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Introduction: Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Methods and Results: Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. [ABSTRACT FROM AUTHOR]

IntroductionOsteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis.Methods and ResultsMurine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors.DiscussionOur findings define integrin α2β1 as an early marker of OC differentiation.

The amino acid composition of collagens from different animal species was determined and their binding capacities to integrin α2β1 and HT1080 cells were studied by enzyme-linked immunosorbent assay (ELISA) and cell adhesion experiments. Furthermore, the effect of amino acid composition on collagen binding to α2β1 was discussed. The results showed that there were significant differences among the binding capacities of collagens from different animal species to integrin α2β1 and HT1080 cells, and the binding capacity of mammalian collagen was higher than that of fish collagen. The effects of amino acid composition on the binding capacities of collagen to integrin α2β1 and HT1080 cells were similar. The binding capacities were positively correlated with the contents of hydroxyproline, polar uncharged amino acids, imino acids and the degree of hydroxylation of proline, but negatively correlated with the contents of glutamate, glycine, polar charged amino acids and acidic amino acids (P < 0.01). Based on the content of informative amino acids at the high-affinity binding site between collagen and integrin α2β1, a mathematical model between the contents of hydroxyproline and arginine and collagen-integrin α2β1 binding was established by multivariate step-wise regression analysis, which had good predictive accuracy.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In Alport mice, activation of the endothelin A receptor (ETAR) in mesangial cells results in sub‐endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF‐κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild‐type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA‐seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA‐seq. Results were validated in vivo using RNA‐seq from RNA isolated from wild‐type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up‐ or down‐regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2β1 integrin/DDR1 co‐receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2β1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Abstract Background Ferritin, the natural iron storage protein complex, self-assembles into a uniform cage-like structure. Human H-ferritin (HFn) has been shown to transverse the blood–brain barrier (BBB) by binding to transferrin receptor 1 (TfR1), which is abundant in endothelial cells and overexpressed in tumors, and enters cells via endocytosis. Ferritin is easily genetically modified with various functional molecules, justifying that it possesses great potential for development into a nanocarrier drug delivery system. Results In this study, a unique integrin α2β1-targeting H-ferritin (2D-HFn)-based drug delivery system was developed that highlights the feasibility of receptor-mediated transcytosis (RMT) for glioma tumor treatment. The integrin targeting α2β1 specificity was validated by biolayer interferometry in real time monitoring and followed by cell binding, chemo-drug encapsulation stability studies. Compared with naïve HFn, 2D-HFn dramatically elevated not only doxorubicin (DOX) drug loading capacity (up to 458 drug molecules/protein cage) but also tumor targeting capability after crossing BBB in an in vitro transcytosis assay (twofold) and an in vivo orthotopic glioma model. Most importantly, DOX-loaded 2D-HFn significantly suppressed subcutaneous and orthotopic U-87MG tumor progression; in particular, orthotopic glioma mice survived for more than 80 days. Conclusions We believe that this versatile nanoparticle has established a proof-of-concept platform to enable more accurate brain tumor targeting and precision treatment arrangements. Additionally, this unique RMT based ferritin drug delivery technique would accelerate the clinical development of an innovative drug delivery strategy for central nervous system diseases with limited side effects in translational medicine. Graphic Abstract

Integrin α2β1 plays an important role in cellular migration and metastasis processes associated with prostate cancer. The aim of this study was to assess whether selective inhibition of integrin α2β1 is an effective strategy to target metastatic prostate cancer cells. In this regard, we examined the effects of the inhibitor BTT‐3033, which selectively interferes with the connection between integrin a2b1 and its ligand, on migration, epithelial–mesenchymal transition (EMT), cell cycle arrest, apoptosis, and specific intracellular signaling pathways using LNcap‐FGC and DU‐145 prostate cancer cell lines. Western blot analysis and immunocytochemistry assays showed that inhibition of integrin a2b1 inhibits EMT, through the increased expression of E‐cadherin and decreased expression of N‐cadherin and vimentin. Scratch wound healing assays revealed a direct effect on integrin α2β1 in the migration capacity of cells. In addition, treatment with BTT‐3033 induced a reduction in cell viability and proliferation, as assessed by MTT and BrdU assays. In addition, the results show that BTT‐3033 inhibits cell proliferation by inducing G1 cell cycle arrest. Moreover, inhibition of integrin α2β1 induces apoptosis through the activation of ROS, Bax protein upregulation, caspase‐3 activation, and depletion of ΔΨm. Molecular signaling studies showed that integrin α2β1 was a positive regulator of MKK7 phosphorylation. In conclusion, our results reveal a critical role for integrin a2b1 in the proliferation of prostate cancer cells, as demonstrated by EMT inhibition, cell cycle arrest, and apoptosis induction in response to treatment with its specific inhibitor BT‐3033. [ABSTRACT FROM AUTHOR]

