β2 Integrin/ICAM Expression in Crohn's Disease

We have previously reported on the expression of the β2 integrin family of adhesion molecules and their ligands, the ICAM molecules, in the normal human intestine. These molecules likely have a role to play in the inflammatory response and, therefore, were studied in a group of patients with Crohn's disease. A comprehensive study was undertaken in both colon (n= 8) and ileum (n= 10) specimens from 15 patients who underwent surgical resections. Immunohistochemistry was performed for CD18, CD11a, CD11b, CD11c, αd, ICAM-1, ICAM-2, and ICAM-3. Each of the mucosal, submucosal, muscle, and adventitial layers were scored for expression. Specimens from normal colon (n= 15), normal ileum (n= 6), and ulcerative colitis (n= 7) were used for comparisons. Compared with normal, the expression in the colon mucosa and submucosa in Crohn's disease was increased for all β2 integrins. Mucosal CD11c expression was significantly greater in Crohn's disease than in ulcerative colitis. In the colon muscle and adventitial layers the expression in Crohn's disease was similar to normal but increased compared with ulcerative colitis. In Crohn's disease ileum, the β2 integrin mucosal and submucosal expression was similar to normal; however, muscle and adventitial expression was increased, particularly for CD11c. Colon ICAM-1, ICAM-2, and ICAM-3 expression in Crohn's disease was similar to that seen in ulcerative colitis. ICAM-1 was predominantly expressed on endothelium but in the inflammatory bowel diseases was also evident on mucosal mononuclear cells. ICAM-1 and ICAM-2 expression was increased in Crohn's disease colon and ileum compared with normals. This was most notable in ileal mucosa since ICAM-2 is typically absent in normal ileal mucosa. In summary, we are reporting a comprehensive immunohistochemical study of the differential expression of β2 integrins, including the newly described αd molecule, and the ICAM molecules in all layers of the colon and ileum from patients with Crohn's disease. The increased expression of these molecules may have implications for therapeutic interventions in Crohn's disease.     

Integrin α1/β1 and α2/β1 as a receptor for IgA1 in human glomerular mesangial cells in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of IgA1 and galactose-deficient IgA1 is expected to play a pathogenic role. However, the identity of the receptor for IgA1 is still controversial. Hence, the aim of this study was to explore the receptor for galactose-deficient IgA1. Human monoclonal IgA1 was treated with exoglycosidase and FITC-conjugated control, asialo- and agalactosyl-IgA1 was used as a probe to detect the receptor in cultured human mesangial cells. Tumor necrosis factor-α or transforming growth factor-β1 treatment accelerated IgA1-binding on mesangial cells, and these effects were diminished by the addition of dexamethasone, whereas these changes were not dependent on galactose-deficiency of IgA1. According to comprehensive gene expression analysis, we focused on integrin β1. Pre-treatment by Mn(2+), which activates integrin by changing its structure, enhanced the binding of IgA1 in cultured mesangial cells. Furthermore, pre-incubation with collagens specifically enhanced binding of IgA1 in the cultured human mesangial cells without activation by Mn(2+). Collagen type IV distributed in the mesangial region of the glomeruli as well as Bowman's capsule and tubular basal membrane in IgAN patients, and the IgA1 with collagen type IV induced proliferative signals on mesangial cells by phosphorylating extracellular signal-regulated kinase more effectively than the IgA1 alone. Immunoprecipitation assay revealed the binding of IgA1 and integrin α1/β1 and α2/β1 heterodimer and down-regulation of integrin α1, α2 and β1 expression in human mesangial cells induced by each specific small interfering RNA diminished the ability to bind IgA1 probe. Integrin α1/β1 and α2/β1 would be a candidate receptor for IgA1.     

Integrin α5β1: a potent inhibitor of experimental lung metastasis

The integrin alpha5beta1 seems to be the most relevant receptor of tumor cells for binding to fibronectin. Although numerous studies suggest a role of tumor cell fibronectin interaction in tumor metastasis, differential integrin expression on tumor cells has, however, not been correlated with metastatic capabilities. We addressed this question by transfection of the integrin alpha5beta1 cDNA into HT-29 human colon carcinoma cells which led to de novo expression of functional integrin alpha5beta1. Similar to other reports, expression of the integrin alpha5beta1 in HT-29 tumor cells exerted an inhibitory action on cell proliferation as indicated in our study by formation of fewer colonies in soft agar. The tumor growth inhibitory property of the integrin alpha5beta1 was also shown by reduction of subcutaneous xenograft growth in nude mice to approximately 50% of that of control transfectants. For the first time, we found that several clones of integrin alpha5 subunit transfectants displayed dramatically reduced formation of lung colonies and cutaneous metastasis after intravenous injection into nude mice. While most animals inoculated with control transfectant cells formed macroscopically visible lung colonies ranging from 12.6 +/- 2.6 to 22.0 +/- 6.6 (mean colony number +/- SEM), mice inoculated with HT-29 cell clones expressing the integrin alpha5beta1 were almost completely free of lung colonies (ranging from 0.0 +/- 0 to 0.2 +/- 0.1). Our results imply that integrin alpha5beta1 expression inhibits circulating tumor cells in pursuing late steps of the metastatic process as represented by the artificial metastasis (lung colonisation) model.     

