Ritva Heljasvaara

• University of Oulu

The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII is markedly upregulated in human breast cancer (BC) and is clos...

The globally increasing prevalence of obesity is associated with the development of metabolic diseases such as type 2 diabetes, dyslipidemia, and fatty liver. Excess adipose tissue (AT) often leads to its malfunction and to a systemic metabolic dysfunction because, in addition to storing lipids, AT is an active endocrine system. Adipocytes are embe...

Despite having been characterized for several decades, the development of therapeutically useful compounds based on integrins has shown little success. In cancer biology, previous studies mainly focused on the roles of integrins in tumor cells and tumor vasculature, but currently, the role of integrins in cancer-associated fibroblasts (CAFs) in the...

There is currently an increased interest in understanding the role of the tumor microenvironment (TME) in tumor growth and progression. In this context the role of integrins in cancer-associated fibroblasts (CAFs) will need to be carefully re-evaluated. Fibroblast-derived cells are not only in the focus in tumors, but also in tissue fibrosis as wel...

Hypoxia-inducible factors (HIFs) induce hundreds of genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling the location of dermal papilla (DP) alternates between the dermis and hypo...

Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast c...

Key points Extracellular matrix is highly remodelled in obesity and associates with the development of metabolic disorders, such as insulin resistance. Previously, we have shown that the lack of specific collagen XVIII isoforms impairs adipocyte differentiation in mice. Here, we show that mice lacking the medium and long isoforms of collagen XVIII...

The extracellular matrix (ECM) is ubiquitously involved in neoplastic transformation, tumour growth and metastatic dissemination, and the interplay between tumour and stromal cells and the ECM is now considered crucial for the formation of a tumour-supporting microenvironment. The 28 different collagens (Col) form a major ECM protein family and dis...

Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRβ+ CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11-deficiency led to a drastic reduction of tumor progression and met...

The efficient healing of skin wounds is crucial for securing the vital barrier function of the skin, but pathological wound healing and scar formation are major medical problems causing both physiological and psychological challenges for patients. A number of tightly coordinated regenerative responses, including haemostasis, the migration of variou...

The microenvironment plays a central role in cancer, and neoplastic cells actively shape it to their needs by complex arrays of extracellular matrix (ECM) proteins, enzymes, cytokines and growth factors collectively referred to as the matrisome. Studies on the cancer matrisome have been performed for single or few neoplasms, but a more systematic a...

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specif...

Collagen XVIII (ColXVIII) is a non-fibrillar collagen and proteoglycan that exists in three isoforms: short, medium and long. The medium and long isoforms contain a unique N-terminal domain of unknown function, DUF959, and our sequence-based secondary structure predictions indicated that DUF959 could be an intrinsically disordered domain. Recombina...

Fig. 3C did not indicate the borders between different sections of the same gel for the calprotectin, S100A8, and S100A9 immunoblots from day 8. The actin immunoblots were electrophoresed on parallel gels. This error has now been corrected and does not affect the results or conclusions of this work. (Figure presented). © 2018 by The American Societ...

Here we describe a protocol to utilize human benign leiomyoma tissue in in vitro 3D model that enables an assessment of cell invasion. The chapter also describes detailed instructions for image analysis to quantify the results. Leiomyoma is a benign tumor of the uterus which mimics authentic components of the tumor microenvironment including fibrob...

Relapse of acute myeloid leukemia (AML) is still dramatically frequent, imposing the need for early markers to quantify such risk. Recent evidence point to a prominent role for extracellular matrix (ECM) in AML, but its prognostic value has not yet been investigated. Here we have investigated whether the expression of a 15-ECM gene signature could...

Collagen XVII and integrin α6β4 have well-established roles as epithelial adhesion molecules. Their binding partner laminin 332 as well as integrin α6β4 are largely recognized to promote invasion and metastasis in various cancers, and collagen XVII is essential for the survival of colon and lung cancer stem cells. We have studied the expression of...

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal keratinocytes (n = 11) with real-time quantitative PCR and Western...

