Scleraxis-lineage cells are required for correct muscle patterning.

Movement of the vertebrate body is supported by the connection of muscle, tendon and bone. Each skeletal muscle in the vertebrate body has a unique shape and attachment site; however, the mechanism that ensures reproducible muscle patterning is incompletely understood. In this study, we conducted targeted cell ablation using scleraxis (Scx)-Cre to examine the role of Scx-lineage cells in muscle morphogenesis and attachment in mouse embryos. We found that muscle bundle shapes and attachment sites were significantly altered in embryos with Scx-lineage cell ablation. Muscles in the forelimb showed impaired bundle separation and limb girdle muscles distally dislocated from their insertion sites. Scx-lineage cells were required for post-fusion myofiber morphology, but not for the initial segregation of myoblasts in the limb bud. Furthermore, muscles could change their attachment site, even after formation of the insertion. Lineage tracing suggested that the muscle patterning defect was primarily attributed to the reduction of tendon/ligament cells. Our study demonstrates an essential role of Scx-lineage cells in the reproducibility of skeletal muscle attachment, in turn revealing a previously unappreciated tissue-tissue interaction in musculoskeletal morphogenesis.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression.

Impairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4-/- mice. Upon AML exposure, Lama4-/- mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4-/- MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4-/- mice post-cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging.

Tissue stem cells (SCs) divide infrequently as a protective mechanism against internal and external stresses associated with aging. Here, we demonstrate that slow- and fast-cycling SCs in the mouse skin epidermis undergo distinct aging processes. Two years of lineage tracing reveals that Dlx1+ slow-cycling clones expand into the fast-cycling SC territory, while the number of Slc1a3+ fast-cycling clones gradually declines. Transcriptome analysis further indicate that the molecular properties of each SC population are altered with age. Mice lacking fibulin 7, an extracellular matrix (ECM) protein, show early impairments resembling epidermal SC aging, such as the loss of fast-cycling clones, delayed wound healing, and increased expression of inflammation- and differentiation-related genes. Fibulin 7 interacts with structural ECM and matricellular proteins, and the overexpression of fibulin 7 in primary keratinocytes results in slower proliferation and suppresses differentiation. These results suggest that fibulin 7 plays a crucial role in maintaining tissue resilience and epidermal SC heterogeneity during skin aging.

Timosaponin AIII Disrupts Cell-Extracellular Matrix Interactions through the Inhibition of Endocytic Pathways.

Timosaponin AIII (TAIII), a steroidal saponin isolated from the root of Anemarrhena asphodeloides Bunge, exhibits various pharmacological activities, including anti-cancer properties. TAIII inhibits the migration and invasion of various cancer cell types. However, the mechanism underlying how TAIII regulates the motility of cancer cells remains incompletely understood. In this study, we demonstrate that TAIII disrupted cell-extracellular matrix (ECM) interactions by inhibiting internalization of cell surface proteins, such as integrins. We found that TAIII inhibited cell adhesion on various ECMs. Structure-activity relationship analysis demonstrated that TAIII exhibited unique activity among the saponins from Anemarrhena asphodeloides Bunge and that the number and position of saccharide moieties were important for TAIII to exert its activity. Time lapse imaging revealed that TAIII also suppressed cell spreading on the ECM, membrane ruffling, and lamellipodia formation. Furthermore, we examined integrin ß1 behaviors in response to TAIII treatment and found that TAIII blocked its internalization. These findings contribute to delineating the potential molecular mechanisms by which TAIII exerts anti-metastatic activity.

Development of a novel purification method for AAV vectors using tangential flow filtration.

Adeno-associated virus (AAV) vector can efficiently transduce therapeutic genes in various tissue types with less side effects; however, owing to complex multistep processes during manufacture, there have been surges in the pricing of recently approved AAV vector-based gene therapy products. This study aimed to develop a simple and efficient method for high-quality purification of AAV vector via tangential flow filtration (TFF), which is commonly used for concentration and diafiltration of solutions during AAV vector purification. We established a novel purification method using TFF and surfactants. Treatment with two classes of surfactants (anionic and zwitterionic) successfully inhibited the aggregation of residual proteins separated from the AAV vector in the crude product by TFF, obtaining a clearance of 99.5% residual proteins. Infectivity of the AAV vector purified using the new method was confirmed both in vitro and in vivo, and no remarkable inflammation or tissue damage was observed in mouse skeletal muscle after local administration. Overall, our proposed method could be used to establish a platform for the purification of AAV vector.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active ß1-integrin in adult tendon cells.

