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Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with a poor prognosis and few treatment options. In the most widely used experimental model for this disease, bleomycin is administered into the lungs of mice, causing a reaction of inflammation and consequent fibrosis that resembles the progression of human IPF. The inflammation and fibrosis together induce changes in gene expression that can be analyzed with reverse transcription quantitative real-time PCR (RT-qPCR), in which accurate normalization with a set of stably expressed reference genes is critical for obtaining reliable results. This work compares ten commonly used candidate reference genes in the late, fibrotic phase of bleomycin-induced pulmonary fibrosis and ranks them from the most to the least stable using NormFinder and geNorm. Sdha, Polr2a and Hprt were identified as the best performing and least variable reference genes when alternating between normal and fibrotic conditions. In order to validate the findings, we investigated the expression of Tnf and Col1a1, representing the hallmarks of inflammation and fibrotic changes, respectively. With the best three genes as references, both were found to be upregulated relative to untreated controls, unlike the situation when analyzed solely with Gapdh, a commonly used reference gene. We therefore recommend Sdha, Polr2a and Hprt as reference genes for RT-qPCR in the 4-week bleomycin challenge that represents the late fibrotic phase.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5β1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. Significance: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer. See related commentary by Kamiyama and Augustin, p. 35

Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11β1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11β1 both have a role in the regulation of bone homeostasis. To study whether α11β1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11β1 as their only collagen receptor. The interaction between collagen XIII and integrin α11β1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11β1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11β1-collagen XIII interaction by crossbreeding α11 null mice (Itga11−/−) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11β1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.

The modification of genes in animal models has evidently and comprehensively improved our knowledge on proteins and signaling pathways in human physiology and pathology. In this review, we discuss almost 40 monogenic rare diseases that are enriched in the Finnish population and defined as the Finnish disease heritage (FDH). We will highlight how gene-modified mouse models have greatly facilitated the understanding of the pathological manifestations of these diseases and how some of the diseases still lack proper models. We urge the establishment of subsequent international consortiums to cooperatively plan and carry out future human disease modeling strategies. Detailed information on disease mechanisms brings along broader understanding of the molecular pathways they act along both parallel and transverse to the proteins affected in rare diseases, therefore also aiding understanding of common disease pathologies.

Both transmembrane and extracellular cues, one of which is collagen XIII, regulate the formation and function of the neuromuscular synapse, and their absence results in myasthenia. We show that the phenotypical changes in collagen XIII knock-out mice are milder than symptoms in human patients, but the Col13a1-/- mice recapitulate major muscle findings of congenital myasthenic syndrome type 19 and serve as a disease model. In the lack of collagen XIII neuromuscular synapses do not reach full size, alignment, complexity and function resulting in reduced muscle strength. Collagen XIII is particularly important for the preterminal integrity, and when absent, destabilization of the motor nerves results in muscle regeneration and in atrophy especially in the case of slow muscle fibers. Collagen XIII was found to affect synaptic integrity through binding the ColQ tail of acetylcholine esterase. Although collagen XIII is a muscle-bound transmembrane molecule, it also undergoes ectodomain shedding to become a synaptic basal lamina component. We investigated the two forms' roles by novel Col13a1tm/tm mice in which ectodomain shedding is impaired. While postsynaptic maturation, terminal branching and neurotransmission was exaggerated in the Col13a1tm/tm mice, the transmembrane form's presence sufficed to prevent defects in transsynaptic adhesion, Schwann cell invagination/retraction, vesicle accumulation and acetylcholine receptor clustering and acetylcholinesterase dispersion seen in the Col13a1-/- mice, pointing to the transmembrane form as the major conductor of collagen XIII effects. Altogether, collagen XIII secures postsynaptic, synaptic and presynaptic integrity, and it is required for gaining and maintaining normal size, complexity and functional capacity of the neuromuscular synapse.

Osteoporosis is the most common degenerative bone disease that occurs when the balance of bone production and resorption is perturbed. Loss of bone mass or alteration in its quality leads to significant weakening of the bones and subsequently to higher fracture risk. Collagen XIII (ColXIII) is a conserved transmembrane protein expressed in many mesenchymal tissues. Here we show that ColXIII is a regulator of bone remodeling niche. In this study, we found that ColXIII expression is significantly upregulated in osteoporotic patients. In view of that, we studied bone homeostasis in ColXIII-overexpressing mice (Col13a1oe) up to 72 weeks of age and observed a cortical bone overgrowth followed by a drastic bone loss, together with increased bone vascularization. Moreover, our results demonstrate that the ColXIII-derived ectodomain enhances angiogenesis through β1-integrins and the JNK pathway. Consequently, these data suggest that ColXIII has a role in age-dependent cortical bone deterioration with possible implications for osteoporosis and fracture risk.