Collagens are key structural components of basement membranes, providing a scaffold for other components or adhering cells. Collagens and collagen-derived active fragments contribute to biological activities such as cell growth, differentiation and migration. Here, we report that collagen XV knock-out (ColXV KO) mice are resistant to experimental i...

Collagen XVIII is a ubiquitous basement membrane (BM) proteoglycan produced in three tissue-specific isoforms that differ in their N-terminal non-collagenous sequences, but share collagenous and C-terminal non-collagenous domains. The collagenous domain provides flexibility to the large collagen XVIII molecules on account of multiple interruptions...

Supplementary Figures and Tables

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. We monitored the effects of VEGF-D in cutaneous squamous cell carcinoma (cSCC) by subjecting transgenic mice overexpressing VEGF-D in the skin (...

Fibrosis is one of the most prevalent features of age-related diseases like obesity, diabetes, non-alcoholic fatty liver disease, chronic kidney disease, or cardiomyopathy and affects millions of people in all countries. Although the understanding about the pathophysiology of fibrosis has improved a lot during the recent years, a number of mechanis...

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and its incidence is increasing globally. We have examined the role of complement component C3 and complement factor B (CFB) in the progression of cSCC. Significantly elevated mRNA levels for C3 and CFB were detected din cSCC cell lines (n = 8) compared to normal hum...

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely under...

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells an...

Collagen type XV and XVIII are proteoglycans found in the basement membrane zones of endothelial and epithelial cells, and known for their cryptic anti-angiogenic domains named restin and endostatin, respectively. Mutations or deletions of these collagens are associated with eye, muscle and microvessel phenotypes. We now describe a novel role for t...

Significance A previously unrecognized role is described for collagen XVIII—a ubiquitous, structurally complex basement membrane proteoglycan—in supporting preadipocyte differentiation and the maintenance of this differentiated state, and hence the size and lipid-clearing/storage functions of white adipose tissue depots. Specific lack of medium and...

Key points Collagen XV and XVIII occur in muscle and connective tissue capillaries and are needed for maintaining a normal circulatory phenotype. Lack of collagen XV in mice leads to increased vascular permeability, increased extraction fraction, and increased extravascular extracellular space in striated muscle. Lack of collagen XVIII in mice lead...

Purpose: Collagen XVIII deficiency leads to anterior and posterior eye defects in Col18a1(-/-) mice, and overexpression of its C-terminal endostatin domain under a K14 promoter leads to cataract. We studied the consequences of K14-driven overexpression of the thrombospondin-1 (Tsp-1)-like domain, and also the roles of the three collagen XVIII isof...

Background: The objective of this study was to assess the possibility of predicting histological characteristics of meningiomas on the basis of preoperative MRI and the correlation of the expression of vascular endothelial growth factor (VEGF) and collagen XVIII with histological parameters already established as predictive of the course of these...

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement...

Conditioned Arr-HSC culture medium inhibits HSC-3 cell migration in co-culture experiments. Conditioned media from Ctrl-HSC (CtrlCM) and Arr-HSC (ArrCM) clones were collected at 24 h and administered to Ctrl-HSC cells. A. Cell migration was assayed with a Transwell assay in which 30 000 cells were allowed to migrate through Transwell inserts and th...

Characterization of stable HSC-3 and MDA-MB-435 cell clones overexpressing arresten by Western blotting. The secretion of recombinant arresten in stably transfected HSC-3 and MDA-MB-435 cells was verified by Western blotting. The cell culture medium was concentrated by acetone precipitation and the proteins were separated by SDS-PAGE and immunoblot...

Overexpression of arresten reduces the viability of HSC-3 cells in 2D culture. A. Proliferative cells were detected by BrdU labeling. 5000 Ctrl-HSC and Arr-HSC cells were allowed to attach and the cell proliferation was measured after 24 h using the colorimetric cell proliferation ELISA BrdU assay at 450 nm (n = 10 wells). B. Viable cells were dete...

