Β1 Integrin Research Articles

Leucine-Rich Repeat Containing 15-Mediated Cell Adhesion Is Essential for Integrin Signaling in TGF-β1-Induced PDL Fibroblastic Differentiation.

Human periodontal ligament cells (hPDLCs) cultured from periodontal ligament (PDL) tissue contain postnatal stem cells that can be differentiated into PDL fibroblasts. We obtained PDL fibroblasts from hPDLCs by treatment with low concentrations of TGF-β1. Since the extracellular matrix and cell surface molecules play an important role in differentiation, we had previously developed a series of monoclonal antibodies against PDL fibroblast-specific cell surface molecules. One of these, the anti-PDL51 antibody, recognized a protein that was significantly upregulated in TGF-β1-induced PDL fibroblasts and highly accumulated in the PDL region of the tooth root. Mass spectrometry revealed that the antigen recognized by the anti-PDL51 antibody was leucine-rich repeat containing 15 (LRRC15), and this antibody specifically recognized the extracellular glycosylated moiety of LRRC15. Experiments presented here show that as fibroblastic differentiation progresses, increased amounts of LRRC15 localized at the cell surface and membrane. Inhibition of LRRC15 by siRNA-mediated depletion and by antibody blocking resulted in downregulation of the representative PDL fibroblastic markers. Moreover, following LRRC15 inhibition, the directed and elongated cell phenotypes disappeared, and the long processes of the end of the cell body were no longer found. Through a specific interaction between integrin β1 and LRRC15, the focal adhesion kinase signaling pathway was activated in PDL fibroblasts. Furthermore, it was shown that increased LRRC15 was important for the activation of the integrin-mediated cell adhesion signal pathway for regulation of cellular functions, including fibroblastic differentiation, proliferation, and cell migration arising from the expression of PDL-related genes in TGF-β1-induced PDL fibroblastic differentiation.

Single-cell RNA sequencing unveils Lrg1's role in cerebral ischemia‒reperfusion injury by modulating various cells

Background and purposeCerebral ischemia‒reperfusion injury causes significant harm to human health and is a major contributor to stroke-related deaths worldwide. Current treatments are limited, and new, more effective prevention and treatment strategies that target multiple cell components are urgently needed. Leucine-rich alpha-2 glycoprotein 1 (Lrg1) appears to be associated with the progression of cerebral ischemia‒reperfusion injury, but the exact mechanism of it is unknown.MethodsWild-type (WT) and Lrg1 knockout (Lrg1−/−) mice were used to investigate the role of Lrg1 after cerebral ischemia‒reperfusion injury. The effects of Lrg1 knockout on brain infarct volume, blood‒brain barrier permeability, and neurological score (based on 2,3,5-triphenyl tetrazolium chloride, evans blue dye, hematoxylin, and eosin staining) were assessed. Single-cell RNA sequencing (scRNA-seq), immunofluorescence, and microvascular albumin leakage tests were utilized to investigate alterations in various cell components in brain tissue after Lrg1 knockout.ResultsLrg1 expression was increased in various cell types of brain tissue after cerebral ischemia‒reperfusion injury. Lrg1 knockout reduced cerebral edema and infarct size and improved neurological function after cerebral ischemia‒reperfusion injury. Single-cell RNA sequencing analysis of WT and Lrg1−/− mouse brain tissues after cerebral ischemia‒reperfusion injury revealed that Lrg1 knockout enhances blood‒brain barrier (BBB) by upregulating claudin 11, integrin β5, protocadherin 9, and annexin A2. Lrg1 knockout also promoted an anti-inflammatory and tissue-repairing phenotype in microglia and macrophages while reducing neuron and oligodendrocyte cell death.ConclusionsOur results has shown that Lrg1 mediates numerous pathological processes involved in cerebral ischemia‒reperfusion injury by altering the functional states of various cell types, thereby rendering it a promising therapeutic target for cerebral ischemia‒reperfusion injury.

