SCO-spondin-derived peptide NX210 rescues neurons from cerebral ischemia/reperfusion injury through modulating the Integrin-ß1 mediated PI3K/Akt pathway.

Ischemic stroke is a common condition with high morbidity and mortality, causing irreversible neuronal damage and seriously affecting neurological function. There has been no ideal effective treatment so far. The NX210 peptide is derived from the thrombospondin type 1 repeat (TSR) sequence of SCO-spondin, and has been reported to exert various neurogenic properties. This study investigated whether NX210 had therapeutic effects and possible underlying mechanisms against cerebral ischemia/reperfusion (I/R). Therefore, primary embryonic rat cortical neurons and Sprague-Dawley (SD) rats that were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion/reperfusion (MCAO/R) injuries, respectively, were treated with or without NX210. We found that NX210 reduced OGD/R-induced cell viability loss and cytotoxicity. NX210 decreased cerebral infarct volume and brain edema, ameliorated neurological dysfunction, attenuated oxidative stress damage, and diminished neuronal apoptosis in MCAO/R rats. Furthermore, western blot analysis shown that treatment with NX210 up-regulated the expression of Integrin-ß1, phosphorylated-PI3K (p-PI3K) and phosphorylated-Akt (p-Akt). The Integrin-ß1 specific inhibitor, ATN-161, was used to identify pathways involved. The anti-oxidation activities and anti-apoptosis of NX210 was reversed by treatment with ATN-161. Overall, our results indicated that NX210 prevents oxidative stress and neuronal apoptosis in cerebral I/R via upregulation of the Integrin-ß1/PI3K/Akt signaling pathway. These results indicated that NX210 may be a promising therapeutic candidate for ischemic stroke.

The interactions between integrin α5ß1 of liver cancer cells and fibronectin of fibroblasts promote tumor growth and angiogenesis.

Hepatocellular carcinoma (HCC) progression is closely related to pathological fibrosis, which involves heterotypic intercellular interactions (HIIs) between liver cancer cells and fibroblasts. Here, we studied them in a direct coculture model, and identified fibronectin from fibroblasts and integrin-α5ß1 from liver cancer cells as the primary responsible molecules utilizing CRISPR/Cas9 gene-editing technology. Coculture led to the formation of 3D multilayer microstructures, and obvious fibronectin remodeling was caused by upregulated integrin-α5ß1, which greatly promoted cell growth in 3D microstructures. Integrin-α5 was more sensitive and specific than integrin-ß1 in this process. Subsequent mechanistic exploration revealed the activation of integrin-Src-FAK, AKT and ERK signaling pathways. Importantly, the growth-promoting effect of HIIs was verified in a xenograft tumor model, in which more blood vessels were observed in bigger tumors derived from the coculture group than that derived from monocultured groups. Hence, we conducted triculture by introducing human umbilical vein endothelial cells, which aligned to and differentiated along multilayer microstructures in an integrin-α5ß1 dependent manner. Furthermore, fibronectin, integrin-α5, and integrin-ß1 were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α5, and integrin-ß1 as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment.

Uremic toxin indoxyl sulfate induces dysfunction of vascular smooth muscle cells via integrin-ß1/ERK signaling pathway.

