Integrin Β1 Overexpression Research Articles

Luteolin inhibits angiogenesis and enhances radiotherapy sensitivity of laryngeal cancer via downregulating Integrin β1

AimTo demonstrate the role and mechanism of luteolin in radio-sensitization and angiogenesis of laryngeal cancer. MethodsFirstly, we analyzed the cytotoxicity of Luteolin and radiation sensitive cytotoxicity through CCK8, and selected subsequent radiation doses and Luteolin concentrations. Next, we further analyzed the effects of Luteolin on radiation sensitivity and neovascularization of laryngeal cancer, and conducted CCK8, plate cloning, and angiogenesis experiments, respectively. At the same time, the effects of individual treatment and combination treatment on the expression of Integrin β1 and VEGFA were analyzed through immunofluorescence analysis. We also analyzed the regulation of Integrin β1 protein expression by Luteolin through Western blot. To investigate the mechanism of Integrin β1, we transfected overexpressed and silenced Integrin β1 vectors and analyzed the role of Integrin β1 in Luteolin enhancing radiation sensitivity of laryngeal cancer by repeating the above experiments. We have also constructed an in vivo subcutaneous tumor transplantation model to further validate the cell experimental results. The expression of Integrin, KI67, VEGFA, and CD31 was analyzed through Western blot and immunohistochemistry experiments. ResultsRadiation inhibited cell proliferation and decreased Integrin β1 expression, and increased the radiosensitivity through inhibiting cell proliferation, and inhibit angiogenesis during radiation. Overexpression of Integrin β1 weakened radiotherapy sensitivity on the basis of cells treated with combined administration. Integrin β1 is considered as the downstream molecule of luteolin, participating in radiosensitivity of luteolin to FaDu cells. Animal experiments also demonstrated that luteolin strengthened tumor suppression and anti-angiogenesis during radiation via Integrin β1. ConclusionIn summary, our results manifested that radio-sensitivity effect of luteolin depended on downregulating Integrin β1 in laryngocarcinoma.

The Sall2 transcription factor promotes cell migration regulating focal adhesion turnover and integrin β1 expression.

SALL2/Sall2 is a transcription factor associated with development, neuronal differentiation, and cancer. Interestingly, SALL2/Sall2 deficiency leads to failure of the optic fissure closure and neurite outgrowth, suggesting a positive role for SALL2/Sall2 in cell migration. However, in some cancer cells, SALL2 deficiency is associated with increased cell migration. To further investigate the role of Sall2 in the cell migration process, we used immortalized Sall2 knockout (Sall2 -/- ) and Sall2 wild-type (Sall2 +/+ ) mouse embryonic fibroblasts (iMEFs). Our results indicated that Sall2 positively regulates cell migration, promoting cell detachment and focal adhesions turnover. Sall2 deficiency decreased cell motility and altered focal adhesion dynamics. Accordingly, restoring Sall2 expression in the Sall2 -/- iMEFs by using a doxycycline-inducible Tet-On system recovered cell migratory capabilities and focal adhesion dynamics. In addition, Sall2 promoted the autophosphorylation of Focal Adhesion Kinase (FAK) at Y397 and increased integrin β1 mRNA and its protein expression at the cell surface. We demonstrated that SALL2 increases ITGB1 promoter activity and binds to conserved SALL2-binding sites at the proximal region of the ITGB1 promoter, validated by ChIP experiments. Furthermore, the overexpression of integrin β1 or its blockade generates a cell migration phenotype similar to that of Sall2 +/+ or Sall2 -/- cells, respectively. Altogether, our data showed that Sall2 promotes cell migration by modulating focal adhesion dynamics, and this phenotype is associated with SALL2/Sall2-transcriptional regulation of integrin β1 expression and FAK autophosphorylation. Since deregulation of cell migration promotes congenital abnormalities, tumor formation, and spread to other tissues, our findings suggest that the SALL2/Sall2-integrin β1 axis could be relevant for those processes.

