Immunohistochemical characterization of integrin alfa 6 in uterus and cervix of the cynomolgus monkeys (Macaca fascicularis) with MfPV3 infection.

BACKGROUND: Cervical cancer is an abnormal growth of cervical tissue epithelial cells due to persistent human papilloma virus (HPV) infection. Cynomolgus monkeys (Macaca fascicularis) can be naturally and spontaneously infected with M. fascicularis Papillomavirus Type 3 (MfPV3), a virus that is phylogenetically closely related to human oncogenic HPV (HPV-16 and HPV-34), and therefore a potentially beneficial for modeling HPV disease. This study aims to evaluate the expression of the integrin alpha 6 (ITGα6) receptor in cynomolgus monkeys spontaneously infected with MfPV3, which this receptor also found in human infected with HPV. METHODS: The study was done on archived Formalin-fixed Paraffin-Embedded (FFPE) samples of uterine and cervix tissue of cynomolgus monkeys. Immunohistochemistry was also performed to quantify the expression levels of ITGα6. RESULTS: The results showed 80% of the samples positive Cervical Intraepithelial Neoplasia (CIN) and increased expression of ITGα6 significantly in Positive-MfPV3 group than negative-MfPV3 group. CONCLUSIONS: This indicated the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection type.

The pathophysiological role of circulating adhesion molecules in schizophrenia: A systematic review and meta-analysis.

BACKGROUND: Increasing evidence suggests an association between schizophrenia and atherosclerosis. We conducted a systematic review and meta-analysis of cell adhesion molecules, critically involved in early atherosclerosis, in schizophrenia. METHODS: We searched electronic databases from inception to 11 November 2023 for case-control studies assessing vascular cell, VCAM-1, intercellular, ICAM-1, platelet endothelial cell, PECAM-1, neural cell, NCAM, and Down syndrome cell, DSCAM, adhesion molecules, selectins (E-, L-, and P-selectin), integrins, and cadherins in patients with schizophrenia and healthy controls. Risk of bias and certainty of evidence were assessed using the JBI checklist and GRADE, respectively. RESULTS: In 19 eligible studies, there were non-significant between-group differences in the concentrations of cell adhesion molecules, barring higher P-selectin in patients with schizophrenia (standard mean difference, SMD = 2.05, 95 % CI 0.72 to 3.38, p = 0.003; I2 = 97.2 %, p<0.001; very low certainty of evidence). Limited or no information was available regarding PECAM-1, DSCAM, ESAM, integrins, and cadherins. In meta-regression and subgroup analysis, there were significant associations between the SMD of ICAM-1 and matrix used (plasma or serum) and pharmacological treatment of schizophrenia, and between the SMD of VCAM-1 and pharmacological treatment, but not with other study and patient characteristics. CONCLUSIONS: The results of our systematic review and meta-analysis do not support a significant role of immunoglobulin-like adhesion molecules, selectins, integrins, or cadherins in mediating the associations between schizophrenia, atherosclerosis, and cardiovascular disease. Further studies are warranted to investigate these associations in patients with different cardiovascular risk and the effects of antipsychotic treatments on cell adhesion molecules and surrogate markers of atherosclerosis (PROSPERO registration number: CRD42023463916).

Development of a Ratiometric Tension Sensor Exclusively Responding to Integrin Tension Magnitude in Live Cells.

Integrin tensions are critical for cell mechanotransduction. By converting force to fluorescence, molecular tension sensors image integrin tensions in live cells with a high resolution. However, the fluorescence signal intensity results collectively from integrin tension magnitude, tension dwell time, integrin density, sensor accessibility, and so forth, making it highly challenging to specifically monitor the molecular force level of integrin tensions. Here, a ratiometric tension sensor (RTS) was developed to exclusively monitor the integrin tension magnitude. The RTS consists of two tension-sensing units that are coupled in series and always subject to the same integrin tension. These two units are activated by tension to fluoresce in separate spectra and with different activation rates. The ratio of their activation probabilities, reported by fluorescence ratiometric measurement, is solely determined by the local integrin tension magnitude. RTS responded sensitively to the variation of integrin tension magnitude in platelets and focal adhesions due to different cell plating times, actomyosin inhibition, or vinculin knockout. At last, RTS confirmed that integrin tension magnitude in platelets and focal adhesions decreases monotonically with the substrate rigidity, verifying the rigidity dependence of integrin tensions in live cells and suggesting that integrin tension magnitude could be a key biomechanical factor in cell rigidity sensing.

