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A hallmark of ageing is the redistribution of body fat. Particularly, subcutaneous fat decreases paralleled by a decrease of skin collagen I are typical for age-related skin atrophy. In this paper, we hypothesize that collagen I may be a relevant molecule stimulating the differentiation of adipose-derived stem cells (ASCs) into adipocytes augmenting subcutaneous fat. In this context lipogenesis, adiponectin, and collagen I receptor expression were determined. Freshly isolated ASCs were characterized by stemness-associated surface markers by FACS analysis and then transdifferentiated into adipocytes by specific medium supplements. Lipogenesis was evaluated using Nile Red staining and documented by fluorescence microscopy or quantitatively measured by using a multiwell spectrofluorometer. Expression of adiponectin was measured by real-time RT-PCR and in cell-free supernatants by ELISA, and expression of collagen I receptors was observed by western blot analysis. It was found that supports coated with collagen I promote cell adhesion and lipogenesis of ASCs. Interestingly, a reverse correlation to adiponectin expression was observed. Moreover, we found upregulation of the collagen receptor, discoidin domain-containing receptor 2; receptors of the integrin family were absent or downregulated. These findings indicate that collagen I is able to modulate lipogenesis and adiponectin expression and therefore may contribute to metabolic dysfunctions associated with ageing.

β1 integrins are critical for T cell migration, survival and costimulation. The integrin α2β1, which is a receptor for collagen, also named VLA-2, is a major costimulatory pathway of effector T cells and has been implicated in arthritis pathogenesis. Herein, we have examined its ability to promote methotrexate (MTX) resistance by enhancing effector T cells survival. Our results show that attachment of anti-CD3-activated human polarized Th17 cells to collagen but not to fibronectin or laminin led to a significant reduction of MTX-induced apoptosis. The anti-CD3+collagen-rescued cells still produce significant amounts of IL-17 and IFNγ upon their reactivation indicating that their inflammatory nature is preserved. Mechanistically, we found that the prosurvival role of anti-CD3+collagen involves activation of the MTX transporter ABCC1 (ATP Binding Cassette subfamily C Member 1). Finally, the protective effect of collagen/α2β1 integrin on MTX-induced apoptosis also occurs in memory CD4 + T cells isolated from rheumatoid arthritis (RA) patients suggesting its clinical relevance. Together these results show that α2β1 integrin promotes MTX resistance of effector T cells, and suggest that it could contribute to the development of MTX resistance that is seen in RA.

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors., Competing Interests: None., (Schattauer GmbH Stuttgart.)

Blocking the expression of integrin α2β1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2β1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2β1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the "reversed" (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2β1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2β1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2β1 integrin, these cadherins promote integrin β1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2β1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2β1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2β1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor α (PDGFRα) and laminin beta 1 (LAMB1) expression. PDGFRα has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFRα-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFRα expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFRα expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFRα-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of α2β1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFRα signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFRα-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFRα-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression.

Adverse immune reactions prevent clinical translation of numerous implantable devices and materials. Although inflammation is an essential part of tissue regeneration, chronic inflammation ultimately leads to implant failure. In particular, macrophage polarity steers the microenvironment toward inflammation or wound healing via the induction of M1 and M2 macrophages, respectively. Here, this paper demonstrates that macrophage polarity within biomaterials can be controlled through integrin-mediated interactions between human monocytic THP-1 cells and collagen-derived matrix. Surface marker, gene expression, biochemical, and cytokine profiling consistently indicate that THP-1 cells within a biomaterial lacking cell attachment motifs yield proinflammatory M1 macrophages, whereas biomaterials with attachment sites in the presence of interleukin-4 (IL-4) induce an anti-inflammatory M2-like phenotype and propagate the effect of IL-4 in induction of M2-like macrophages. Importantly, integrin α2β1 plays a pivotal role as its inhibition blocks the induction of M2 macrophages. The influence of the microenvironment of the biomaterial over macrophage polarity is further confirmed by its ability to modulate the effect of IL-4 and lipopolysaccharide, which are potent inducers of M2 or M1 phenotypes, respectively. Thus, this study represents a novel, versatile, and effective strategy to steer macrophage polarity through integrin-mediated 3D microenvironment for biomaterial-based programming., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Myocardial infarction is the leading cause of morbidity and mortality in developed countries. It causes a left ventricular dysfunction, mainly due to the loss of functional tissue, resulting in heart failure. New therapies are being developed, using a tissue engineering approach, with the ultimate goal of restoring cardiac function by regenerating and repairing the damaged myocardium. In the present study we investigated the behaviour of a specific population of c-kit positive human cardiac stem cells, called Multipotent Adult Stem Cells (MASCs), grown within three-dimensional collagen scaffolds (3D), to establish whether they could be used in post-infarction cardiac regeneration. We also evaluated the expression levels of the Granulocyte Macrophage-Colony Stimulating Factor Receptor (GM-CSFR) and endoglin, a component of the Transforming Growth Factor beta (TGF-ß) receptor complex. Finally, we also evaluated the expression of the α2β1integrin. MASCs cultured within 3D collagen matrices are able to proliferate and migrate even in the absence of chemotactic agents and express high levels of factors involved in cell proliferation and migration, such as GM-CSFRα chain and integrins. They therefore represent a promising approach to tissue engineering aimed to restore cardiac function. Our results also suggest a role of GM-CSF in cell proliferation, while TGF-β does not seem to be relevant.