Targeting the JAK2V617F Hematopoietic Stem Cell through Beta1 Integrin

The major barrier to progress in treatment of primary myelofibrosis (PMF) is the lack of effective therapies targeting the malignant JAK2 V617F hematopoietic stem cell (HSC), the source of disease progression and relapse. Expression of JAK2 V617F induces abnormal proliferation and differentiation of HSC, affecting all blood lineages but most prominently the erythro-megakaryocytic lineage, leading to clonal megakaryocyte proliferation, erythrocytosis and thrombocytosis. In our previously published work ( Matsuura et al, Blood 2020) we identified elevated expression of the α5 subunit of the α5β1 integrin receptor as a driver of JAK2 V617F megakaryocyte proliferation. Expression of β1 integrin subunit was similar in wild-type (WT) and JAK2 V617F megakaryocytes, but activation of β1 integrin, detected by the conformation-specific 9EG7 antibody, was elevated in JAK2 V617F megakaryocytes. Following up on those findings, we detected elevated β1 integrin activation in long-term hematopoietic stem cells (HSC LT, LSKCD48-CD150+) from JAK2 V617F transgenic mice ( Xing et al, Blood 2008) when compared to WT controls. To determine whether inhibition of β1 integrin in vivo reduces the number of HSC LT in JAK2 V617F transgenic mice, we examined HSC LT 24 hours after intravenous administration of 2mg/Kg of anti-β1 integrin inhibitory antibody HMβ1-1. This regimen led to a sharp reduction in number and percentage of HSC LT in JAK2 V617F transgenic mice, to 45% of control antibody-treated animals. The effect on WT HSC LT was negligible. HSC ST (LSKCD48-CD150-) and MPP2 (LSKCD48+CD150+) were also reduced, but the reduction was not statistically significant. MPP3/4 (LSKCD48+CD150-) and LSK were not affected. Megakaryocyte progenitors (LKCD41+CD150+) were significantly reduced. Inhibition of β1 integrin also sharply reduced peripheral blood platelet numbers in WT animals. The reduction in JAK2 V617F transgenic mice was not statistically significant. A longer observation time of 7 days demonstrated that while WT animals were able to mount a robust regenerative response with slight but significant increases in HSC LT and normalization of platelet numbers, in JAK2 V617F transgenic mice the number of HSC LT remained depressed, as did downstream stem/progenitor populations at various significance levels. To gain insight into the mechanisms that lead to reduction in HSC LT, we examined apoptosis, cell cycle and HSC mobilization, all processes known to be affected by β1 integrin. Although basal expression of the apoptotic marker annexin V was decreased in JAK2 V617F HSC LT compared to WT, no differences were detected upon administration of HMβ1-1 in both genotypes at 4 hours post-administration. Similarly, peripheral blood colony assays revealed a basally higher level of stem/progenitor mobilization in JAK2 V617F, which did not significantly increase upon HMβ1-1 administration. In contrast, the cell cycle profile, examined at 4 hours post-administration to account for the earliest effects, demonstrated a decrease in G0 phase cells and increase in S/G2/M cells indicating cell cycle induction and exit from quiescence in both WT and JAK2 V617F HSC LT. The results indicate that inhibition of β1 integrin disrupts the adhesion-mediated cell cycle suppression in both WT and JAK2 V617F HSC LT leading to exit from quiescence. However, the downstream consequences of cell division in HSC LT differ significantly, with an apparent loss of regenerative potential in JAK2 V617F HSC LT, at least at the observed timepoints. Longer observation times are required to determine whether continuous β1 integrin inhibition can lead to JAK2 V617F HSC LT exhaustion. More sensitive assays to monitor mobilization are warranted to quantify possible minute amounts of mobilized HSC LT. Our preclinical studies indicate that β1 integrin is an attractive target to specifically reduce the number of JAK2 V617F HSC LTin vivo. The role of β1 integrin in HSC LT has not been thoroughly studied due to lack of phenotype in β1 integrin-deficient HSC ( Brakebush et al, Immunity 2002). Our studies using the anti-β1 integrin antibody in WT HSC LT confirm previous findings. However, β1 integrin seems to be critical for maintenance of JAK2 V617F HSC LT, offering a possible therapeutic window. Understanding how β1 integrin supports quiescence in HSC LT will bring us a step closer to understanding the mechanisms that drive malignancy and fitness in JAK2 V617F HSC LT.

