In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines.

Papaverine (PPV) is a benzylisoquinoline alkaloid isolated from Papaver somniferum that exerts antiproliferative activity. However, several questions remain regarding the biochemical pathways affected by PPV in tumourigenic cells. In this study, the influence of PPV on cell migration (light microscopy), expression of vascular endothelial growth factor (VEGF) B, VEGF R1, VEGF R2, and phosphorylated focal adhesion kinase (pFAK) were investigated using spectrophotometry in MDA-MB-231-, A549- and DU145 cell lines. The migration assay revealed that, after 48 h, PPV (100 µM) reduced cell migration to 81%, 91%, and 71% in MDA-MB-231-, A549-, and DU145 cells, respectively. VEGF B expression was reduced to 0.79-, 0.71-, and 0.73-fold after 48 h of exposure to PPV in MDA-MB-231-, A549- and DU145 cells, while PPV exposure of 48 h increased VEGF R1 expression in MDA-MB-231- and DU145 cells to 1.38 and 1.46. A fold decrease in VEGF R1 expression was observed in A549 cells to 0.90 after exposure to 150 µM. No statistically significant effects were observed on VEGF R2- and FAK expression after exposure to PPV. This study contributes to the understanding of the effects of a phytomedicinal alkaloid compound in cancer cells and may provide novel approaches to the application of non-addictive alkaloids.

miR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors.

The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR-125a-5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR-125a-5p mimic, imatinib-resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST-1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fisher's exact test or log-rank test. Over-expression of miR-125a-5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib-sensitive parental cells. Treatment with FAKi in imatinib-resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR-125a-5p over-expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR-125a-5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST.

Insulin promotes cell migration by regulating PSA-NCAM.

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration.

PEGylated TiO2 nanoparticles mediated inhibition of cell migration via integrin beta 1.

Nanoparticles (NPs) elicit various physiological responses in cellular environment, and the effect of NPs on cell migration is of high interest. In this work, the effects of NPs on cell migration and their possible mechanisms were studied. Here, we showed that after exposure to pegylated titanium dioxide nanoparticles (TiO2-PEG NPs, where PEG stands for the polyethylene glycol), NCI-H292 cells exhibited slower migration than control cells. Furthermore, larger NPs inhibited cell migration much stronger than smaller NPs. Following NP exposure, the cells showed decreased expression of integrin beta 1 and phosphorylated focal adhesion kinase (pFAK), and disrupted F-actin structures. We demonstrated that a possible mechanism involved NP-mediated promotion of the lysosomal degradation of integrin beta 1, thus leading to reduced expression of pFAK and cytoskeletal disruption and inhibited cell migration. Therefore, our results showed that inhibition of NCI-H292 cell migration by NPs is mediated through integrin beta 1, which provides useful information for the application of NPs in cancer therapy and related fields.

Ca(2+) and EGF induce the differentiation of human embryo mesenchymal stem cells into epithelial-like cells.

The mesenchymal to epithelial transition (MET) occurs in organ development and anti-tumorigenesis. We have investigated the effects of calcium (Ca(2+)) and epidermal growth factor (EGF) on human mesenchymal stem cell (hMSCs) differentiation into epithelial-like cells. hMSCs lost their biological characteristics after EGF transfection, and MET was achieved by adding 0.4 mmol Ca(2+). Western blotting and immunofluorescence showed expression of EGF, keratin, keratin 19 (K19), ß1-integrin, E-cadherin and phosphorylated focal adhesion kinase (p-FAK, Ser-910) increased in hMSCs infected with EGF and exposed to Ca(2+), although Smad3 activation was downregulated. hMSCs co-stimulated with EGF transfection and Ca(2+) can therefore differentiate into epithelial-like cells in vitro.

Integrin αv mediates contractility whereas integrin α4 regulates proliferation of human bladder smooth muscle cells via FAK pathway under physiological stretch.