Expression and diagnostic significance of integrin beta-2 in synovial fluid of patients with osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by synovial cartilage degeneration and is the leading cause of disability and pain worldwide. This study sought to investigate the expression of integrin beta-2 (ITGB2) in synovial fluid of OA patients and its clinical significance. METHODS: A total of 110 OA patients were enrolled, who were classified into grade I (N = 35), II (N = 42), and III (N = 33) according to the Kellgren-Lawrence classification, with 110 healthy subjects as controls, and their clinical data were compared. ITGB2 level was detected by RT-qPCR. The receiver operating characteristic curve was used to analyze the predictive value of ITGB2 on OA occurrence. The correlation between ITGB2 and bone metabolism indexes procollagen type I N-terminal peptide (PINP), bone glaprotein (BGP), bone alkaline phosphatase (BALP), and ß-collagen I telopeptide (ß-CTX) was analyzed by the Pearson method. Logistic regression model was performed to analyze the influencing factors of OA. RESULTS: The content of red blood cells, white blood cells, PINP, BGP, and BALP was lowered in OA patients, while ß-CTX was elevated. ITGB2 was highly-expressed in OA patients, negatively-correlated with PINP, BGP, and BALP, but positively-correlated with ß-CTX. ITGB2 level increased with the elevation of OA grade. The ITGB2 level >1.375 had certain diagnostic values for OA. ITGB2 level is related to OA severity and may be a biomarker for OA classification. ITGB2 was an independent risk factor for OA. CONCLUSION: High expression of ITGB2 in synovial fluid can assist OA diagnosis and may be a biomarker for OA grade.

Differential Regulation of Integrin α5 and ß4 in Normal and Psoriatic Epidermal Keratinocytes

Abstract Psoriasis is a chronic skin inflammation, characterized by impaired differentiation, hyperproliferation of keratinocytes involving pro-inflammatory factors interleukin (IL)-13/17A, tumor necrosis factor (TNF)-α, interferon (IFN)-γ. Among the integrin family, α5 is important for blood vessel formation, and ß4 for proliferation, differentiation of keratinocytes. To investigate the expression and regulation of integrin α5 and ß4 in psoriatic keratinocytes. Skin biopsies were obtained from 14 psoriatic patients and 12 normal volunteers. We compared the immunolocalization and regulation of α5 and ß4 between the psoriatic and normal ones, before and after incubation with MEK/ERK pathway inhibitor U0126 by immunohistochemistry and western blot separately. Immunohistochemistry showed psoriatic keratinocytes had higher α5 than normal ones. According to western blot, IL-17A and IL-13 increased normal keratinocytes' α5 and ß4 respectively, but psoriatic keratinocytes were the exact opposite. Incubated with U0126, normal keratinocytes' α5 was enhanced by the 5 cytokines ; while IL-13/17A, IFN-γ suppressed ß4. Psoriatic keratinocytes' α5 was increased by IL-13/17A, decreased by IFN-γ; but ß4 increased by IL-17A, IFN-γ. IL-13/17A, TNF-α, IFN-γ regulate α5 and ß4 through ERK pathway whether normal or psoriasis. The normal and psoriatic keratinocytes respond to the same cytokines differently

Integrins: An Important Link between Angiogenesis, Inflammation and Eye Diseases.

Integrins belong to a group of cell adhesion molecules (CAMs) which is a large group of membrane-bound proteins. They are responsible for cell attachment to the extracellular matrix (ECM) and signal transduction from the ECM to the cells. Integrins take part in many other biological activities, such as extravasation, cell-to-cell adhesion, migration, cytokine activation and release, and act as receptors for some viruses, including severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). They play a pivotal role in cell proliferation, migration, apoptosis, tissue repair and are involved in the processes that are crucial to infection, inflammation and angiogenesis. Integrins have an important part in normal development and tissue homeostasis, and also in the development of pathological processes in the eye. This review presents the available evidence from human and animal research into integrin structure, classification, function and their role in inflammation, infection and angiogenesis in ocular diseases. Integrin receptors and ligands are clinically interesting and may be promising as new therapeutic targets in the treatment of some eye disorders.