Non-Enzymatic Role of Platelet 12-LOX in Platelet Activation

Background 12-lipoxygenase (12-LOX) plays an essential role in regulation of vascular disease and human platelet function through oxylipin generation such as 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE) and 12-hydroxyeicosatetraenoic acid (12-HETE). Previous literature has demonstrated that 12-LOX enzymatic function such as oxylipin generation is important for collagen- or thrombin-mediated signaling events. However, the addition of 12(S)-HETE does not completely restore the inhibitory effect of pharmacologically or genetically inhibiting 12-LOX on platelet activation, indicating that 12-LOX may have a non-enzymatic function in platelets. Therefore, we tested the non-enzymatic role of 12-LOX in platelet activation. Method 12 LOX was co-immunoprecipitated (co-IP) from washed human platelets following FcγRIIa or thrombin stimulation. The bound proteins were eluted by laemmli buffer and the elutes were analyzed on SDS-PAGE. Proteins co-IP with 12-LOX at various times were identified by mass spectrometry and confirmed by western blot analysis. Result Silver-stained gel of 12-LOX co-IP indicated the presence of several protein bands in complex association with 12-LOX at different time points. These bands were analyzed by mass spectroscopy and several proteins observed in complex with 12-LOX were identified including integrin β3, Talin-1, Myh9, Nexilin, Gelsolin, Actin, cytoplasmic 2, and Actin related protein 2/3 (ARP 2/3) . To confirm the mass spectroscopy identified protein, these proteins were immunoblotted following co-IP of 12-LOX. to confirm their association with 12-LOX prior to and following platelet activation. These interactions were further confirmed with a reverse co-IP. Finally, to determine if the association pattern of these proteins with 12-LOX is disrupted in the presence of a 12-LOX inhibitor already shown to limit platelet activation and which is currently in clinical trials for treatment of HIT (ML355/VLX-1005), we were able to demonstrate that the inhibitor appears to lock the protein complex into place. Conclusion Our study identified several proteins that are present in 12-LOX complex such as integrin β3, talin-1, Myh9 and ARP 2/3. 12-LOX interacts with Integrin β3, Talin-1 and cytoskeletal proteins. Furthermore, the dynamic movement of the proteins in this complex into and out of association with 12-LOX is essential for normal platelet activation suggesting 12-LOX plays an important non-enzymatic role in regulation of agonist-mediated platelet activation.

PACSIN2 Orchestrates Actin Reorganization during Megakaryocyte Maturation

Thrombocytopenia, defined by a platelet count below 150,000/µL, poses significant mortality risks, primarily due to bleeding. Blood platelets are produced by bone marrow megakaryocytes (MKs) in unique cellular processes that require polyploidization and extensive intracellular membrane rearrangements leading to the formation of the demarcation membrane system (DMS), the surface-connected membrane reservoir for future platelets. The membrane-deforming F-BAR protein PACSIN2 is one of the most abundant and conserved BAR proteins in platelets, and exomechip analysis and genome-wide association studies have associated PACSIN2 single nucleotide variants with platelet count and mean platelet volume in humans. Our previous work has shown that PACSIN2 localizes with the initiating DMS in MKs in a process regulated by the cytoskeletal and scaffolding protein filamin A (FlnA). Recently, we have found that Pacsin2 -/- mice display mild thrombocytopenia with slightly enlarged platelets and severe thrombus formation defects due to increased activation of platelet integrin β1. Here we investigated the role of PACSIN2 in MK maturation and platelet production. Cultured fetal liver cell-derived Pacsin2 -/- MKs formed proplatelets normally compared to controls. Pacsin2 -/- bone marrow sections displayed MK count comparable to control mice, and freshly isolated bone marrow Pacsin2 -/- MKs had normal ploidy, indicating normal MK maturation. The observed effects of PACSIN2 deficiency led to a defective formation of the DMS within MKs. In PACSIN2 deficient MKs, the DMS exhibited a less distinct structure, making it challenging to delineate clear platelet territories. Interestingly, the lack of PACSIN2 also impacted the spatial distribution of GPIbα and integrin β1 membrane receptors. These receptors, typically dispersed within the cytoplasm in normal MKs, were concentrated at the cell periphery in Pacsin2 -/-MKs. A similar relocation was observed for polymerized actin. This finding underscores the pivotal role of PACSIN2 in actin reorganization within MKs, which in turn influences the rearrangement of GPIbα and integrin β1. The data imply that receptor and actin cytoskeleton rearrangements are critical to DMS formation and subsequent platelet production in vivo.