PURPOSE: The requirement of integrins for mechanotransduction has been recognized for some time. We investigated the role of integrin subunits and their pathway in the physiological stretch induced contractility and proliferation of human bladder smooth muscle cells. MATERIALS AND METHODS: Human bladder smooth muscle cells were seeded on silicone membrane and subjected to stretch, simulating bladder cycles of various stretches and times, as controlled by customized software on a modified BioDynamic bioreactor. Cell proliferation, viability and cycle were determined by BrdU incorporation assay, the Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Haimen, People's Republic of China) and flow cytometry, respectively. Cell contractility was determined using a collagen gel contraction assay. RESULTS: Physiological stretch increased cell contractility, proliferation and viability. Knockdown of integrin αv but not α4 in the cells disrupted the enhanced contractility induced by stretch. Under physiological stretch conditions, the integrin αv level and phospho-FAK/FAK ratio correlated positively with cell stretch induced enhanced contractility. Further examination revealed that contractile marker expression was associated with integrin αv activation through the FAK pathway. At the same time integrin α4 but not integrin αv mediated stretch induced cell proliferation and viability. CONCLUSIONS: These data revealed that different integrins have different roles in the contractility and proliferation of human bladder smooth muscle cells under physiological stretch. This suggests that different integrins may become specific therapeutic targets in patients with voiding dysfunction. They may also be used to design a specific microenvironment for optimal bladder tissue regeneration.

High expression of phosphorylated focal adhesion kinase predicts a poor prognosis in human colorectal cancer.

Background: Phosphorylated Focal adhesion kinase (FAK) has been reported to be intimately involved in various malignant tumors. The effect of p-FAK on colorectal cancer (CRC) is still disputable. The purpose of this study is to investigate the role of p-FAK in the prognosis of colorectal cancer. Methods: The clinical significance of p-FAK expression in CRC was evaluated by immunohistochemistry in a large cohort, including carcinoma and para-carcinoma tissues from 908 patients, and normal tissues, adenoma, and metastasis tissues. The correlation between p-FAK expression and CRC occurrence was investigated in tumor and other tissues. Factors contributing to prognosis were evaluated using Kaplan-Meier survival analysis and Cox regression model. Results: p-FAK is apparently overexpressed in CRC and metastasis tissues. Compared with low p-FAK expression, patients with high p-FAK expression had shorter overall survival [hazard ratio (HR), 2.200; 95% confidence interval (CI), 1.265-3.452; p < 0.01] and disease-free survival (HR, 2.004; 95% CI 1.262-3.382; p < 0.01) in multivariate Cox analysis after adjusting other prognostic factors. High p-FAK expression was also related to a worse chemotherapeutic response in patients who achieved adjuvant chemotherapy (p < 0.01). Conclusion: Expression level of p-FAK is an independent risk factor and can serve as a prognostic biomarker for CRC. High p-FAK expression predicts an unfavorable prognosis of CRC as well as poor chemotherapeutic response.

Anti-Cancer Activity of the Combinational Treatment of Noozone Cold Plasma with p-FAK Antibody-Conjugated Gold Nanoparticles in OSCC Xenograft Mice.

Oral squamous cell cancer (OSCC) is the most common type of oral cancer (about 80-90% of cases) and various research is being done to cure the disease. This paper aims to verify whether treatment with no-ozone cold plasma (NCP), which is designed for safe usage of the plasma on oral cavities, in combination with gold nanoparticles conjugated with p-FAK antibody (p-FAK/GNP) can trigger the selective and instant killing of SCC-25 cells both in vitro and in vivo. When SCC25 and HaCaT cells are exposed to p-FAK/GNP+NCP, the instant cell death was observed only in SCC25 cells. Such p-FAK/GNP+NCP-mediated cell death was observed only when NCP was directly treated on SCC25 harboring p-FAK/GNP. During NCP treatment, the removal of charged particles from NCP using grounded electric mesh radically decreased the p-FAK/GNP+NCP-mediated cell death. This p-FAK/GNP+NCP-mediated selective cell death of OSCC was also observed in mice xenograft models using SCC25 cells. The mere treatment of p-FAK/GNP and NCP on the xenograft tumor slowly decreased the size of the tumor, and only about 50% of the tumor remained at the end of the experiment. On the other hand, 1 week of p-FAK/GNP+NCP treatment was enough to reduce half of the tumor size, and most of tumor tissue had vanished at the end. An analysis of isolated tissues showed that in the case of individual treatment with p-FAK/GNP or NCP, the cancer cell population was reduced due to apoptotic cell death. However, in the case of p-FAK/GNP+NCP, apoptotic cell death was unobserved, and most tissues were composed of collagen. Thus, this paper suggests the possibility of p-FAK/GNP+NCP as a new method for treating OSCC.

