Multimerin-2 maintains vascular stability and permeability.

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2-/- (Mmrn2-/-) mice. Although Mmrn2-/- mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2-/- mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2-/- vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2-/- mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

Abrogation of EMILIN1-ß1 integrin interaction promotes experimental colitis and colon carcinogenesis.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9ß1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis.

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1-/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9ß1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain.

The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth.

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

The α4ß1/EMILIN1 interaction discloses a novel and unique integrin-ligand type of engagement.

EMILIN1, a homo-trimeric adhesive ECM glycoprotein, interacts with the α4ß1 integrin through its gC1q domain. Uniquely among the C1q family members, the EMILIN1 gC1q presents only nine-stranded ß-sandwich fold and the missing strand is substituted by a disordered 19-residue long segment spanning from Y927 to G945 at the apex of the gC1q domain. This unstructured loop exposes to the solvent the acidic residue E933, which plays a key role in the α4ß1 integrin mediated interaction. Here, we experimentally determined that the three E933 residues (one from each monomer) are all required for ligand binding. By docking the NMR structure of the gC1q to a virtual α4ß1 crystal structure based on the known structures of α4ß7 and α5ß1 integrins we built a model of α4ß1-gC1q complex where three E933 residues are smoothly forced to coordinate the Mg2+ ion at the ßI MIDAS site of the integrin. By bringing the three E933 close in space, the trimeric supramolecular organization of gC1q allows the formation of a proper 3D geometry and suggests a quaternary-structure-dependent mode of interaction. Furthermore, we experimentally identified R904 as a synergistic residue for cell adhesion. Accordingly, the model showed that this residue is able to form potential stabilizing intra-chain salt bridges with residues E928 and E930. This mode of interaction likely accounts for a more stable and durable α4ß1-gC1q interaction in comparison with the prototypic CS1 ligand. To our knowledge, this is the first report describing the simultaneous involvement of all the three acidic residues of a trimeric ligand in the formation of a dimeric complex with the integrin ßI domain.

Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4ß1 integrin interaction, cell adhesion and anti-proliferative activity.

The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4ß1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4ß1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4ß1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4ß1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.

In Vitro Biocompatibility Assessment and In Vivo Behavior of a New Osteoconductive ßTCP Bone Substitute.

OBJECTIVE: Beta-tricalcium phosphate (ßTCP) granules (OsproLife) exhibit a pure crystalline phase and a rough microporous surface for promoting cell adhesion and microsized intragranule porosity for improving wettability and resorption necessary for bone regeneration. OsproLife is a fully resorbable, space-maintaining, and osteoconductive synthetic material for the filling of bone defects. To asses OsproLife properties, a similar synthetic biomaterial, already on the market, has been chosen as reference: Cerasorb has the same chemical composition, but different crystal structure, surface morphology, and granule size. The aim of this study is to compare the properties of OsproLife and Cerasorb. METHODS: Chemical purity, composition and physical properties, in vitro cytotoxicity, and in vivo bone performance in a rabbit model were analyzed. ßTCP OsproLife granules (test) were compared with Cerasorb (control). Histological and µCT analyses were performed at 6, 12, and 56 weeks after implantation. RESULTS: ßTCP OsproLife and Cerasorb granules result to be both biocompatible and characterized by the same osteoconductivity and resorption rate. CONCLUSION: ßTCP OsproLife granules are a promising bone substitute for dental and orthopedic applications.

Diagnostic Exome Sequencing Identifies a Novel Gene, EMILIN1, Associated with Autosomal-Dominant Hereditary Connective Tissue Disease.

Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio-exome sequencing of a 55-year-old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN-1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN-1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomal-dominant connective tissue disorder.

Characterization and cytocompatibility of a new injectable multiphasic bone substitute based on a combination of polysaccharide gel-coated OSPROLIFE(®) HA/TTCP granules and bone marrow concentrate.

The purpose of this study was to examine the in vitro cytocompatibility of a novel injectable multiphasic bone substitute (MBS) based on polysaccharide gel-coated OSPROLIFE(®) hydroxyapatite (HA)/tetracalcium phosphate (TTCP) granules combined with bone marrow concentrate (BMC). Polysaccharide gel-coated granules loaded in syringe were combined with BMC diluted in ionic crosslinking solution. The product was then maintained in culture to investigate the cytocompatibility, distribution, and osteogenic differentiation function of cells contained in the BMC. The in vitro cytocompatibility was assessed after 0, 24, and 96 h from the injectable MBS preparation using the LIVE/DEAD(®) staining kit. The results highlighted that cells remained viable after combination with the polysaccharide gel-coated granules; also, viability was maintained over time. The distribution of the cells in the product, observed using confocal microscopy, showed viable cells immersed in the polysaccharide gel formed between the granules after ionic crosslinking. The mesenchymal stromal cells (MSC) contained in the injectable MBS, the basic elements for bone tissue regeneration, were able to differentiate toward osteoblasts, producing an osteogenic matrix as evidenced by alizarin red-s (AR-S) staining. In conclusion, we found that the injectable MBS may have the potential to be used as a bone substitute by applying a "one-step" procedure in bone tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 894-902, 2016.

A new HA/TTCP material for bone augmentation: an in vivo histological pilot study in primates sinus grafting.

OBJECTIVE: Synthetic calcium phosphate bone substitutes are widely used in sinus graft procedures due to their osteoconductive and biocompatible properties. Hydroxyapatite (HA), beta-tricalcium phosphate (ß-TCP), and HA/ß-TCP composite are the most applied materials. The aim of this study was to propose a new mineralogical formulation, HA/tetracalcium phosphate (TTCP), as biomaterial for bone regeneration in the maxillary sinus. METHODS: Sinus grafts were performed by using granules of a HA/TTCP blend and a collagen membrane. Bone response at time points of 14 and 17 weeks was histologically evaluated. RESULTS: After 14 weeks of healing, histomorphometric analysis showed the formation of new bone trabeculae among HA/TTCP granules. After 17 weeks, the bone trabeculae were thicker and HA/TTCP granules were still present. Histomorphometric analysis revealed a bone graft contact (BGC) of 64%. CONCLUSIONS: After 17 weeks from implantation, HA/TTCP synthetic bone graft performed very well as osteoconductive material: BGC was found very high, and bone volume and vital bone showed an ideal bone density for implant placement. HA/TTCP granules are accounted for to accelerate new bone formation and to reduce the time needed for the graft healing, thus achieving high quantity of the new bone formed.

Perspectives of the Si3N4-TiN ceramic composite as a biomaterial and manufacturing of complex-shaped implantable devices by electrical discharge machining (EDM).

PURPOSE: In this work we investigated the suitability of electroconductive silicon nitride/titanium nitride composite for biomedical implantable devices with particular attention on the processing route that allows the net-shaping of complex components by electrical discharge machining (EDM). METHODS: The composite, constituted mainly of a beta-Si3N4, dispersed TiN grains and a glassy grain boundary phase, exhibited a low density and high hardness, strength and toughness. Bulk, surface characteristics and properties of the Si3N4-TiN composite were analyzed. After the EDM process, the microstructure of the machined surface was examined. RESULTS AND CONCLUSIONS: The obtained results showed that the Si3N4-TiN ceramic composite together with the EDM manufacturing process might potentially play a key role in implantable load-bearing prosthesis applications.

The solution structure of EMILIN1 globular C1q domain reveals a disordered insertion necessary for interaction with the alpha4beta1 integrin.

The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded beta-sandwich fold of the gC1q domain is reduced to nine beta strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the alpha4beta1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of alpha4beta1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu(933)). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-alpha4beta1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site.