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

Project leaders
Prof. Taina Pihlajaniemi. M.D., Ph.D.
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. Alterations in the vasculature contribute to 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 ligand-mediated translocation of Tie2 into 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

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. 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.

To gain a better understanding of the roles of the multiplexins in tumourigenesis, we studied their expression and localisation at different stages of human cutaneous squamous cell carcinoma (cSCC). Immunohistochemical analyses of a large human skin tissue microarray in collaboration with Prof. Veli-Matti Kähäri, University of Turku, showed that collagen XVIII expression significantly increases in advanced cSCC and associates strongly with poor prognosis, while keratinocytes in normal skin showed negative staining for it. Collagen XV appeared instead as deposits in the tumour stroma. Altogether, our results demonstrated for the first time that the multiplexins may contribute in distinct manners to epithelial tumourigenesis.

In collaboration with Prof. Kai Kaarniranta, University of Eastern Finland, we showed that the collagen XVIII knock-out mouse represents a novel model with which to study the role of proteostasis in the degeneration of retinal pigment epithelium (RPE), and the autophagy-related proteins as therapeutic targets in the search for novel ways to combat age-related damage to RPE cells. Moreover, with Prof. Michael Goligorsky, New York Medical College, USA, we showed that kidney fibrosis in aging appears to be a result of cumulative stresses occurring on the background of synergistically acting geronic (aging) proteins, which include collagen XVIII-derived endostatin and the enzyme transglutaminase 2.

Accumulation of collagen is the hallmark of fibrosis, which is a direct or indirect cause of many chronic diseases. Early detection of fibrosis is important in e.g. in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We contributed to the work of the groups of Prof. Elina Ikonen and Prof. Hannele Yki-Järvinen, University of Helsinki, to test whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. It was demonstrated that SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Collaborative studies with Prof. Miikka Vikkula’s group, de Duve Institute, Brussels, Belgium, have revealed that majority of venous malformations (VMs) are caused by somatic gain-of-function mutations in TIE2/PIK3CA signalling pathway causing structurally aberrant ECM and strong induction of pericellular proteolytic pathways implicated in ECM remodelling. Interestingly, we have also found that specific TIE2 mutations can cause a spectrum of venous anomalies that vary in terms of heredity and the ensuing complications. These include blue rubber bleb nevus syndrome characterized by multiple VM lesions. Genetic studies indicated the same somatic TIE2 double mutation in all lesions from a given patient, suggesting a common cellular origin from the primary lesion, a hypothesis that was supported by in vitro experiments.

Vascular endothelial growth factor D (VEGF-D) promotes the lymph node metastasis of cancer by inducing the growth of lymphatic vasculature, but its specific roles in tumorigenesis have not been elucidated. In collaboration with Prof. Kari Alitalo, University of Helsinki, we monitored the effects of VEGF-D in mouse cSCC. Our data suggest that VEGF-D may be beneficial in early-stage tumors since it suppresses the pro-tumorigenic inflammation, while at later stages VEGF-D-induced tumor lymphatics provide a route for metastasis.

The adipose tissue regulates fat metabolism and thermogenesis and can rapidly be remodelled to meet physiological requirements. It is also highly vascularized and ECs have roles to control adipose tissue. Collaborative studies with the Alitalo group using novel transgenic mice expressing VEGF-B specifically in the adipose tissue showed increased angiogenesis and improved glucose metabolism in high-fat diet suggesting novel therapeutic use for VEGF-B in metabolic complications.

We have also contributed to the work of the group of Dr. Lari Lehtiö, University of Oulu, on the characterization of properties and the development of selective inhibitors of human diphtheria toxin-like ADP-ribosyltransferase (ARTD or PARP) family members.

Future Goals

We will unravel the roles and mechanisms of action of collagen XVIII in bone marrow, breast, skin, kidney and adipose tissue stem cell microenvironments, and define the functions of collagen XV in mesenchymal stem cells and in bone. We will pursue the roles of collagen XIII in the motor synapse, the cardiovascular and skeletal systems, and the lungs. We will also assess the significance of the multiplexins and transmembrane collagens in tumourigenesis. The medical significance will be addressed in terms of target identification and validation in order to develop new diagnostic, prognostic and therapeutic measures.

We aim to investigate EC-ECM interplay involving the synthesis and modifications of ECM constituents and EC interactions with perivascular mural and other stromal cells in developmental angiogenesis and to identify pathways causing pathological ECM remodelling and its importance for pathogenesis in vascular anomalies.

