Cell-extracellular matrix interactions in stem cell niches and vascular structures

Project leaders
Prof. Taina Pihlajaniemi. M.D., Ph.D.
Prof. Lauri Eklund Ph.D.

Oulu Center for Cell–Matrix Research
Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine
University of Oulu

Background and Significance

We have found that two conserved subgroups of collagens, the basement membrane (BM) collagens XV and XVIII forming the multiplexin subfamily, and the structurally homologous collagens XIII and XXIII, which belong to the subfamily of transmembrane collagens, have important regulatory roles by virtue of affecting the structure and properties of the ECM, and by binding growth factors and cellular receptors. Both collagen families also associate with malignant processes.

We have identified collagen XIII as a novel muscle-derived regulator of motor synapse differentiation, maturation and functional efficacy. Recently the first human disease was identified with mutations in COL13A1 – congenital myasthenic syndrome type 19 with impaired neuromuscular transmission. Despite the similarities and localization at the BM zones of collagens XV and XVIII, we have shown that these collagens have distinct biological functions. For example, whereas collagen XV functions in skeletal and cardiac muscle and microvessels, collagen XVIII has important roles in the eye and the brain. Mutations in COL18A1 lead to Knobloch syndrome, characterized by ocular abnormalities and a spectrum of other defects. Collagen XVIII has also gained much attention on account of its anti-angiogenic and anti-tumourigenic C-terminal endostatin domain.

The network of arteries, capillaries and veins forms the functional vasculature needed for normal development and tissue homeostasis. Structural and functional alterations in the vasculature contribute to many common diseases from cancer to the complications of diabetes, and mutations in genes regulating blood vessel morphogenesis cause vascular anomalies. Angiopoietin growth factors (Ang1–4) and tyrosine kinase receptors Tie1 and Tie2 form an endothelial signalling system. Our studies have shown unique, ligand-mediated translocation of Tie2 into endothelial cell (EC)–ECM contact sites. Disturbed Ang/Tie signalling in cultured cells and in vasculature causes changes in EC–ECM interactions and in the structure of the perivascular ECM, suggesting important roles for Ang/Tie signalling in regulating the vasculature via the EC–ECM axis.

Recent Progress

We have demonstrated, for the first time, an ECM-specific transcriptional program, which establishes leukemic stem cells and has a significant prognostic impact on patients. We have identified a specific ECM signature that predicts favourable outcome in acute myeloid leukemia (AML). We have also identified a pantetheinase enzyme, vanin-1, as a crucial factor determining AML patients’ outcome. In collaboration with Profs S. Cinti, P. Leoni and A. Poloni, Università Politecnica delle Marche, Italy, we have studied adipocyte plasticity and functional specification, and reported the supporting role of bone marrow adipocytes in hematopoietic microenvironment and sustaining hematopoietic stem cell survival.

With Prof. S. Goerdt, Heidelberg University, Germany, we found that GATA4-dependent transcriptional program determines the specification and functional competence of the hepatic sinusoidal endothelial cells, and its deletion in these cells leads to defects in embryonic development, including in hematopoiesis. In collaboration with Profs. Peppi Koivunen and Johanna Myllyharju, we have also contributed to understanding the role of hypoxia-inducible factor prolyl-4-hydroxylases in hematopoiesis and inflammatory signaling.

To gain a better understanding of the roles of distinct collagens in epithelial carcinogenesis, we have studied expression and roles of a transmembrane collagen XIII, and a basement membrane collagen and multiplexin XVIII in breast cancer. Ongoing work suggests that collagen XVIII is strongly upregulated in human breast cancer and associates with poor prognosis, and increased collagen XIII expression is associated with certain breast cancer lines. Experiments with gene-modified mice and cellular models are ongoing to clarify the mechanisms of action of these collagens in breast cancer. In addition, in collaboration with Prof. Daniel Greenspan, University of Wisconsin, we showed that α3 chains of the fibril-forming collagen V support proliferation of mammary tumour cells by enhancing signaling via glypican-1 receptor. With Prof. Kaisa Tasanen, University of Oulu, we showed that collagen XVII and integrin β4 regulate migration and invasion of cutaneous squamous cell carcinoma (cSCC) cells. With Prof. Veli-Matti Kähäri, University of Turku, we identified the C3 and C3b components of the complement system as novel tumour cell-associated biomarkers of recurrent and metastatic cSCC, and showed that they regulate proliferation and migration of cSCC cells.