Background: Ferritin, the natural iron storage protein complex, self-assembles into a uniform cage-like structure. Human H-ferritin (HFn) has been shown to transverse the blood–brain barrier (BBB) by binding to transferrin receptor 1 (TfR1), which is abundant in endothelial cells and overexpressed in tumors, and enters cells via endocytosis. Ferritin is easily genetically modified with various functional molecules, justifying that it possesses great potential for development into a nanocarrier drug delivery system. Results: In this study, a unique integrin α2β1-targeting H-ferritin (2D-HFn)-based drug delivery system was developed that highlights the feasibility of receptor-mediated transcytosis (RMT) for glioma tumor treatment. The integrin targeting α2β1 specificity was validated by biolayer interferometry in real time monitoring and followed by cell binding, chemo-drug encapsulation stability studies. Compared with naïve HFn, 2D-HFn dramatically elevated not only doxorubicin (DOX) drug loading capacity (up to 458 drug molecules/protein cage) but also tumor targeting capability after crossing BBB in an in vitro transcytosis assay (twofold) and an in vivo orthotopic glioma model. Most importantly, DOX-loaded 2D-HFn significantly suppressed subcutaneous and orthotopic U-87MG tumor progression; in particular, orthotopic glioma mice survived for more than 80 days. Conclusions: We believe that this versatile nanoparticle has established a proof-of-concept platform to enable more accurate brain tumor targeting and precision treatment arrangements. Additionally, this unique RMT based ferritin drug delivery technique would accelerate the clinical development of an innovative drug delivery strategy for central nervous system diseases with limited side effects in translational medicine. [ABSTRACT FROM AUTHOR]

Skeletal muscle and bone are highly interrelated, and previous proteomic analyses suggest that lumican is one of muscle-derived factors. To further understand the role of lumican as a myokine affecting adjacent bone metabolism, we investigated the effects of lumican on osteoblast biology. Lumican expression was significantly higher in the cell lysates and conditioned media (CM) of myotubes than those of undifferentiated myoblasts, and the known anabolic effects of myotube CM on osteoblasts were reduced by excluding lumican from the CM. Lumican stimulated preosteoblast viability and differentiation, resulting in increased calvaria bone formation. The expression of osteoblast differentiation markers was consistently increased by lumican. Lumican increased the phosphorylation of ERK, whereas ERK inhibitors completely reversed lumican-mediated stimulation of Runx2 and ALP activities in osteoblasts. Results of a binding ELISA experiment in osteoblasts show that transmembrane integrin α2β1 directly interacted with lumican, and an integrin α2β1 inhibitor attenuated the stimulation of ERK and ALP activities by lumican. Taken together, the results indicate that muscle-derived lumican stimulates bone formation via integrin α2β1 and the downstream ERK signal, indicating that this is a potential therapeutic target for metabolic bone diseases.

The T-2 toxin exerts a variety of toxic effects on both experimental animals and humans. The integrin family plays a major role in mediating cell-ECM interactions. Therefore, the present study aimed to investigate the involvement of integrin α2β1 in T-2 toxin-induced C28/I2 chondrocyte damage. The pathological damage of articular cartilage injury induced by T-2 toxin was observed by H&E staining. The expression levels of collagen 2 and MMP-13 (Matrix metalloproteinases 13) were detected using immunohistochemistry in articular cartilage tissues and western blotting in the cells. The blocking effect of integrin α2β1 inhibitor on T-2 toxin-induced chondrocyte matrix degradation was examined by western blotting. Further, the effect of integrin α2β1 inhibitor on T-2 toxin-induced chondrocyte apoptosis was analyzed. About 100 ng/g body weight (BW)/day T-2 toxin was fed to Sprague-Dawley (SD) rats, T-2 toxin treatment (0, 6, 12, and 24 ng/mL) induced C28/I2 chondrocytes. Both in vivo and in vitro , chondrocyte survival was inhibited, and the production of type II collagen was significantly reduced (p < 0.05). However, the level of MMP-13 was up-regulated (p < 0.05). Matrix degradation was effectively blocked after the pre-treatment by integrin α2β1 inhibitor (p < 0.05). Conclusively, Integrin α2β1 is a critical signaling pathway for communication between cells and the extracellular matrix, the present study provides a new clue to elucidate the mechanism of T-2 toxin-induced chondrocyte damage. • T-2 toxin had obvious toxic effects on chondrocytes and cartilage tissue. • T-2 toxin blocked the synthesis of proteoglycans and collagen in the cartilage extracellular matrix. • Integrin α2β1 signaling pathway may be involved in the degradation of type II collagen. [ABSTRACT FROM AUTHOR]