Purpose: Growing evidence has demonstrated that aberrant expression of integrin α2β1 might contribute to the invasion, metastasis and drug resistance of non-small cell lung cancer (NSCLC). Thus, the integrin α2β1 targeting 68 Ga-DOTA-A2B1 tracer was validated in NSCLC in contrast to accumulation of the clinically used 18 F-FDG PET tracer to see if 68 Ga-DOTA-A2B1-PET imaging can offer a valuable and critical diagnostic imaging criterion for the identification of phenotypes of aggressive lung cancer., Methods: To verify the prognostic value of integrin α2β1, several quantitative and functional in vitro assays were validated in different NSCLC cell lines (CL1-0, CL1-5, A549 and selected A549 ++ cells). Positron emission tomography (PET) imaging studies using both standard 18 F-FDG and a newly developed 68 Ga-labeled integrin α2β1 ( 68 Ga-DOTA-A2B1) tracer were sequentially performed on mice with lung tumor xenografts in different anatomic locations (subcutaneous, orthotopic and osseous) to validate the targeting capability of the 68 Ga-DOTA-A2B1 tracers. Treatment responses were monitored by injecting animals with metastatic bone tumors with 5 mg/kg doxorubicin. All in vivo treatment responses in each treatment subgroup were monitored with a PET imaging system to evaluate the up-regulation of integrin expression at the earliest stage of treatment (6 h)., Results: The PET and computed tomography (CT) images from NSCLC xenograft animals unambiguously demonstrated accumulation of the integrin tracer 68 Ga-DOTA-A2B1 in the tumor lesions at all locations. The average tumor uptake and tumor-to-normal (T/N) ratio were 2.51 ± 0.56 %ID/g and T/N = 2.82, 3.40 ± 0.42 %ID/g and T/N = 1.52, and 1.58 ± 0.108 %ID/g and T/N = 2.31 in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). The xenograft tumors were all clearly visible. In contrast, the accumulation of 18 F-FDG reached 3.6 ± 0.76 %ID/g, 1.39 ± 0.075 %ID/g and 3.78 ± 0.73 %ID/g in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). However, due to the high background uptake by normal tissue, the T/N values were less than or close to 1, making the tumors almost indistinguishable in the PET imaging analysis. Furthermore, 68 Ga-DOTA-A2B1-PET imaging of the treated osseous tumor model demonstrated more than 19% tracer uptake in A549 lesions (1.72 ± 0.95 %ID/g vs. pretreatment 1.44 ± 0.12 %ID/g, p = 0. 015) 6 h post-treatment with doxorubicin. The elevated intensity of tracer uptake was in accordance with the results of in vitro Western blot and ex vivo integrin staining, demonstrating elevated integrin α2β1 expression., Conclusion: In this study, integrin α2β1 was identified as a biomarker of aggressive malignant NSCLC. Thus, efforts should be devoted to validating integrin α2β1 as a potential target for non-invasive diagnosis and as a predictive marker for monitoring treatment responses using a preclinical PET imaging system., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objective: The wound bed score is a validated tool to monitor wound healing in chronic wounds, and depends on visual examination by trained personnel. This study describes the feasibility of adding some biochemical and immunohistochemical parameters to increase the objectivity and specificity of the wound bed score Method: Patients with chronic wounds on the lower leg with different durations were enrolled to assess the correlation between the wound bed score and specific wound-related biomarkers, namely MMP-9, MMP-2, NGAL, albumin, integrin α2/β1, and other histochemical (CD68, PK1, CD32, fractalkine, periostin) and immunocytochemical markers from biopsies and smears taken from wound edges and bed., Results: The study examined samples from 10 patients. Patients with an unfavourable wound bed score had a low expression of periostin and fractalkine in the wound bed tissue. CD68 PK1 showed a low or negative expression in the majority of the samples. Patients negative for CD68 PK1 were also negative for CD32. Principal component analysis revealed that the albumin level and the amount of proteins were associated with a high wound bed score. Two different subsets of patients could be discriminated either by integrin α2/β1 and albumin percentages or the MMP-9 and MMP-2 activities Conclusion: These preliminary results pave the way towards an improved wound status diagnosis and an advanced quality of wound care and management. These findings need confirming with a large number of patients and at different time points.

The platelet receptors glycoprotein (Gp)VI, integrin α 2 β 1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca 2+ ([Ca 2+ ] i ) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca 2+ ] i measurements using confocal imaging. All three collagen receptors coupled to [Ca 2+ ] i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca 2+ ] i signals leading to real-time increases in integrins α 2 β 1 - and α IIb β 3 -mediated platelet adhesion. α IIb β 3 -dependent platelet aggregation was dependent on P 2 Y 12 signalling. Co-engagement of α 2 β 1 and GpIb/V/IX generated transient [Ca 2+ ] i spikes and low amplitude [Ca 2+ ] i responses that potentiated GpVI-dependent [Ca 2+ ] i signalling. Therefore α 2 β 1 , GpIb/V/IX and GpVI synergise to generate [Ca 2+ ] i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for α IIb β 3 in stable platelet adhesion and aggregation.

Collagen III is critical to the integrity of blood vessels and distensible organs, and in hemostasis. Examination of the human collagen III interactome reveals a nearly identical structural arrangement and charge distribution pattern as for collagen I, with cell interaction domains, fibrillogenesis and enzyme cleavage domains, several major ligand-binding regions, and intermolecular crosslink sites at the same sites. These similarities allow heterotypic fibril formation with, and substitution by, collagen I in embryonic development and wound healing. The collagen III fibril assumes a "flexi-rod" structure with flexible zones interspersed with rod-like domains, which is consistent with the molecule's prominence in young, pliable tissues and distensible organs. Collagen III has two major hemostasis domains, with binding motifs for von Willebrand factor, α2β1 integrin, platelet binding octapeptide and glycoprotein VI, consistent with the bleeding tendency observed with COL3A1 disease-causing sequence variants.