In characterization of the physiological functions of collagen XIII in the nervous system we have found it to affect both pre- and postsynaptic integrity of the neuromuscular synapse. We demonstrated that Col13a1 knockout mice recapitulate major muscle findings of congenital myasthenic syndrome type 19 and can serve as a novel model for this disease. Pre-existing defects of the neuromuscular system in knockout mice were shown to compromise regeneration and functional recovery in a peripheral nerve injury model. Moreover, we demonstrated the presence of collagen XIII autoantibodies in myasthenia gravis patients with no or low levels of acetylcholine receptor autoantibodies; however, further studies are needed to evaluate whether collagen XIII autoantibodies are pathogenic. Our ongoing work with mouse models also suggest functional roles for collagen XIII in bone, skin and the cardiovascular system, and distinct roles for the homologous collagens XIII and XXIII in skin tumourigenesis and post-injury regeneration of the neuromuscular system.

We have also contributed to the work of the group of Dr. Lari Lehtiö, University of Oulu, on the development of selective inhibitors of human tankyrases, an of professor Jari Koistinaho, University of Eastern Finland, on the significance of multiplexin collagen XV in ischemic stroke.

Collaborative studies with Prof. Miikka Vikkula, de Duve Institute, Brussels, Belgium, revealed the genetic cause, and molecular and cellular abnormalities in the Blue Rubber Bleb Nevus syndrome (BRBN). BRBN is caused by somatic, allelic double mutations in TIE2 resulting in dozens-to-hundreds widely distributed cutaneous and internal venous malformations that increase in number with age. Genetic studies indicated the same mutation in all lesions from a given patient, suggesting a common origin of diseased cells from the primary lesion, a hypothesis that was supported by in vitro experiments. Mutant endothelial cell translocation and ability to form new colonies resembles metastatic dissemination in cancer, but has not before been demonstrated for a non-malignant human disorder. In collaboration with Prof. Janne Heikkilä, University of Oulu, we utilized hyperspectral-imaging technology in analysis of non-stained lung sections and we also contributed in identification a new molecular mechanism controlling excessive vascular leakage in collaboration with Prof. Kari Alitalo. During his post-doctoral visit in University of Queensland, Brisbane, Australia in Prof. Parton´s laboratory, Dr. Mika Kaakinen studied the cardiac phenotype of mice lacking cavin-1. This led to weak membrane resistance to mechanical stretch, attenuated stretch signalling, pressure development, and uncoupling of eNOS that increased bioavailable NO.

Future Goals

We will unravel the roles and mechanisms of action of collagen XVIII isoforms in stem cell microenvironments, and those of collagen XV in the cardiovascular system. We will pursue the roles of collagen XIII in the motor synapse development, the cardiovascular system, and in bone formation and properties.

We have initiated a bioinformatics study to characterize transcription factor-regulated ECM modules in human cancers. We will build an atlas of ECM-based regulation of cancer formation and validate selected candidate modules/molecules in patient materials. We will assess the significance of multiplexins and transmembrane collagens in neoplasms by using experimental cancer models, and study their mechanisms of action in cultured tumour cells. The medical significance of cell–ECM homeostasis will be addressed in terms of target identification and validation to develop new diagnostic, prognostic and therapeutic measures.