Downregulation of glycoprotein non-metastatic melanoma protein B prevents high glucose-induced angiogenesis in diabetic retinopathy.

Diabetic retinopathy (DR), a microvascular complication characterized by abnormal angiogenesis, is the most common reason for irreversible blindness. Glycoprotein non-metastatic melanoma protein B (GPNMB), as a transmembrane protein, was found to be associated with angiogenesis. This study aims to investigate the role of GPNMB in DR. The levels of GPNMB and Integrin β1 were detected by real-time PCR and western blot and were found to be increased in human retinal microvascular endothelial cells (HRMECs) with high glucose (HG, 25mmol/L) treatment. Knockdown of GPNMB was mediated by lentivirus carrying shRNA targeting GPNMB in vivo and in vitro. Functional experiments, including cell counting kit-8 (CCK-8), scratch, and tube formation assays, showed the anti-proliferative, anti-migrative, and anti-angiogenic roles of GPNMB knockdown in HRMECs using the lentivirus system following HG challenge. Additionally, increased GPNMB levels were detected in the retina of DR rats induced by a single intraperitoneal injection of streptozotocin (60mg/kg) using real-time PCR, western blot, and immunofluorescence assays. Downregulation of GPNMB inhibited the angiogenesis and vascular endothelial growth factor production in the retina of rats with DR. Furthermore, overexpression of Integrin β1 led to increased angiogenesis in DR. Integrin β1 was indicated as a target protein of GPNMB. Upregulated-Integrin β1 restored GPNMB knockdown-induced inhibition of cell viability, migration, and tube formation in HRMECs. In conclusion, we provide evidence for the angiogenic role of GPNMB and demonstrate that silencing GPNMB may represent a therapeutic potential in the treatment of DR.

LGL1 binds to Integrin β1 and inhibits downstream signaling to promote epithelial branching in the mammary gland.

SUMMARYBranching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.

Integrin β1 subunit regulates cellular and secreted MUC5AC and MUC5B production in NCI–H292 human lung epithelial cells

The surface of the human respiratory tract is covered with a mucus layer containing mucin 5AC (MUC5AC) and mucin 5B (MUC5B) as the main components. This layer contributes to biological defense by eliminating irritants, but excessive MUC5AC secretion by the airway epithelial cells exacerbates asthma. Therefore, regulating mucin production is important for asthma treatment. In this study, the effects of integrin β1 subunit on MUC5AC and MUC5B production were examined in NCI–H292 human lung cancer epithelial cells. When integrin β1 was overexpressed, cellular and secreted MUC5AC levels were decreased, whereas cellular MUC5B production was increased. Conversely, integrin β1 depletion using siRNA increased cellular and secreted MUC5AC production, but decreased cellular MUC5B production. Further, the activity of extracellular signal-regulated kinase (ERK), which promotes MUC5AC production, was decreased by integrin β1 overexpression and increased by its depletion. These results suggest that integrin β1 suppresses MUC5AC production and promotes MUC5B production by downregulating ERK.

Circ_0085495 knockdown reduces adriamycin resistance in breast cancer through miR-873-5p/integrin β1 axis.

Circular RNAs (circRNAs) are reported to be related to cancer chemoresistance. However, the role of circ_0085495 in adriamycin (ADM) and its action mechanism has not been elucidated in breast cancer. Cell counting kit-8 was employed to detect cell viability. Quantitative real-time-PCR and western blot were performed to examine the gene and protein expression level. Flow cytometry and colony formation assay were conducted to measure cell apoptosis and proliferation. Cell migration and invasion were evaluated via transwell assay. The target association between molecules was confirmed by dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Tumor xenograft assay was implemented to explore the role of circ_0085495 in vivo. Circ_0085495 and Integrin β1 were upregulated, while miR-873-5p was downregulated in ADM-resistant cells. Circ_0085495 was a stable circRNA, mainly located in the cytoplasm. Depletion of circ_0085495 repressed ADM resistance, proliferation and metastasis of ADM-resistant breast cancer cells, which was weakened by miR-873-5p inhibition or integrin β1 overexpression. Circ_0085495 sponged miR-873-5p to positively regulate integrin β1 expression. Integrin β1 knockdown also inhibited ADM resistance. Furthermore, circ_0085495 knockdown inhibited tumor growth in vivo. Circ_0085495 knockdown reduced ADM resistance in ADM-resistant cells through modulating miR-873-5p/integrin β1 axis, indicating circ_0085495 as a promising target for overcoming ADM resistance in breast cancer patients.

