TY - JOUR A1 - Tvorogov, Denis A1 - Anisimov, Andrey A1 - Zheng, Wei A1 - Leppánen, Veli-Matti A1 - Tammela, Tuomas A1 - Laurinavicius, Simonas A1 - Holnthoner, Wolfgang A1 - Helotera, Hanna A1 - Holopainen, Tanja A1 - Jeltsch, Michael A1 - Kalkkinen, Nisse A1 - Lankinen, Hikka A1 - Ojala, Päivi M. A1 - Alitalo, Kari T1 - Effective Suppression of Vascular Network Formation by Combination of Antibodies Blocking VEGFR Ligand Binding and Receptor Dimerization JF - Cancer Cell KW - Vascular Network Y1 - 2019 IS - 18(6) SP - 630 EP - 640 ER - TY - GEN A1 - Mühleder, Severin A1 - Fuchs, Christiane A1 - Bassilio, Jose A1 - Sczwarc, Dorota A1 - Pill, Karoline A1 - Slezak, Paul A1 - Labuda, Krystina A1 - Siehs, Christian A1 - Pröll, Johannes A1 - Priglinger, Eleni A1 - Redl, Heinz A1 - Holnthoner, Wolfgang T1 - The purinergic receptor P2Y2 modulates endothelial sprouting and angiogenesis KW - Angiogenesis Y1 - 2018 ER - TY - JOUR A1 - Teuschl, Andreas A1 - Holnthoner, Wolfgang A1 - Monforte, Xavier T1 - Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination JF - Acta Biomater. N2 - Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of "AuriScaff", an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. STATEMENT OF SIGNIFICANCE: After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches. KW - Auricular cartilage KW - Cartilage Regeneration KW - Human adipose derived stromal/stem cells KW - Tissue Engineering KW - Decellularization Y1 - 2020 VL - 2019 IS - Mar/86 SP - 207 EP - 222 ER - TY - JOUR A1 - Priglinger, Eleni A1 - Schuh, Christina A1 - Steffenhagen, Carolin A1 - Wurzer, Christoph A1 - Maier, Julia A1 - Nürnberger, Sylvia A1 - Holnthoner, Wolfgang A1 - Fuchs, Christiane A1 - Suessner, Susanne A1 - Rünzler, Dominik A1 - Redl, Heinz A1 - Wolbank, Susanne T1 - Improvement of adipose tissue-derived cells by low-energy extracorporeal shock wave therapy. JF - Cytotherapy N2 - BACKGROUND: Cell-based therapies with autologous adipose tissue-derived cells have shown great potential in several clinical studies in the last decades. The majority of these studies have been using the stromal vascular fraction (SVF), a heterogeneous mixture of fibroblasts, lymphocytes, monocytes/macrophages, endothelial cells, endothelial progenitor cells, pericytes and adipose-derived stromal/stem cells (ASC) among others. Although possible clinical applications of autologous adipose tissue-derived cells are manifold, they are limited by insufficient uniformity in cell identity and regenerative potency. METHODS: In our experimental set-up, low-energy extracorporeal shock wave therapy (ESWT) was performed on freshly obtained human adipose tissue and isolated adipose tissue SVF cells aiming to equalize and enhance stem cell properties and functionality. RESULTS: After ESWT on adipose tissue we could achieve higher cellular adenosine triphosphate (ATP) levels compared with ESWT on the isolated SVF as well as the control. ESWT on adipose tissue resulted in a significantly higher expression of single mesenchymal and vascular marker compared with untreated control. Analysis of SVF protein secretome revealed a significant enhancement in insulin-like growth factor (IGF)-1 and placental growth factor (PLGF) after ESWT on adipose tissue. DISCUSSION: Summarizing we could show that ESWT on adipose tissue enhanced the cellular ATP content and modified the expression of single mesenchymal and vascular marker, and thus potentially provides a more regenerative cell population. Because the effectiveness of autologous cell therapy is dependent on the therapeutic potency of the patient's cells, this technology might raise the number of patients eligible for autologous cell transplantation. KW - Shockwave Therapy KW - Tissue Regeneration KW - Regenerative Medicine Y1 - SP - 1079 EP - 1095 ER - TY - JOUR A1 - Bachmann, Barbara A1 - Spitz, Sarah A1 - Rothbauer, Mario A1 - Jordan, Christian A1 - Purtscher, Michaela A1 - Zirath, Helene A1 - Schuller, Patrick A1 - Eilenberger, Christoph A1 - Ali, Syed Faheem A1 - Mühleder, Severin A1 - Priglinger, Eleni A1 - Harasek, Michael A1 - Redl, Heinz