Current therapeutic interventions in bone defects are mainly focused on finding the best bioactive materials for inducing bone regeneration via activating the related intracellular signaling pathways. Integrins are trans‐membrane receptors that facilitate cell‐extracellular matrix (ECM) interactions and activate signal transduction. To develop a suitable platform for supporting human bone marrow mesenchymal stem cells (hBM‐MSCs) differentiation into bone tissue, electrospun poly L‐lactide (PLLA) nanofiber scaffolds were coated with nano‐hydroxyapatite (PLLA/nHa group), gelatin nanoparticles (PLLA/Gel group), and nHa/Gel nanoparticles (PLLA/nHa/Gel group) and their impacts on cell proliferation, expression of osteoblastic biomarkers, and bone differentiation were examined and compared. MTT data showed that proliferation of hBM‐MSCs on PLLA/nHa/Gel scaffolds was significantly higher than other groups (P <.05). Alkaline phosphatase activity was also more increased in hBM‐MSCs cultured under osteogenic media on PLLA/nHa/Gel scaffolds compared to others. Gene expression evaluation confirmed up‐regulation of integrin α2β1 as well as the osteogenic genes BGLAP, COL1A1, and RUNX2. Following use of integrin α2β1 blocker antibody, the protein level of integrin α2β1 in cells seeded on PLLA/nHa/Gel scaffolds was decreased compared to control, which confirmed that most of the integrin receptors were bound to gelatin molecules on scaffolds and could activate the integrin α2β1/ERK axis. Collectively, PLLA/nHa/Gel scaffold is a suitable platform for hBM‐MSCs adhesion, proliferation, and osteogenic differentiation in less time via activating integrin α2β1/ERK axis, and thus it might be applicable in bone tissue engineering. [ABSTRACT FROM AUTHOR]

Thrombosis represents the leading cause of death and disability upon major adverse cardiovascular events (MACEs). Numerous pathological conditions such as COVID-19 and metabolic disorders can lead to a heightened thrombotic risk; however, the underlying mechanisms remain poorly understood. Our study illustrates that 2-methylbutyrylcarnitine (2MBC), a branched-chain acylcarnitine, is accumulated in patients with COVID-19 and in patients with MACEs. 2MBC enhances platelet hyperreactivity and thrombus formation in mice. Mechanistically, 2MBC binds to integrin α2β1 in platelets, potentiating cytosolic phospholipase A2 (cPLA2) activation and platelet hyperresponsiveness. Genetic depletion or pharmacological inhibition of integrin α2β1 largely reverses the pro-thrombotic effects of 2MBC. Notably, 2MBC can be generated in a gut-microbiota-dependent manner, whereas the accumulation of plasma 2MBC and its thrombosis-aggravating effect are largely ameliorated following antibiotic-induced microbial depletion. Our study implicates 2MBC as a metabolite that links gut microbiota dysbiosis to elevated thrombotic risk, providing mechanistic insight and a potential therapeutic strategy for thrombosis. [Display omitted] • 2MBC accumulation leads to increased thrombotic risk • 2MBC directly binds to integrin α2β1 and potentiates platelet hyperreactivity • Inhibition of integrin α2β1 ameliorates 2MBC-induced heightened thrombotic risk • 2MBC is a co-metabolite bridging gut microbiota dysbiosis and thrombosis Huang et al. demonstrate that 2MBC, a host and gut microbial co-metabolite, is positively associated with thrombotic risk in humans. Their study elucidates that 2MBC potentiates platelet hyperreactivity through integrin α2β1 and indicates that 2MBC is a metabolite that potentially links gut microbiota dysbiosis to an increased risk of thrombosis. [ABSTRACT FROM AUTHOR]

Information about mechanical strain in the extracellular space is conducted along collagen fibers connected with integrins and then transmitted within cells. An aim of the study is to verify the hypothesis that the stiffness of cardiac human fibroblast substrates exerts a regulatory effect on collagen metabolism via integrin α2β1 and downstream signaling. The experiments were performed on human cardiac fibroblasts cultured on stiff or soft polyacrylamide gels. Extracellular and intracellular collagen content, metalloproteinase-1 (MMP-1), metalloproteinase-9 (MMP-9) and expression of the α1 chain of the procollagen type I gene (Col1A1) were elevated in cultures settled on soft substrate. The substrate stiffness did not modify tissue inhibitors of matrix metalloproteinase capacity (TIMPs 1–4). Integrin α2β1 inhibition (TC-I 15) or α2 subunit silencing resulted in augmentation of collagen content within the culture. Expression of Col1A1 and Col3A1 genes was increased in TC-I 15-treated fibroblasts. Total and phosphorylated levels of both FAK and Src kinases were elevated in fibroblasts cultured on stiff substrate. Inhibition of FAK (FAK kinase inhibitor 14) or Src kinase (AZM 47527) increased collagen content within the culture. The substrate stiffness exerted a regulatory influence on collagen metabolism via integrin α2β1 and its downstream signaling (FAK and Src kinases) in cardiac fibroblasts.