Cationic solid lipid nanoparticles loaded by integrin β1 plasmid DNA attenuates IL-1β-induced apoptosis of chondrocyte.

Aging-related inflammation is tightly linked with the development of osteoarthritis (OA). As the pro-inflammatory cytokine, IL-1β has been associated with physical dysfunction and frailty. It is still elusive whether and how IL-1β blockade improves the outcome of OA. Here we develop a cationic solid lipid nanoparticles (SLNs) system that effectively mediate non-viral delivery of plasmid DNA (pDNA) into cells. Compared with other DNA transfer technologies including lipofetamin 2000, SLNs-pDNA system is less toxic and exerts identical effectiveness on DNA transfer. Loaded with integrin β1 overexpression pDNA, the SLNs-pDNA mainly localized in cytoplasm and enforced expression of integrin β1 in rat chondrocytes. Moreover, upon exposure to IL-1β stimulation, SLNs-pDNA treatment attenuates the apoptosis rat chondrocytes and augments tissue repair. Our data thus demonstrate that SLNs-pDNA functions as a potential therapeutic nanomedicine in the treatment of osteoarthritis.

Activin A increases human trophoblast invasion by upregulating integrin β1 through ALK4.

Activin A promotes human trophoblast invasion during the first trimester of pregnancy and is associated with preeclampsia and pregnancy-induced hypertension (PE/PIH) in naturally conceived pregnancies. However, whether integrin β1 mediates activin A-increased trophoblast invasion remains unknown and the evidence is limited regarding the predictive value of activin A for PE/PIH in women receiving in vitro fertilization (IVF) treatment. Here, we studied the role and underlying molecular mechanisms of integrin β1 in activin A-promoted invasion in immortalized (HTR8/SVneo) and primary human extravillous trophoblast (EVT) cells. A nest case-control study was designed to investigate the predictive/diagnostic value of activin A in IVF pregnancies. Results showed that integrin β1 expression increased after activin A treatment and knockdown of integrin β1 significantly decreased both basal and activin A-increased HTR8/SVneo cell invasion. SB431542 (TGF-β type I receptors inhibitor) abolished activin A-induced SMAD2/SMAD3 phosphorylation and integrin β1 overexpression. Activin A-upregulated integrin β1 expression was attenuated after the depletion of ALK4 or SMAD4 in both HTR8/SVneo and primary EVT cells. Furthermore, we found similar first-trimester activin A levels in IVF patients with or without subsequent PE/PIH. These results reveal that integrin β1 mediates activin A-promoted trophoblast invasion through ALK4-activated SMAD2/3-SMAD4 pathway, and the predictive/diagnostic value of first-trimester maternal serum activin A for hypertensive disorders of pregnancy might be different in IVF population.

Effect of Wenxia Changfu Formula Combined With Cisplatin Reversing Non-Small Cell Lung Cancer Cell Adhesion-Mediated Drug Resistance.