Spectroscopic Analysis of a Library of DNA Tension Probes for Mapping Cellular Forces at Fluid Interfaces.

Oligonucleotide-based probes offer the highest spatial resolution, force sensitivity, and molecular specificity for cellular tension sensing and have been developed to measure a variety of molecular forces mediated by individual receptors in T cells, platelets, fibroblasts, B-cells, and immortalized cancer cell lines. These fluorophore-oligonucleotide conjugate probes are designed with a stem-loop structure that engages cell receptors and reversibly unfolds due to mechanical strain. With the growth of recent work bridging molecular mechanobiology and biomaterials, there is a need for a detailed spectroscopic analysis of DNA tension probes that are used for cellular imaging. In this manuscript, we conducted an analysis of 19 DNA hairpin-based tension probe variants using molecular dynamics simulations, absorption spectroscopy, and fluorescence imaging (epifluorescence and fluorescence lifetime imaging microscopy). We find that tension probes are highly sensitive to their molecular design, including donor and acceptor proximity and pairing, DNA stem-loop structure, and conjugation chemistry. We demonstrate the impact of these design features using a supported lipid bilayer model of podosome-like adhesions. Finally, we discuss the requirements for tension imaging in various biophysical contexts and offer a series of experimental recommendations, thus providing a guide for the design and application of DNA hairpin-based molecular tension probes.

3D Imaging and Quantification of the Integrin at a Single-Cell Base on a Multisignal Nanoprobe and Synchrotron Radiation Soft X-ray Tomography Microscopy.

The development of three-dimensional (3D) single-cell imaging and protein quantitative methods can provide more comprehensive information for diagnoses. We report the design and synthesis of a multisignal nanoprobe (AuGdNC@BSA-CV) for single-cell 3D imaging and quantifying the integrin αIIbß3 using correlated synchrotron radiation soft X-ray tomography microscopy and an iterative tomographic algorithm termed equally sloped tomography for the first time. Moreover, on the basis of the Au or Gd content of our nanoprobe, the number of integrin αIIbß3 on a single cell also can be accurately quantified (1.5 × 107 per cell) via inductively coupled plasma mass spectrometry.

Click-Chemistry (CuAAC) Trimerization of an αv ß6 Integrin Targeting Ga-68-Peptide: Enhanced Contrast for in-Vivo PET Imaging of Human Lung Adenocarcinoma Xenografts.

αv ß6 Integrin is an epithelial transmembrane protein that recognizes latency-associated peptide (LAP) and primarily activates transforming growth factor beta (TGF-ß). It is overexpressed in carcinomas (most notably, pancreatic) and other conditions associated with αv ß6 integrin-dependent TGF-ß dysregulation, such as fibrosis. We have designed a trimeric Ga-68-labeled TRAP conjugate of the αv ß6 -specific cyclic pentapeptide SDM17 (cyclo[RGD-Chg-E]-CONH2 ) to enhance αv ß6 integrin affinity as well as target-specific in-vivo uptake. Ga-68-TRAP(SDM17)3 showed a 28-fold higher αv ß6 affinity than the corresponding monomer Ga-68-NOTA-SDM17 (IC50 of 0.26 vs. 7.4 nM, respectively), a 13-fold higher IC50 -based selectivity over the related integrin αv ß8 (factors of 662 vs. 49), and a threefold higher tumor uptake (2.1 vs. 0.66 %ID/g) in biodistribution experiments with H2009 tumor-bearing SCID mice. The remarkably high tumor/organ ratios (tumor-to-blood 11.2; -to-liver 8.7; -to-pancreas 29.7) enabled high-contrast tumor delineation in PET images. We conclude that Ga-68-TRAP(SDM17)3 holds promise for improved clinical PET diagnostics of carcinomas and fibrosis.

The role of integrins in melanoma: a review.