ITGB1-Expressing Effector Memory CD4 + T-Cell Response Associates with Clinical Recovery in Childhood Immune Thrombocytopenia

Background: The autoimmune bleeding disorder childhood immune thrombocytopenia (ITP) features antibody and T-cell immune responses against platelet self-antigens. This involves activation of B-cells, CD4 + T-cell help, and CD8 + T-cells. Novel prognostic biomarkers are needed to predict recovery and treatment responses. Compared to healthy children, changes in total lymphocytes, CD4 +, regulatory CD4 +, CD8 +T-cells, CD19 + B-cells, and NK cells were described in childhood ITP. Children with transient ITP were also found to have a fewer CD25 hi CD4 + T-cells than those who develop chronic ITP. In contrast, the platelet-stimulated peripheral blood mononuclear cells (PBMC) of children with chronic ITP produced more IL-2 than those of transient ITP. At present, none of these biomarkers have been validated for ITP prognosis, and consequently they are not in clinical use. This emphasizes the need for clinical-grade biomarkers to predict clinical responses in childhood ITP. Aims: To predict spontaneous recovery and IVIg response in newly diagnosed ITP by (1) validating previously suggested changes in immune cell frequencies, and (2) identifying novel immune cell subsets as predictors. Methods: Children with newly diagnosed ITP were randomized 1:1 to observation or IVIg treatment (TIKI trial). Recovery was determined by platelet counts according to IWG criteria 1, 4, 13, 26 and 52 weeks after the diagnosis. For validation, the CD4 +, regulatory CD4 + (CD25 +/CD127 lo), CD8 +, CD19 +, and NK cells were quantified at the time of diagnosis in a centralized laboratory by flow cytometry. For identification of novel predictors, CD4 + central (CD45RO + CD27 +) and effector memory (CD45RO + CD27 -) frequencies were determined. The primary clinical outcome was longitudinal recovery, either spontaneous or after IVIg (adjusted Cox-proportional hazard model). Secondary outcomes included the mean age-adjusted difference between transient/persistent and chronic ITP by multivariate regression, and comparison to age-appropriate healthy control data. Additionally, PBMC (N=6) were analyzed at diagnosis by single-cell RNA sequencing (scRNA-seq) combined with T- and B- cell receptor sequencing (scTCR-seq and scBCR-seq). Results: For validation of previously suggested predictors, the absolute CD3 +, CD4 +, CD8 +, CD19 +, and NK cell counts of newly diagnosed ITP patients were within the age-appropriate healthy reference range (N=158); they were not associated with recovery, and there were no age-adjusted differences between transient/persistent (N=139) and chronic ITP (N=19). The regulatory CD4 + T cell frequency was not reduced compared to age-appropriate reference data; not associated with recovery, and not different between transient/persistent and chronic ITP patients. For identification of novel predictors, we found that high CD4 +effector memory cells numbers were associated with a reduced recovery rate, with an adjusted hazard ratio for complete recovery over a one-year follow-up to be 0.55 (95% CI, 0.35 - 0.85; ≥ median; adjusted for age and treatment; N=150). Chronic ITP patients displayed a mean age-adjusted increase in effector memory CD4 +cells of 1.4 % (95% CI, 0.4 - 2.4; P= 0.005). The association with recovery was independent of a preceding infection, total leukocyte and lymphocyte counts, and the presence of anti-platelet IgG or IgM autoantibodies. ScRNA-seq analysis of 7965 PBMC also showed an effector memory CD4 + T-cell cluster that was expanded in chronic ITP, present in at a frequency of 8.1 ± 1.4 % (mean ± sem) in transient vs 14.8 ± 1.2 % in chronic ITP. This cluster also expressed high levels of fibronectin receptor integrin b1 ( ITGB1) interleukin 32 (IL32) interleukin 7 receptor (IL7R) andlymphotoxin beta (LTB). The effector memory phenotype of CD4 + ITGB1 + T-cells was confirmed by flow cytometry analysis in healthy individuals and ITP patients. Conclusions: Previously suggested changes in T-, B-, or NK cell frequencies could not be validated as predictors of spontaneous recovery or IVIg response. However, we identified the frequency of ITGB1-expressing effector memory CD4 + T cells as an independent predictor of spontaneous recovery and IVIg response. Thus, T cell phenotyping at the time of diagnosis may be suitable to determine prognosis and personalize treatment decisions in newly diagnosed childhood ITP.