BACKGROUND: Protein-bound uremic toxins (PBUTs) are reported to be one of the major culprits in chronic kidney disease-cardiovascular disease (CKD-CVD) development, yet its mechanism is not fully clear. Our previous study confirmed elevated expression of integrin-ß1 (ITGß1) in vascular smooth muscle cells of uremic patients. Thus, this study aimed to explore the relationship between PBUTs and ITGß1 in uremic vasculature injury. METHODS: Human umbilical vein smooth muscle cells (HUVSMCs) and endothelial cells (HUVECs) were treated with two representative PUBTs, indoxyl sulfate (IS) and p-cresyl sulfate (PC). Both cells were measured for the expression of ITGß1 and downstream signaling pathways and assayed for proliferation, migration, adhesion and apoptosis. RESULTS: The IS treatments were observed with significantly up-regulated ITGß1 in HUVSMCs but not in HUVECs, while PC did not induce ITGß1 alteration in either HUVSMCs or HUVECs. Furthermore, overexpression of ITGß1 revealed activated downstream signal-regulated kinase (ERK) signaling pathway with promoted focal adhesion, migration, proliferation but no apoptosis in HUVSMCs by IS. These functional and pathway alterations could be significantly suppressed by RNA interference of ITGß1. More importantly, the application of ERK1/2 inhibitor significantly suppressed the focal adhesion, migration and proliferation of HUVSMCs. CONCLUSION: We first demonstrated that ITGß1/ERK signaling pathway mediated abnormal focal adhesion, migration and proliferation of vascular smooth muscle cells stimulated by IS. ITGß1/ERK signaling may serve as a novel therapeutic target for CKD-CVD.

Common and Distinctive Intercellular Communication Patterns in Human Obstructive and Nonobstructive Hypertrophic Cardiomyopathy.

Hypertrophic Cardiomyopathy (HCM) is a common inherited disorder characterized by unexplained left ventricular hypertrophy with or without left ventricular outflow tract (LVOT) obstruction. Single-nuclei RNA-sequencing (snRNA-seq) of both obstructive and nonobstructive HCM patient samples has revealed alterations in communication between various cell types, but no direct and integrated comparison between the two HCM phenotypes has been reported. We performed a bioinformatic analysis of HCM snRNA-seq datasets from obstructive and nonobstructive patient samples to identify differentially expressed genes and distinctive patterns of intercellular communication. Differential gene expression analysis revealed 37 differentially expressed genes, predominantly in cardiomyocytes but also in other cell types, relevant to aging, muscle contraction, cell motility, and the extracellular matrix. Intercellular communication was generally reduced in HCM, affecting the extracellular matrix, growth factor binding, integrin binding, PDGF binding, and SMAD binding, but with increases in adenylate cyclase binding, calcium channel inhibitor activity, and serine-threonine kinase activity in nonobstructive HCM. Increases in neuron to leukocyte and dendritic cell communication, in fibroblast to leukocyte and dendritic cell communication, and in endothelial cell communication to other cell types, largely through changes in the expression of integrin-ß1 and its cognate ligands, were also noted. These findings indicate both common and distinct physiological mechanisms affecting the pathogenesis of obstructive and nonobstructive HCM and provide opportunities for the personalized management of different HCM phenotypes.

Schwann cells-derived exosomes promote functional recovery after spinal cord injury by promoting angiogenesis.

Exosomes are small vesicles that contain diverse miRNA, mRNA, and proteins that are secreted by multiple cells, and play a vital function in cell-cell communication. Numerous exosomes produced by cells have been demonstrated to be protective against spinal cord injury (SCI). This study aims to investigate the neuroprotective effect of Schwann cells-derived exosomes (SCs-Exos) on spinal cord injury. We found that SCs-Exos can be taken directly by brain-derived endothelial cells.3 (bEnd.3 cells) and promoted to proliferate, migrate, and form bEnd.3 tube. Additionally, our results showed that the pro-angiogenesis molecules, Integrin-ß1, were highly expressed in SCs-Exos. Moreover, we used special shRNA technology to investigate the role of Integrin-ß1 in mediating the effect of SCs-Exos-induced angiogenesis on bEnd.3 cells. We observed that the pro-angiogenic effect of SCs-Exos on bEnd.3 cells was suppressed by inhibiting the expression of integrin-ß1 in SCs-Exos. In the SCI model, we found that SCs-Exos attenuated tissue damage and improved functional recovery after SCI. Using immunofluorescence staining, we observed that SCs-Exos treatment promoted angiogenesis in SCI, and integrin-ß1 was required to promote angiogenesis. In conclusion, our results indicate that SCs-Exos promote angiogenesis by delivering integrin-ß1 and may serve as a promising novel therapeutic agent for enhancing neurological functional recovery after SCI.