Conformational activation of integrins is generally required for ligand binding and cellular signalling. However, we have previously reported that the nonactivated conformation of α2β1 integrin can also bind to large ligands, such as human echovirus 1. In this study, we show that the interaction between the nonactivated integrin and a ligand resulted in the activation of focal adhesion kinase (FAK) in a protein kinase C dependent manner. A loss-of-function mutation, α2E336A, in the α2-integrin did not prevent the activation of FAK, nor did EDTA-mediated inactivation of the integrin. Full FAK activation was observed, since phosphorylation was not only confirmed in residue Y397, but also in residues Y576/7. Furthermore, initiation of downstream signaling by paxillin phosphorylation in residue Y118 was evident, even though this activation was transient by nature, probably due to the lack of talin involvement in FAK activation and the absence of vinculin in the adhesion complexes formed by the nonactivated integrins. Altogether these results indicate that the nonactivated integrins can induce cellular signaling, but the outcome of the signaling differs from conventional integrin signaling.

Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α 1 β 1 , α 2 β 1 , α 10 β 1 and α 11 β 1 , were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α 1 and α 2 subunits. Integrin α 2 β 1 -mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance., Statement of Significance: Carbodiimide stabilised collagen is employed extensively for the fabrication of biologically active materials. Despite this common usage, the effect of carbodiimide crosslinking on cell-collagen interactions is unclear. Here we have found that carbodiimide crosslinking of collagen inhibits native-like, whilst increasing non-native like, cellular interactions. We propose a mechanistic model in which carbodiimide modifies the carboxylic acid groups on collagen that are essential for cell binding. As such we feel that this research provides a crucial, long awaited, insight into the bioactivity of carbodiimide crosslinked collagen. Through the ubiquitous use of collagen as a cellular substrate we feel that this is fundamental to a wide range of research activity with high impact across a broad range of disciplines., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Many studies have shown that α 2 β 1 integrin plays an important role in the development of cardiac fibrosis. However, the mechanism of how α 2 β 1 integrin regulates the differentiation and proliferation of myofibroblasts in cardiac fibrosis through fibrillar collagen (FC) remains uncertain. We established that FC mimicked the 3-dimensional extracellular matrix (ECM) of fibroblasts from post-myocardial infarction (MI) patients in vivo. This allowed us to explore the differentiation and proliferation of cardiac fibroblasts on FC. Here, we report that low expression of α 2 β 1 integrin increased protein kinase B (AKT) activation and α -smooth muscle actin ( α -SMA) expression. This occurred due to the instability of phosphatase and tensin homolog (PTEN) in myofibroblasts on FC. We also demonstrated that FC reduced protein phosphatase type 2A (PP2A) activity of myofibroblasts, which was coincident with low α 2 β 1 integrin expression and activation of AKT, but not mitogen-activated protein kinase (ERK). In addition, knock-down of both β 1 integrin and PP2A in fibroblasts promoted differentiation and proliferation via AKT activation and increased α -SMA expression. In summary, our study demonstrated that low α 2 β 1 integrin expression regulated its downstream targets PTEN and AKT via crosstalk with PP2A, a critical cell signaling pathway that permits aberrant differentiation and proliferation of myofibroblasts on FC., Competing Interests: The authors have no potential conflict of interests to declare.

Integrins are transmembrane receptors composed of one α subunit and one β subunit and are involved in cellular growth, differentiation, and apoptosis. The collagen-binding integrins α1β1 and α2β1 have been shown to regulate wound and tumor vascularization by different mechanisms. In this study, we assessed wound and tumor vascularization in mice with genetic ablation of both integrin subunits α1 and α2, which resulted in loss of integrins α1β1 and α2β1. Wound angiogenesis was investigated in excisional wounds that were inflicted on the back skin of control and mice lacking integrin α1β1 and α2β1. Mutant mice displayed reduced wound angiogenesis, which correlated with decreased macrophage numbers at 3 and 7 days after injury, and showed significantly attenuated vascularization of sponge implants. Angiogenesis induced by tumors arising from intradermal injection of B16 F1 melanoma cells was also reduced in comparison to controls 7 days after injection. This reduction in angiogenesis correlated with increased levels and activity of circulating matrix metalloproteinase 9 and elevated angiostatin levels in plasma of mutant mice, which reduced endothelial cell proliferation. Ex vivo mutant aortic ring explants developed significantly fewer and thinner aortic sprouts with fewer branch points than controls because of impaired endothelial cell proliferation. In conclusion, the loss of integrins α1β1 and α2β1 in mice results in reduced wound and tumor angiogenesis by cell-autonomous and extrinsic mechanisms., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is characterized by neuronal death, neurofibrillary tangles, and senile plaques. Amyloid-beta (Aβ) is the major component of plaques and consists of two prominent isoforms, Aβ40 and Aβ42. As many risk factors for AD are vascular in origin and blood vessel defects in clearing Aβ from the brain are a potential key component of AD pathology, we have focused on the neuron-blood vessel interface, and in particular, the vascular basement membrane, which coats blood vessels and physically separates them from neurons. A prominent component of the vascular basement membrane is the extracellular matrix proteoglycan perlecan. Domain V (DV) is the C-terminal domain and is generated by perlecan proteolysis. DV interacts with the α2 integrin and Aβ is a ligand for both α2β1 and αvβ1. Due to the known interaction of DV with α2β1 and α2β1's requirement for Aβ deposition and neurotoxicity, we hypothesized that DV and/or its C-terminal domain, LG3, might alter neurotoxic signaling pathways by directly blocking or otherwise interfering with α2β1 binding by Aβ. Our study suggests that α2β1 mediates Aβ-induced activation of c-Jun and caspase-3, key components of the neurotoxic pathway, in primary cortical and hippocampal neurons. We further demonstrate that DV and/or LG3 may therapeutically modulate these α2β1 mediated neurotoxic effects suggesting that they or other α2β1 integrin modulators could represent a novel approach to treat AD. Finally, our results suggest different neurotoxicity susceptibilities between cortical and hippocampal neurons to Aβ40 and Aβ42 as further underscored by differing neuroprotective potencies of LG3 in each cell type.