A1 - Holnthoner, Wolfgang A1 - Ertl, Peter T1 - Engineering of three-dimensional pre-vascular networks within fibrin hydrogel constructs by microfluidic control over reciprocal cell signaling JF - Biomicrofluidics KW - Microfluidic KW - Vascularization KW - Tissue Engineering Y1 - 2019 ER - TY - GEN A1 - Schneider, Karl Heinrich A1 - Aigner, Petra A1 - Monforte Vila, Xavier A1 - Holnthoner, Wolfgang A1 - Teuschl, Andreas A1 - Bergmeister, Helga A1 - Redl, Heinz T1 - Naturally derived acellular small diameter vascular grafts from human placenta for reconstructive surgery KW - Placenta KW - Grafting KW - Surgery Y1 - 2018 ER - TY - JOUR A1 - Schneider, Jaana A1 - Pultar, Marianne A1 - Oesterreicher, Johannes A1 - Bobbili, Madhusudhan Reddy A1 - Mühleder, Severin A1 - Priglinger, Eleni A1 - Redl, Heinz A1 - Spittler, Andreas A1 - Grillari, Johannes A1 - Holnthoner, Wolfgang T1 - Cre mRNA Is Not Transferred by EVs from Endothelial and Adipose-Derived Stromal/Stem Cells during Vascular Network Formation JF - Int J Mol Sci. N2 - Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs. KW - Tissue Engineering KW - Stem Cells KW - Vascular Network Formation KW - EVs KW - Endothelial Cells Y1 - VL - 2021 IS - 22(8) SP - 4050 ER - TY - JOUR A1 - Nürnberger, Sylvia A1 - Schneider, Cornelia A1 - van Osch, Gerjo A1 - Keibl, Claudia A1 - Rieder, Bernhard A1 - Monforte, Xavier A1 - Teuschl, Andreas A1 - Mühleder, Severin A1 - Holnthoner, Wolfgang A1 - Schädl, Barbara A1 - Gahleitner, Christoph A1 - Redl, Heinz A1 - Wolbank, Susanne T1 - Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination. JF - Acta Biomaterialia KW - Tissue Engineering KW - Decellularization KW - Cartilage Y1 - ER - TY - JOUR A1 - Verstraeten, Valerie A1 - Holnthoner, Wolfgang A1 - Van Steensel, Maurice A1 - Veraart, Joep C J M A1 - Bladergroen, Reno S A1 - Heckmann, Caronline A A1 - Keskitalo, Salla A1 - Frank, Jorge A1 - Alitalo, Kari A1 - van Geel, Michel A1 - Steijlen, Peter M T1 - Functional analysis of FLT4 mutations associated with Nonne-Milroy lymphedema JF - Journal of Investigative Dermatology KW - Nonne-Milroy iymphedema KW - Mutation Y1 - 2019 IS - 129(2) SP - 509 EP - 512 ER - TY - JOUR A1 - Holnthoner, Wolfgang T1 - Adipose-tissue-derived therapeutic cells in their natural environment as an autologous cell therapy strategy: the microtissue-stromal vascular fraction JF - Eur Cell Mater. N2 - The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31+, smooth muscle actin+). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix. KW - Adipose-tissue KW - autologous cell therapy KW - microtissue KW - stromal vascular fraction KW - vascularization Y1 - 2020 VL - 2019 IS - Feb, 37 SP - 113 EP - 133 ER - TY - JOUR A1 - Schneider, Karl Heinrich A1 - Aigner, Petra A1 - Holnthoner, Wolfgang A1 - Monforte Vila, Xavier A1 - Nürnberger, Sylvia A1 - Rünzler, Dominik A1 - Redl, Heinz A1 - Teuschl, Andreas T1 - Decellularized human placenta chorion matrix as a favorable source of small-diameter vascular grafts JF - Acta Biomaterialia KW - Grafting KW - Tissue Engineering Y1 - 2018 ER - TY - JOUR A1 - Strohmeier, Karin A1 - Hofmann, Martina A1 - Jacak, Jaroslaw A1 - Narzt, Marie-Sophie A1 - Wahlmueller, Marlene A1 - Mairhofer, Mario A1 - Schädl, Barbara A1 - Holnthoner, Wolfgang A1 - Barsch, Martin A1 - Sandhofer, Matthias A1 - Wolbank, Susanne A1 - Priglinger, Eleni T1 - Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema JF - Biomedicines N2 - Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease. KW - Tissue Engineering KW - Adipose Tissue KW - Lipedema KW - Endothelial Cells Y1 - VL - 2022 IS - 10(5) SP - 1163 ER - TY - JOUR A1 - Rohringer, Sabrina A1 - Holnthoner, Wolfgang A1 - Hackl, Matthias A1 - Weihs, Anna A1 - Rünzler, Dominik A1 - Skalicky, Susanna A1 - Karbiener, Michael A1 - Scheideler, Marcel A1 - Pröll, Johannes A1 - Gabriel, Christian A1 - Schweighofer, Bernhard A1 - Gröger, Marion A1 - Spittler, Andreas A1 - Grillari, Johannes A1 - Redl, Heinz T1 - Molecular and cellular effects of in vitro shockwave treatment on lymphatic endothelial cells. JF - PLoS one KW - Shockwave