In the development of biomaterials with specific structural domains associated with various cellular activities, the limited integrin specificity of commonly used adhesion sequences, such as the RGD tripeptide, has resulted in an inability to precisely control cellular responses. To overcome this limitation, we conducted multiple replications of the integrin α 2 β 1 -specific ligand, the collagen hexapeptide Gly-Phe-Pro-Gly-Glu-Arg (GFPGER) in Pichia pastoris . This enabled the development of recombinant collagen with high biological activity, which was subsequently expressed, isolated, and purified for structural and functional analysis. The proteins carrying the multiple replications GFPGER sequence demonstrated significant bioactivity in cells, leading to enhanced cell adhesion, osteoblast differentiation, and mineralization when compared to control groups. Importantly, these effects were mediated by integrin α 2 β 1 . The new collagen constructed in this study is expected to be a biomaterial for regulating specific cell functions and fates.

Previous reports indicated that integrins associated signals are tightly related to tumor progression. Here, we observed elevated expression of integrin α2β1 in tumor tissues from microtubule‐directed chemotherapeutic drugs (MDCDs) resistant patients compared with the samples from chemosensitive patients. More importantly, we sorted the integrin α2β1+ tumor cells and found those cells revealed high MDCDs resistance, whereas MDCDs shows effective cytotoxicity to those integrin α2β1− tumor cells in vitro and in vivo. Mechanistically, we demonstrated that integrin α2β1 could induce MDCDs resistance through the activation of the PI3K/AKT pathway. Applying MPEG‐PLA to co‐encapsulate the integrin α2β1 inhibitor E7820 and MDCDs could effectively reverse MDCDs resistance, resulting in enhanced anticancer effects while avoiding potential systemic toxicity in vitro and in vivo. In conclusion, the expression of integrin α2β1 contributes to MDCDs resistance, while applying E7820 combination treatment by MPEG‐PLA nanoparticles could reverse the resistance. [ABSTRACT FROM AUTHOR]

The tumor microenvironment is associated with various tumor progressions, including cancer metastasis, immunosuppression, and tumor sustained growth. Tumor‐associated macrophages (TAMs) are considered an indispensable component of the tumor microenvironment, participating in the progression of tumor microenvironment remodeling and creating various compounds to regulate tumor activities. This study aims to observe enriched TAMs in tumor tissues during bladder cancer development, which markedly facilitated the proliferation of bladder cancer cells and promoted tumor growth in vivo. We determined that TAMs regulate tumor sustained growth by secreting type I collagen, which can activate the prosurvival integrin α2β1/PI3K/AKT signaling pathway. Furthermore, traditional chemotherapeutic drugs combined with integrin α2β1 inhibitor showed intensive anticancer effects, revealing an innovative approach in clinical bladder cancer treatment. [ABSTRACT FROM AUTHOR]

Integrin α2β1, also known as very late antigen (VLA)-2, is a collagen-binding molecule expressed constitutively on platelets. Vatelizumab, a monoclonal antibody targeting the α2 subunit (CD49b) of VLA-2, was recently investigated for its safety and efficacy during a Phase 2 clinical study in multiple sclerosis patients, as integrin-mediated collagen binding at the site of inflammation is central to a number of downstream pro-inflammatory events. In the course of this study, we could show that VLA-2 is expressed ex vivo on platelets, platelet–T-cell aggregates, as well as a small population of highly activated memory T cells. Even though the clinical trial did not meet its primary clinical end-point (reduction in the cumulative number of new contrast-enhancing lesions on magnetic resonance imaging (MRI)), we observed enhanced frequencies of regulatory T cells (TREG) following vatelizumab treatment. Elevated TREG frequencies might be explained by the inhibition of p38 mitogen-activated protein kinase (MAPK) signaling, which is critically involved in the polarization of T helper 17 (TH17) cells and is activated by the α2 integrin cytoplasmic domain. Our findings suggest that blockade of VLA-2 might be a way to safely shift the TH17/TREG balance by inducing TREG in vivo. [ABSTRACT FROM AUTHOR]

Currently, the same first-line chemotherapy is administered to almost all patients suffering from primary ovarian cancer. The high recurrence rate emphasizes the need for precise drug treatment in primary ovarian cancer. Being crucial in ovarian cancer progression and chemotherapeutic resistance, integrins became promising therapeutic targets. To evaluate its prognostic and predictive value, in the present study, the expression of integrin α2β1 was analyzed immunohistochemically and correlated with the survival data and other therapy-relevant biomarkers. The significant correlation of a high α2β1-expression with the estrogen receptor alpha (ERα; p = 0.035) and epithelial growth factor receptor (EGFR; p = 0.027) was observed. In addition, high α2β1-expression was significantly associated with a low number of tumor-infiltrating immune cells (CD3 intratumoral, p = 0.017; CD3 stromal, p = 0.035; PD-1 intratumoral, p = 0.002; PD-1 stromal, p = 0.049) and the lack of PD-L1 expression (p = 0.005). In Kaplan–Meier survival analysis, patients with a high expression of integrin α2β1 revealed a significant shorter progression-free survival (PFS, p = 0.035) and platinum-free interval (PFI, p = 0.034). In the multivariate Cox regression analysis, integrin α2β1 was confirmed as an independent prognostic factor for both PFS (p = 0.021) and PFI (p = 0.020). Dual expression of integrin α2β1 and the hepatocyte growth factor receptor (HGFR; PFS/PFI, p = 0.004) and CD44v6 (PFS, p = 0.000; PFI, p = 0.001; overall survival [OS], p = 0.025) impaired survival. Integrin α2β1 was established as a prognostic and predictive marker in primary ovarian cancer with the potential to stratify patients for chemotherapy and immunotherapy, and to design new targeted treatment strategies.