The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: : The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair., Significance: Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair., (©AlphaMed Press.)

Unlabelled: The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2β1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment., Statement of Significance: Natural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Lysyl oxidase (LOX) catalyzes crosslink formation between fibrillar collagens and elastins and an increase in LOX activity has been associated with cardiac fibrosis following myocardial infarction (MI). It has been previously reported that LOX expression is regulated by growth factors and cytokines including transforming growth factor (TGF-β1); however, it is unclear how the biophysical and biochemical properties of the cellular microenvironment affect LOX expression. In this study, we isolated rat cardiac fibroblasts (CF) and infarct cardiac fibroblasts (ICF), from healthy and 1-week post-MI left ventricular tissue respectively, and cultured them under varied substrate conditions in vitro to assess their influence on LOX expression. Culture of ICF on collagen I-coated plates increased LOX expression versus uncoated plates with an additional increase observed with the presence of TGF-β1. To further investigate the effect of integrin interactions with collagen I on LOX expression, we inhibited the α2β1 integrin from binding to collagen I and found gene and protein expression of LOX to be downregulated. Together, this demonstrates that the interaction of α2β1 integrin to collagen I in the cellular microenvironment can regulate expression of LOX. Further studies investigating additional integrin interactions may identify therapeutic targets for treating cardiac fibrosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

High expression of the collagen receptor, α2β1 integrin, on platelets of ITGA2 807T-allele carriers has been identified as a risk factor for thromboembolic conditions, and α2β1 inhibitors are considered to be potential therapeutic agents. In 59 genotyped individuals, we measured α2 expression levels on platelets and analyzed platelet adhesion to collagen under flow conditions. A sulfonamide-type small-molecule inhibitor of α2β1 integrin decreased average platelet adhesion in individuals with the C/T807T genotype but not in those harboring C807C. Thus, genotype can be used to select a human subpopulation that has the highest probability of showing a positive response to α2β1 inhibitors.

An antithrombotic nanoconjugate was designed in which a designed biomimetic peptide LWWNSYY was immobilized to the surface of poly(glycidyl methacrylate) nanoparticles (PGMA NPs). Our previous work has demonstrated LWWNSYY to be an effective inhibitor of integrin α2β1-collagen interaction and subsequent thrombus formation, however its practical application suffered from the formation of clusters in physiological environment caused by its high hydrophobicity. In our present study, the obtained LWWNSYY-PGMA nanoparticles (L-PGMA NPs) conjugate, with an improved dispersibility of LWWNSYY by PGMA NPs, have shown binding to collagen receptors with a Kd of 3.45 ± 1.06 μM. L-PGMA NPs have also proven capable of inhibiting platelet adhesion in vitro with a reduced IC50 of 1.83 ± 0.29 μg/mL. High inhibition efficiency of L-PGMA NPs in thrombus formation was further confirmed in vivo with a 50% reduction of thrombus weight. Therefore, L-PGMA NPs were developed as a high-efficiency antithrombotic nanomedicine targeted for collagen exposed on diseased blood vessel wall.

Variants in CIB2 can underlie either Usher syndrome type I (USH1J) or nonsyndromic hearing impairment (NSHI) (DFNB48). Here, a novel homozygous missense variant c.196C>T and compound heterozygous variants, c.[97C>T];[196C>T], were found, respectively, in two unrelated families of Dutch origin. Besides, the previously reported c.272 T>C functional missense variant in CIB2 was identified in two families of Pakistani origin. The missense variants are demonstrated not to affect subcellular localization of CIB2 in vestibular hair cells in ex vivo expression experiments. Furthermore, these variants do not affect the ATP-induced calcium responses in COS-7 cells. However, based on the residues affected, the variants are suggested to alter αIIβ integrin binding. HI was nonsyndromic in all four families. However, deafness segregating with the c.272T>C variant in one Pakistani family is remarkably less severe than that in all other families with this mutation. Our results contribute to the insight in genotype-phenotype correlations of CIB2 mutations.