In a new consortium we will investigate hydrodynamic brain clearance mechanisms including the recently discovered glymphatic system and how this could be manipulated as a novel treatment for neurodegenerative disorders.

We will develop advanced imaging methodologies to acquire structural and functional information of ECM and vasculature in living mice and investigate developmental vessel maturation and pathophysiological mechanisms and perivascular ECM remodelling in vascular diseases.

Publications 2017-

Bayramoglu N, Kaakinen M, Eklund L, Heikkilä J. Virtual H&E Staining of Hyperspectral Lung Histology Images Using Conditional Generative Adversarial Networks. Bioimage Computing ICCV, accepted 2017.

Dhungana H, Huuskonen MT, Pihlajaniemi T, Heljasvaara R, Vivien D, Kanninen KM, Malm T, Koistinaho J, Lemarchant S. Lack of collagen XV is protective after ischemic stroke in mice. Cell Death Dis J 8: e2541, 2017.
Eklund L, Kangas J, Saharinen P. Angiopoietin-Tie signalling in the cardiovascular and lymphatic systems. Clin Sci 131: 87-103, 2017.

Géraud C, Koch PS, Zierow J, Klapproth K, Busch K, Olsavszky V, Leibing T, Demory A, Ulbrich F, Diett M, Singh S, Sticht C, Breitkopf-Heinlein K, Richter K, Karppinen SM, Pihlajaniemi T, Arnold B, Rodewald H-R, Augustin H, Schledzewski K, Goerdt S. GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis. J Clin Invest 127: 1099-1114, 2017.

Heinolainen K, Karaman S, D'Amico G, Tammela T, Sormunen R, Eklund L, Alitalo K, Zarkada G. VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling. Circ Res 120: 1414-1425, 2017.

Heljasvaara R, Aikio M, Ruotsalainen H, Pihlajaniemi T. Collagen XVIII in tissue homeostasis and dysregulation – lessons learned from model organisms and human patients. Matrix Biol 57-58: 55-75, 2017.

Huang G, Ge G, Izzi V, Greenspan DS. α3 chains of type V collagen regulate breast tumour growth via glypican-1. Nat Commun 8:14351, 2017.

Härönen H, Zainul Z, Tu H, Naumenko N, Sormunen R, Miinalainen I, Shakirzyanova A, Oikarainen T, Abdullin A, Martin P, Santoleri S, Koistinaho J, Silman I, Giniatullin R, Fox MA, Heikkinen A, Pihlajaniemi T. Collagen XIII secures pre- and postsynaptic integrity of the neuromuscular synapse. Hum Mol Genet 26: 2076-2090, 2017.

Izzi V, Lakkala J, Devarajan R, Ruotsalainen H, Savolainen E-R, Koistinen P, Heljasvaara R, Pihlajaniemi T. An extracellular matrix signature in leukemia precursor cells and acute myeloid leukemia. Haematologica 102: e245-e248, 2017.

Izzi V, Lakkala J, Devarajan R, Savolainen E-R, Koistinen P, Heljasvaara R, Pihlajaniemi T. Vanin 1 (VNN1) levels predict poor outcome in acute myeloid leukemia. Am J Hematol 93: E4-E7, 2018.

Izzi V, Lakkala J, Devarajan R, Savolainen E-R, Koistinen P, Heljasvaara R, Pihlajaniemi T. Expression of a specific extracellular matrix signature is a favourable prognostic factor in acute myeloid leukemia. Leuk Res Rep 9: 9-13, 2017.

Izzi V, Heljasvaara R, Pihlajaniemi T. Editorial: Understanding the extracellular matrix in acute myeloid leukemia. Haematologica 102:1807-1809, 2017.

Kaakinen M, Reichelt ME, Ma Z, Ferguson C, Martel N, Porrello ER, Hudson JE, Thomas WG, Parton RG, Headrick JP. Cavin-1 deficiency modifies myocardial and coronary function, stretch responses and ischaemic tolerance: roles of NOS over-activity. Basic Res Cardiol 112: 24, 2017.