Collagen XIII occurs as both a transmembrane-bound and a shed extracellular protein and is able to regulate the formation and function of neuromuscular synapses. Its absence results in myasthenia: presynaptic and postsynaptic defects at the neuromuscular junction (NMJ), leading to destabilization of the motor nerves, muscle regeneration and atrophy. Mutations inCOL13A1have recently been found to cause congenital myasthenic syndrome, characterized by fatigue and chronic muscle weakness, which may be lethal. We show here that muscle defects in collagen XIII-deficient mice stabilize in adulthood, so that the disease is not progressive until very late. Sciatic nerve crush was performed to examine how the lack of collagen XIII or forced expression of its transmembrane form affects the neuromuscular synapse regeneration and functional recovery following injury. We show that collagen XIII-deficient male mice are unable to achieve complete NMJ regeneration and functional recovery. This is mainly attributable to presynaptic defects that already existed in the absence of collagen XIII before injury. Shedding of the ectodomain is not required, as the transmembrane form of collagen XIII alone fully rescues the phenotype. Thus, collagen XIII could serve as a therapeutic agent in cases of injury-induced PNS regeneration and functional recovery. We conclude that intrinsic alterations at the NMJ inCol13a1−/−mice contribute to impaired and incomplete NMJ regeneration and functional recovery after peripheral nerve injury. However, such alterations do not progress once they have stabilized in early adulthood, emphasizing the role of collagen XIII in NMJ maturation.SIGNIFICANCE STATEMENTCollagen XIII is required for gaining and maintaining the normal size, complexity, and functional capacity of neuromuscular synapses. Loss-of-function mutations inCOL13A1cause congenital myasthenic syndrome 19, characterized by postnatally progressive muscle fatigue, which compromises patients' functional capacity. We show here in collagen XIII-deficient mice that the disease stabilizes in adulthood once the NMJs have matured. This study also describes a relevant contribution of the altered NMJ morphology and function to neuromuscular synapses, and PNS regeneration and functional recovery in collagen XIII-deficient mice after peripheral nerve injury. Correlating the animal model data on collagen XIII-associated congenital myasthenic syndrome, it can be speculated that neuromuscular connections in congenital myasthenic syndrome patients are not able to fully regenerate and restore normal functionality if exposed to peripheral nerve injury.

Transmembrane collagen XIII has been linked to maturation of the musculoskeletal system. Its absence in mice (Col13a1-/- ) results in impaired neuromuscular junction (NMJ) differentiation and function, while transgenic overexpression (Col13a1oe ) leads to abnormally high bone mass. Similarly, loss-of-function mutations in COL13A1 in humans produce muscle weakness, decreased motor synapse function and mild dysmorphic skeletal features. Here, analysis of the exogenous overexpression of collagen XIII in various muscles revealed highly increased transcript and protein levels, especially in the diaphragm. Unexpectedly, the main location of exogenous collagen XIII in the muscle was extrasynaptic, in fibroblast-like cells, while some motor synapses were devoid of collagen XIII, possibly due to a dominant negative effect. Concomitantly, phenotypical changes in the NMJs of the Col13a1oe mice partly resembled those previously observed in Col13a1-/- mice. Namely, the overall increase in collagen XIII expression in the muscle produced both pre- and postsynaptic abnormalities at the NMJ, especially in the diaphragm. We discovered delayed and compromised acetylcholine receptor (AChR) clustering, axonal neurofilament aggregation, patchy acetylcholine vesicle (AChV) accumulation, disrupted adhesion of the nerve and muscle, Schwann cell invagination and altered evoked synaptic function. Furthermore, the patterns of the nerve trunks and AChR clusters in the diaphragm were broader in the adult muscles, and already prenatally in the Col13a1oe mice, suggesting collagen XIII involvement in the development of the neuromuscular system. Overall, these results confirm the role of collagen XIII at the neuromuscular synapses and highlight the importance of its correct expression and localization for motor synapse formation and function.

Collagen XIII and the homologous collagens XXIII and XXV form a subgroup of type II transmembrane proteins within the collagen superfamily. Collagen XIII consists of a short cytosolic domain, a transmembrane domain and a large extracellular ectodomain, which may be shed into the pericellular matrix. It has been proposed that collagen XIII may function as an adhesion molecule, due to its cellular localization at focal contacts, numerous interactions with basement membrane (BM) and other extracellular matrix (ECM) proteins and expression at various cell-cell and cell-matrix junctions. Recent in vivo studies highlight its involvement in the development, differentiation and maturation of musculoskeletal tissues and vessels and in maintaining tissue integrity.

Human subjects consuming fish oil showed a significant suppression of cyclooxygenase-2 (COX-2) expression in blood monocytes when stimulated in vitro with lipopolysaccharide (LPS), an agonist for Toll-like receptor 4 (TLR4). Results with a murine monocytic cell line (RAW 264.7) stably transfected with COX-2 promoter reporter gene also demonstrated that LPS-induced COX-2 expression was preferentially inhibited by docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), the major n-3 polyunsaturated fatty acids (PUFAs) present in fish oil. Additionally, DHA and EPA significantly suppressed COX-2 expression induced by a synthetic lipopeptide, a TLR2 agonist. These results correlated with the preferential suppression of LPS- or lipopeptide-induced NF kappa B activation by DHA and EPA. The target of inhibition by DHA is TLR itself or its associated molecules, but not downstream signaling components. In contrast, COX-2 expression by TLR2 or TRL4 agonist was potentiated by lauric acid, a saturated fatty acid. These results demonstrate that inhibition of COX-2 expression by n-3 PUFAs is mediated through the modulation of TLR-mediated signaling pathways. Thus, the beneficial or detrimental effects of different types of dietary fatty acids on the risk of the development of many chronic inflammatory diseases may be in part mediated through the modulation of TLRs.