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). [ABSTRACT FROM AUTHOR]

Copyright of West China Journal of Stomatology is the property of Sichuan University, West China College of Stomatology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective: The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting galvanotaxis and the effects of electrical stimulation on integrin α2β1 and actin filaments in human dermal fibroblasts. Methods: Human dermal fibroblasts were treated with the monophasic pulsed microcurrent of 0, 100, 200, or 300 μA for 8 hours, and cell migration and cell viability were measured 24 hours after starting monophasic pulsed microcurrent stimulation. Polarization of integrin α2β1 and lamellipodia formationwere detected by immunofluorescent staining 10 minutes after starting monophasic pulsed microcurrent stimulation. Results: The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P < .01) without any change in cell viability; treatment with 300-μA monophasic pulsed microcurrent did not alter the migration ratio. The electrostimulus of 200 μA also promoted integrin α2β1 polarization and lamellipodia formation at the cathode edge (P < .05). Conclusion: The results show that 200 μA is an effective monophasic pulsed microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts. [ABSTRACT FROM AUTHOR]

Multifunctional nanoprobes open exciting possibilities for accurate diagnosis and therapy. In this research, we developed a 64 Cu-labeled GdVO 4 :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 GdVO 4 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 a 2 ß 1 targeting. After initial evaluation of the cytotoxicity and targeting capability with PC-3 cells, the obtained multifunctional nanoprobes ( 64 Cu-DOTA-GdVO 4 :4%Eu-DGEA) were further explored for targeted positron emission tomography (PET) and T 1 -weighted magnetic resonance imaging (MRI) of PC-3 tumor (prostate cancer, high integrin a 2 ß 1 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, GdVO 4 :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. [ABSTRACT FROM AUTHOR]

Abstract: Background: Collagen is the most abundant protein in human skin and is responsible for its resilience. In particular during photoaging, collagen homeostasis is out of balance leading to a continuous loss of intact collagen and to the observed signs of aged skin such as diminished tensile strength and wrinkle development. The process of collagen turnover is very slow and the relevance of cellular uptake of damaged collagen, most likely mediated via Endo180 or integrin α2β1, still remains a matter of investigation. Objective: We investigated the role of different collagen receptors on dermal fibroblasts for collagen internalization and their impact on collagen homeostasis during photoaging. Methods: TaqMan Real-Time PCR, flow cytometry, UV irradiation, knockdown experiments and immunostaining. Results: We show that Endo180 and integrin α2 are regulated in photoaged skin and after acute UV stress in vivo and in vitro. Knockdown experiments revealed that Endo180 is essential for cellular uptake of collagen fragments by dermal fibroblasts, whereas integrin α2 is important for initial binding of collagen. UV irradiation decreases collagen endocytosis. This correlates with reduced Endo180 expression and pericellular accumulation of collagen fragments during photoaging. Conclusion: Our findings correlate for the first time impaired collagen uptake via Endo180 with the pericellular accumulation of collagen fragments during photoaging. We assume an altered pericellular niche of fibroblasts in photoaged skin that has an impact on collagen homeostasis. [Copyright &y& Elsevier]

Abstract: Integrins are receptors of the extracellular matrix (ECM), playing a vital role in pathophysiological processes. They bind to ECM ligands like collagens and can mediate wound healing as well as tumor metastasis and thrombosis, thus being a part of cell adhesion and migration as well as platelet aggregation. For this reason, identifying α2β1 integrin-specific antagonists can assist in the development of drugs to treat tumor progression, angiogenesis, and cardiovascular diseases. Snake venoms have been shown to contain antagonists which target collagen-binding integrins. EMS16, rhodocetin, and VP12 are three toxins belonging to the C-type lectin-related protein family and have been proven to inhibit the α2β1 integrin, specifically the α2 integrin A domain. To specifically isolate antagonists targeting the α2β1 integrin A domain, we developed a protocol based on affinity chromatography. Using this novel approach, the toxin VP-i was isolated from Vipera palaestinae venom. We show that VP-i binds to the α2 integrin A domain and that it successfully inhibits adhesion of various cells to type I collagen as well as cell migration. Moreover, our results indicate that VP-i differs structurally from the previously purified VP12, although not functionally, and therefore is a further venom compound which can be utilized for drug development. [Copyright &y& Elsevier]