Living tissues or organ modules consist of different types of highly organized cells and extracellular matrices (ECMs) in a hierarchical manner, such as the multilayered structure of blood vessels and the radial structures of hepatic lobules. Due to animal examinations being banned in the EU since 2013 and a shortage in the demand for tissue repair or organ transplantation, the creation of artificial 3D tissues possessing specific structures and functions similar to natural tissues are key challenges in tissue engineering. To date, we have developed a simple but unique bottom-up approach, a hierarchical cell manipulation technique, with a nanometer-sized ECM matrix consisting of fibronectin (FN) and gelatin (G) on cell surfaces. About 10 nm thick FN/G ECM films on cell surfaces were coated successfully by using layer-by-layer coating methodology. Various 3D constructs with higher cell density with different types of cells were successfully constructed. In addition to the construction of tissues with higher cell densities, other tissues, such as cartilage or skin tissues, with different cell densities are also important tissue models for tissue engineering and pharmaceutical industries. Thus, we recently developed other methodologies, the collagen coating method and multiple coating method, to fabricate micrometer-sized level ECM layers on cell surfaces. Various micro- or millimeter-sized 3D constructs with lower cell densities were constructed successfully. By using these two methods, cell distances in 2D or 3D views can be controlled by different thicknesses of ECM layers on cell surfaces at the single-cell level. Both FN/G and the collagen coating method resulted in homogenous 3D tissues with a controlled layer numbers, cell type, cell location, and properties; these will be promising to achieve different goals in tissue engineering., (© 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vascularization of bone defects is considered a crucial component to the successful regeneration of large bone defects. Although vascular endothelial growth factor (VEGF) has been delivered to critical-size bone defect models to augment blood vessel infiltration into the defect area, its potential to increase bone repair remains ambiguous. In this study, we investigated whether integrin-specific biomaterials modulate the effects of VEGF on bone regeneration. We engineered protease-degradable, VEGF-loaded poly(ethylene glycol) (PEG) hydrogels functionalized with either a triple-helical, α2 β1 integrin-specific peptide GGYGGGP(GPP)5 GFOGER(GPP)5 GPC (GFOGER) or an αv β3 integrin-targeting peptide GRGDSPC (RGD). Covalent incorporation of VEGF into the PEG hydrogel allowed for protease degradation-dependent release of the protein while maintaining VEGF bioactivity. When applied to critical-size segmental defects in the murine radius, GFOGER-functionalized VEGF-free hydrogels exhibited significantly increased vascular volume and density and resulted in a larger number of thicker blood vessels compared to RGD-functionalized VEGF-free hydrogels. VEGF-loaded RGD hydrogels increased vascularization compared to VEGF-free RGD hydrogels, but the levels of vascularization for these VEGF-containing RGD hydrogels were similar to those of VEGF-free GFOGER hydrogels. VEGF transiently increased bone regeneration in RGD hydrogels but had no effect at later time points. In GFOGER hydrogels, VEGF did not show an effect on bone regeneration. However, VEGF-free GFOGER hydrogels resulted in increased bone regeneration compared to VEGF-free RGD hydrogels. These findings demonstrate the importance of integrin-specificity in engineering constructs for vascularization and associated bone regeneration., (© 2016 Wiley Periodicals, Inc.)

Gleditsia sinensis thorns (GST) have been used as a traditional medicine for carbuncles and skin diseases. The purpose of this study was to decide whether non-toxicological levels of water extract of GST (WEGST) are effective in inhibiting the progress of prostate cancer formation and to identify the target molecule involved in the WEGST-mediated inhibitory process of prostate cancer cell migration and in vivo tumor formation. Through the Boyden chamber migration assay, we found that non-toxic levels of WEGST could not attenuate the PC3 migration to the bottom area coated with serum but significantly inhibited PC3 cell migration to the collagen-coated bottom area. We also found that non-toxic levels of WEGST significantly attenuated collagen against adhesion. Interestingly, ectopic administration of WEGST could not affect the expression of α2β1 integrin, which is known as a receptor of collagen. However, when the PC3 cells adhered to a collagen-coated plate, the expression of α2 integrin but not that of β1 integrin was significantly inhibited by the administration of non-toxic levels of WEGST, leading to the inhibition of focal adhesion kinase (FAK) phosphorylation. Furthermore, oral administration of WEGST (25 mg/kg/day) significantly inhibited the size of a PC3 cell-xenografted tumor. Taken together, these results suggest a novel molecular mechanism for WEGST to inhibit prostate cancer progression at particular stages, such as collagen-mediated adhesion and migration, and it might provide further development for the therapeutic use of WEGST in the treatment of prostate cancer progression.

Cancer metastasis is the major cause of cancer-related death. Chemoprevention is defined as the use of natural or synthetic substances to prevent cancer formation or cancer progress. In the present study, we investigate the antitumor activity of esculin and its oligomer fractions in U87 glioblastoma cells. We showed that esculin and its oligomers reduced U87 cell growth in a dose dependent manner. They also inhibited cell adhesion to collagen IV and vitronectin by interfering with the function of their respective receptors α2β1 and αvβ5 integrins. Furthermore, the tested samples were able to reduce migration of U87 cells towards another extracellular matrix fibronectin. Moreover, esculin and its oligomer fractions inhibited in vitro angiogenesis of endothelial cells (HMEC-1). In summary, our data provide the first evidence that esculin and its oligomer fractions are able to reduce adhesion, migration of glioblastoma cells and in vitro angiogenesis. Esculin and its oligomers may thus exert multi-target functions against cancer cells.

Background: Viperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential., Methods: Bar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function., Results: Bar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC50=1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation., Conclusion: A non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function., General Significance: This study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The question of how cells sense substrate mechanical cues has gained increasing attention among biologists. By introducing contour-based data analysis to single-cell force spectroscopy, we identified a loading-rate threshold for the integrin α2β1-DGEA bond beyond which a dramatic increase in bond lifetime was observed. On the basis of mechanical cues (elasticity or topography), the effective spring constant of substrates k is mapped to the loading rate r under actomyosin pulling speed v, which, in turn, affects the lifetime of the integrin-ligand bond. Additionally, downregulating v with a low-dose blebbistatin treatment promotes the neuronal lineage specification of mesenchymal stem cells on osteogenic stiff substrates. Thus, sensing of the loading rate is central to how cells sense mechanical cues that affect cell-extracellular matrix interactions and stem cell differentiation.