Y1 - ER - TY - JOUR A1 - Holnthoner, Wolfgang A1 - Szwarc, Dorota T1 - Purinergic P2Y 2 receptors modulate endothelial sprouting JF - Cell Mol Life Sci. N2 - Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth. KW - Angiogenesis KW - Endothelial KW - Purinergic KW - P2Y2 KW - Cell Sprouting Y1 - 2020 VL - 2020 IS - Mar. SP - 885 EP - 901 ER - TY - JOUR A1 - Trisko, Johanna A1 - Fleck, Johanna A1 - Kau, Silvio A1 - Oesterreicher, Johannes A1 - Holnthoner, Wolfgang T1 - Lymphatic and Blood Endothelial Extracellular Vesicles: A Story Yet to Be Written JF - Life N2 - Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development. KW - Tissue Engineering KW - Endothelial Cells KW - vascularization KW - extracellular vesicles KW - lymphatic endothelial cells Y1 - VL - 2022 IS - 12(5) SP - 654 ER - TY - JOUR A1 - Ghalamkarpour, A. A1 - Holnthoner, Wolfgang A1 - Saharinen, Pipsa A1 - Boon, Laurence M A1 - Mulliken, J B A1 - Alitalo, Kari T1 - Recessive primary congenital lymphoedema caused by a VEGFR3 mutation JF - Journal of Medical Genetics KW - Lymphoedema Y1 - 2019 IS - 46(6) SP - 399 EP - 404 ER - TY - JOUR A1 - Sayer, Simon A1 - Zandrini, Tommaso A1 - Markovic, Marica A1 - Van Hoorick, Jasper A1 - Van Vlierberghe, Sandra A1 - Baudis, Stefan A1 - Holnthoner, Wolfgang A1 - Ovsianikov, Aleksandr T1 - Guiding cell migration in 3D with high-resolution photografting JF - Scientific Reports N2 - Multi-photon lithography (MPL) has proven to be a suitable tool to precisely control the microenvironment of cells in terms of the biochemical and biophysical properties of the hydrogel matrix. In this work, we present a novel method, based on multi-photon photografting of 4,4′-diazido-2,2′-stilbenedisulfonic acid (DSSA), and its capabilities to induce cell alignment, directional cell migration and endothelial sprouting in a gelatin-based hydrogel matrix. DSSA-photografting allows for the fabrication of complex patterns at a high-resolution and is a biocompatible, universally applicable and straightforward process that is comparably fast. We have demonstrated the preferential orientation of human adipose-derived stem cells (hASCs) in response to a photografted pattern. Co-culture spheroids of hASCs and human umbilical vein endothelial cells (HUVECs) have been utilized to study the directional migration of hASCs into the modified regions. Subsequently, we have highlighted the dependence of endothelial sprouting on the presence of hASCs and demonstrated the potential of photografting to control the direction of the sprouts. MPL-induced DSSA-photografting has been established as a promising method to selectively alter the microenvironment of cells. KW - Tissue Engineering KW - Cell migration KW - photografting Y1 - VL - 2022 IS - 12(1), 10196 SP - 8626 ER - TY - JOUR A1 - Hromada, Carina A1 - Hartmann, Jaana A1 - Oesterreicher, Johannes A1 - Stoiber, Anton A1 - Daerr, Anna A1 - Schädl, Barbara A1 - Priglinger, Eleni A1 - Teuschl-Woller, Andreas H. A1 - Holnthoner, Wolfgang A1 - Heinzel, Johannes Christoph A1 - Hercher, David T1 - Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro JF - Biomolecules N2 - Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction. KW - Tissue Engineering KW - peripheral nerve regeneration KW - lymphangiogenesis KW - Schwann cells KW - lymphatic endothelial cells Y1 - VL - 2022 IS - 12, 6 SP - 820 ER - TY - JOUR A1 - Rothbauer, Mario A1 - Byrne, Ruth A. A1 - Schobesberger, Silvia A1 - Olmos Calvo, Isabel A1 - Fischer, Anita A1 - Reihs, Eva I. A1 - Spitz, Sarah A1 - Bachmann, Barbara A1 - Sevelda, Florian A1 - Holinka, Johannes A1 - Holnthoner, Wolfgang A1 - Redl, Heinz A1 - Toegel, Stefan A1 - Windhager, Reinhard A1 - Kiener, Hans P. A1 - Ertl, Peter T1 - Establishment of a human three-dimensional chip-based chondro-synovial coculture joint model for reciprocal cross talk studies in arthritis research JF - Lab on a Chip N2 - Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases. KW - Tissue Engineering KW - coculture joint model KW - arthritis KW - human three-dimensional chip Y1 - VL - 2021 IS - 21 SP - 4128 EP - 4143 ER -