Extracellular Matrix Adhesion Proteins Research Articles

Identification of Rare Genetic Variants in Familial Spontaneous Coronary Artery Dissection and Evidence for Shared Biological Pathways.

Rare familial spontaneous coronary artery dissection (SCAD) kindreds implicate genetic disease predisposition and provide a unique opportunity for candidate gene discovery. Whole-genome sequencing was performed in fifteen probands with non-syndromic SCAD who had a relative with SCAD, eight of whom had a second relative with extra-coronary arteriopathy. Co-segregating variants and associated genes were prioritized by quantitative variant, gene, and disease-level metrics. Curated public databases were queried for functional relationships among encoded proteins. Fifty-four heterozygous coding variants in thirteen families co-segregated with disease and fulfilled primary filters of rarity, gene variation constraint, and predicted-deleterious protein effect. Secondary filters yielded 11 prioritized candidate genes in 12 families, with high arterial tissue expression (n = 7), high-confidence protein-level interactions with genes associated with SCAD previously (n = 10), and/or previous associations with connective tissue disorders and aortopathies (n = 3) or other vascular phenotypes in mice or humans (n = 11). High-confidence associations were identified among 10 familial SCAD candidate-gene-encoded proteins. A collagen-encoding gene was identified in five families, two with distinct variants in COL4A2. Familial SCAD is genetically heterogeneous, yet perturbations of extracellular matrix, cytoskeletal, and cell-cell adhesion proteins implicate common disease-susceptibility pathways. Incomplete penetrance and variable expression suggest genetic or environmental modifiers.

Urinary microRNA in Diabetic Kidney Disease: A Literature Review.

Diabetic kidney disease is the most common primary disease of end-stage kidney disease globally; however, a sensitive and accurate biomarker to predict this disease remains awaited. microRNAs are endogenous single-stranded noncoding RNAs that have intervened in different post-transcriptional regulations of various cellular biological functions. Previous literatures have reported its potential role in the pathophysiology of diabetic kidney disease, including regulation of Transforming Growth Factor-β1-mediated fibrosis, extracellular matrix and cell adhesion proteins, cellular hypertrophy, growth factor, cytokine production, and redox system activation. Urinary microRNAs have emerged as a novel, non-invasive liquid biopsy for disease diagnosis. In this review, we describe the available experimental and clinical evidence of urinary microRNA in the context of diabetic kidney disease and discuss the future application of microRNA in routine practice.

Collective rotational motion of freely expanding T84 epithelial cell colonies

Coordinated rotational motion is an intriguing, yet still elusive mode of collective cell migration, which is relevant in pathological and morphogenetic processes. Most of the studies on this topic have been carried out on epithelial cells plated on micropatterned substrates, where cell motion is confined in regions of well-defined shapes coated with extracellular matrix adhesive proteins. The driver of collective rotation in such conditions has not been clearly elucidated, although it has been speculated that spatial confinement can play an essential role in triggering cell rotation. Here, we study the growth of epithelial cell colonies freely expanding (i.e. with no physical constraints) on the surface of cell culture plates and focus on collective cell rotation in such conditions, a case which has received scarce attention in the literature. One of the main findings of our work is that coordinated cell rotation spontaneously occurs in cell clusters in the free growth regime, thus implying that cell confinement is not necessary to elicit collective rotation as previously suggested. The extent of collective rotation was size and shape dependent: a highly coordinated disc-like rotation was found in small cell clusters with a round shape, while collective rotation was suppressed in large irregular cell clusters generated by merging of different clusters in the course of their growth. The angular motion was persistent in the same direction, although clockwise and anticlockwise rotations were equally likely to occur among different cell clusters. Radial cell velocity was quite low as compared to the angular velocity, in agreement with the free expansion regime where cluster growth is essentially governed by cell proliferation. A clear difference in morphology was observed between cells at the periphery and the ones in the core of the clusters, the former being more elongated and spread out as compared to the latter. Overall, our results, to our knowledge, provide the first quantitative and systematic evidence that coordinated cell rotation does not require a spatial confinement and occurs spontaneously in freely expanding epithelial cell colonies, possibly as a mechanism for the system.