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal-regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active ß1-integrin-containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp-mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5ß1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

Abnormalities in esophageal smooth muscle induced by mutations in collagen XIX.

Collagen XIX is a nonfibrillar collagen that localizes in restricted tissues at very low amounts. A previous study on Col19a1 null mice revealed that collagen XIX is involved in esophageal muscle physiology and morphogenesis. Here, we use histological analysis to show that mice with a Col19a1 mutant lacking the NC3 domain and seven collagen triplets display abnormal transition of smooth to striated muscle in the abdominal segment of esophagus, and a widened esophagus with age. With two newly prepared antibodies, we analyzed the expression of collagen XIX in the mouse esophagus and show that collagen XIX colocalizes with α-smooth muscle actin. By immunoelectron microscopy, we confirmed the localization of collagen XIX in esophageal smooth muscle cells. Col19a1 mutant mice contained reduced levels of mutated Col19a1 mRNA. Interestingly, hepatocyte growth factor, which has an important role in esophageal striated muscle development, was reduced in the esophagus of the Col19a1 mutant mice. These findings suggest that collagen XIX may be critical for the function of esophageal smooth muscle cells as a scaffold for anteroposterior migration of esophagus-striated muscle cells.

Fibulin-1 Integrates Subendothelial Extracellular Matrices and Contributes to Anatomical Closure of the Ductus Arteriosus.

OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.

Mammalian hemicentin 1 is assembled into tracks in the extracellular matrix of multiple tissues.

BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals. RESULTS: Hmcn1 -/- mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency. CONCLUSION: That Hmcn1 -/- , Hmcn2 -/- , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage.

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-ß gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice.

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP-tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)-positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor ß (TGF-ß) signaling. scx-deficient mice had migration of Sca-1-positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx-null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro, and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx-null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body.

Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, ß1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

The pro-α1(V) collagen gene (Col5a1) is coordinately regulated by miR-29b with core promoter in cultured cells.

AIMS: Col5a1 encodes the α1 chain of type V collagen, a quantitatively minor fibrillar collagen that is critical for the formation and function of the organs in the body. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate biological functions by binding to the 3'-untranslated region (3'UTR) of specific target mRNA. In this study, we investigated the posttranscriptional regulation of miRNAs on the Col5a1 gene expression. MATERIALS AND METHODS: We cultured osteoblasts and fibroblasts of cell lines. To examine the 3'UTR activity of the Col5a1 gene, chimeric plasmids constructs containing the core promoter and 3'UTR of Col5a1 were generated and luciferase assays were performed. We also evaluated the role of miRNA using constructs that were mutated at the putative binding sites of miRNA. In addition, we evaluated the endogenous mRNA and protein, and luciferase activity of the Col5a1 gene after miRNA overexpression/knockdown or CRISPR/Cas9-induced knockout. RESULTS: The luciferase assay showed a decreased activity of the 3'UTR of Col5a1 gene. However, the expression of the mutant constructs of miRNA-binding sites was restored. The overexpression of miRNA inhibited the Col5a1 gene not only with regard to the luciferase activity and endogenous mRNA but also at the protein level. In contrast, the RNAi-mediated knockdown or CRISPR/Cas9 system increased the expression of the Col5a1 gene. CONCLUSION: These results provided evidence that miR-29b regulates the Col5a1 gene expression through binding to the 3'UTR, which might play an important role in the pathogenesis of disease related to bone metabolism and fibrogenic reactions.

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis.

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-ß activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-ß1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-ß1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-ß bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.

Role of the extracellular matrix-located Mac-2 binding protein as an interactor of the Wnt proteins.