The PI3K/mTOR inhibitor Gedatolisib eliminates dormant breast cancer cells in organotypic culture, but fails to prevent metastasis in preclinical settings.

Dormant, disseminated tumor cells (DTCs) are thought to be the source of breast cancer metastases several years or even decades after initial treatment. To date, a selective therapy that leads to their elimination has not been discovered. While dormant DTCs resist chemotherapy, evidence suggests that this resistance is driven not by their lack of proliferation, but by their engagement of the surrounding microenvironment, via integrin-ß1-mediated interactions. Because integrin-ß1-targeted agents have not been translated readily to the clinic, signaling nodes downstream of integrin-ß1 could serve as attractive therapeutic targets in order to sensitize dormant DTCs to therapy. By probing a number of kinases downstream of integrin-ß1, we determined that PI3K inhibition with either a tool compounds or a compound (PF-05212384; aka Gedatolisib) in clinical trials robustly sensitizes quiescent breast tumor cells seeded in organotypic bone marrow cultures to chemotherapy. These results motivated the preclinical study of whether Gedatolisib-with or without genotoxic therapy-would reduce DTC burden and prevent metastases. Despite promising results in organotypic culture, Gedatolisib failed to reduce DTC burden or delay, reduce or prevent metastasis in murine models of either triple-negative or estrogen receptor-positive breast cancer dissemination and metastasis. This result held true whether analyzing Gedatolisib on its own (vs. vehicle-treated animals) or in combination with dose-dense doxorubicin and cyclophosphamide (vs. animals treated only with dose-dense chemotherapies). These data suggest that PI3K is not the node downstream of integrin-ß1 that confers chemotherapeutic resistance to DTCs. More broadly, they cast doubt on the strategy to target PI3K in order to eliminate DTCs and prevent breast cancer metastasis.

Discoidin Domain Receptor 2 Regulates AT1R Expression in Angiotensin II-Stimulated Cardiac Fibroblasts via Fibronectin-Dependent Integrin-ß1 Signaling.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-ß1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-ß1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-ß1.

Apoptosis induction by siRNA targeting integrin-ß1 and regorafenib/DDAB-mPEG-PCL hybrid nanoparticles in regorafenib-resistant colon cancer cells.

Colorectal cancer (CRC) is regarded as the third most common cancer worldwide. Although Regorafenib as a receptor tyrosine kinase inhibitor (RTKI) disrupts tumor growth and angiogenesis in metastatic CRC (mCRC) patients, drug resistance leads to poor prognosis and survival. Integrin-ß1 overexpression has been proposed to be the major player in this regard. Herein, the Regorafenib-resistant human colon cancer cell line (SW-48) was induced, and the Integrin-ß1 gene expression, as well as apoptosis, was assessed through the combination of small interfering RNA (siRNA) targeting Integrin-ß1 and Regorafenib/Dimethyldioctadecylammonium bromide (DDAB)-methoxy poly (ethylene glycol) (mPEG)-poly-ε-caprolactone (PCL) hybrid nanoparticles (HNPs). In the current study, Regorafenib-resistant SW-48 cell line was generated in which the Regorafenib half-maximal inhibitory concentration (IC50) for non-resistant and resistant cells was 13.5±1.5 µM and 55.1±0.8 µM, respectively. The results of DLS also demonstrated that the size and the charge of the HNPs were equal to 66.56±0.5 nm and +29.5±1.2 mv, respectively. In addition, the Integrin-ß1 gene expression was significantly higher in resistant cells than in non-resistant ones (P<0.05). The siRNA/HNP complexes in combination with Regorafenib/HNPs were accordingly identified as the most effective treatment to decrease the Integrin-ß1 gene expression and to enhance the apoptosis rate in resistant cells (P<0.001). Overall, the study indicated that combination therapy using siRNA/HNP and Regorafenib/HNPs complex could down-regulate the Integrin-ß1 gene expression and consequently trigger apoptosis, and this may potentially induce drug sensitivity.