Integrins are the major family of cell adhesion receptors in humans and essential for a wide range of normal physiology, including formation and maintenance of tissue structure integrity, cell migration, proliferation, and differentiation. Integrins also play a prominent role in tumor growth and metastasis. Translational research has tried to define the contribution of integrins to the phenotypic aggressiveness of melanoma because such knowledge is clinically useful. For example, differential expression of integrins in primary cutaneous melanoma can be used to distinguish indolent from aggressive, prometastatic melanoma. Recent studies have shown that gene expression-based testing of patient-derived melanoma tissue is feasible, and molecular tests may fully replace interventional surgical methods such as sentinel lymph node biopsies in the future. Because of their central role in mediating invasion and metastasis, integrins are likely to be useful biomarkers. Integrins are also attractive candidate targets for interventional therapy. This article focuses on the role of integrins in melanoma and highlights recent advances in the field of translational research.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.

OBJECTIVE: The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). METHODOLOGY: Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. RESULTS: FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. CONCLUSIONS: Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Integrin α11ß1 is expressed in breast cancer stroma and associates with aggressive tumor phenotypes.

Cancer-associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen-binding integrin α11ß1 has been proposed to be upregulated in a pro-tumorigenic subtype of cancer-associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11ß1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin-fixed paraffin-embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co-expression patterns of integrin α11 in relation to αSMA and cytokeratin-14 were also investigated. Integrin α11 was expressed to varying degrees in spindle-shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co-localized with αSMA in stromal cells, and with αSMA and cytokeratin-14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple-negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.

Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvß6 integrin-positive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin αVß6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets αVß6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvß6 integrin, as a probe scaffold, and 68Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with 68Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVß6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([natGa]CG6 and [natGa]Ac-CG6) showed high affinity to αVß6 integrin. Both probes could be labeled by 67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [67Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [67Ga]CG6. The αVß6 integrin-positive xenografts were clearly visualized by PET imaging with [68Ga]Ac-CG6. The intratumoral distribution of [68Ga]Ac-CG6 coincided with the αVß6 integrin-positive regions detected by immunohistochemistry. Thus, [68Ga]Ac-CG6 is a useful peptide probe for the imaging of αVß6 integrin in PDAC.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Integrated Transcriptomics, Proteomics, and Glycomics Reveals the Association between Up-regulation of Sialylated N-glycans/Integrin and Breast Cancer Brain Metastasis.

Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.

Antitumoral Potential of Lansbermin-I, a Novel Disintegrin from Porthidium lansbergii lansbergii Venom on Breast Cancer Cells.

BACKGROUND: Disintegrins from snake venoms bind with high specificity cell surface integrins, which are important pharmacological targets associated with cancer development and progression. OBJECTIVE: In this study, we isolated a disintegrin from the Porthidium lansbergii lansbergii venom and evaluated its antitumoral effects on breast cancer cells. METHODS: The isolation of the disintegrin was performed on RP-HPLC and the inhibition of platelet aggregation was evaluated on human platelet-rich plasma. The inhibition of cell adhesion was also evaluated in vitro on cultures of cell lines by the MTT method as well as the inhibition of breast cancer cell migration by the wound healing assay. The binding of the disintegrin to integrin subunits was verified by flow cytometry and confocal microscopy. Finally, inhibition of angiogenesis was assessed in vitro on HUVEC cells and the concentration of VEGF was measured in the cellular supernatants. RESULTS: The disintegrin, named Lansbermin-I, is a low molecular weight protein (< 10 kDa) that includes an RGD on its sequence identified previously. Lansbermin-I showed potent inhibition of ADP and collagen-induced platelet aggregation on human plasma and also displayed inhibitory effects on the adhesion and migration of breast cancer MCF7 and MDA-MB 231cell lines, without affecting nontumorigenic breast MCF-10A and lung BEAS cells. Additionally, Lansbermin-I prevented MCF7 cells to adhere to fibronectin and collagen, and also inhibited in vitro angiogenesis on human endothelial HUVEC cells. CONCLUSION: Our results display the first report on the antitumor and anti-metastatic effects of an RGDdisintegrin isolated from a Porthidium snake venom by possibly interfering with α2 and/or ß1-containing integrins. Thus, Lansbermin-I could be an attractive model to elucidate the role of disintegrins against breast cancer development.

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control.