Continual proteomic divergence of HepG2 cells as a consequence of long-term spheroid culture

Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may influence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to define the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the influence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profiling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confidently identified, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.

Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.

Nanostructured scaffolds based on bioresorbable polymers and graphene oxide induce the aligned migration and accelerate the neuronal differentiation of neural stem cells

Within the field of neural tissue engineering, there is a huge need for the development of materials that promote the adhesion, aligned migration and differentiation of stem cells into neuronal and supportive glial cells. In this study, we have fabricated bioresorbable elastomeric scaffolds combining an ordered nanopatterned topography together with a surface functionalization with graphene oxide (GO) in mild conditions. These scaffolds allowed the attachment of murine neural stem cells (NSCs) without the need of any further coating of its surface with extracellular matrix adhesion proteins. The NSCs were able to give rise to both immature neurons and supporting glial cells over the nanostructured scaffolds in vitro, promoting their aligned migration in cell clusters following the nanostructured grooves. This system has the potential to reestablish spatially oriented neural precursor cell connectivity, constituting a promising tool for future cellular therapy including nerve tissue regeneration.

Three-Dimensional Human Liver-Chip Emulating Premetastatic Niche Formation by Breast Cancer-Derived Extracellular Vesicles.

The liver is one of the most common sites of breast cancer metastasis and is associated with high lethality. Although the interaction between tumor cells and their microenvironment at metastatic sites has been recognized as a key regulator of tumor progression, the underlying mechanism is not fully elucidated. Here, we describe a three-dimensional (3D) microfluidic human liver-on-a-chip (liver-chip) that emulates the formation of a premetastatic niche to investigate the roles of breast cancer-derived extracellular vesicles (EVs) in liver metastasis. We demonstrate that breast cancer-derived EVs activate liver sinusoidal endothelial cells (LSECs) in the liver-chip, inducing endothelial to mesenchymal transition and destruction of vessel barriers. In addition, we show that transforming growth factor β1 (TGFβ1) in breast cancer-derived EVs upregulates fibronectin, an adhesive extracellular matrix protein, on LSECs, which facilitates the adhesion of breast cancer cells to the liver microenvironment. Furthermore, we observed that EVs isolated from triple-negative breast cancer (TNBC) patients with liver metastasis contain higher TGFβ1 levels and induce adhesion of more breast cancer cells to the 3D human liver-chip than do EVs isolated from healthy donors or nonmetastatic TNBC patients. These findings provide a better understanding of the mechanisms through which breast cancer-derived EVs guide secondary metastasis to the liver. Furthermore, the 3D human liver-chip described in this study provides a platform to investigate the mechanisms underlying secondary metastasis to the liver and possible therapeutic strategies.

523 MITF-mediated extracellular matrix changes control intratumour heterogeneity in melanoma

Differential tumour cell behaviour caused by environmental conditions, termed dynamic heterogeneity, is a prime cause of drug resistance and understanding its underlying mechanisms is crucial to design effective therapies. We have demonstrated cell-cycling heterogeneity in both melanoma xenograft tumours and melanoma spheroids. This was characterized by the presence within the same tumour/spheroid of clusters of proliferating cells and clusters of G1-arrested cells. The location of the quiescent zones supported oxygen/nutrient deprivation as the cause of cell cycle arrest and the G1-arrested cells reversed to cycling when cultured under normoxia in 2D cultures. Here we demonstrate that this heterogeneity is consistently decreased in vivo and in vitro by high expression of MITF, a transcription factor strongly associated with melanoma development, progression and therapy response. This phenomenon is not associated with reduced hypoxia, indicating that high MITF expression allows a portion of hypoxic cells to maintain cell cycling despite oxygen deprivation. Conversely, modulation of MITF expression lead to changes of spheroid architecture and in extracellular matrix (ECM) and cell-ECM adhesion and crosstalk proteins. Furthermore, ECM swapping and inhibition of the Rho/ROCK signalling pathway, respectively, rescues and mimics the morphology and cell cycle effects of high MITF expression. These findings support a novel role of MITF in controlling intratumour melanoma heterogeneity through changes in cell-ECM crosstalk.