Mattiucci D, Maurizi G, Izzi V, Cenci L, Ciarlantini M, Mancini S, Mensà E, Pascarella R, Vivarelli M, Olivieri A, Leoni P, Poloni A. Bone marrow adipocytes support hematopoietic stem cell survival. J Cell Physiol 233: 1500-1511, 2018.

Maurizi G, Poloni A, Mattiucci D, Santi S, Maurizi A, Izzi V, Giuliani A, Mancini S, Zingaretti MC, Perugini J, Severi I, Falconi M, Vivarelli M, Rippo MR, Corvera S, Giordano A, Leoni P, Cinti S. Human white adipocytes convert into "rainbow" adipocytes in vitro. J Cell Physiol 232:2887-2889, 2017.

Maurizi G, Petäistö T, Maurizi A, Della Guardia L. Key genes regulating the liposecretion process of mature adipocytes. J Cell Physiol, Sep 19, 2017. [Epub ahead of print].

Moilanen JM, Löffek S, Kokkonen N, Salo S, Väyrynen JP, Hurskainen T, Manninen A, Riihilä P, Heljasvaara R, Franzke CW, Kähäri VM, Salo T, Mäkinen MJ, Tasanen K. Significant role of collagen XVII and integrin β4 in migration and invasion of the less aggressive squamous cell carcinoma cells. Sci Rep 7: 45057, 2017.  

Myllymäki MN, Määttä J, Dimova EY, Izzi V, Väisänen T, Myllyharju J, Koivunen P, Serpi R. Notch downregulation and extramedullary erythrocytosis in hypoxia-inducible factor prolyl 4-hydroxylase 2-deficient mice. Mol Cell Biol 37, 2017.

Nathubhai A, Haikarainen T, Koivunen J, Murthy S, Koumanov F, Lloyd MD, Holman GD, Pihlajaniemi T, Tosh D, Lehtiö L, Threadgill MD. Highly potent and isoform selective dual site binding tankyrase/Wnt signaling inhibitors that increase cellular glucose uptake and have antiproliferative activity. J Med Chem 60: 814-820, 2017.

Nkizinkiko Y, Desantis J, Koivunen J, Haikarainen T, Murthy S, Sancineto L, Massari S, Ianni F, Obaji E, Loza M, Pihlajaniemi T, Brea J, Tabarrini O, Lehtiö L. 2-Phenylquinazolinones as dual-activity tankyrase-kinase inhibitors. Sci Rep, in press.

Riihilä P, Nissinen L, Farshchian M, Kallajoki M, Kivisaari A, Meri S, Grénman R, Peltonen S, Peltonen J, Pihlajaniemi T, Heljasvaara R, Kähäri V-M. Complement component C3 and complement factor B promote growth of cutaneous squamous cell carcinoma. Am J Pathol 187: 1186-1197, 2017.

Saharinen P, Eklund L, Alitalo K. Therapeutic targeting of the angiopoietin-Tie pathway. Nat Rev Drug Discov 16: 635-661, 2017.

Soblet J, Kangas J, Nätynki M, Mendola A, Helaers R, Uebelhoer M, et al. (Kaakinen M, Eklund L). Blue rubber bleb nevus (BRBN) syndrome is caused by somatic TEK (TIE2) mutations. J Invest Dermatol 137:207-216, 2017.

Tu H, Pirskanen-Matell R, Heikkinen A, Oikarainen T, Risteli J, Pihlajaniemi T. Autoimmune antibodies to collagen XIII in myasthenia gravis patients. Muscle and Nerve, Sept 8, 2017 [Epub ahead of print].

Ullah K, Rosendahl A-H, Izzi V, Bergmann U, Pihlajaniemi T, Mäki JM, Myllyharju, J. Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways. Sci Rep 7:17220, 2017.