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-ß1 and focal adhesion kinase activation.

Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin ß1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin ß1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin ß1 and promotes neuroblastoma cell migration.

Polymeric siRNA delivery targeting integrin-ß1 could reduce interactions of leukemic cells with bone marrow microenvironment.

Uncontrolled proliferation of the myeloid cells due to BCR-ABL fusion has been successfully treated with tyrosine kinase inhibitors (TKIs), which improved the survival rate of Chronic Myeloid Leukemia (CML) patients. However, due to interactions of CML cells with bone marrow microenvironment, sub-populations of CML cells could become resistant to TKI treatment. Since integrins are major cell surface molecules involved in such interactions, the potential of silencing integrin-ß1 on CML cell line K562 cells was explored using short interfering RNA (siRNA) delivered through lipid-modified polyethyleneimine (PEI) polymers. Reduction of integrin-ß1 in K562 cells decreased cell adhesion towards human bone marrow stromal cells and to fibronectin, a major extracellular matrix protein for which integrin-ß1 is a primary receptor. Interaction of K562 cells with fibronectin decreased the sensitivity of the cells to BCR-ABL siRNA treatment, but a combinational treatment with integrin-ß1 and BCR-ABL siRNAs significantly reduced colony forming ability of the cells. Moreover, integrin-ß1 silencing enhanced the detachment of K562 cells from hBMSC samples (2 out of 4 samples), which could make them more susceptible to TKIs. Therefore, the polymeric-siRNA delivery targeting integrin-ß1 could be beneficial to reduce interactions with bone marrow microenvironment, aiding in the response of CML cells to therapeutic treatment.

Thymoquinone Inhibits Proliferation and Migration of MDA-MB-231 Triple Negative Breast Cancer Cells by Suppressing Autophagy, Beclin-1 and LC3.

BACKGROUND: Triple Negative Breast Cancer (TNBC) is an aggressive and highly heterogeneous subtype of breast cancer associated with poor prognosis. A better understanding of the biology of this complex cancer is needed to develop novel therapeutic strategies for the improvement of patient survival. We have previously demonstrated that Thymoquinone (TQ), the major phenolic compound found in Nigella sativa, induces anti-proliferative and anti-metastatic effects and inhibits in vivo tumor growth in orthotopic TNBC models in mice. Also, we have previously shown that Beclin-1 and LC3 autophagy genes contributes to TNBC cell proliferation, migration and invasion, suggesting that Beclin-1 and LC3 genes provide proto-oncogenic effects in TNBC. However, the role of Beclin-1 and LC3 in mediating TQ-induced anti-tumor effects in TNBC is not known. OBJECTIVE: To investigate the effects of TQ on the major autophagy mediators, Beclin-1 and LC3 expression, as well as autophagic activity in TNBC cells. METHODS: Cell proliferation, colony formation, migration and autophagy activity were evaluated using MTS cell viability, colony formation assay, wound healing and acridine orange staining assays, respectively. Western blotting and RT-PCR assays were used to investigate LC3 and Beclin-1 protein and gene expressions, respectively, in MDA-MB-231 TNBC cells in response to TQ treatments. RESULTS: TQ treatment significantly inhibited cell proliferation, colony formation, migration and autophagic activity of MDA-MB-231 cells and suppressed LC3 and Beclin-1 expressions. Furthermore, TQ treatment led to the inhibition of Integrin-ß1, VEGF, MMP-2 and MMP-9 in TNBC cells. CONCLUSION: TQ inhibits autophagic activity and expression of Beclin-1 and LC3 in TNBC cells and suppresses pathways related to cell migration/invasion and angiogenesis, including Integrin-ß1, VEGF, MMP-2 and MMP- 9, suggesting that TQ may be used to control autophagic activity and oncogenic signaling in TNBC.