Non-small cell lung cancer (NSCLC), the major form of primary lung cancer, is a common cause of cancer-related death worldwide. Cell adhesion-mediated drug resistance (CAM-DR), a form of chemotherapy resistance, has been reported to confer resistance to various chemotherapeutic agents. Integrin β1 signaling plays an important role in CAM-DR and has been proposed as a potential target for NSCLC. Wenxia Changfu Formula (WCF) is a Traditional Chinese Compound Prescription for the intervention treatment of NSCLC combined with cisplatin (DDP). This study aims to investigate the effect and mechanism of WCF combined with DDP in reversing CAM-DR. Firstly, the chemical profile of WCF was characterized by UPLC/Q-TOF-MS analysis. A total of 237 compounds with mzCloud Best Match of greater than 70 were identified by using the online database mzCloud. Secondly, we established A549 three-dimensional(3D) cells cultured in vitro and nude mice xenografts models of the A549 cell line with Integrin β1 overexpression. In vitro, the cell viability, migration and adhesion were measured though MTT Assay, Wound Healing Assay and Cell Adhesion Assay, the Integrin β1 expression of the A549 cells was assessed through immunocytochemistry; in vitro, the transplanted tumor morphology and the colocalization of Integrin β1 and its ligands were tested by HE staining and immunofluorescence. As a result, we found that the combination effectively reduced cell viability, suppressed migration and adhesion, and downregulated the protein level of Integrin β1 in three-dimensional cultured A549 cells. And the combination of WCF with DDP significantly inhibited tumor growth, increased organelle vacuolations and decreased colocalization of Integrin β1 and its ligands including fibulin-2 and laminin. Taken together, our results confirm that the combination of WCF with DDP could reverse the lung cancer CAM-DR through the Integrin β1 signaling pathway.

Integrin β1 in Adipose-Derived Stem Cells Accelerates Wound Healing via Activating PI3K/AKT Pathway.

This study aims to investigate the effect of integrin β1 on wound healing induced by adipose-derived stem cells (ADSCs), as well as the corresponding mechanism. Integrin β1 was overexpressed in ADSCs. Thereafter, flow cytometry and transwell chambers technology were used to measure the endothelial-like differentiation (CD31 as a biomarker of endothelial cell) and cell migration, respectively. Western blot was used to detect the activation of PI3K/AKT, NF-κB and ERK signaling pathways. The effects of integrin β1 overexpression on healing time, healing rate and fibroblast number were further evaluated in the rat models of chronic refractory wound. The overexpression of integrin β1 increased CD31+ endothelial-like cells (about 3.6-fold), promoted cell migration (about 1.9-fold) and enhanced the activation of PI3K (p-PI3K; about 2.1-fold) and AKT (p-AKT; about 2.2-fold). These effects were all weakened when PI3K/AKT pathway was inhibited by LY294002 treatment. In addition, the experiments in rat wound models showed that integrin β1 overexpression obviously shortened healing time (approximately 0.41-fold), increased healing rate (about 2.7-fold, 2.8-fold and 1.6-fold at day 7, 14 and 21) and increased the number of fibroblasts (approximately 3.1-fold at day 21). All of the above differences were statistically significant (p < 0.05). Integrin β1 can promote the migration and endothelial-like differentiation of ADSCs by activating PI3K/AKT pathway and then enhance the function of ADSCs in promoting wound healing.

Chinese Herbal Medicine Wenxia Changfu Formula Reverses Cell Adhesion-Mediated Drug Resistance via the Integrin β1-PI3K-AKT Pathway in Lung Cancer.

In the treatment of lung cancer, the multidrug resistance to chemotherapeutic drugs is one of the reasons of low rates for cure and treatment failure, the combination of chemotherapeutic drugs and traditional Chinese medicine can increase the sensitivity of chemotherapy and reduce its adverse effects. Our previous study has proved that Chinese herbal medicine (CHM) Wenxia Changfu Formula (WCF for short) effectively enhances chemotherapeutic efficacy in lung cancer treatment and reverses multidrug resistance in lung cancer cells in vitro. The present study aims to investigate the effect and mechanism of WCF in reversing cell adhesion-mediated drug resistance of lung cancer by using A549 three-dimensional cell culture and nude mouse model of the A549 cell line with Integrin β1 overexpression. We show that the combination of WCF with DDP can decrease proliferation of lung cancer cells by inducing cell cycle arrest and apoptosis. Moreover, we find that the combination of WCF with DDP suppresses the expression of certain molecules which regulate cell cycle and apoptosis. Mechanistically, we show that the Integrin β1, FAK, PI3K, and AKT protein expressions are suppressed by DDP and even more responses are observed when DDP and WCF are combined, showing WCF treatment enhances the effect of commonly used anticancer drugs. In line with the above findings, our results confirm that WCF reverses cell adhesion-mediated drug resistance of lung cancer via inactivating Integrin β1/PI3K/AKT and apoptosis induction.