The Wnt proteins constitute a conserved family of secreted palmitoleate-containing signaling proteins that play important roles in development and tissue homeostasis. Their hydrophobic nature has raised the question of how the proteins are transported outside the cells. Accumulating evidence suggests that several different mechanisms, including transport by lipoprotein particles and exosomes, may contribute to this process. Here, we expressed epitope-tagged Wnt4 in HEK293 cells, and identified Mac-2 binding protein (Mac-2BP) as its binding partner in the serum-free conditioned medium. Serine-to-alanine substitution at the conserved fatty acid-conjugation site did not affect Mac-2BP binding. Subsequent studies showed that Mac-2BP may be a general Wnt interactor. It is found in the extracellular matrix (ECM) of various tissues, where it forms unusual oligomeric ring-like structures. Its functions appear to include interactions with cells and certain ECM components. Intriguingly, both Wnt signaling and Mac-2BP expression are upregulated in many types of cancer. Our studies on the four-domain Mac-2BP indicate a crucial role in Wnt binding for the C-terminal domain that bears no sequence similarity to any other protein. Mac-2BP may have a role in regulating the extracellular spreading and storage of the Wnts, thereby modulating their bioavailability and stability.

Effects of GABA on the expression of type I collagen gene in normal human dermal fibroblasts.

We examined the effects of GABA on type I collagen gene expression in normal human dermal fibroblasts. Real-time PCR analysis indicated GABA increased the level of type I collagen transcripts, and suppressed the expression of matrix metalloproteinase-1, which is a collagen-degrading enzyme. These results suggest GABA improves the skin elasticity by regulating type I collagen expression.

GABA promotes elastin synthesis and elastin fiber formation in normal human dermal fibroblasts (HDFs).

The multiple physiological effects of γ-aminobutyric acid (GABA) as a functional food component have been recently reported. We previously reported that GABA upregulated the expression of type I collagen in human dermal fibroblasts (HDFs), and that oral administration of GABA significantly increased skin elasticity. However, details of the regulatory mechanism still remain unknown. In this study, we further examined the effects of GABA on elastin synthesis and elastin fiber formation in HDFs. Real-time PCR indicated that GABA significantly increased the expression of tropoelastin transcript in a dose-dependent manner. Additionally, the expression of fibrillin-1, fibrillin-2, and fibulin-5/DANCE, but not lysyl oxidase and latent transforming factor-ß-binding protein 4, were also significantly increased in HDFs. Finally, immunohistochemical analysis confirmed that treatment with GABA dramatically increased the formation of elastic fibers in HDFs. Taken together, our results showed that GABA improves skin elasticity in HDFs by upregulating elastin synthesis and elastin fiber formation.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Fibulin-2 is essential for angiotensin II-induced myocardial fibrosis mediated by transforming growth factor (TGF)-ß.

Fibrosis is an ominous pathological process in failing myocardium, but its pathogenesis is poorly understood. We recently reported that loss of an extracellular matrix (ECM) protein, fibulin-2, protected against ventricular dysfunction after myocardial infarction (MI) in association with absence of activation of transforming growth factor (TGF)-ß signaling and suppressed upregulation of ECM protein expression during myocardial remodeling. Here we investigated the role of fibulin-2 in the development of myocardial hypertrophy and fibrosis induced by continuous pressor-dosage of angiotensin II (Ang II) infusion. Both wild type (WT) and fibulin-2 null (Fbln2KO) mice developed comparable hypertension and myocardial hypertrophy by Ang II infusion. However, myocardial fibrosis with significant upregulation of collagen type I and III mRNA was only seen in WT but not in Fbln2KO mice.Transforming growth factor (TGF)-ß1 mRNA and its downstream signal, Smad2, were significantly upregulated in WT by Ang II, whereas there were no Ang II-induced changes in Flbn2KO, suggesting fibulin-2 is necessary for Ang II-induced TGF-ß signaling that induces myocardial fibrosis. To test whether fibulin-2 is sufficient for Ang II-induced TGF-ß upregulation, isolated Flbn2KO cardiac fibroblasts were treated with Ang II after transfecting with fibulin-2 expression vector or pretreating with recombinant fibulin-2 protein. Ang II-induced TGF-ß signaling in Fbln2KO cells was partially rescued by exogenous fibulin-2, suggesting that fibulin-2 is required and probably sufficient for Ang II-induced TGF-ß activation. Smad2 phosphorylation was induced just by adding recombinant fibulin-2 to KO cells, suggesting that extracellular interaction between fibulin-2 and latent TGF-ß triggered initial TGF-ß activation. Our study indicates that Ang II cannot induce TGF-ß activation without fibulin-2 and that fibulin-2 has an essential role in Ang II-induced TGF-ß signaling and subsequent myocardial fibrosis. Fibulin-2 can be considered as a critical regulator of TGF-ß that induces myocardial fibrosis.