Pilot study: alteration of deleted in liver cancer1 and phosphorylated focal adhesion kinase Y397 cytoplasmic expression and the prognostic value in advanced epithelial ovarian carcinoma.

BACKGROUND: Deletion in liver cancer gene (DLC1) and phosphorylated focal adhesion kinase (p-FAK) have recently been reported as metastasis-related genes. However, the roles and prognostic values of their expression in epithelial ovarian carcinomas (EOCs) remain unclear. METHODS: The expression and prognostic value of DLC1 and p-FAK Y397 in EOC were evaluated by immunohistochemistry and multivariate analysis. RESULTS: Low expression of DLC1 and high expression of p-FAK Y397 were found in the 76 cases of EOC. The expression of DLC1 and p-FAK Y397 were negatively correlated. Multivariate analysis showed that the combination of them was an independent prognostic marker of EOC (P = 0.0319). CONCLUSIONS: DLC1 and pFAK Y397 had an association with the clinicopathologic characteristics of EOC. Expression of neither of these genes was a prognostic factor alone, but the combination revealed a significant prognostic value in the 60 cases of advanced stage EOC.

Substrate stiffness effects on SH-SY5Y: The dichotomy of morphology and neuronal behavior.

Like many other cell types, neuroblastoma cells are also known to respond to mechanical cues in their microenvironment in vitro. They were shown to have mechanotransduction pathways, which result in enhanced neuronal morphology on stiff substrates. However, in previous studies, the differentiation process was monitored only by morphological parameters. Motivated by the lack of comprehensive studies that investigate the effects of mechanical cues on neuroblastoma differentiation, we used SH-SY5Y cells differentiated on polyacrylamide (PA) gels as a model. Cells differentiated on the surface of PA hydrogels with three different elastic moduli (0.1, 1, and 50 kPa) were morphologically evaluated and their electrophysiological responsiveness was probed using calcium imaging. Immunodetection of neural marker TUJ1 and p-FAK was used for biochemical characterization. Groups with defined stiffness that are matching and nonmatching to neural tissue extracellular matrix were used to distinguish biomimetic results from other effects. Results show that while cells display morphologies that do not resemble neurons on soft substrates, they are in fact electrophysiologically more responsive and abundant in neuronal marker TUJ1. Our findings suggest that while neuronal differentiation occurs more efficiently in microenvironments mechanically mimicking neural tissue, the SH-SY5Y model demonstrates morphologies that conflict with neuronal behavior under these conditions. These results are expected to contribute considerable input to researchers that use SH-SY5Y as a neuron model.

P0-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin.

P0-related protein (PZR), a Noonan and Leopard syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and Leopard syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with ß1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(α5ß1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.

The expressions of Hedgehog and PI3K-AKT pathway components correlate with invasion and metastasis in hepatocellular carcinoma.

OBJECTIVE: To investigate the expression and clinical significance of Shh, Gli1, FAK, p-FAK and p-AKT in HCC. METHODS: Immunohistochemistry was used to measure Shh, Gli1, FAK, p-FAK, and p-AKT expressions in 50 cases of HCC and paracancerous tissues. The Shh, Gli1, and FAK mRNA levels were determined by qRT-PCR in 20 HCCs. The correlations between the expressions of these target genes and the clinicopathological factors were analyzed in HCC. RESULTS: The immunohistochemical results showed that the expressions of Shh, Gli1, FAK, p-FAK, and p-AKT in 50 HCC tissues were significantly higher than those of the paracancerous tissues (P < 0.05). Shh and p-FAK expressions were associated with portal vein invasion, capsular integrity, and distant metastasis (P < 0.05). Gli1, FAK, and p-AKT expressions were closely related to tumor diameter, tumor differentiation, portal vein invasion, capsular integrity, TNM stage and distant metastasis (P < 0.05). Shh was related to Gli1 and p-FAK (r = 0.67, 0.30; P = 0.00, 0.03), Gli1 was positively related to p-FAK and p-AKT (r = 0.52, 0.49; P = 0.00, 0.00), and there was a positive correlation between p-FAK and p-AKT (r = 0.36, P = 0.00). Furthermore, the Shh, Gli1, and FAK mRNA levels in the HCC tissues were significantly higher than those in the paracancerous tissues (P < 0.0001), and the high TNM stages (III and IV) or distant metastasis were significantly higher than those in the low TNM stages (I and II) (P < 0.05) or without distant metastasis (P < 0.05). CONCLUSION: In HCC, the Hh and PI3K-AKT signaling pathways are both abnormally activated, and Shh, Gli1, FAK, p-FAK and p-AKT can serve as indicators to predict the prognosis of liver cancer.