Expression and clinical significance of CD151 and integrin α3β1 in colorectal tumors

目的:探讨CD151和整合素α3β1在直肠腺瘤及结直肠腺癌组织中的表达及其与临床各个病理因素之间的关系,并且研究两者的相关性.方法:应用免疫组织化学双染方法对正常结直肠黏膜、结直肠腺瘤及结直肠腺癌组织各120例进行CD151和整合素α3β1检测,并进行Kaplan-Meier生存分析.采用Spearman等级相关分析CD151和整合素αβ1之间的相关性.结果:CD151结直肠正常黏膜、腺瘤、腺癌组织的阳性率分别为21.7％、52.5％、72％,腺瘤和腺癌组织分别与正常黏膜比较均具有统计学意义.整合素α3β1在结直肠正常黏膜、腺瘤、腺癌组织的阳性率分别为34.2％、55％、70％,腺瘤和腺癌组织分别与正常黏膜比较均具有统计学意义.在结直肠腺癌中,CD151和整合素α3β1的表达与患者年龄、性别和肿瘤的部位、大小无相关性,与肿瘤的分化程度、浸润深度、淋巴结转移及Duke's分期有关.CD151和整合素α3β1在大肠正常黏膜、腺瘤及腺癌组织中的表达经双变量相关分析,表达呈正相关.从图的Kaplan-Meier生存曲线及Log-Rank检验可知,CD151+、α3β1+、CD151+α3β1+与大肠癌患者5年生存期密切相关,是影响大肠癌预后的因素.结论:CD151和整合素α3β1在大肠癌的表达密切相关,提示CD151与整合素α3β1复合物存在于大肠癌,其表达对预后产生明显的影响.CD151与整合素α3β1联合表达是临床预后判断的可靠指标.     

Targeting Integrin α4β7 in Steroid-Refractory Intestinal Graft-versus-Host Disease

Steroid refractory acute graft-versus-host-disease of the gut is a serious complication associated with high mortality after allogeneic stem cell transplantation. Treatment options are limited and not predictably effective. We describe the treatment of steroid-refractory acute graft-versus-host-disease with vedolizumab, an antibody directed against integrin α4β7, in 6 patients. All patients responded, and 4 of 6 patients are alive with a median follow-up of 10 months.     

Crucial Role for Endothelial Cell α2β1 Integrin Receptor Clustering in Collagen-Induced Angiogenesis

Angiogenesis is a crucial mechanism of vascular growth and regeneration that requires biosynthesis and cross-linking of collagens in vivo and is induced by collagen in vitro. Here, we use an in vitro model in which apical Type I collagen gels rapidly induce angiogenesis in endothelial monolayers. We extend previous studies demonstrating the importance of the endothelial α2β1 integrin, a key collagen receptor, in angiogenesis by investigating the roles of receptor clustering and conformational activation. Immunocytochemical localization of α2β1 integrins in endothelial monolayers showed a concentration of integrins along cell–cell borders. After inducing angiogenesis with collagen, the receptors redistributed to apical cell surfaces, aligning with collagen fibers, which were also redistributed during angiogenesis. Levels of conformationally activated α2β1 integrins were unchanged during angiogenesis and undetected on endothelial cells binding collagen in suspension. We mimicked the polyvalency of collagen fibrils using antibody-coated polystyrene beads to cluster endothelial cell surface α2β1 integrins, which induced rapid angiogenesis in the absence of collagen gels. Clustering of αvβ3 integrins and PECAM-1 but not of α1 integrins also induced angiogenesis. Soluble antibodies alone had no effect. Thus, the angiogenic property of collagen may reside in its ability to ligate and cluster cell surface receptors such as α2β1 integrins. Furthermore, synthetic substrates that promote the clustering of select endothelial cell surface receptors mimic the angiogenic properties of Type I collagen and may have applications in promoting vascularization of engineered tissues. Anat Rec, 2019. © 2019 American Association for Anatomy     