Overexpression of Integrin β7 in Newly Diagnosed Multiple Myeloma with High Risk Cytogenetic Abnormality and Poorer Prognosis

Introduction The second most prevalent malignant blood malignancy is multiple myeloma (MM), but the condition is still incurable. In the interaction between MM cells and the bone marrow microenvironment, adhesion molecules are known to mediate adhesion and promote the survival and proliferation of MM cells. Integrin β7 (ITGB7) is a member of the adhesion molecule family. However, there are few clinical studies on ITGB7 in MM and there is no prognostic analysis. By analyzing the relationship between ITGB7 expression and clinical signs and prognosis, this study thus provides a basis for evaluating the clinical and prognostic importance of ITGB7 expression in MM patients. Methods In 39 patients with newly diagnosed multiple myeloma from 2021.1 to 2022.9, we assessed the expression of ITGB7 in relation to clinical characteristics and prognosis. Flow cytometry was used to detect the median fluorescence of ITGB7, detecting that ITGB7 median fluorescence intensity (MFI) in abnormal plasma cell was significantly higher than that in mature B cells (18.76 vs 5.62, P<0.001). ROC curve defines that ITGB7 best indicator for predicting MM disease is the abnormal plasma cell to mature B cells median fluorescence intensity ratio (MFI P/B), with a cut-off value of 4.025. Results Compared to patients with low expression of ITGB7, patients with high expression of ITGB7 showed a significant increase in mSMART stratification risk (82.60% vs 50.00%, P=0.041), while those with IgH/FGFR3 showed a significant increase (38.9% vs 6.30%, P=0.043). Patients with high expression of ITGB7 had poorer prognostic trends in PFS and OS, (PFS: P=0.071; OS: P=0.070). Conclusion Overexpression of ITGB7 is prone to merging with high risk cytogenetic abnormality and poorer prognosis in newly diagnosed MM patients.

Stiffened tumor microenvironment enhances perineural invasion in breast cancer via integrin signaling.

Accumulating studies have shown that tumors are regulated by nerves, and there is abundant nerve infiltration in the tumor microenvironment. Many solid tumors including breast cancer (BRCA) have different degrees of perineural invasion (PNI), which is closely related to the tumor occurrence and progression. However, the regulatory mechanism of PNI in BRCA remains largely unexplored. PNI-related molecular events are analyzed by the RNAseq data of BRCA samples deposited in The Cancer Genome Atlas (TCGA) database. Extracellular matrix (ECM) components within the tumor microenvironment are analyzed by immunohistochemical staining of α-SMA, Sirius red staining, and Masson trichrome staining. Soft and stiff matrix gels, living cell imaging, and dorsal root ganglion (DRG) coculture assay are used to monitor cancer cell invasiveness towards nerves. Western blotting, qRT-PCR, enzyme-linked immunosorbent assay combined with neutralizing antibody and small molecular inhibitors are employed to decode molecular mechanisms. Comparative analysis that the ECM was significantly associated with PNI status in the TCGA cohort. BRCA samples with higher α-SMA activity, fibrillar collagen, and collagen content had higher frequency of PNI. Compared with soft matrix, BRCA cells cultured in stiff matrix not only displayed higher cell invasiveness to DRG neurons but also had significant neurotrophic effects. Mechanistically, integrin β1 was identified as a functional receptor to the influence of stiff matrix on BRCA cells. Moreover, stiffened matrix-induced activation of integrin β1 transduces FAK-YAP signal cascade, which enhances cancer invasiveness and the neurotrophic effects. In clinical setting, PNI-positive BRCA samples had higher expression of ITGB1, phosphorylated FAK, YAP, and NGF compared with PNI-negative BRCA samples. Our findings suggest that stiff matrix induces expression of pro-metastatic and neurotrophic genes through integrin β1-FAK-YAP signals, which finally facilitates PNI in BRCA. Thus, our study provides a new mechanism for PNI in BRCA and highlights nerve-based tumor treatment strategies.

Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells.

A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA-MB-231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle-like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.

Restoring Synaptic Function: How Intranasal Delivery of 3D-Cultured hUSSC Exosomes Improve Learning and Memory Deficits in Alzheimer's Disease.

Memory problems are often the first signs of cognitive impairment related to Alzheimer's disease (AD), and stem cells and stem cell-derived exosomes (EXOs) have been studied for their therapeutic potential to improve the disease signs. While many studies have shown the anti-inflammatory and immunomodulatory effects of stem cells and exosomes on improving memory in different AD models, there is still insufficient data to determine how they modulate neural plasticity to enhance spatial memory and learning ability. Therefore, we conducted a study to investigate the effects of exosomes derived from 3D-cultured human Unrestricted Somatic Stem Cells (hUSSCs) on spatial memory and neuroplasticity markers in a sporadic rat model of AD. Using male Wistar rats induced by intracerebral ventricle injection of streptozotocin, we demonstrated that intranasal administration of hUSSC-derived exosomes could decrease Aβ accumulation and improve learning and memory in the Morris water maze test. We also observed an increase in the expression of pre-synaptic and post-synaptic molecules involved in neuronal plasticity, including NMDAR1, integrin β1, synaptophysin, pPKCα, and GAP-43, in the hippocampus. Our findings suggest that intranasal administration of exosomes can ameliorate spatial learning and memory deficits in rats, at least in part, by increasing the expression of neuroplasticity proteins. These results may encourage researchers to further investigate the molecular pathways involved in memory improvement after stem cell and exosome therapy, with the goal of increasing the efficacy and safety of exosome-based treatments for AD.

High expression of long noncoding RNA UCA1 promotes invasion, migration and epithelial-mesenchymal transition of trophoblasts in vitro

To investigate the role of urothelial carcinoma antigen 1 (UCA1) in regulation of invasion, migration and epithelial-mesenchymal transition (EMT) of trophoblast HTR-8/SVneo cells and its association with tubal pregnancy. Cultured HTR- 8/SVneo cells stimulated with interleukin-6 (IL-6) were examined for changes in UCA1 expression and cell migration ability using qRT-PCR and scratch assay, respectively. A HTR-8/SVneo cell model with UCA1 silencing was constructed by transient transfection, and the migration and invasion abilities of the cells were assessed using Scratch assay and Transwell assay; qRT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of EMT markers. HTR-8/SVneo cells stimulated with IL-6 exhibited significantly increased migration ability and up-regulated expression of UCA1 (P < 0.01). UCA1 silencing obviously suppressed migration and invasion abilities of HTR-8/SVneo cells (P < 0.01), significantly up-regulated the mRNA and protein expressions of EMT epithelial marker E-cadherin (P < 0.01), and down-regulated the expressions of the mesenchymal markers integrin β3, vimentin and N-cadherin (P < 0.05). UCA1 may be a key gene that promotes the occurrence of tubal pregnancy and thus provides a new therapeutic target for tubal pregnancy.

Cerebral arterioles express the laminin subunits α4 and α5 in conjunction with α6β4 integrin, but strongly downregulate laminin α4 during hypoxia-induced arteriogenic remodeling