NGF/FAK signal pathway is implicated in angiogenesis after acute cerebral ischemia in rats.

Neurogenesis in the cerebral infarction after an ischemic event is important to the rehabilitation of patients. However, the mechanism of angiogenesis around cerebral ischemia is not clear. Our study designed to test whether the nerve growth factor (NGF)-P-focal adhesion kinase (FAK) signaling pathway for associations with angiogenesis plays a key role in post-acute cerebral ischemia of rats. Firstly, we implanted the Matrigel, a carrier of basement membrane matrix, into the abdominal skin of rats to identify the relevant components of the NGF-P-FAK signaling pathway related to angiogenesis. Secondly, we used a model established by ligation of the middle cerebral artery (MCA) to observe the effect of the same signal pathway on angiogenesis in the subventricular and subgranular zones of the dentate gyrus(SVG and SGZ). The results showed that the tissue scores was significantly increased by NGF. However, the tissue scores was signifcaintly decreased by FAK inhibitor TAE226. Furthermore, CD31 and α-SMA were significantly increased by NGF and were decreased by anti-NGF and TAE226 in Matrigel. The P-FAK protein expression in Matrigel was markedly increased by NGF and decreased by TAE226. In the SVZ and SVG of cerebral ischemia, the numbers of BrdU-positive cells were significantly increased by NGF and decreased by TAE226, respectively. Our findings suggest that the therapy targeting the NGF-P-FAK signaling pathway may be an option for patients suffering from cerebral ischemia.

PDIA6 regulation of ADAM17 shedding activity and EGFR-mediated migration and invasion of glioblastoma cells.

OBJECTIVE In patients with glioblastoma, local invasion of tumor cells causes recurrence and shortens survival. The goal of this study was to determine whether protein disulfide isomerase (PDI) A6 regulates migration and invasion of glioblastoma cells and the associated factors. METHODS U87MG cells were treated with either PDIA6 or ADAM17 small interfering RNA (siRNA) fragments or with both types of siRNA fragments, and expression was confirmed by reverse transcription-polymerase chain reaction and Western blot. Migration and invasion were assessed using a wound-healing assay, a Matrigel assay, and an organotypic culture system. After the U87MG cells were treated with siRNAs and epidermal growth factor receptor (EGFR) inhibitors, the expression of matrix metalloproteinase-2 (MMP-2), membrane Type 1-matrix metalloproteinase (MT1-MMP), integrin, phosphorylated focal adhesion kinase (pFAK), and phosphorylated EGFR (pEGFR) was detected by Western blotting and zymography. RESULTS U87MG cell migration and invasion increased significantly after inhibition of PDIA6. The MMP-2 activation ratio and ADAM17 activity (as a sheddase of the proligand) increased, and expression of pEGFR, pFAK, integrin α5ß3, and MT1-MMP was induced, compared with control levels. Furthermore, heparin-binding epidermal growth factor (EGFR signaling ligand) was highly expressed in PDIA6-knockdown cells. After siPDIA6-transfected U87MG cells were treated with EGFR signaling inhibitors, expression of pFAK, MMP-2, and MT1-MMP decreased and invasion decreased significantly. Simultaneous double-knockdown of PDIA6 and ADAM17 reduced pEGFR and pFAK expression, compared with control levels. CONCLUSIONS The authors propose that inhibiting PDIA6 could transduce EGFR signaling by activating and inducing ADAM17 during migration and invasion of U87MG glioblastoma cells. The results of this study suggest that PDIA6 is an important component of EGFR-mediated migration and invasion of U87MG cells. This is the first report of the effects of PDIA6 on migration and invasion in glioblastoma.