The effect of physiological stretch and the valvular endothelium on mitral valve proteomes.

This work is important to the field of heart valve pathophysiology as it provides new insights into molecular markers of mechanically induced valvular degeneration as well as the protective role of the valvular endothelium. These discoveries reported here advance our current knowledge of the valvular endothelium and how its removal essentially takes valve leaflets into an environmental shock. In addition, it shows that static conditions represent a mild pathological state for valve leaflets, while 10% cyclic stretch provides valvular cell quiescence. These findings impact the field by informing disease stages and by providing potential new drug targets to reverse or slow down valvular change before it affects cardiac function.

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMβ2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-β-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMβ2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 μg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 μg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

Zwitterionic PEG-PC Hydrogels Modulate the Foreign Body Response in a Modulus-Dependent Manner.

Reducing the foreign body response (FBR) to implanted biomaterials will enhance their performance in tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are increasingly popular for this application due to their low cost, ease of use, and the ability to tune their compliance via molecular weight and cross-linking densities. PEG hydrogels can elicit chronic inflammation in vivo, but recent evidence has suggested that extremely hydrophilic, zwitterionic materials and particles can evade the immune system. To combine the advantages of PEG-based hydrogels with the hydrophilicity of zwitterions, we synthesized hydrogels with comonomers PEG and the zwitterion phosphorylcholine (PC). Recent evidence suggests that stiff hydrogels elicit increased immune cell adhesion to hydrogels, which we attempted to reduce by increasing hydrogel hydrophilicity. Surprisingly, hydrogels with the highest amount of zwitterionic comonomer elicited the highest FBR. Lowering the hydrogel modulus (165 to 3 kPa), or PC content (20 to 0 wt %), mitigated this effect. A high density of macrophages was found at the surface of implants associated with a high FBR, and mass spectrometry analysis of the proteins adsorbed to these gels implicated extracellular matrix, immune response, and cell adhesion protein categories as drivers of macrophage recruitment. Overall, we show that modulus regulates macrophage adhesion to zwitterionic-PEG hydrogels, and demonstrate that chemical modifications to hydrogels should be studied in parallel with their physical properties to optimize implant design.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

New Type of Antitumor Agent Targeting the Cell Adhesion Molecule, Integrin.

The extracellular matrix (ECM) provides a variety of biological signals for cell regulation. It is apparent that some of these signals are derived from functional sites, which are concealed in the higher structure of ECM protein molecules. Previously, we found that fibronectin, a ubiquitous cell adhesive ECM protein, harbors a cryptic functional site termed FNIII14, which can be exposed through the processing of fibronectin by inflammatory proteinases, including matrix metalloproteinase-2 (MMP-2). FNIII14, once exposed, induces a conformational change in beta1-integrins necessary for their functional inactivation, resulting in weakened cell adhesion to the ECM. Interestingly, eukaryotic peptide elongation factor 1A (eEF1A) was recently identified as a membrane receptor mediating this anti-adhesive effect of FNIII14. Here, we show that exposure of FNIII14 from the fibronectin matrix, and its interaction with the membrane receptor eEF1A, contributes to the migration and invasion of cancer cells. Furthermore, an in vivo experiment using a mouse xenograft model shows that FNIII14 could be a promising target for preventing lung metastasis of melanoma.

Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse.

Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell-ECM adhesion proteins, and ECM degrading and maturating enzymes. Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P < .05); in POP-HVCs, MMP1, MMP3, and MMP10, ADAMTS8 and 13, tissue inhibitor of metalloproteinases (TIMPs) 1 to 3, ITGA2, ITGA4, ITGA6, ITGB1, contactin (CNTN1), catenins (A1 and B1), and laminins (A3 and C1) were significantly upregulated, whereas COLs (1, 4, 5, 6, 11, and 12) and LOXL1 were downregulated. Human vaginal cells massively secrete MMPs and TIMPs proteins; MMP1, MMP8, MMP9 protein expression and MMP2 gelatinase activity were increased, whereas TIMP2 decreased in SCM from POP-HVCs compared to control-HVCs. Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro mechanical stretch. Risk factors that induce stretch may alter ECM composition and cell-ECM interaction in pelvic floor tissue leading to the abatement of pelvic organ support and subsequent POP development.