Previous studies have shown that expression of the endothelial laminin receptor α6β4 integrin in the brain is uniquely restricted to arterioles. As exposure to chronic mild hypoxia (CMH, 8 % O2) stimulates robust angiogenic and arteriogenic remodeling responses in the brain, the goal of this study was to determine how CMH influences cerebrovascular expression of the β4 integrin as well as its potential ligands, laminin 411 and 511, containing the α4 and α5 laminin subunits respectively, and then define how aging impacts this expression. We observed the following: (i) CMH launched a robust arteriogenic remodeling response both in the young (10 weeks) and aged (20 months) brain, correlating with an increased number of β4 integrin+ vessels, (ii) while the laminin α4 subunit is expressed evenly across all cerebral blood vessels, laminin α5 was highly expressed preferentially on β4 integrin+ arterioles, (iii) CMH-induced arteriolar remodeling was associated with strong downregulation of the laminin α4 subunit but no change in the laminin α5 subunit, (iv) in addition to its expression on arterioles, β4 integrin was also expressed at lower levels on capillaries specifically in white matter (WM) tracts but not in the grey matter (GM), and (v), these observations were consistent in both the brain and spinal cord, and age had no obvious impact. Taken together, our findings suggest that laminin 511 may be a specific ligand for α6β4 integrin and that dynamic switching of the laminin subunits α4 and α5 might play an instructive role in arteriogenic remodeling. Furthermore, β4 integrin expression differentiates WM from GM capillaries, highlighting a novel and important difference.

Integrin β Expression as a New Diagnostic Marker for Arteriovenous Thrombosis: A Single-Center Prospective Study.

Integrin β plays an important role in the pathogenesis of thrombosis and inflammation, and it may be a shared pathogenic mechanism between arterial and venous thromboses. With the goal of identifying new treatment targets for thrombotic diseases and specific diagnostic markers for venous thromboembolism (VTE), this prospective clinical study was performed to clarify the relationship between integrin and thrombosis. The levels of integrin β1-3, interleukin-6 (IL-6), and C-reactive protein were significantly higher in patients with acute myocardial infarction (AMI; n = 44) and acute VTE (n = 43) compared to healthy controls (n = 33). The IL-6 and integrin β1-3 levels were also significantly higher in the AMI group compared to the VTE and control groups. Logistic regression analysis identified IL-6 and integrin β1-3 levels as independent risk factors for thrombotic disease. Based on the receiver-operating characteristic curve, Youden index, sensitivity, and specificity, the diagnostic accuracy value for VTE was greater than 0.8 when integrins β1, β2, and β3 were combined. Overall, these results suggest that integrin β levels can contribute to improving the diagnosis and treatment of arteriovenous thrombosis.

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

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

GMI, a Ganoderma microsporum protein, abolishes focal adhesion network to reduce cell migration and metastasis of lung cancer

BackgroundCancer metastasis is a major cause of cancer-related deaths, emphasizing the urgent need for effective therapies. Although it has been shown that GMI, a fungal protein from Ganoderma microsporum, could suppress primary tumor growth in a wide spectrum of cancer types, it is still unclear whether GMI exhibits anti-metastasis properties, particularly in lung cancers. Further investigation is needed. Aims and objectivesThe objective of this study is to investigate the potential inhibitory effects of GMI on lung cancer metastasis in vivo. Utilizing systematic and comprehensive approaches, our research aims to elucidate the underlying molecular mechanisms responsible for the anti-metastatic effects. Materials and methodsIn vitro migration and cell adhesion assays addressed the epithelial-to-mesenchymal transition (EMT)-related phenotype. Proteomic and bioinformatic analyses identified the GMI-regulated proteins and cellular responses. GMI-treated LLC1-bearing mice were analyzed using IVIS Spectrum to assess the anti-metastatic effect. Key findingsGMI inhibits EMT as well as cell migration. GMI disrupts cell adhesion and downregulates integrin, resulting in inhibition of phosphorylated FAK. GMI induces macropinocytosis and lysosome-mediated degradation of integrin αv, α5, α6 and β1. GMI downregulates Slug via inhibition of FAK activity, which in turn enhances expressions of epithelial-related markers and decreases cell mobility. Mechanistically, GMI-induced FAK inhibition engenders MDM2 expression and enhances MDM2/p21/Slug complex formation, leading to Slug degradation. GMI treatment reduces the metastatic pulmonary lesion and prolongs the survival of LLC1-bearing mice. SignificanceOur findings highlight GMI as a promising therapeutic candidate for metastatic lung cancers, offering potential avenues for further research and drug development.