Integrin αvβ6 predicts poor prognosis and promotes resistance to cisplatin in hilar cholangiocarcinoma

ObjectiveIntegrin αvβ6 is associated with an extremely aggressive cancer phenotype. However, little is known about the clinicopathological significance and prognostic value of integrin αvβ6 in human hilar cholangiocarcinoma.MethodsIn the present study, bioinformatics analysis demonstrated a significant increase of integrin β6 gene expression in cholangiocarcinoma tissues compared to non-tumorous tissues, which was further validated in clinical samples through RT-qPCR and western blotting analyses. Integrin αvβ6 was observed to be expressed in 48.6% of tumors, and its expression was related to a poor tumor differentiation (p = 0.002), lymph node metastasis (p<0.001) and advanced TNM stage (p=0.001). Furthermore, patients who were αvβ6-positive showed a significantly shorter overall survival period than those who were αvβ6-negative (p=0.004). Multivariate analysis confirmed that integrin αvβ6 was an independent prognostic factor (p=0.002). In addition, loss- and gain-of-function assays showed integrin αvβ6 not only played an important role in colony formation, but also protected cholangiocarcinoma cells from cisplatin-induced growth inhibition and apoptosis. ERK/MAPK signaling pathway was involved in integrin αvβ6-mediated resistance of cholangiocarcinoma cells to cisplatin.ConclusionsTaken together, the present findings revealed that integrin αvβ6 could serve as a potential prognostic predictor and contribute to cisplatin resistance, which might prove to be a promising target candidate for the clinical intervention of human hilar cholangiocarcinoma.     

A Key Role for the Integrin α2β1 in Experimental and Developmental Angiogenesis

The α2β1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin α2 I or β1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a β1 I-like domain inhibitor and by function-blocking anti-α2β1 but not -α1β1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish α2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin α2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of α2β1 integrin. Collectively, our results provide strong biochemical and genetic evidence of a central role for α2β1 integrin in experimental and developmental angiogenesis.Angiogenesis is the formation of new capillaries from pre-existing blood vessels and is essential for human development, wound healing, and tissue regeneration.¹ Angiogenesis is dependent on interactions of endothelial cells with growth factors and extracellular matrix components.^2,3 Endothelial cell-collagen interactions are thought to play a role in angiogenesis in vivo and in vitro and require the function of the α1β1 and α2β1 integrins,³ two receptors known to cross talk.⁴ Thus, vascular endothelial growth factor (VEGF)-induced angiogenesis in Matrigel plugs implanted in mice is markedly inhibited by anti-α1β1 and -α2β1 integrin antibodies.^5,6 Studies using various collagen-induced angiogenesis assays also suggest a critical role for endothelial cell α2β1 integrin^2,7,8 binding to the GFPGER_502–507 sequence of the collagen triple helix.⁹ Consistent with these findings, endorepellin, a potent anti-angiogenic molecule derived from the C terminus of perlecan^10,11 disrupts α2β1 integrin function,^{12,13,14,15,16} and some of the affected gene products have been associated with the integrin-mediated angiogenesis.¹⁷ Endothelial cell-collagen interactions may also contribute to tumor-associated angiogenesis.¹⁸ For example, gene products up-regulated in tumor-associated endothelial cells include types I, III, and VI collagens,¹⁹ and tumor-associated angiogenesis is sensitive to endorepellin treatment.^15,20,21Interestingly, α2β1 integrin-null mice show no overt alteration in either vasculogenesis or angiogenesis but display only a mild platelet dysfunction phenotype and altered branching morphogenesis of the mammary glands.^22,23 This observation suggests that in mammals, there is functional compensation during development, but that α2β1 integrin might be required for postnatal angiogenesis. Indeed, when adult α2β1-null mice are experimentally challenged, they show an enhanced angiogenic response during wound healing²⁴ and tumor xenograft development.^15,25The α1β1 and α2β1 integrins include inserted domains (I domains) in their α subunits that mediate ligand binding.^26,27 The α2 I domain is composed of a Rossman fold and a metal ion coordination site (MIDAS), proposed to ligate the GFPGER_502–507 sequence of collagen, thereby inducing receptor activation.^26,28 Other integrin domains may also play a role in ligand binding and receptor activation. For example, the β1 I-like domain seems to allosterically modulate collagen ligation by the α2 I domain, and, intracellularly, the cytoplasmic sequence of the α2 subunit functions as a hinge, locking the receptor in an inactive conformation, and membrane-soluble peptide mimetics of this sequence were shown to promote α2β1 receptor activation.²⁹ Recently, a family of small molecule inhibitors (SMIs)² targeting the function of the α2β1 integrin were designed.³⁰ Specifically, inhibitors of α2β1 integrin function were prepared using modular synthesis, enabling substitutions of arylamide scaffold backbones with various functional groups, creating SMIs targeted to the I domain or the intact integrin.^30,31,32 In this study, we tested the activities of a group of SMIs on endothelial cell-collagen interactions and angiogenesis in vitro and in vivo. We provide evidence that SMI496, which binds between the I domains of β1 and α2 subunits,³² interferes with α2β1 integrin activity on endothelial cells both in vitro and in vivo, suggesting a potential therapeutic modality to interfere with angiogenesis. Moreover, interference with α2 integrin expression in embryonic zebrafish caused a vascular phenotype characterized by abnormal angiogenesis.     