Integrin Ligands with α/β-Hybrid Peptide Structure: Design, Bioactivity, and Conformational Aspects.

Integrins are cell surface receptors for proteins of the extracellular matrix and plasma-borne adhesive proteins. Their involvement in diverse pathologies prompted medicinal chemists to develop small-molecule antagonists, and very often such molecules are peptidomimetics designed on the basis of the short native ligand-integrin recognition motifs. This review deals with peptidomimetic integrin ligands composed of α- and β-amino acids. The roles exerted by the β-amino acid components are discussed in terms of biological activity, bioavailability, and selectivity. Special attention is paid to the synthetic accessibility and efficiency of conformationally constrained heterocyclic scaffolds incorporating α/β-amino acid span.

Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of cholesterol-fed rabbits.

The cholesterol-fed rabbit is commonly used as a model to study the vascular effects of hypercholesterolemia and resulting atherosclerotic lesions. Here we undertook a proteomic case-control investigation of ascending aortas from male New Zealand White rabbits after 10 weeks on a high-cholesterol (2% w/w) diet (HCD, n = 5) or control diet (n = 5), in order to determine the changes in response to the HCD. Histology confirmed intimal thickening in the HCD group consistent with atherosclerosis, and LC-MS/MS analysis of individually-obtained ascending aortic extracts labelled with isobaric (iTRAQ) tags enabled the identification and quantitation of 453 unique proteins above the 1% false discovery rate threshold. Of 67 proteins showing significant differences in relative abundance (p < 0.05), 62 were elevated and five decreased in ascending aortas from HCD-fed rabbits compared to controls. Six proteins were selected for validation using Multiple Reaction Monitoring, which confirmed the iTRAQ results. Many of the observed protein changes are consistent with known molecular perturbations in the ascending aorta that occur in response to hypercholesterolemia, e.g. elevation of tissue levels of apolipoproteins, extracellular matrix adhesion proteins, glycolytic enzymes, heat shock proteins and proteins involved in immune defense. We also made a number of novel observations, including a 15-fold elevation of glycoprotein (trans-membrane) nmb-like (Gpnmb) in response to HCD. Gpnmb has previously been linked to angiogenesis but not to atherosclerosis. This and additional novel observations merit further investigation as these perturbations may play important and as yet undiscovered roles in the pathogenesis of atherosclerosis in rabbits as well as humans.

Modeling of hypo/hyperglycemia and their impact on breast cancer progression related molecules.

Breast cancer (BC) arises commonly in women with metabolic dysfunction. The underlying mechanism by which glycemic load can exert its action on tumor metastasis is under investigated. In this study we showed that glycemic microenvironment alters the expression of three classes of proteins, VEGF and its receptors, cell to cell, and cell to extracellular matrix (ECM) adhesion proteins in MDA-MB-231 parental cells and its two metastatic variants to the bone and brain (MDA-MB-231BO and MDA-MB-231BR, respectively). Using western blotting, we showed that VEGFR2 levels were higher in these variant cells and persisted in the cells under extreme hypoglycemia. Hypoglycemia did not alter VEGFR2 expression per se but rather suppressed its posttranslational glycosylation. This was reversed rapidly upon the restoration of glucose, and cyclohexamide (CHX) treatment demonstrated that this deglycosylated VEGFR2 was not a product of de-novo protein synthesis. VEGFR2 co-receptor Neuropilin-1 was up-regulated four-fold in all MDA-MB-231 cells (parental and two variants) compared to VEGFR2 expression, and was also susceptible to glycemic changes but resistant to CHX treatment for up to 72 hrs. Hypoglycemia also resulted in a significant decrease in specific catenin, cadherin, and integrin proteins, as well as cellular proliferation and colony forming ability. However, MDA-MB-231BR cells showed a unique sensitivity to hypo/hyperglycemia in terms of morphological changes, colony formation ability, integrin β3 expression and secreted VEGF levels. In conclusion, this study can be translated clinically to provide insight into breast cancer cell responses to glycemic levels relevant for our understanding of the interaction between diabetes and cancer.