Background/aims: Plasma gelsolin (pGSN) is an actin-binding protein that plays a critical role in the pathogenesis of rheumatoid arthritis. However, whether pGSN is involved in other immunological diseases remains unknown. This study focused on the relationship between pGSN and immunoglobulin A (IgA) nephropathy (IgAN)., Methods: Two hundred patients with IgAN, 200 patients each with several other types of nephropathy and healthy controls (HCs) who underwent kidney biopsies between 2000 and 2014 were enrolled in the study. The Oxford classification system was used to predict the risk of disease progression. Serum and renal tissue were used to detect pGSN, and the correlations between pGSN and IgA, galactose-deficient IgA1 (Gd-IgA1), transforming growth factor beta1 (TGF-β1), fibronectin (FN) content, clinical symptoms, and kidney function were analyzed., Results: We found that the pGSN levels were significantly decreased in sera from IgAN patients compared to sera from patients with other forms of glomerular nephritis and HCs. Furthermore, the serum pGSN levels were negatively correlated with the serum IgA1, FN, and TGF-β1 levels, and positively correlated with the estimated glomerular filtration rate. Conversely, the glomerular pGSN content was significantly elevated in the IgAN patients and was positively correlated with TGF-β1 and FN levels. In renal tissue, the pGSN levels were significantly higher in IgAN patients with M1 and S1 compared to patients with M0 and S0 (p < 0.05). Meanwhile, pGSN promoted human mesangial cell (HMC) proliferation by facilitating cell mitosis in vitro. pGSN also promoted integrin α2β1 expression in HMCs and enhanced the integrin α2β1-pGSN interaction., Conclusion: Our study suggested that pGSN may play an important role in the development of IgAN by promoting the proliferation of mesangial cells and that serum and glomerular pGSN levels may be new markers for predicting IgAN progression and prognosis., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background and Objective: Burn is a public health problem, it causes physical disability even death. Treatment of burn wound has been conducted in various ways, but the satisfactory healing has not been provided. Bone marrow-derived mesenchymal stem cells (BM-MSCs) treatment is one of attempt to burn recovery, accelerate wound healing and angiogenesis. This study aimed to investigate the effect of allogeneic BM-MSC treatment on the expression of collagen type I and integrin α2β1 in burn skin tissue of rat observed on day 14., Materials and Methods: Twelve Wistar rats divided into two groups, control group (injected with phospate buffer solution) and treatment group (injected with BM-MSC). Rat was anaesthetized with xylazine and ketamine (ratio 1:1), fur of rat's back was shaved and full thickness burn was made by boiling plate in hot water for 30 min and patched on the back for 20 min. The burns were covered by tegaderm film and elastomult haft. Antalgin as an analgetic was injected to rats during observation process. Burns of rat was observed on day 14. In this study one-way analysis of variance test and Tukey as a further test were analyzed., Results: The results showed that the healing time of allogeneic BM-MSC treatment on burn skin tissue rats was faster, the thickness of collagen type I in burn skin tissue of rats was thicker (0.977 μm) than controls (0.475 μm) and statistically demonstrated significant differences (p = 0.000). The average percentage of integrin α2β1 expression was higher (2.94%) than control group (2.34%), but the differences were not statistically significant (p = 0.176)., Conclusion: The study concluded that BM-MSC treatment was able to accelerate the healing process of burns by increasing the thickness of the collagen and the percentage of integrin α2β1, thus accelerated the cell migration involved during wound healing.

Extracellular matrix, via its receptors the integrins, has emerged as a crucial factor in cancer development. The α2β1 integrin is a major collagen receptor that is widely expressed and known to promote cell migration and control tissue homeostasis. Growing evidence suggests that it can be a key pathway in cancer. Recent studies have shown that α2β1 integrin is a regulator of cancer metastasis either by promoting or inhibiting the dissemination process of cancer cells. The α2β1 integrin signaling can also enhance tumor angiogenesis. Emerging evidence supports a role for α2β1 integrin in cancer chemoresistance especially in hematological malignancies originating from the T cell lineage. In addition, α2β1 integrin has been associated with cancer stem cells. In this review, we will discuss the complex role of α2β1 integrin in these processes. Collagen is a major matrix protein of the tumor microenvironment and thus, understanding how α2β1 integrin regulates cancer pathogenesis is likely to lead to new therapeutic approaches and agents for cancer treatment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Atherothrombosis underlies acute coronary syndromes, including unstable angina and acute myocardial infarction. Within the unstable plaque, monocytes express collagenolytic matrix metalloproteinases (MMPs), including MMP-13, which degrades fibrous collagen. Following rupture, vessel wall components including degraded collagen are exposed to circulating platelets. Platelet receptors then mediate the recruitment and activation of platelets to form a thrombus, blocking blood flow and resulting in myocardial infarction and sudden death., Objectives: Here we aim to provide information on the effects of collagen degradation on platelet adhesion and thrombus formation., Methods: Using increasing concentrations of MMP-13, we induced progressive degradation of fibrous and monomeric collagen I, visualized by electrophoresis, and then investigated the capacity of the resulting fragments to support static platelet adhesion and thrombus formation in whole flowing blood., Results: Both integrin and glycoprotein VI-dependent interactions with fibrous collagen underpin high levels of platelet adhesion under both conditions, with little obvious effect of MMP-13 treatment. Static platelet adhesion to monomeric collagen was strongly α2β1-dependent regardless of degradation status. Under flow conditions, partially degraded monomeric collagen supported increased thrombus deposition at 10 μg mL(-1) MMP-13, falling close to background when collagen degradation was complete (100 μg mL(-1) MMP-13)., Conclusions: New binding activities come into play after partial digestion of collagen monomers, and net platelet-reactivity through all axes is abolished as degradation becomes more complete., (© 2015 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: Malignant transformation of melanocytes frequently coincides with an alteration in the expression of cell-cell adhesion molecules (cadherins) and cell-extracellular matrix proteins (integrins). How these two adhesion systems interplay to impact on cell invasion remains to be described in melanoma., Methods: Cell adhesion networks were localised by immunofluorescence in human primary cutaneous melanoma, metastatic melanoma in the lymph nodes, and melanoma cell lines. The role of these cell adhesion networks was assessed both in vivo, by analysing their impact on tumour growth in mice, and in vitro, with the use of functional tests including cell aggregation and cell migration., Results: We found that α2β1 integrin associates with both E-cadherin and N-cadherin to form two adhesive networks, distinguishable by the interaction-or not-of α2β1 integrin with type I collagen. N-cadherin/α2β1 integrin and E-cadherin/α2β1 integrin networks differently participated towards tumour growth in mice. The N-cadherin/α2β1 integrin network showed specific involvement in melanoma cell invasion and migration towards type I collagen. On the other hand, the E-cadherin/α2β1 network regulated cell-cell adhesion., Conclusions: This suggests that different signalling environments can be generated, depending on the type and/or local concentration of cadherin present in the adhesion complex, which potentially leads to differential cell responses. Further clarification of how these adhesive networks are regulated is fundamental to understanding important physiological and pathological processes such as morphogenesis, wound healing, tumour invasion and metastasis.