Hypothermia Attenuates β1 Integrin Expression on Extravasated Neutrophils in an Animal Model of Meningitis

Brain injury in meningitis occurs in part as a consequence of leukocyte migration and activation. Leukocyte integrins are pivotal in the inflammatory response by mediating adhesion to vascular endothelium and extracellular matrix proteins. We have demonstrated that moderate hypothermia early in the course of meningitis decreases leukocyte sequestration within the brain parenchyma. This study examines whether hypothermia alters neutrophil integrin expression in a rabbit model of bacterial meningitis. Prior to the induction of meningitis, peripheral blood samples were obtained and the neutrophils isolated. Sixteen hours after inducing group B streptococcal meningitis, animals were treated with antibiotics, IV fluids, and mechanically ventilated. Animals were randomized to hypothermia (32–33°C) or normothermia conditions. After 10 hours of hypothermia or normothermia, neutrophils were isolated from the blood and cerebral spinal fluid (CSF), stained for 1 and 2 integrins, and analyzed using flow cytometry. Cerebral spinal fluid neutrophil 1 integrin expression was significantly decreased in hypothermic animals. Beta-1 integrins can assume a higher affinity or "activated" state following inflammatory stimulation. Expression of "activated" 1 integrins was also significantly decreased in hypothermic animals. Beta2 CSF neutrophil integrin expression was decreased in hypothermic animals, but failed to reach significance. These data suggest hypothermia may attenuate extravasated leukocyte expression of both total and "activated" 1 integrins.     

Integrin α7 Binds Tissue Inhibitor of Metalloproteinase 3 to Suppress Growth of Prostate Cancer Cells

Integrin α7 (ITGA7) is a tumor-suppressor gene that is critical for suppressing the growth of malignant tumors; however, the mechanisms allowing ITGA7 to suppress the growth of cancer cells remain unclear. Herein, we show that ITGA7 binds to tissue inhibitor of metalloproteinase 3 (TIMP3) in prostate cancer cells. The ITGA7-TIMP3 binding led to a decreased protein level of tumor necrosis factor α, cytoplasmic translocation of NF-κB, and down-regulation of cyclin D1. These changes led to an accumulation of cells in G₀/G₁ and a dramatic suppression of cell growth. Knocking down TIMP3 or ITGA7/TIMP3 binding interference largely abrogated the signaling changes induced by ITGA7, whereas a mutant ITGA7 lacking TIMP3 binding activity had no tumor-suppressor activity. Interestingly, knocking down ITGA7 ligand laminin β1 enhanced ITGA7-TIMP3 signaling and the downstream tumor-suppressor activity, suggesting the existence of a counterbalancing role between extracellular matrix and integrin signaling. As a result, this report demonstrates a novel and critical signaling mechanism of ITGA7, through the TIMP3/NF-κB/cyclin D1 pathway.Integrin α7 (ITGA7) is a member of the extracellular matrix binding proteins. As a major class of cell adhesion molecules in mammalian cells, integrins are involved in many cellular processes, including development, immune responses, leukocyte trafficking, and hemostasis.¹ The integrin superfamily consists of 24 members, each of which mediates a unique function in mammals. The regulation of integrin expression is critical for certain aspects of tissue differentiation and regeneration (eg, keratinocyte differentiation, hair follicle formation, and skeletal muscle development),^2–4 and abnormal integrin expression is associated with several human diseases (eg, muscular dystrophy, Glanzmann’s thrombasthenia, and congenital cardiac myopathy).^4–6ITGA7 forms a heterodimer with integrin β1 in the plasma membrane and is responsible for communication between the extracellular matrix and cells.⁷ Itga7-deficient mice display significant hyperplasia and hypertrophy of arteries and arterioles and a malformation of skeletal muscles.^4,8 Recent mutational analysis revealed ITGA7 mutations in prostate cancer, hepatocellular carcinoma, soft tissue leiomyosarcoma, and glioblastoma multiforme, with frequencies ranging from 25% to 83%.⁹ Many of these mutations resulted in truncation, microdeletion, or frameshift of the protein. Interestingly, patients with prostate cancer or hepatocellular carcinoma harboring ITGA7 mutations also had a higher rate of clinical relapse.A meta-analysis of previously published microarray data^10–15 indicated that ITGA7 was down-regulated in nonmetastatic prostate cancer and leiomyosarcoma, but the magnitude of the down-regulation was larger in metastatic cancers. Also, prostate cancer and soft tissue leiomyosarcoma, with focal or no ITGA7 expression, were associated with a shorter metastasis-free survival time. The forced expression of normal ITGA7 in prostate cancer and leiomyosarcoma cell lines suppressed tumor growth and cancer cell migration in vitro. A mouse model of PC3 and DU145 xenograft prostate tumors showed a dramatic reduction in tumor volume, metastatic rate, and mortality rate when ITGA7 expression was restored. However, the molecular mechanism of ITGA7-mediated tumor-suppressor activity remains unclear. Herein, we report that the tissue inhibitor of metalloproteinase 3 (TIMP3), a matrix proteinase and a tumor suppressor, interacts with the C-terminus of ITGA7. We further show that the activation of ITGA7 leads to redistribution of NF-κB to the cytoplasm, the down-regulation of cyclin D1, and cell growth arrest.     