Purpose: The ability of PET to aid in the diagnosis and management of recurrent and/or disseminated metastatic prostate cancer may be enhanced by the development of novel prognostic imaging probes. Accumulating experimental evidence indicates that overexpression of integrin αβ may correlate with progression in human prostate cancer. In this study, Cu-labeled integrin αβ-targeted PET probes were designed and evaluated for the imaging of prostate cancer. Methods: DGEA peptides conjugated with a bifunctional chelator (BFC) were developed to image integrin αβ expression with PET in a subcutaneous PC-3 xenograft model. The microPET images were reconstructed by a two-dimensional ordered subsets expectation maximum algorithm. The average radioactivity accumulation within a tumor or an organ was quantified from the multiple region of interest volumes. Results: The PET tracer demonstrated prominent tumor uptake in the PC-3 xenograft (integrin αβ-positive). The receptor specificity was confirmed in a blocking experiment. Moreover, the low tracer uptake in a CWR-22 tumor model (negative control) further confirmed the receptor specificity. Conclusion: The sarcophagine-conjugated DGEA peptide allows noninvasive imaging of tumor-associated αβ expression, which may be a useful PET probe for evaluating the metastatic potential of prostate cancer. [ABSTRACT FROM AUTHOR]

Platelets in flowing blood at high-shear stress are recruited to exposed subendothelial collagen of injured vessels by GPIb–von Willebrand factor (vWf) and integrin α2β1 (α2β1)–collagen interactions. Platelet adhesion to type I collagen depends mainly on the α2β1–collagen interaction and that to type III collagen depends on the GPIb–vWf interaction due to vWf's weak affinity for type I collagen. Contributions of these two interactions would differ depending on expressions of α2β1, vWf, or GPIb. We quantitated platelet adhesion to low- and high-density collagen under high-shear flow conditions in the presence of anti-α2β1 (Gi9) and anti-GPIb (NNKY5-5) antibodies to determine if their inhibitory effects were correlated with the amounts of α2β1, GPIb and vWf. Gi9 inhibition of adhesion to type I collagen was decreased in platelets with more integrin α2β1. Gi9 and NNKY5-5 are more inhibitory against adhesion to low-density type III and I, respectively. Higher α2β1 expression decreases adhesion to low-density type III and increases Gi9 inhibition of adhesion to high-density type III, suggesting crosstalk between the α2β1–collagen and GPIb–vWf interactions in adhesion to type III. Integrin α2β1–collagen and GPIb–vWf interactions both contribute to platelet adhesion to collagen under high-shear flow. In adhesion under high-shear stress, the two interactions would compensate for each other, when there is a deficiency in one or the other. The α2β1–collagen interaction was also suggested to have an inhibitory effect on platelet adhesion to type III collagen, through a yet undefined mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Antiangiogenic activity can be elicited by the kringle domains 1 and 2 of tissue-type plasminogen activator (TK1–2), or the kringle 2 domain alone. In a previous report, we showed that the anti-migratory effect of TK1–2 is mediated in part by its interference with integrin α2β1. Since integrin α2β1 interacts with collagen type I through the DGEA (Asp-Gly-Glu-Ala) amino acid sequence, and a similar sequence, DGDA (Asp-Gly-Asp-Ala), exists in the kringle 2 domain, we investigated whether the DGDA sequence has a role in antiangiogenic activity of TK1–2. In an adhesion assay, the DGDA peptide inhibited adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized TK1–2. Pretreatment of the DGDA peptide also blocked anti-migratory activity of TK1–2. When the DGDA peptide alone was tested for antiangiogenic activity, it effectively inhibited VEGF-induced migration of HUVECs and tube formation on Matrigel. In addition, the DGDA peptide decreased differentiation of endothelial progenitor cells on collagen type I matrix. These data suggest that the DGDA sequence presents a functional epitope of TK1–2 and that it can be used as a potential novel antiangiogenic peptide. [Copyright &y& Elsevier]

Background and Purpose: During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin alpha2beta1 agonist.Experimental Approach: Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.Key Results: Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin alpha2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-beta. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by alpha2beta1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-beta mAb, or a phosphoinositide-3 kinase inhibitor, LY294002. Aggretin stimulated the phosphorylation of PDGFR-beta, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, U73122 and PD98059.Conclusions and Implications: Aggretin induces VSMC proliferation and migration mainly through binding to integrin alpha2beta1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production. [ABSTRACT FROM AUTHOR]

Abstract: The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin α2β1 polarized strongly in the fibril direction. In contrast, α2β1-deficient cells adhered but polarized less well, suggesting a role of integrin α2β1 in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. [Copyright &y& Elsevier]

Abstract: Platelet collagen receptors, such as Gp VI, are attractive targets for antithrombotic therapy. In this paper, I discuss the current knowledge regarding collagen–platelet interactions, including the role of platelet receptors, the recognition of collagen by platelets, the effect of the interaction on platelet activation and thrombosis and the effect of collagen structure on the platelet interaction, and highlight the areas in which additional information is required to pursue the goal of antithrombotic therapy, using the collagen–platelet interaction as the site of intervention. Understanding the detail of the receptor recognition motifs within collagen not only may reveal new antithrombotic targets within collagen, but will almost certainly lead to the development of defined reagents that can be used in vitro and ex vivo to explore thrombus formation further. [Copyright &y& Elsevier]