RACK1 promotes the invasive activities and lymph node metastasis of cervical cancer via galectin-1.

Cervical cancer remains the first leading cause of cancer-related mortality among female reproductive system malignancies worldwide, and invasion and lymph node metastasis of cervical cancer represent the major reason for its poor prognosis. In this study, we found that RACK1 facilitated tumor cell invasion and lymphatic tube formation in vitro, as well as promoted lymphangiogenesis and lymph node metastasis in vivo in a galectin-1-dependent manner. Mechanism studies revealed that RACK1 promoted the expression and secretion of galectin-1 by reducing miR-1275 levels. Additionally, RACK1 also augmented galectin-1-induced downstream MEK/ERK, FAK, and AKT signaling via integrin-ß1 in cervical cancer cells. Tissue microarray confirmed that RACK1 was upregulated in squamous intraepithelial lesion and cancer, and RACK1 was positively correlated with invasion/metastasis phenotype, galectin-1 expression, and unfavorable prognosis in cervical cancer cases. Human papillomavirus E6 oncogene contributes to increased expression of RACK1 via the enhancement of its O-GlcNAcylation and protein stability. Together, our results demonstrate that RACK1 stimulates tumor invasion and lymph node metastasis of cervical cancer via galectin-1 and imply that targeting RACK1/galectin-1 axis provides promising means for cervical cancer treatment.

PFN1 and integrin-ß1/mTOR axis involvement in cornea differentiation of fibroblast limbal stem cells.

Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re-epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune-modulatory of fibroblast limbal stem cells (f-LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton-remodelling proteins, including Profilin-1 (PFN1). In this study we postulate that pfn-1 knock down promotes epithelial lineage by inhibiting the integrin-ß1(CD29)/mTOR pathway and subsequent NANOG down-expression. We showed that it is possible modulate pfn1 expression levels by treating f-LSCs with Resveratrol (RSV), a natural compound: pfn1 decline is accompanied with up-regulation of the specific differentiation epithelial genes pax6 (paired-box 6), sox17 (sex determining region Y-box 17) and ΔNp63-α (p63 splice variant), consistent with drop-down of the principle stem gene levels. These results contribute to understand the molecular biology of corneal epithelium development and suggest that pfn1 is a potential molecular target for the treatment of corneal blindness based on epithelial cell dysfunction.

Histopathological and proteomic analyses identify integrin-ß1 as a potential mediator of phlebosclerosis in uremic patients.

BACKGROUND: Patients with uremia have an excessive mortality from cardiovascular disease (CVD). Arterial remodeling is mainly responsible for uremia-induced CVD and has been well studied, yet venous remodeling is poorly understood. Here we investigate the histopathology and proteomic profiles of venous remodeling in uremic patients. METHODS: Forearm cephalic veins were isolated from nine uremic patients during surgeries for arteriovenous fistula, and from nine healthy controls when applying surgical debridement. Hematoxylin-eosin, Masson's trichrome, von Kossa, and immunohistochemistry (IHC) against proliferating cell nuclear antigen were stained for histopathology. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was executed to explore the proteome of the veins. The core regulatory protein was validated by western blot, IHC, and immunofluorescence. RESULTS: Phlebosclerosis, characterized by intimal rarefaction and medial thickening with disordered proliferation of vascular smooth muscle cells (VSMCs), was the prominent pathological manifestation of peripheral veins in uremic patients, while inflammatory cell infiltration, atherosclerosis or calcification were not obviously detected. iTRAQ analysis showed that 350 proteins were significantly changed in phlebosclerosis of uremic patients compared with healthy controls, of which integrin-ß1 (ITGß1) exhibited the strongest regulatory ability by intermolecular interaction network analysis. The enhanced ITGß1 expression was mainly co-expressed with the disordered proliferation of VSMCs while a little with vascular endothelial cells in the forearm cephalic veins of uremic patients. CONCLUSIONS: Phlebosclerosis is the prominent pathological manifestation in peripheral veins of uremic patients. This pathological alteration mainly attributes to the disordered proliferation of VSMCs, which is potentially mediated by ITGß1.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Semaphorin 7A modulates cytokine-induced memory-like responses by human natural killer cells.