Targeting nanoparticles to desired intracellular compartments is a major challenge. Integrin-type adhesion receptors are connected to different endocytosis routes in a receptor-specific manner. According to our previous observations, the internalization of an α2β1-integrin-echovirus-1 complex takes place via a macropinocytosis-like mechanism, suggesting that the receptor could be used to target nanoparticles to this specific entry route. Here, silica-based nanoparticles, carrying monoclonal antibodies against the α2β1 integrin as address labels, were synthesized. Studies with flow cytometry, atomic force microscopy and confocal microscopy showed the particles to attach to the cell surface via the α2β1 integrin. Furthermore, quantitative analysis of nanoparticle trafficking inside the cell performed with the BioImageXD software indicated that the particles enter cells via a macropinocytosis-like process and end up in caveolin-1 positive structures. Thus, we suggest that different integrins can guide particles to distinct endocytosis routes and, subsequently, also to specific intracellular compartments. In addition, we show that with the BioImageXD software it is possible to conduct sensitive and complex analyses of the behavior of small fluorescent particles inside cells, using basic confocal microscopy images.

Background: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFβ signaling and angiogenesis in vitro and in vivo., Methods: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFβ pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided., Results: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2β1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFβ signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFβ markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFβ markers, n = 6; VEGF, n = 21). Inhibition of TGFβ receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTβRII = 29.7, SD = 6.1, P = .02)., Conclusions: Type VII collagen suppresses TGFβ signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

To investigate E-cadherin, β-catenin, and α2β1 and α3β1 integrins in 40 samples of non-metastatic and metastatic oral squamous cell carcinoma (OSCC) with positive cervical lymph nodes (LN). Immunohistochemistry was used to evaluate expression in the lesion center (LC) and invasive tumor front (ITF) of non-metastatic (n=18) and metastatic (n=22) OSCC and in the LN on the metastatic neoplastic cells (MNC; n=22). In metastatic OSCC, E-cadherin and β-catenin presented significantly lower cytoplasmic membrane expression in the ITF and MNC when compared to the LC and lower cytoplasmic expression in MNC when compared to the LC and ITF (p<0.05). Integrins α2β1 and α3β1 showed high cytoplasmic expression in the LC, ITF and MNC (p>0.05). A positive correlation was observed between E-cadherin cytoplasmic expression and α2β1 (p=0.860) and α3β1 (p=0.975) expression. When comparing the primary sites of metastatic and non-metastatic disease, β-catenin presented lower cytoplasmic membrane (p=0.013) expression in metastatic OSCC. E-cadherin presented low expression and the integrins high expression in both groups. Abnormal expression of β-catenin and E-cadherin associated with high expression of α2β1 and α3β1 integrins contribute to LN metastasis in OSCC.

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I., (Copyright © 2015. Published by Elsevier B.V.)

Unlabelled: Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg . mL(-1) and 10 μg . mL(-1) of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy., Trial Registration: ClinicalTrials.gov NCT00763893.

Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbβ3 and α2β1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.

Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin α2β1 was examined. The results showed β1 and α2β1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin α2β1., (© 2014 Wiley Periodicals, Inc.)

Transplant vasculopathy is associated with neointimal accumulation of recipient-derived mesenchymal stem cells. Increased circulating levels of LG3, a C-terminal fragment of perlecan, were found in renal transplant patients with vascular rejection. Here, we evaluated whether LG3 regulates the migration and homing of mesenchymal stem cells and the accumulation of recipient-derived neointimal cells. Mice were transplanted with a fully-MHC mismatched aortic graft followed by intravenous injection of recombinant LG3. LG3 injections increased neointimal accumulation of α-smooth muscle actin positive cells. When green fluorescent protein (GFP)-transgenic mice were used as recipients, LG3 injection favored accumulation of GFP+ cells to sites of neointima formation. LG3 increased horizontal migration and transmigration of mouse and human MSC in vitro and led to increased ERK1/2 phosphorylation. Neutralizing β1 integrin antibodies or use of mesenchymal stem cells from α2 integrin-/- mice decreased migration in response to recombinant LG3. Reduced intima-media ratios and decreased numbers of neointimal cells showing ERK1/2 phosphorylation were found in α2-/- recipients injected with recombinant LG3. Collectively, our results suggest that LG3, through interactions with α2β1 integrins on recipient-derived cells leading to activation of ERK1/2 and increased migration, favors myointimal thickening., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Purpose: Many epidemiological studies have evaluated associations of platelet glycoprotein receptor alpha2beta1 integrin (ITGA2) and glycoprotein IIIa (ITGB3) gene polymorphisms with diabetic retinopathy (DR), but the published data are inconclusive. The aim of the present study was to assess the associations by using meta-analysis., Methods: A comprehensive electronic search (PubMed, EMBASE, Elsevier Science Direct, CNKI and Wanfang) was carried out until 31 August 2013. Odds ratios (ORs) and its 95% confidence intervals (CIs) were used to assess the strength of the associations., Results: Nine studies including 1678 cases and 1329 controls were included in the meta-analysis. Meta-analysis was performed for ITGA2 gene BgI II polymorphism (7 studies including 758 cases and 570 controls) and ITGB3 gene PlA1/A2 polymorphism (4 studies including 1047 cases and 861 controls). Significant associations were found for BgI II (+ versus -: OR = 1.42, 95% CI = 1.06-1.90, p = 0.02; +/- + +/+ versus -/-: OR = 1.46, 95% CI = 0.99-2.15, p = 0.06; +/+ versus -/- + +/-: OR = 1.90, 95% CI = 1.35-2.67, p = 0.0003) and PlA1/A2 (A2 versus A1: OR = 0.74, 95% CI = 0.52-1.07, p = 0.11; A1A2 + A2A2 versus A1A1: OR = 0.80, 95% CI = 0.64-0.99, p = 0.04; A2A2 versus A1A1 + A1A2: OR = 0.45, 95% CI = 0.27-0.75, p = 0.002) polymorphisms., Conclusion: This meta-analysis demonstrates that DR is associated with ITGA2 BgI II and ITGB3 PlA1/A2 polymorphisms.