A role for activated Cdc42 in glioblastoma multiforme invasion.

Cdc42 is a Rho-GTPase which plays a major role in regulating cell polarity and migration by specifying the localization of filopodia. However, the role of Cdc42 in GBM invasion has not been thoroughly investigated. We generated stable doxycycline-inducible clones expressing wild type (WT)-, constitutively active (CA)-, and dominant negative (DN)-Cdc42 in three different human glioma cell lines. Expression of CA-Cdc42 significantly increased the migration and invasive properties of malignant glioma cells compared to WT and DN-Cdc42 cell clones, and this was accompanied by a greater number of filopodia and focal adhesion structures which co-localize with phosphorylated focal adhesion kinase (FAK). By mass spectrometry and immunoprecipitation studies, we demonstrated that activated Cdc42 binds to IQGAP1. When implanted orthotopically in mice, the CA-Cdc42 expressing glioma cells exhibited enhanced local migration and invasion, and led to larger tumors, which significantly reduced survival. Using the Cancer Genome Atlas dataset, we determined that high Cdc42 expression is associated with poorer progression free survival, and that Cdc42 expression is highest in the proneural and neural subgroups of GBM. In summary, our studies demonstrate that activated Cdc42 is a critical determinant of the migratory and invasive phenotype of malignant gliomas, and that its effect may be mediated, at least in part, through its interaction with IQGAP1 and phosphorylated FAK.

Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration.

AIMS: After injury, the adult zebrafish can regenerate the heart. This requires the activation of the endocardium and epicardium as well as the proliferation of pre-existing cardiomyocytes to replace the lost tissue. However, the molecular mechanisms involved in this process are not completely resolved. In this work, we aim to identify the proteins involved in zebrafish heart regeneration and to explore their function. METHODS AND RESULTS: Using a proteomic approach, we identified Hyaluronan-mediated motility receptor (Hmmr), a hyaluronic acid (HA) receptor, to be expressed following ventricular resection in zebrafish. Moreover, enzymes that produce HA, hyaluronic acid synthases (has), were also expressed following injury, suggesting that this pathway may serve important functions in the regenerating heart. Indeed, suppression of HA production, as well as depletion of Hmmr, blocked cardiac regeneration. Mechanistically, HA and Hmmr are required for epicardial cell epithelial-mesenchymal transition (EMT) and their subsequent migration into the regenerating ventricle. Furthermore, chemical inhibition of Focal Adhesion Kinase (FAK) or inhibition of Src kinases, downstream effectors of Hmmr, also prevented epicardial cell migration, implicating a HA/Hmmr/FAK/Src pathway in this process. In a rat model of myocardial infarction, both HA and HMMR were up-regulated and localized in the infarct area within the first few days following damage, suggesting that this pathway may also play an important role in cardiac repair in mammals. CONCLUSION: HA and Hmmr are required for activated epicardial cell EMT and migration involving the FAK/Src pathway for proper heart regeneration.

The glycophorin A transmembrane sequence within integrin αvß3 creates a non-signaling integrin with low basal affinity that is strongly adhesive under force.

Integrin heterodimeric cell adhesion and signaling receptors bind ligands of the extracellular matrix and relay signals bidirectionally across cell membranes. Thereby, integrins adopt multiple conformational and functional states that control ligand binding affinity and linkage to cytosolic/cytoskeletal proteins. Here, we designed an integrin chimera encompassing the strongly dimerizing transmembrane domain (TMD) of glycophorin A (GpA) in the context of the otherwise unaltered integrin αvß3. We hypothesized that this chimera should have a low basal affinity to soluble ligand but should be force-activatable. By cellular expression of this chimera, we found a decreased integrin affinity to a soluble peptide ligand and inhibited intracellular signaling. However, under external forces applied by an atomic force microscope or by a spinning disc device causing shear forces, the mutant caused stronger cell adhesion than the wild-type integrin. Our results demonstrate that the signaling- and migration-incapable integrin αvß3-TMD mutant TMD-GpA shows the characteristics of a primed integrin state, which is of low basal affinity in the absence of forces, but may form strong bonds in the presence of forces. Thus, TMD-GpA may mimic a force-activatable signaling intermediate.