Abstract: Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short α and β cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin α2β1 is a major collagen receptor but to date, only few proteins have been shown to interact with the α2 cytoplasmic tail or with the α2β1 complex. In order to identify novel binding partners of a α2β1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-α2 and GST-Jun α2 bound His-tagged calreticulin while GST-β1 and GST-Fos β1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun α2/GST-Fos β1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with αvβ3-positive focal contacts. Here, we provide evidence that this interaction also occurs with α2β1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen. [Copyright &y& Elsevier]

Objective(s). The purpose was to study the interrelationship between cell surface integrin receptor (α2β1) and matrixmetalloproteinases.Methods. Immunoprecipitation and cell adhesion assay were done to assay α2β1 and α3β1 on SiHa cell surface. Zymogram was developed to assay secreted MMP activity of cells grown in presence of α2 monoclonal antibody. Immunoblot was developed to assay expression of MMP-2, FAK, and p-FAK. Plasma membrane-dependent activation of MMP2 was performed by incubating pure MMP-2 with membrane-enriched fraction isolated from SiHa cells.Results. Immunoprecipitation and cell adhesion assay results confirmed the presence of α2β1 receptor on SiHa cells. Zymographic analysis of serum-free media collected at different time points from SiHa cells grown on α2 monoclonal antibody-coated culture dishes showed the expression and activation of MMP-2 within 2–4 h, confirmed by immunoblot. Western blot of cells grown on α2-coated dishes for 30 min–4 h showed increased phosphorylation of FAK. Membrane-enriched fraction isolated from SiHa cells was found to specifically activate proMMP-2 to its activated forms within 30 min.Conclusion(s). The experimental findings strongly indicate that SiHa cell surface α2β1 regulates MMP-2 expression. Increased phosphorylation of focal adhesion kinase (FAK) strongly indicates the possible role of FAK in signaling cascade. Incubation of SiHa cell membrane fraction with pure MMP-2 strongly confirms the cell membrane-dependent activation of proMMP2. [Copyright &y& Elsevier]

The role of the phospholipase C (PLC)γ2 isotype in platelet activation was evaluated by studying PLCγ2 −/− mice. These mice have a prolonged bleeding time but their platelets respond normally to non-collagenous agonists. PLCγ2-null platelets show residual aggregation response to collagen fibres (6% versus 74% for wild-type) with minimal granule secretion and no shape change. A delayed shape change is observed at later aggregation times. Specific activation by glycoprotein (GP)VI agonists (convulxin, collagen-related peptide and GPVI crosslinking) is, however, abolished. Antibodies against integrin α2β1 and GPVI each inhibit the residual collagen response, implying a role of α2β1 in platelet activation and a functional association with GPVI. These responses are also prevented by blocking integrin αIIbβ3 and phosphoinositide 3-kinase, whereas aspirin treatment and ADP receptor blockade only inhibit shape change. These results provide evidence for a PLCγ2-independent collagen activation pathway requiring cooperation between GPVI and α2β1 leading to αIIbβ3-dependent aggregation and shape change by released ADP and thromboxane A2. [Copyright &y& Elsevier]

Adhesion of blood platelets to fibrillar collagens plays a crucial role in haemostasis. Collagen type II is a homotrimeric member of the fibrillar collagen family, whose ability to interact with platelets has been poorly investigated. In this work, we analysed platelet adhesion to the whole collagen type II molecule, as well as to its CNBr peptides. We found that collagen type II is as efficient as collagen type I in supporting platelet adhesion. Platelet binding sites on collagen type II were identified in two different CNBr-derived peptides, CB8 and CB11. The ability of these peptides to support platelet adhesion required the triple helical conformation. Interaction of platelets with CB8 and CB11 peptides was totally dependent on the presence of Mg2+ ions, and was completely inhibited by the anti-integrin α2β1 antibody P1E6. Upon adhesion to CB8 and CB11, a significant increase in intracellular protein tyrosine phosphorylation was observed. The pattern of tyrosine phosphorylated proteins in CB8- and CB11-adherent platelets was very similar to that observed in platelets adherent to the whole collagen molecule. By immunoprecipitation experiments, we identified two substrates that were tyrosine phosphorylated in adherent platelets as the tyrosine kinase Syk and the PLCγ2 isozyme. By contrast, platelet adhesion to CB8 and CB11 did not promote tyrosine phosphorylation of FcR γ-chain. Finally, we found that collagen type II, but not the CNBr-derived peptides, was able to induce cell aggregation associated to protein tyrosine phosphorylation when added to a platelet suspension. These results identify the CNBr peptides from collagen type II CB8 and CB11 as ligands for platelet integrin α2β1, and recognise their ability to support platelet adhesion and activation. [Copyright &y& Elsevier]