Åström P, Heljasvaara R, Nyberg P, Al-Samadi A, Salo T. Human tumor tissue-based 3D in vitro invasion assats. Methods Mol Biol 1731:213-221, 2018.

Research Group Members

Project Leaders:
Taina Pihlajaniemi, M.D., Ph.D., Professor, Vice Rector (Vice President), Research (University of Oulu)
Lauri Eklund, Ph.D., Professor (University of Oulu and Biocenter Oulu)

Senior and Post-doctoral Investigators:
Harri Elamaa, Ph.D. (Academy of Finland)
Anne Heikkinen, Ph.D. (Academy of Finland and Biocenter Oulu)
Ritva Heljasvaara, Ph.D. (University of Oulu, Academy of Finland and University of Bergen, Norway)
Valerio Izzi, Ph.D. (University of Oulu and Academy of Finland)
Mika Kaakinen, Ph.D. (Biocenter Oulu, Academy of Finland and University of Oulu)
Sanna-Maria Karppinen, Ph.D. (Academy of Finland), leave of absence until May 2017
Jarkko Koivunen, Ph.D. (Academy of Finland)
Marjut Nätynki, Ph.D. (University of Oulu and Academy of Finland)
Heli Ruotsalainen, Ph.D. (Academy of Finland and University of Oulu)
Sabrina Santoleri, Ph.D. (Academy of Finland)

Ph.D. Students:
Miki Aho, Med.Cand., leave of absence
Raman Devarajan, M.Sc. (Academy of Finland)
Charlotta Henriksson, Med. Cand. (Sigrid Jusélius Foundation and Biocenter Oulu)
Heli Härönen, M.D. (Academy of Finland and Sigrid Jusélius Foundation)
Riku Kallunki, Med. Cand. (Jalmari and Rauha Ahokas Foundation)
Inderjeet Kaur, Ph.D. (chemistry) (Biocenter Oulu), until September 2017  
Antti Kemppainen, Med.Cand. (Academy of Finland and Biocenter Oulu)
Minna Kihlström, M.Sc. (Adacemy of Finland)
Juho Lakkala, Med. Cand. (Research Foundation of Blood Diseases and Sigrid Jusélius Foundation)
Oula Norman, Med. Student (Sigrid Jusélius Foundation)
Noora Petrelius, Med. Student (Sigrid Jusélius Foundation)
Tiina Petäistö, M.Sc. (University of Oulu), leave of absence from June 2017
Mia Rinta-Jaskari, Med. Cand. (Finnish Cultural Foundation)
Saad Ullah Akram, M.Sc. (Biocenter Oulu)
Jeena Ainikkal Velayudhan, M.Sc. (I4Future H2020 MSCA COFUND)
Zarin Zainul, M.Sc. (Biocenter Oulu)

Laboratory Technicians, 6 (University of Oulu, Academy of Finland, Finnish Cancer Foundation)

Foreign Scientists, 7

National and International Activities

Centre of Excellence in Cell-Extracellular Matrix Research, Academy of Finland Program for 2012-2017

Taina Pihlajaniemi, Director, Johanna Myllyharju, Vice director, other Group leaders Lauri Eklund, Aki Manninen, Seppo Vainio and Robert Winqvist

Group Members Who Spent More Than Two Weeks in Foreign Laboratories During 2017

Ph.D. Ritva Heljasvaara, University of Bergen, Norway
Ph.D. M.D. Jaakko Kangas, Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Japan  

EU Projects (present and progress)

I4Future H2020 MSCA COFUND, PhD co-supervisor

Co-operation With Finnish and Foreign Companies

Paras Biopharmaceuticals Finland Oy, Oulu, Finland, assistance in developing diagnostics and therapeutic products
UPM Biochemicals, Helsinki, Finland, testing of 3D cellulose nanofibril hydrogel
Senop Oy, development of microscopic imaging technologies

Last updated: 15.10.2018