Cytokine-induced memory-like (CIML) NK cells are endowed with the capacity to mediate enhanced effector functions upon cytokine or activating receptor restimulation for several weeks following short-term preactivation with IL-12, IL-15, and IL-18. Promising results from a first-in-human clinical trial highlighted the clinical potential of CIML NK cells as adoptive immunotherapy for patients with hematologic malignancies. However, the mechanisms underlying CIML NK cell differentiation and increased functionality remain incompletely understood. Semaphorin 7A (SEMA7A) is a potent immunomodulator expressed in activated lymphocytes and myeloid cells. In this study, we show that SEMA7A is substantially upregulated on NK cells stimulated with cytokines, and specifically marks activated NK cells with a strong potential to release IFN-γ. In particular, preactivation of NK cells with IL-12+IL-15+IL-18 resulted in greater than tenfold upregulation of SEMA7A and enhanced expression of the ligand for SEMA7A, integrin-ß1, on CIML NK cells. Strikingly, preactivation in the presence of antibodies targeting SEMA7A lead to significantly decreased IFN-γ production following restimulation. These results imply a novel mechanism by which cytokine-enhanced SEMA7A/integrin-ß1 interaction promotes CIML NK cell differentiation and maintenance of increased functionality. Our data suggest that targeting SEMA7A/integrin-ß1 signaling might provide a novel immunotherapeutic approach to potentiate antitumor activity of CIML NK cells.

Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

Aging and neurodegenerative diseases share a condition of neuroinflammation entailing the production of endogenous cell debris in the CNS that must be removed by microglia ( i.e., resident macrophages), to restore tissue homeostasis. In this context, extension of microglial cell branches toward cell debris underlies the mechanisms of microglial migration and phagocytosis. Amoeboid morphology and the consequent loss of microglial branch functionality characterizes dysregulated microglia. Microglial migration is assisted by another glial population, the astroglia, which forms a dense meshwork of cytoplasmic projections. Amoeboid microglia and disrupted astrocyte meshwork are consistent traits in aged CNS. In this study, we assessed a possible correlation between microglia and astroglia morphology in rat models of chronic neuroinflammation and aging, by 3-dimensional confocal analysis implemented with particle analysis. Our findings suggest that a microglia-astroglia interaction occurs in rat hippocampus via cell-cell contacts, mediating microglial cell branching in the presence of inflammation. In aged rats, the impairment of such an interaction correlates with altered distribution, morphology, and inefficient clearance by microglia. These data support the idea that generally accepted functional boundaries between microglia and astrocytes should be re-evaluated to better understand how their functions overlap and interact.-Lana, D., Ugolini, F., Wenk, G. L., Giovannini, M. G., Zecchi-Orlandini, S., Nosi, D. Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

NF-κB signaling and integrin-ß1 inhibition attenuates osteosarcoma metastasis via increased cell apoptosis.