Gingipains disrupt F-actin and cause osteoblast apoptosis via integrin β1.

The aim of this study was to explore the cellular mechanisms underlying gingipain-caused changes in cell morphology and apoptosis of osteoblasts. Human calvarial osteoblasts and mouse osteoblasts MC3T3-E1 were treated with gingipain extracts from Porphyromonas gingivalis stain W83. Apoptosis was detected with annexin V and propidium iodide flow cytometry analysis or terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling staining. F-actin was determined by immunostaining. Western blotting was used to detect protein expression. Knocking down and overexpressing approaches were used to determine the role of integrin β1. Osteoblasts exposed to gingipain extracts displayed increased apoptosis, accompanied by loss of F-actin integrity and cell shrinkage. The effects of gingipain extracts were abolished by the cysteine protease inhibitor N-tosyl-l-lysyl chloromethyl-ketone. Notably, gingipain extracts resulted in reduction of integrin β1, accompanied by diminished active RhoA whereas without effect on the total RhoA. Knockdown of integrin β1 resembled those seen in gingipain-treated osteoblasts. By contrast, the effects of gingipain extracts were abrogated by either overexpression of integrin β1 or presence of RhoA agonist CN03. Gingipain-induced F-actin disruption and apoptosis are mediated by the degradation of integrin β1 and inhibition of RhoA activity, which account for osteoblast apoptosis.

Abstract 5938: In vivo targeting of laminin-411-β1 integrin-Notch signaling pathway using nanobioconjugate alters glioma microenvironment for effective treatment

Abstract Introduction: Brain gliomas have been extensively characterized by genomic and molecular marker analysis. These studies have underscored glioblastoma multiforme (GBM) heterogeneity, which may underlie failure of even the newest drugs to improve patient survival. For this reason, we turned to genetic modulation of glioma microenvironment using a novel nanobioconjugate. Methods/Results: Clinical material from 107 GBM patients was analyzed, and tumors with overexpression of “malignant” tumor vascular basement membrane laminin-411 were found to have higher recurrence rate and shorter patient survival. In 92% of human GBM samples, overexpression of laminin-411 was associated with high expression of cancer stem cell (CSC) markers. To examine the interaction mechanisms between GBM cells and their extracellular matrix microenvironment, blood-brain barrier (BBB) passing nanobioconjugates based on poly(β-L-malic acid) (PMLA) were synthesized that specifically inhibit α4 and β1 chains of trimeric laminin-411. In vitro. Normal brain endothelial cells and astrocytes had higher expression of β1 integrin, Notch-1, and Notch ligands when seeded on “malignant” laminin-411 as compared with “normal” laminin-421. All these markers were downregulated in two GBM cell lines treated with antisense oligonucleotides against laminin α4 and β1 chains, suggesting regulation of Notch pathway by laminin-411 through integrin β1. In vivo. In GBM xenograft mouse models increased expression of laminin-411 correlated with overexpression of integrin β1 and Notch signaling pathway members. In two mouse models with intracranial human LN229 and U87MG GBMs, treatment with PMLA-based nanobioconjugate against tumor microenvironment protein laminin-411 led to significantly increased animal survival, associated with marked suppression of laminin-411- β1 integrin-Dll4-Notch axis and CSC markers CD133, Nestin, and c-Myc. Conclusion: BBB crossing and brain tumor-targeted nanodrug therapy using laminin-411 suppression provided a unique tool to study mechanistic interactions between tumor microenvironment and signaling pathways, and showed promise for efficient GBM treatment affecting both the bulk of tumor cells and CSC. Support: NIH grants U01 CA151815, R01 CA136841, R01 CA188743, R01 CA209921 Citation Format: Julia Y. Ljubimova. In vivo targeting of laminin-411-β1 integrin-Notch signaling pathway using nanobioconjugate alters glioma microenvironment for effective treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5938. doi:10.1158/1538-7445.AM2017-5938