For detail characterization of ECM and vasculature we are setting up advanced imaging techniques to dissect cellular and molecular events in physiology and in pathologies using fixed samples and in living mice.

Our studies are expected to provide novel insights into vascular morphogenesis and pathophysiological mechanisms in vascular disease helping to develop evidence-based therapies in the patients in the future.

Publications 2016-

Biocenter Publications:

Akram SA, Kannala J, Eklund L, Heikkilä J. Joint cell segmentation and tracking using cell proposals. Proc IEEE International Symposium on Biomedical Imaging (ISBI) 920-924, 2016.

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, epub ahead of print, 2016.

Honkanen H-K, Izzi V, Petäistö T, Holopainen T, Harjunen V, Pihlajaniemi T, Alitalo K, Heljasvaara R. Elevated VEGF-D modulates tumor inflammation and reduces the growth of carcinogen-induced skin tumors. Neoplasia 18(7): 436-446, 2016.

Karppinen SM, Honkanen H-K, Heljasvaara R, Riihilä P, Autio-Harmainen H, Sormunen R, Harjunen V, Väisänen M-R, Väisänen T, Hurskainen T, Tasanen-Määttä K, Kähäri V-M, Pihlajaniemi T. Collagens XV and XVIII show different expression and localization in cutaneous squamous cell carcinoma: type XV appears in tumour stroma while XVIII becomes upregulated in tumour cells and lost from microvessels. Exp Dermatol 25: 348-354, 2016.

Kivinen N, Felszeghy S, Kinnunen AI, Setälä N, Aikio M, Kinnunen K, Sironen R, Pihlajaniemi T, Kauppinen A, Kaarniranta K. Absence of collagen XVIII in mice causes age-related insufficiency in retinal pigment epithelium proteostasis. Biogerontology 17:749-761, 2016.

Lin CH, Chen J, Zhang Z, Johnson GV, Cooper AJ, Feola J, Bank A, Shein J, Ruotsalainen HJ, Pihlajaniemi TA, Goligorsky MS. Endostatin and transglutaminase 2 are involved in fibrosis of aging kidney. Kidney Int 89: 1281–1292, 2016.

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, epub ahead of print, 2016.

Pirhonen J, Arola J, Sädevirta S, Luukkonen P, Karppinen SM, Pihlajaniemi T, Isomäki A, Hukkanen M, Yki-Järvinen H, and Ikonen E. Continuous grading of early fibrosis in NAFLD using label-free imaging: a proof-of-concept study. PLoS One 11: e0147804, 2016.

Robciuc MR, Kivelä R, Williams IM, de Boer JF, van Dijk TH, Elamaa H, Tigistu-Sahle F, Molotkov D, Leppänen VM, Käkelä R, Eklund L, Wasserman DH, Groen AK, Alitalo K. VEGF-B/VEGFR1-Induced expansion of adipose microvasculature counteracts obesity and related metabolic complications. Cell Metab 23:712-724, 2016.

Venkannagari H, Verheugd P, Koivunen J, Haikarainen T, Obaji E, Yashwanth A, Narwal M, Pihlajaniemi T, Lüscher B, and Lehtiö L. Chemical probe rescues cells from ARTD10/PARP10 induced apoptosis and sensitizes cancer cells to DNA damage. Cell Chem Biol 23: 1251-1260, 2016.

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 signaling in the cardiovascular and lymphatic systems. Clin Sci 131: 87-103, 2017.

Huang G, Ge1 G, Izzi V, and Greenspan DS. a3(V) Collagen regulates breast tumor growth via glypican-1-mediated effects. Nat Commun 8:14351, 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.

Soblet J, Kangas J, Nätynki M, Mendola A, Helaers R, Uebelhoer M, Kaakinen M, Cordisco M, Dompmartin A, Enjolras O, Holden S, Irvine AD, Kangesu L, Léauté-Labrèze C, Lanoel A,  Lokmic Z, Maas S, McAleer MA, Penington A, Rieu P, Syed S, van der Vleuten C, Watson R, Fishman SJ, Mulliken JB, Eklund L, Limaye N,  Boon LM, and Vikkula M. Blue Rubber Bleb Nevus (BRBN) Syndrome is caused by Somatic TEK (TIE2) Mutations. J Invest Dermatol 137:207-216, 2017.