Nanoscale organisation of receptor ligands has become an important approach to study the clustering behaviour of cell-surface receptors. Biomimetic substrates fabricated via different nanopatterning strategies have so far been applied to investigate specific integrins and cell types, but without multivalent control. Here we use DNA origami to surpass the limits of current approaches and fabricate nanoarrays to study different cell adhesion processes, with nanoscale spatial resolution and single-molecule control. Notably, DNA nanostructures enable the display of receptor ligands in a highly customisable manner, with modifiable parameters including ligand number, ligand spacing and most importantly, multivalency. To test the adaptability and robustness of the system we combined it with focused ion beam and electron-beam lithography nanopatterning to additionally control the distance between the origami structures (i.e. receptor clusters). Moreover, we demonstrate how the platform can be used to interrogate two different biological questions: (1) the cooperative effect of integrin and growth factor receptor in cancer cell spreading, and (2) the role of integrin clustering in cardiomyocyte adhesion and maturation. Thereby we find previously unknown clustering behaviour of different integrins, further outlining the importance for such customisable platforms for future investigations of specific receptor organisation at the nanoscale.

Effect of plasma-nitrided titanium surfaces on the differentiation of pre-osteoblastic cells.

A titanium surface nitrided by plasma contains nitrogen ions that guarantee resistance to corrosion and biocompatibility. Despite this, no descriptions concerning the influence of the expression of cell adhesion proteins and their influence on osteogenic cell differentiation are available. Thus, the present study aimed to assess the response of murine pre-osteoblastic cells (MC3T3-E1) cultured on nitrided titanium surfaces. Pre-osteoblastic cells were grown on polished titanium discs, used as controls, and on previously characterized plasma-nitrided titanium discs. Cells from both groups were submitted to the MTT cell viability test. The expressions of α5, α2, and ß1 integrin were assessed by flow cytometry and immunofluorescence, while osteocalcin expression was assessed by flow cytometry. The nitrided surface presented higher α2 and ß1 integrin expressions, as well as osteocalcin expression, when compared to the polished surface, with no alterations in cell viability. These findings seem to suggest that the plasma nitriding treatment produces a titanium surface with the potential for effective in vitro osseointegration.

Peptide-Functionalized Hydrogels Modulate Integrin Expression and Stemness in Adult Human Epidermal Keratinocytes.

The extracellular matrix (ECM) controls keratinocyte proliferation, migration, and differentiation through ß-integrin signaling. Wound-healing research requires expanding cells in vitro while maintaining replicative capacity; however, early terminal differentiation under traditional culture conditions limits expansion. Here, a design of experiments approach identifies poly(ethylene glycol)-based hydrogel formulations with mechanical properties (elastic modulus, E = 20.9 ± 0.56 kPa) and bioactive peptide sequences that mimic the epidermal ECM. These hydrogels enable systematic investigation of the influence of cell-binding domains from fibronectin (RGDS), laminin (YIGSR), and collagen IV (HepIII) on keratinocyte stemness and ß1 integrin expression. Quantification of 14-day keratin protein expression shows four hydrogels improve stemness compared to standard techniques. Three hydrogels increase ß1 integrin expression, demonstrating a positive linear relationship between stemness and ß1 integrin expression. Multifactorial statistical analysis predicts an optimal peptide combination ([RGDS] = 0.67 mm, [YIGSR] = 0.13 mm, and [HepIII] = 0.02 mm) for maintaining stemness in vitro. Best-performing hydrogels exhibit no decrease in Ki-67-positive cells compared to standards (15% decrease, day 7 to 14; p < 0.05, Tukey Test). These data demonstrate that precisely designed hydrogel biomaterials direct integrin expression and promote proliferation, improving the regenerative capability of cultured keratinocytes for basic science and translational work.

Analyzing Integrin-Dependent Adhesion.

In this unit, methods for the analysis of integrin-dependent adhesion are described. Two major types of assays are commonly used for this analysis. The first are cell adhesion assays. A key application of this type of assay is to identify which integrin(s) mediate cell-substrate interactions; a comprehensive list of antibodies suitable for this purpose is detailed. The second are solid-phase assays in which purified integrins and integrin ligands are used. These assays can be used, e.g., to measure apparent affinities of integrins for different ligands and IC50 values of pharmacological inhibitors. Curr. Protoc. Cell Biol. 53:9.4.1-9.4.17. © 2018 by John Wiley & Sons, Inc.