Expression of Nerve Growth Factor in Human Pancreatic β Cells

Nerve growth factor (NGF) is an important modulator of rat pancreatic β-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy.

We found that in normal human pancreas, insulin and NGF are co-expressed in β cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in β cells.

Pancreatic β cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor.

In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate.

We demonstrate that human β cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat β cells.     

All-trans-Retinoic Acid Imprints Expression of the Gut-Homing Marker α4β7 while Suppressing Lymph Node Homing of Dendritic Cells

Tissue-directed trafficking of dendritic cells (DCs) as natural adjuvants and/or direct vaccine carriers is highly attractive for the next generation of vaccines and immunotherapeutics. Since these types of studies would undoubtedly be first conducted using nonhuman primate models, we evaluated the ability of all-trans-retinoic acid (ATRA) to induce gut-homing α4β7 expression on rhesus macaque plasmacytoid and myeloid DCs (pDCs and mDCs, respectively). Induction of α4β7 occurred in both a time-dependent and a dose-dependent manner with up to 8-fold increases for mDCs and 2-fold increases for pDCs compared to medium controls. ATRA treatment was also specific in inducing α4β7 expression, but not expression of another mucosal trafficking receptor, CCR9. Unexpectedly, upregulation of α4β7 was associated with a concomitant downregulation of CD62L, a marker of lymph node homing, indicating an overall shift in the trafficking repertoire. These same phenomena occurred with ATRA treatment of human and chimpanzee DCs, suggesting a conserved mechanism among primates. Collectively, these data serve as a first evaluation for ex vivo modification of primate DC homing patterns that could later be used in reinfusion studies for the purposes of immunotherapeutics or mucosa-directed vaccines.     

Expression of Retrovirally Transduced IL-1 α in IL-6-Dependent B Cells: A Murine Model of Aggressive Multiple Myeloma

Abstract

Retroviral-mediated gene transfer was employed to introduce an IL-1α cDNA into an IL-6-dependent murine B-cell line. Bone marrow metastases and bone lesions were frequently observed following intravenous injection of these B cells into syngeneic mice. Because the retroviral vector also contained the neomycin phosphotransferase gene, metastatic cells could be easily recovered from bone marrow by addition of G418 to the culture medium. Interestingly, the metastatic B cells were found to retain their IL-6 dependency through several transplant generations. By comparison, intravenous injection of autonomously-growing B-cell lines generated in vitro by retroviral introduction of an IL-6 cDNA rarely resulted in bone marrow metastases. These results demonstrate that abrogation of growth factor dependency is neither necessary nor sufficient for the in vivo growth and dissemination of tumor cells in this experimental system. It is proposed that the increased metastasis of the IL-1 α-producing B-cells to bone marrow is due to alterations in cell adhesion molecules. The B-cell bone marrow metastasis model described here may be useful for studies of bone marrow homing and for evaluation of therapeutic regimens for multiple myeloma.