E-cadherin is a 120-kDa transmembrane glycoprotein expressed mainly on the surface of epithelial cells. The best characterised function of E-cadherin is homotypic, calcium-dependent cell–cell adhesion; however, the observation that E-cadherin is also capable of interacting with the αEβ7 integrin to mediate leukocyte cell–cell adhesion [Nature 372 (1994) 190] suggests that it also participates in heterotypic interactions. To investigate the possibility that E-cadherin may interact with integrins expressed on non-leukocytic cells, cell adhesion and solid-phase receptor–ligand binding experiments were performed using a pentameric E-cadherin construct designed to detect low affinity, high avidity interactions. HT1080 human fibrosarcoma cells specifically adhered to pentameric E-cadherin, and this adhesion was inhibited by anti-functional monoclonal antibodies directed against the integrin α2 and β1 subunits, but not by a series of antibodies recognising other subunits. This suggested that the E-cadherin receptor was α2β1, a previously characterised collagen/laminin receptor. Pentameric E-cadherin, but not monomeric E-cadherin, specifically bound, in a divalent cation-dependent manner, to both purified α2β1 and to a recombinant form of the A-domain of the α2 subunit, which has been shown to be a major ligand-binding site within this and other integrins. These findings demonstrate that E-cadherin can interact with α2β1 and suggest that heterotypic interactions between E-cadherin and integrins may be more common than originally thought. [Copyright &y& Elsevier]

Abstract: Objectives : The expression density of GPIaIIa, the primary platelet collagen receptor (integrin α2β1), is linked to two polymorphisms (GPIa-807C/T and HPA-5a/b). During evolution a gene shift from the genotypes GPIa-807CC-HPA-5bb to the genotypes GPIa-807CT-HPA-5aa has taken place. The aim of the study was to assess whether iron deficiency anaemia (e.g. increased blood loss) in young women could be associated with a specific genotype, indicating a role as potential evolutionary selection factor. Study design : Women between 18 and 40 yr of age presenting for their first blood donation were asked about alimentary habits and use of oral contraception. Haemoglobin and serum ferritin were measured and the GPIa-C807T and HPA-5 genotypes were determined. Results : Two hundred women were included and grouped according to the WHO definition for iron deficiency anaemia (haemoglobin <121 g L -1 and ferritin <15 µg L -1 ). Eight women fulfilled both WHO-criteria for iron deficiency anaemia, 145 women fulfilled none. No differences regarding age, use of oral contraceptives, alimentary habits, and HPA-5 were found between the groups. The GPIa-807CC genotype was strongly over-represented in the WHO-anaemic women as compared to the non-WHO-anaemic women (87.5% vs. 33.1%, P =0.003). Conclusion : Iron deficiency anaemia in young women might have been the evolutionary disadvantage causing the gene shift from GPIa-807CC to 807CT. [ABSTRACT FROM AUTHOR]

Integrin α2β1, which is a membrane protein consisting of noncovalently bound α2 and β1 chains, mediates cell binding to collagen and plays a role in platelet functions. DNAs encoding the chimeric proteins in which the extracellular domains of each α2 and β1 chain was fused to hinge and Fc regions of human IgG1γ chain were cotransfected into CHO cells. Soluble integrin α2β1 (sα2β1) in which α2 and β1 chains were covalently bound by disulfide bonds was recovered from the culture supernatant. sα2β1 maintained functional characteristics of cell surface α2β1 as indicated by cation-dependent binding to collagen and conformational changes induced by cations or ligand. Intravenously administered sα2β1 in rats colocalized with collagen in inflamed microvessels. Moreover, sα2β1-conjugated liposome administered intravenously reduced bleeding time of the thrombocytopenic mice. These results indicated that sα2β1 has pharmaceutical utilities as an agent for detecting injured vessels and a component of platelet substitute. [Copyright &y& Elsevier]

[Display omitted] An integrin α 2 β 1 -targeted PET probe (68Ga-IABtP) was developed to serve as a supplement and alternative of PSMA imaging for prostate cancer. 68Ga-IABtP was synthesized by labeling the precursor peptide with 68Ga with 93% labeling yield and 4.14 MBq/μg specific radioactivity. 68Ga-IABtP showed no specific uptake in LNCaP prostate cancer cell with low integrin α 2 β 1 expression but significantly increased uptake in PC-3 prostate cancer cell with high integrin α 2 β 1 expression, which could be specifically blocked by the integrin α 2 β 1 monoclonal antibody. The efflux experiments demonstrated that 68Ga-IABtP could rapidly penetrate into PC-3 cell after cell binding, thereby prolonging the residence time in the tumor and allow enough time for probe clearance from the circulation and non-specific organs. The biodistribution study indicated that 68Ga-IABtP showed no specific accumulation in non-target organs and was quickly cleared from the kidney. The in vivo PET-CT imaging demonstrated that 68Ga-IABtP showed no specific uptake in LNCaP tumor but could specifically accumulate in the PC-3 tumor, and was rapidly cleared from spleen, intestine, kidney and liver, resulting in excellent contrast effect with low background signal and high target to non-target ratios. [ABSTRACT FROM AUTHOR]