Biglycan expression in the melanoma microenvironment promotes invasiveness via increased tissue stiffness inducing integrin-β1 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-β1 expression compared to the Bgn−/− fibroblast matrices. Overexpression of integrin-β1 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-β1, 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-β1 expression by melanoma cells in both mice and humans.

Integrin β1 Increases Stem Cell Survival and Cardiac Function after Myocardial Infarction.

Bone mesenchymal stem cells (BMSCs) transplantation is a promising therapeutic approach for myocardial infarction (MI), but its application is limited by poor viability of BMSCs. In this study, we aimed to improve the survival of BMSCs by lentivirus vector mediated overexpression of integrin β1. In vitro study showed that integrin β1 overexpression could facilitate the proliferation of BMSCs under oxygen glucose deprivation condition and regulated the expression of Caspase-3, Bax, Bcl-2, FAK, and ILK in BMSCs. Next, MI was induced in rat model and Igtb1BMSCs, NullBMSCs, or NatBMSCs were transplanted by intramyocardial injection. One week later, the survival of BMSCs was higher in Itgb1 BMSCs group than in other groups. Four weeks after transplantation, heart function was significantly improved in Igtb1BMSCs group compared to other groups. The expression levels of Caspase-3 and Bax were decreased while the expression levels of Bcl-2, FAK, ILK, and VEGF were increased in the cardiomyocytes of Igtb1BMSCs group compared to other groups. In conclusion, integrin β1 overexpression could increase the survival of BMSCs and improve the efficacy of transplanted BMSCs for MI treatment. The beneficial effects may be mediated by inhibiting the apoptosis of both transplanted BMSCs and cardiomyocytes through adhesion-mediated cell survival signaling.

Intravenous Administration of Endothelial Colony-Forming Cells Overexpressing Integrin β1 Augments Angiogenesis in Ischemic Legs.

When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin β1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin β1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin β1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 10(5) cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance: The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin β1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were observed in the ischemic leg tissue, even at the chronic stage. Moreover, the cells appeared functional, as evidenced by the improved blood flow. The cell type used (ECFCs), the route of administration (intravenous, not directly injected into the affected area), and the use of ligand-receptor interactions (extracellular matrix and integrins) for homing represent substantial advantages over previously reported cell therapies for the treatment of peripheral artery disease.

Integrin-β1 regulates chondrocyte proliferation and apoptosis through the upregulation of GIT1 expression.

Chondrocytes play a critical role in the repair process of osteoarthritis, which is also known as degenerative arthritis. Integrins, as the key family of cell surface receptors, are responsible for the regulation of chondrocyte proliferation, differentiation, survival and apoptosis through the recruitment and activation of downstream adaptor proteins. Moreover, G-protein-coupled receptor kinase interacting protein-1 (GIT1) exerts its effects on cell proliferation and migration through interaction with various cytokines. It has been previously suggested that GIT1 acts as a vital protein downstream of the integrin-mediated pathway. In the present study, we investigated the effects of integrin-β1 on cell proliferation and apoptosis, as well as the underlying mechanisms in chondrocytes in vitro. Following transfection with a vector expressing integrin-β1, our results revealed that the overexpression of integrin-β1 enhanced GIT1 expression, whereas the knockdown of integrin-β1 by siRNA suppressed GIT1 expression. However, no significant effect was observed on integrin-β1 expression following the enforced overexpression of GIT1, which suggests that GIT1 is localized downstream of integrin-β1. In other words, integrin-β1 regulates the expression of GIT1. Furthermore, this study demonstrated that integrin-β1 and GIT1 increased the expression levels of aggrecan and type II collagen, thus promoting chondrocyte proliferation; however, they inhibited chondrocyte apoptosis. Taken together, our data demonstrate that integrin-β1 plays a vital role in chondrocyte proliferation, differentiation and apoptosis. GIT1 exerts effects similar to those of integrin-β1 and is a downstream target of integrin-β1.