Tunel-positive cells were detected in keratinized or necrotic areas in HSC-3 xenografts. Apoptotic cells were detected by TUNEL assay (green) in HSC-3 xenografts (blue, DAPI). Scale bar 100 µm. (TIF)

No differences in vimentin staining between Arr-HSC and Ctrl-HSC cells. Immunostaining of vimentin (green) in cultured Ctrl-HSC and Arr-HSC cells (blue, DAPI). Scale bar 100 µm. (TIF)

Cell-cell and cell-substrate interactions, and cell membrane capacitance show changes between Arr-HSC and Ctrl-HSC cells. A. The impedance, reflecting cell adhesion and spreading, was measured for Ctrl-HSC cells treated with ArrCM or CtrlCM using electric cell-substrate impedance sensing (ECIS) (mean of duplicate wells of representative ECIS plates...

Supplemental methods. Cell culture, cloning of arresten into pcDNA3.1 eukaryotic expression vector, transfection and selection of HSC-3 and MDA-MB-435 cells expressing arresten, purification of recombinant arresten, quantitative real-time RT-PCR (qPCR), cell proliferation assay, MTT viability assay, Transwell invasion assay, E-cadherin staining and...

Arresten inhibits migration of HSC-3 and MDA-MB-435 cells, and induces morphological changes in MDA-MB-435 cells in vitro. A. Wound closure in a scratch wound healing assay was markedly slower in both Arr-HSC clones than in the Ctrl-HSC cells. Scale bar 50 µm. B. MDA-MB-435 wounds did not close within 48 h, but both Arr-MDA clones showed reduced mi...

Arresten inhibits cell invasion in vitro. 30 000 Arr-HSC and Ctrl-HSC cells were allowed to invade through the Matrigel-coated Transwell inserts for 22 hours. The invaded cells were stained with hematoxylin and counted under a microscope with 20×magnification. Mann-Whitney U-test, *p<0.05, (n = total number of fields analyzed, 3–5 fields per Transw...

Arresten alters the tissue architecture of HSC-3 xenografts. One million Ctrl-HSC and Arr-HSC cells were injected subcutaneously into the flanks of nude mice (n = 10 per group) and tumor growth was monitored over 16 days. Representative hematoxylin-eosin and E-cadherin stainings of HSC-3 xenografts. A–B. Ctrl-HSC xenografts show an appearance of po...

Relative mRNA expression of arresten and E-cadherin in the HSC-3 and MDA-MB-435 clones. (DOC)

ADAMTSs (A Disintegrin And Metalloprotease with ThromboSpondin domains) are a family of enzymes with both proteolytic and protein interaction functions, which have been implicated in distinct pathologies. In this work, we have investigated the putative role of ADAMTS-12 in inflammation by using a mouse model deficient in this metalloprotease. Contr...

Pulmonary arteriovenous malformations (PAVMs) are a common source of morbidity after bidirectional superior cavopulmonary anastomosis (Glenn). The diversion of hepatic venous effluent away from the pulmonary circulation after Glenn appears to play a significant role in the pathogenesis of PAVMs. Although the liver is known to produce factors that r...

Collagen XVIII (ColXVIII) is a multidomain basement membrane proteoglycan that is expressed as three tissuespecific isoforms. These isoforms share a common C-terminal anti-angiogenic endostatin domain but have distinct N-terminal non-collagenous domains. The longest isoform contains a highly conserved frizzled motif in its N-terminal portion. The f...

Bmx (Bone marrow kinase in chromosome X), also known as Etk, is a member of the Tec family of nonreceptor tyrosine kinases. Bmx is expressed mainly in arterial endothelia and in myeloid hematopoietic cells. Bmx regulates ischemia-mediated arteriogenesis and lymphangiogenesis, but its role in tumor angiogenesis is not known. In this study, we charac...

The incidence of keratinocyte-derived nonmelanoma skin cancers is increasing worldwide because of cumulative recreational exposure to sunlight. At present, no specific molecular markers are available for assessing the progression of premalignant actinic keratoses to invasive cutaneous squamous cell carcinoma (SCC). We examined the role of the Serpi...