Osteosarcoma is a common primary bone malignancy, and distant metastasis limited the cure estimate during last decades. Detailed investigation of osteosarcoma metastasis is valuable for improving therapeutic strategy. Our study indicated increased integrin-ß1 expression and NF-kB signaling activation in metastatic osteosarcoma tissues. Gain-of-function assays showed that integrin-ß1 knockdown significantly inhibited osteosarcoma growth and metastasis, whereas exogenous reintroducing of integrin-ß1 restored cell proliferation and metastasis in vitro and in vivo. NF-κB signaling directly modulated integrin-ß1 expression, which is an effective target for the treatment of osteosarcoma. Mechanically, integrin-ß1 blockage with AIIB2 antibody increased osteosarcoma cell apoptosis. Immunohistochemistry staining of integrin-ß1 revealed that elevated integrin-ß1 expression was correlated with poor prognosis of osteosarcoma patients and acted as an independent detrimental factor for osteosarcoma. Our data showed that integrin-ß1 and NF-κB signaling are promising therapeutic targets to improve the clinical outcome of osteosarcoma patients. The examination of integrin-ß1 expression will also identify patients with high risk of disease progression.

Effect of substrate stiffness on hepatocyte migration and cellular Young's modulus.

Hepatic fibrosis progress accompanied by an unbalanced extracellular matrix (ECM) degradation and deposition leads to an increased tissue stiffness. Hepatocytes interplay with all intrahepatic cell populations inside the liver. However, how hepatocytes migration and cellular Young's modulus influenced by the substrate stiffness are not well understood. Here, we established a stiffness-controllable in vitro cell culture model by using a polyvinyl alcohol (PVA) hydrogel that mimicked the same physical stiffness as a fibrotic liver. Three levels of stiffness were used in our experiment that corresponded to the stiffness levels found in normal liver tissue (4.5 kPa), the early (19 kPa) and late stages (37 kPa) of fibrotic liver tissues. Cytoskeleton of hepatocyte was influenced by substrate stiffness. Soft substrate promoted the cellular migration and directionality. The cellular Young's modulus firstly increased and then decreased with increasing substrate stiffness. Integrin-ß1 and ß-catenin expression on cytomembrane were up-regulated and down-regulated with the increase of substrate stiffness, respectively. Our data not only suggested that hepatocytes were sensitive to substrate stiffness, but also suggested that there may be a potential relationship among substrate stiffness, cellular Young's modulus and the dynamic balance of integrin-ß1 and ß-catenin pathways. These results may provide us a new insight in mechanism investigation of mechano-dependent diseases, especially like fibrosis related diseases.

Biglycan expression in the melanoma microenvironment promotes invasiveness via increased tissue stiffness inducing integrin-ß1 expression.

Novel targeted and immunotherapeutic approaches have revolutionized the treatment of metastatic melanoma. A better understanding of the melanoma-microenvironment, in particular the interaction of cells with extracellular matrix molecules, may help to further improve these new therapeutic strategies.We observed that the extracellular matrix molecule biglycan (Bgn) was expressed in certain human melanoma cells and primary fibroblasts when evaluated by microarray-based gene expression analysis. Bgn expression in the melanoma tissues correlated with low overall-survival and low progression-free-survival in patients. To understand the functional role of Bgn we used gene-targeted mice lacking functional Bgn. Here we observed that melanoma growth, metastasis-formation and tumor-related death were reduced in Bgn-/- mice compared to Bgn+/+ mice. In vitro invasion of melanoma cells into organotypic-matrices derived from Bgn-/- fibroblasts was reduced compared to melanoma invasion into Bgn-proficient matrices. Tissue stiffness as determined by atomic-force-microscopy was reduced in Bgn-/- matrices. Isolation of melanoma cells and fibroblasts from the stiffer Bgn+/+ matrices revealed an increase in integrin-ß1 expression compared to the Bgn-/- fibroblast matrices. Overexpression of integrin-ß1 in B16-melanoma cells abolished the survival benefit seen in Bgn-/- mice. Consistent with the studies performed in mice, the abundance of Bgn-expression in human melanoma samples positively correlated with the expression of integrin-ß1, which is in agreement with results from the organotypic invasion-assay and the in vivo mouse studies.This study describes a novel role for Bgn-related tissue stiffness in the melanoma-microenvironment via regulation of integrin-ß1 expression by melanoma cells in both mice and humans.