Integrin β1 Gene Therapy Enhances in Vitro Creation of Tissue-Engineered Cartilage Under Periodic Mechanical Stress

Background/Aims: Periodic mechanical stress activates integrin β1-initiated signal pathways to promote chondrocyte proliferation and matrix synthesis. Integrin β1 overexpression has been demonstrated to play important roles in improving the activities and functions of several non-chondrocytic cell types. Therefore, in the current study, we evaluated the effects of integrin β1 up-regulation on periodic mechanical stress-induced chondrocyte proliferation, matrix synthesis and ERK1/2 phosphorylation in chondrocyte monolayer culture, and evaluated the quality of tissue-engineered cartilage constructed in vitro under periodic mechanical stress combined with integrin β1 up-regulation. Methods and Results: Our results revealed that under periodic mechanical stress, pre-treatment with integrin β1-wild type vector significantly enhanced chondrocyte proliferation and matrix synthesis and promoted ERK1/2 phosphorylation in comparison to mock transfectants. Furthermore, when chondrocytes were seeded in PLGA scaffolds, more accumulated GAG and type II collagen tissue were detected after Lv-integrin β1 transfection compared with sham controls exposed to periodic mechanical stress. In contrast, in the Lv-shRNA-integrin β1 group, the opposite results were observed. Conclusion: Our findings collectively suggest that in addition to periodic mechanical stress, integrin β1 up-regulation in chondrocytes could further improve the quality of tissue-engineered cartilage.

GW24-e1136 Shear stress affects the uptake of oxidised low-density lipoprotein by human vascular endothelial cells

ObjectivesIn vivo, vascular endothelial cells are constantly subjected to and sensitive to fluid shear stress. Shear stress may be the most crucial local factor affecting atherogenesis. Atherosclerotic lesions preferentially develop...

Identification of integrin β1 as a prognostic biomarker for human lung adenocarcinoma using 2D-LC-MS/MS combined with iTRAQ technology

To discover novel lung adenocarcinoma (AdC) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-tagging combined with 2D-LC-MS/MS analysis was used to identify differentially expressed plasma membrane proteins in lung AdC and paired paraneoplastic normal lung tissues (PNLTs) adjacent to tumors. In this study, significant caveolin-1 downregulation and integrin β1 upregulation was observed in primary lung AdC vs. PNLT. As there has been no report on the association of integrin β1 with lung AdC, immunohistochemical staining was performed to detect the expression of integrin β1 in an independent set of archival tissue specimens including 42 cases of PLNT, 46 cases of without lymph node metastasis primary AdC (non-LNM AdC) and 62 cases of LNM AdC; the correlation of their expression levels with clinicopathological characteristics and clinical outcomes were evaluated. Based on the data, upregulation of integrin β1 was significantly correlated with advanced clinical stage and lymph node metastasis. Integrin β1 overexpression was significantly associated with advanced clinical stage (P<0.05), lymph node metastasis (P<0.05), increased relapse rate (P<0.05) and decreased overall survival (P<0.05) in AdCs. Cox regression analysis indicated that integrin β1 overexpression is an independent prognostic factor. The data suggest that integrin β1 is a potential biomarker for LNM and prognosis of AdC and integrin β1 upregulation may play an important role in the pathogenesis of AdC.