The complex multistage processes of tumour initiation, progression and metastasis challenge the methods that are used in basic cancer biology research and anticancer drug development. Experimental tumour modelling in mice provides means for observing tumour development, identifying target molecules and pathways and designing and testing novel strat...

The extracellular matrix (ECM) is a major determinant of the structural integrity and functional properties of the myocardium in common pathological conditions, and changes in vasculature contribute to cardiac dysfunction. Collagen (Col) XV is preferentially expressed in the ECM of cardiac muscle and microvessels. We aimed to characterize the ECM,...

In acute lung injury, repair of the damaged alveolar-capillary barrier is an essential part of recovery. Endostatin is a 20 to 28 kDa proteolytic fragment of the basement membrane collagen XVIII, which has been shown to inhibit angiogenesis via action on endothelial cells. We hypothesised that endostatin may have a role in inhibiting lung repair in...

To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported m...

Background and aim: Aberrant angiogenesis and defective epithelial repair are key features of idiopathic pulmonary fibrosis (IPF). Endostatin is an antiangiogenic peptide with known effects on endothelial cells. This study aimed to establish the levels of endostatin in the bronchoalveolar lavage fluid (BALF) in IPF and to investigate its actions on...

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous...

3D cell surface reconstruction of V3Nter in vitro. Confocal fluorescence microscopy of mhAT3FS315 mouse liver cancer cells transiently transfected with V3Nter cDNA and probed with anti-DUF-959, followed by anti-rabbit TRITC-labeled IgG (red). Original magnification×1000. Fifteen representative images acquired at 500-nm intervals on the vertical axi...

V3FL localizes at the cell surface, but does not inhibit Wnt/ β-catenin signaling. (A) Schematic of V2FL and V3FL showing DUF-959, FZC18, Tsp-1C18 (thrombospondin-1) and ES (endostatin) modules. Thick horizontal lines indicate the antibodies used. (B–D) Overlays of immunofluorescence and phase contrast confocal microscopy of mhAT3FS315 hepatoma cel...

3D cell surface reconstruction of FZC18 in vivo. Reconstruction of FZC18 staining at the cell surface in a human hepatocellular carcinoma (case TL 03, shown in Figures 8B and S4B). Original magnification×1000. Images were acquired on the vertical axis and stacked on ImageJ 1.37v (NIH, USA). In the light of data on Figure 8B, cell surface signal sho...

Sialylation of V3Nter resides within the DUF-959 module. (A) Schematic of V2Nter (left) and FZC18 (right) cDNA vectors and antibodies. Horizontal color lines below each module denote the antibodies used. (B) Immunoblot of mhAT3FS315 cell conditioned medium after transfection with FZC18 cDNA. Proteins were separated in denaturing 7.5% PAGE-SDS. The...

A 15-amino acid peptide derived from the CRD of FZC18 (RH3 peptide) competes with FZC18 binding to Wnt3a. (A) EBNA-293 cells were cotransfected with mouse V3Nter and with His-tagged mouse Wnt3a. Transfected cells were incubated with 0; 50 or 100 µg×ml−1 of the synthetic peptide RH3 from the CRD domain of FZC18. Cell lysates were analyzed by immunob...

Detection of FZC18 in human liver. (A) Immunoperoxidase staining (brown) in normal human liver shows FZC18 in periportal hepatocytes. In the portal tract (PT), including portal veins (v), bile ducts and basement membrane (arrow) no signal is detected. Case NL 04, shown in Figure 8B. (B) HCC. Speckle-like FZC18 (arrows) is seen on the surface of tum...

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries str...

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and cont...

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteolytic enzymes are implicated in a variety of physiological processes, such as collagen maturation, organogenesis, angiogenesis, reproduction and inflammation. Moreover, deficiency or overexpression of certain ADAMTS proteins is directly involved i...