Characterization of spinal cord glial cells in a model of hindlimb unloading in mice

Exposure to microgravity has been shown to result in damaging alterations to skeletal muscle, bones, and inner organs. In this study, we investigated the effects of microgravity by using a hindlimb unloading model (HUM) in mice. The characteristics of the lumbar spinal cords of HUM mice 30days after hindlimb unloading were examined. Morphometric analysis showed reductions of the total area, gray matter, and white matter by 17%, 20%, and 12%, respectively. Myelinated fibers in the white matter showed prominent myelin destruction. Analysis of the number of glial fibrillary acidic protein (GFAP+)/S100 calcium-binding protein B (S100B−), GFAP+/S100B+, and GFAP−/S100B+ astrocytes in the ventral horn (VH), central channel area (CC), dorsal root entry zone (DREZ), main corticospinal tract (CST), and ventral funiculi (VF) showed that the number of GFAP+/S100B− astrocytes was increased in the DREZ and CST of HUM mice. Additionally, GFAP+/S100B+ cell numbers were significantly decreased in the VH and CST but did not differ in the CC or DREZ of HUM mice, as compared with the control. The numbers of GFAP−/S100B+ cells were significantly reduced only in the VH of HUM mice. Moreover, the number of ionized calcium-binding adaptor molecule 1 (Iba1+) microglia cells was significantly increased in the CC and DREZ of HUM mice. In control mice, homeobox protein HoxB8 (HoxB8+) cells were found only in the CC; in contrast, HoxB8+ cells were observed in all studied areas in HUM mice, with the greatest number found in the CC. Genome-wide transcriptome analysis of the lumbar spinal cords of HUM mice showed decreased expression of genes encoding myelin, extracellular matrix, cytoskeleton, and cell adhesion proteins. Real-time polymerase chain reaction (PCR) confirmed reductions in the expression of mpz, pmp2, pmp22, and prx genes, which are involved in myelination, as well as decreases in the levels of genes encoding extracellular matrix molecules, including glycoproteins (matrix gla protein (MGP), osteoglycin (OGN), microfibrillar associated protein 5 (MFAP), and collagen, type IV, alpha 1 (COL4A)), proteoglycans (perlecan (heparan sulfate proteoglycan) (HSPG)), and metalloproteinases (lysyl oxidase (LOX)). Thus, our results showed that hindlimb unloading caused decreases in gray and white matter areas, changes in gene expression, alterations in myelination, and phenotypic modifications in glial cells in the lumbar spinal cords of mice.

Identification of a novel collagen type І-binding protein from Streptococcus suis serotype 2

Streptococcus suis, a major pathogen of pigs, is an emerging zoonotic agent that causes meningitis and septic shock. cbp40 is a putative virulent gene that has been identified using suppression subtractive hybridization performed on the virulent S. suis serotype 2 strain HA9801 and the avirulent S. suis serotype 2 strain T15. Based on predicted protein features showing a shared conserved domain with the collagen-binding protein Cna of Staphylococcus aureus, Cbp40 is likely to function as a direct mediator of collagen adhesion. Here, the cbp40 gene was cloned and the recombinant protein purified.Western blotting using swine convalescent sera confirmed its role as an immunogenic protein. Collagen binding activity could be detected by western affinity blot and ELISA. Conversely, deletion of the cbp40 gene reduced bacterial adhesion to HEp-2 cells, capacity for biofilm formation, and virulence in a zebrafish infection model. The response of the bEnd.3 cell line to infection with the S. suis serotype 2 strain ZY05719 and the cbp40-knockout strain was evaluated using gene expression arrays. The differentially expressed genes were involved in inflammatory and immune responses, leukocyte adhesion and heterophilic cell adhesion. Collectively, these data suggest that Cbp40 plays an important role as an extracellular matrix adhesion protein that interacts with host cells during infection.

Mena binds α5 integrin directly and modulates α5β1 function

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.