cyclicCHAD is a peptide representing the α2β1 integrin binding sequence of the matrix protein chondroadherin (CHAD), which in our hands proved effective at counteracting bone loss in ovariectomised mice by inhibiting osteoclastogenesis. Given that bone metastases are characterised by exacerbated osteoclast activity as well, we tested this therapy in mice intracardiacally injected with the osteotropic human breast cancer cell line MDA-MB-231. Treatment with cyclicCHAD significantly decreased cachexia and incidence of bone metastases, and induced a trend of reduction of visceral metastasis volume, while in orthotopically injected mice cyclicCHAD reduced tumour volume. In vitro studies showed its ability to impair tumour cell motility and invasion, suggesting a direct effect not only on osteoclasts but also on the tumour cell phenotype. Interestingly, when administered together with a suboptimal, poorly effective, dose of doxorubicin (DXR), cyclicCHAD improved survival and reduced visceral metastases volume to a level similar to that of the optimal dose of DXR alone. Taken together, these preclinical data suggest that cyclicCHAD is a new inhibitor of bone metastases, with an appreciable direct effect also on tumour growth and a synergistic activity in combination with low dose chemotherapy, underscoring an important translational impact., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.

Little is known about the mechanism of integrin activation by cadherin 17 (CDH17). Here we observed the presence of a tri-peptide motif, RGD, in domain 6 of the human CDH17 sequence and other cadherins such as cadherin 5 and cadherin 6. The use of CDH17 RAD mutants demonstrated a considerable decrease of proliferation and adhesion in RKO and KM12SM colon cancer cells. Furthermore, RGD peptides inhibited the adhesion of both cell lines to recombinant CDH17 domain 6. The RGD motif added exogenously to the cells provoked a change in β1 integrin to an active, high-affinity conformation and an increase in focal adhesion kinase and ERK1/2 activation. In vivo experiments with Swiss nude mice demonstrated that cancer cells expressing the CDH17 RAD mutant showed a considerable delay in tumor growth and liver homing. CDH17 RGD effects were also active in pancreatic cancer cells. Our results suggest that α2β1 integrin interacts with two different ligands, collagen IV and CDH17, using two different binding sites. In summary, the RGD binding motif constitutes a switch for integrin pathway activation and shows a novel capacity of CDH17 as an integrin ligand. This motif could be targeted to avoid metastatic dissemination in tumors overexpressing CDH17 and other RGD-containing cadherins., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ-/-) or the complex was depleted. The development of α2β1-/- and GPVI-/- mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro., Approach and Results: To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1-/-, FcRγ-/-, and GPVI-/- mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ-/- platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI-/- and wild type platelets. The difference between FcRγ-/- and GPVI-/- platelet phosphotyrosine levels correlated with the in vivo thrombosis findings., Conclusion: Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings suggest a more complex signaling network downstream of the platelet collagen receptors than previously appreciated.

Cellular uptake of clustered α2β1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.), in contrast to the control MVBs without virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Altogether, the data show marked morphological changes in size and permeability of the endosomes in the infectious entry pathway of this non-enveloped enterovirus and suggest that the formed breakages facilitate the transfer of the genome to the cytoplasm for replication.

Multifunctional nanoprobes open exciting possibilities for accurate diagnosis and therapy. In this research, we developed a (64)Cu-labeled GdVO4:4%Eu two-dimension (2D) tetragonal ultrathin nanosheets (NSs) that simultaneously possess radioactivity, fluorescence, and paramagnetic properties for multimodal imaging. The carboxyl-functionalized Eu(3+)-doped GdVO4 NSs were synthesized by a facile solvothermal reaction, followed by ligand exchange with polyacrylic acid (PAA). With ultrathin thickness of ∼5 nm and width of ∼150 nm, the carboxyl-functionalized NSs were further modified by DOTA chelator for (64)Cu labeling and Asp-Gly-Glu-Ala (DGEA) peptide for integrin α2β1 targeting. After initial evaluation of the cytotoxicity and targeting capability with PC-3 cells, the obtained multifunctional nanoprobes ((64)Cu-DOTA-GdVO4:4%Eu-DGEA) were further explored for targeted positron emission tomography (PET) and T1-weighted magnetic resonance imaging (MRI) of PC-3 tumor (prostate cancer, high integrin α2β1 expression) in vivo. Based on the strong fluorescence of the NSs, the particle distribution in mouse tissues was also determined by fluorescent microscopy. In summary, GdVO4:4%Eu NS is a potential multimodal multiscale nanoprobe that could not only be used for in vivo imaging, but also be tracked in cellular scale and ex vivo due to its fluorescent property., (Copyright © 2014 Elsevier Ltd. All rights reserved.)