Leukocyte infiltration into inflamed tissues is considered to involve sequential steps of rolling over the endothelium, adhesion, and transmigration. In this model, the leukocyte adhesion molecule L-selectin and its ligands expressed on inflamed endothelial cells are involved in leukocyte rolling. We show that upon experimental and human renal isch...

Collagen XVIII is a ubiquitous basement membrane (BM) component and a precursor of endostatin. Using immunohistochemistry and in situ hybridization, we studied the expression and localization of collagen XVIII in different stages of normal oral wound healing, epithelial dysplasia and squamous cell carcinoma (SCC). In mild epithelial dysplasias coll...

The endostatin domain of type XVIII collagen (ColXVIII) inhibits neovascularization and regulates cell migration and matrix turnover. This study was designed to demonstrate the protein and gene expression patterns of ColXVIII/endostatin in the human eye and to ascertain whether endostatin is detectable in ocular fluid samples. Twenty human eyes enu...

This study was designed to clarify the expression of 2 closely related collagen (Col) types XVIII and XV, and the proteolytically derived endostatin fragment of ColXVIII in normal, keratoconus, and scarred human corneas. Immunohistochemistry, in situ hybridization, immunoelectron microscopy, and Western immunoblotting were used for human corneal sa...

Type XVIII collagen is a multidomain protein that contains cleavable C-terminal NC1 and endostatin fragments, which have been shown to either induce or inhibit cell migration. Endostatin is being intensely studied because of its anti-angiogenic activity. Three variants of type XVIII collagen have been reported to be distributed in epithelial and en...

Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in...

Collagen XVIII is a basement membrane (BM) component, whereas MMP-20 (enamelysin) is a matrix metalloproteinase predominantly expressed in teeth. Since MMP-20 was found to degrade collagen XVIII, we studied the co-expression of these proteins in dental tissues. Collagen XVIII surrounded the developing tooth during early and late bell stages and was...

Endostatin, a 20-kDa collagen XVIII fragment, inhibits angiogenesis and tumor growth in vivo, but the mechanisms are still unclear. Matrix metalloproteases (MMPs), a family of extracellular and membrane-associated endopeptidases, collectively digest almost all extracellular matrix and basement membrane components, and thus play an important role in...

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-select...

Matrix metalloproteinase (MMP)-2 and membrane type 1-MMP can process the laminin-5 (Ln-5) gamma2-chain, revealing a cryptic site inducing epithelial cell migration. We investigated whether other MMPs process the Ln-5 gamma2-chain and related their ability to induce epithelial cell migration. The N-terminal sequences of the MMP-3, -12, -13, and -20...

Heparan sulfate proteoglycans (HSPGs) may play a role in the formation and persistence of senile plaques and neurofibrillary tangles in Alzheimer's disease brains. Recently, it has been demonstrated that the human extracellular matrix-associated molecule collagen XVIII is the first collagen carrying heparan sulfate side-chains. Two variants of coll...

A heterologous prime-boost vaccination with DNA vectors and vaccinia virus recombinants (VVr) has been shown to enhance specific cellular immune responses and to elicit significant protection against pathogens in animal models. In this study, we have analyzed, in the leishmaniasis cutaneous murine model, the effectiveness of this prime-boost strate...

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which ex...

Endostatin is an endogenous inhibitor of angiogenesis and tumor growth in mice, which may be generated by proteolytic cleavage of collagen XVIII. In normal tissues, 2 variants of the endostatin precursor, namely the SHORT and LONG forms, regulate tissue specificity. We analyzed 53 human liver biopsies (18 hepatocellular carcinomas, 16 metastases of...

The vaccinia virus (VV) 39-kDa protein, the product of the A4L gene, is a highly antigenic protein of the viral core. Pulse-chase and immunoprecipitation experiments have shown that the 39-kDa protein interacts with p4a (encoded by the A10L gene), the precursor of the most abundant virion protein. This interaction is maintained with the processed 4...

Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both o...

Diss. -- Oulun yliopisto.