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 factor B and C3 regulate growth of cutaneous squamous cell carcinoma. Am J Pathol, in press.

Saharinen P, Eklund L, and Alitalo K. Therapeutic targeting of the vascular angiopoietin-Tie system. Nature Reviews Drug Discovery, in press.

Other Publications:

Gullberg D, Kletsas D, Pihlajaniemi T. Editorial: Wound healing and fibrosis-two sides of the same coin. Cell Tissue Res 365: 449-451, 2016.

Doctoral Theses 2016

Marjut Nätynki, Venous malformation causative mutations affect TIE2 receptor trafficking, downstream signaling and vascular endothelial cell functions. ISBN:978-952-621-139-8

Jaakko Kangas, Molecular dissection of TIE2 and PIK3CA mutations in venous malformations and blue rubber bleb nevus syndrome. ISBN: 978-952-93-6885-3

Guillermo A. Martinez Nieto, Cancer-associated fibroblasts in skin squamous cell carcinoma and breast cancer. Role of fibroblast-derived integrin alpha11 and collagen XV in carcinogenesis. ISBN: 978-952-92-8042-4

Research Group Members

Project Leaders:
Taina Pihlajaniemi, M.D., Ph.D., Professor, Vice President (Vice Rector) Provost for Science and Research (University of Oulu)
Lauri Eklund, Ph.D. Adj.Prof. (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)
Mika Kaakinen, Ph.D. (Academy of Finland and Biocenter Oulu)
Sanna-Maria Karppinen, Ph.D. (Academy of Finland) until July
Jarkko Koivunen, Ph.D. (Biocenter Oulu and Academy of Finland)
Heli Ruotsalainen, Ph.D. (Academy of Finland and University of Oulu)
Sabrina Santoleri, Ph.D. (Academy of Finland)  
Hongmin Tu, Ph.D. (Biocenter Oulu)

Ph.D. Students:
Miki Aho, Med.Cand., leave of absence
Raman Devarajan, M.Sc. (Academy of Finland)
Charlotta Henriksson, Med. Cand. (Sigrid Jusélius Foundation)
Heli Härönen, M.D. (Sigrid Jusélius Foundation)
Riku Kallunki, Med. Cand. (Sigrid Jusélius Foundation)
Jaakko Kangas, M.D. (Academy of Finland) until May
Inderjeet Kaur, Ph.D. (chemistry) (Biocenter Oulu)
Antti Kemppainen, Med.Cand. (Sigrid Jusélius Foundation)
Minna Kihlström, M.Sc. (Academy of Finland)
Juho Lakkala, Med. Cand. (Sigrid Jusélius Foundation)
Guillermo Martinez-Nieto, M.Sc. (CAFFEIN, EU Marie Curie Actions – Initial Training Networks and Sigrid Jusélius Foundation)
Marjut Nätynki, M.Sc. (Finnish Cultural Foundation and Academy of Finland) until July
Tiina Petäistö, M.Sc. (Sigrid Jusélius Foundation and University of Oulu)
Mia Rinta-Jaskari, Med. Cand. (Sigrid Jusélius Foundation)
Zarin Zainul, M.Sc. (Biocenter Oulu)
Saad Ullah Akram, M.Sc. (Biocenter Oulu)

Laboratory Technicians, 6 (University of Oulu and Academy of Finland)

Main source of salary in brackets.

Foreign Scientists, 8

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 2016

Ph.D. Ritva Heljasvaara, University of Bergen, Norway
Ph.D. Mika Kaakinen, University of Queensland, Brisbane, Australia
M.SC. Guillermo A. Martinez Nieto, University of Ghent, Belgium

Visiting Researchers in 2016 (over two weeks)

M.Sc., Irina Primac, University of Liegé, Belgium

EU Projects:

EC Infrafrontier-I3, FP-7-Infrafrontier-2012, grant for 2013-2016. Partner.

Initial training network “Cancer associated fibroblasts (CAF) function in tumor expansion and invasion”. FP7 grant no. 316610. 2012-2016. Partner.

NordForsk Projects

Researcher network project “Nordic Infrastructure in Mouse Models (NorIMM)” NordForsk project no. 69005, 2014-2016. Coordinator Taina Pihlajaniemi.

Researcher network project “Bridging Nordic Imaging - Enabling Discoveries from Atoms to Anatomy”. 2014-2016. Partner Lauri Eklund.

Last updated: 25.4.2017