermediates of APOSECTM were acquired during the manufacturing process and spiked with bovine viral diarrhoea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), pseudorabies virus (PRV), hepatitis A virus (HAV), and porcine parvovirus (PPV). Viral titres were assessed with suitable cell lines.
Results. Methylene blue-assisted viral reduction is mainly effective against enveloped viruses: the minimum log10 reduction factors for BVDV, HIV-1, and PRV were ≥6.42, ≥6.88, and ≥6.18, respectively, with no observed residual infectivity. Viral titres of both HAV and PPV were not significantly reduced, indicating minor inactivation of non-enveloped viruses. Lyophilisation had minor effects on the viability of several enveloped model viruses. Gamma irradiation contributes to the viral safety by reduction of enveloped viruses (BVDV: ≥2.42; HIV-1: 4.53; PRV: ≥4.61) and to some degree of non-enveloped viruses as seen for HAV with a minimum log10 reduction factor of 2.92. No significant reduction could be measured for the non-enveloped virus PPV (2.60).
Discussion. Three manufacturing steps of APOSECTM were evaluated under Good Laboratory Practice conditions for their efficacy at reducing and inactivating potentially present viruses. It could be demonstrated that all three steps contribute to the viral safety of APOSECTM.
January 2019 Paper published in Scientific Reports
Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions
Wagner T1, Traxler D2, Simader E1, Beer L3, Narzt MS4, Gruber F4, Madlener S5, Laggner M1, Erb M6, Vorstandlechner V1, Gugerell A1,2, Radtke C7, Gnecchi M8,9,10, Peterbauer A11, Gschwandtner M4, Tschachler E4, Keibl C12, Slezak P12, Ankersmit HJ13,14,15, Mildner M16
1 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria
2 Department of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
3 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria
4 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria
5 Molecular Neuro-Oncology Research Unit, Department of Pediatrics and Adolescent Medicine and Institute of Neurology, Medical University of Vienna, Vienna, Austria
6 Synlab, Birsfelden, Switzerland
7 Clinical Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria
8 Department of Molecular Medicine, Unit of Cardiology, University of Pavia, Pavia, Italy
9 Coronary Care Unit, Laboratory of Experimental Cardiology for Cell and Molecular Therapy, Fondazione IRCCS Policlinico San Matteo Foundation, Pavia, Italy
10 Department of Medicine, University of Cape Town, Cape Town, South Africa
11 Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria
12 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
13 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria
14 FFG Project 852748 "APOSEC", Medical University of Vienna, Vienna, Austria
15 Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, Austria
16 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria
Abstract
Secretomes from various cell sources exert strong regenerative activities on numerous organs, including the skin. Although secretomes consist of many diverse components, a growing body of evidence suggests that small extracellular vesicles (EVs) account for their regenerative capacity. We previously demonstrated that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs) exhibits wound healing capacity. Therefore, we sought to dissect the molecular composition of EVs present in the secretome and compared wound healing-related activities of these EVs to other subfractions of the secretome and the fully supplemented secretome (MNCaposec). Compared to EVs derived from non-irradiated PBMCs, γ-irradiation significantly increased the size and number and changed the composition of released EVs. Detailed characterization of the molecular components of EVs, i.e. miRNA, proteins, and lipids, derived from irradiated PBMCs revealed a strong association with regenerative processes. Reporter gene assays and aortic ring sprouting assays revealed diminished activity of the subfractions compared to MNCaposec. In addition, we showed that MNCaposec accelerated wound closure in a diabetic mouse model. Taken together, our results suggest that secretome-based wound healing represents a promising new therapeutic avenue, and strongly recommend using the complete secretome instead of purified subfractions, such as EVs, to exploit its full regenerative capacity.
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