2-hydroxy-4-methoxy benzaldehyde from Hemidesmus indicus root extract suppresses toll-like receptor2-mediated migration and invasive mechanisms in rheumatoid arthritis

IntroductionElevated expression of Toll-like receptor2 (TLR2) in synovial fibroblasts, endothelial and other infiltrating cells in rheumatoid arthritis promotes their migration and invasion. Our aim was to isolate a TLR2 inhibitor from Hemidesmus indicus root extract and test its effectiveness in regulating migration and invasion of fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth. MethodFractionation of TLR2 inhibitor by a bioassay, followed by testing the inhibitor in the cultures of isolated fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth in the presence of a TLR2 agonist, Pam3CSK4 was undertaken. ResultsTLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde significantly lowered Pam3CSK4-induced cell migration, invasion, MMP-1, MMP-3, Pro-MMP-2 and Pro-MMP-9 expression in fibroblast-like synovial cells isolated from the discarded biopsy specimens of rheumatoid arthritis patients as well as in ex vivo synovial explant culture. TLR2 inhibition lowered active Rac1 protein and β1 integrin mRNA levels in synovial fibroblasts and suppressed filopodia and lamellipodia formation in response to Pam3CSK4. In endothelial cells, 2-hydroxy-4-methoxy benzaldehyde inhibited Pam3CSK4-stimulated vascular endothelial growth factor (VEGF) and ICAM-1 expression and thereby lowered tube formation and endothelial migration. TLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde also attenuated the Pam3CSK4-evoked outgrowth formation in ex vivo synovial explants. 2-hydroxy-4-methoxy benzaldehyde targeted TLR2-MyD88 interactions and prevented dimerization. Conclusions2-hydroxy-4-methoxy benzaldehyde is the active constituent in the roots of H indicus that appears to act through TLR2 inhibition, followed by modulation of the agonist-induced cell migration, invasion and angiogenesis.

CBX7 is involved in the progression of cervical cancer throughthe ITGβ3/TGFβ1/AKT pathway.

The chromobox protein homolog 7 (CBX7) serves a tumor-suppressive role in human malignant neoplasias. The downregulation of CBX7 is associated with the poor prognosis and aggressiveness of various human cancers. However, the biological functions and underlying mechanisms of CBX7 in cervical cancer remain unclear. The present study investigated the role and mechanism of CBX7 in cervical cancer. Lentivirus and siRNA were used to construct cervical cancer cells with stable CBX7 knockdown and SiHa xenograft models. The cell growth, migration, invasion and apoptosis were observed through in vivo and in vitro experiments. The expression levels of CBX7, integrin β3 (ITGβ3), transforming growth factor β1 (TGFβ1), phosphatidylinositol-3-kinase (PI3K), AKT, E-cadherin (E-cad) and vimentin (VIM) were detected by western blot analysis and reverse transcription-quantitative PCR. The correlation between CBX7 and these genes was analyzed. TGFβ1 was also silenced through shRNA in cells with stable CBX7 knockdown to detect its effect on cell growth, invasion and apoptosis, and on pathway-related gene expression. It was revealed that knockdown of CBX7 promoted the proliferation, migration, and invasion of cervical cancer cells, and inhibited apoptosis. In addition, CBX7 knockdown promoted tumor growth in vivo. Correlation analysis demonstrated that CBX7 was negatively correlated with ITGβ3, TGFβ1, PI3K, AKT, phosphorylated AKT and VIM, but positively correlated with E-cad. Moreover, the knockdown of TGFβ1 reversed the promotion of cell proliferation and inhibition of apoptosis induced by CBX7 knockdown and attenuated the increase of ITGβ3, TGFβ1, PI3K, AKT and VIM caused by CBX7 knockdown. In conclusion, the findings of the present study indicated that the downregulation of CBX7 enhances cell migration and invasion while inhibiting cell apoptosis in cervical cancer by modulating the ITGβ3/TGFβ1/AKT signaling pathways.

MicroRNA-205 represses breast cancer metastasis by perturbing the rab coupling protein [RCP]-mediated integrin β1 recycling on the membrane.

During cancer cell invasion, integrin undergoes constant endo/exocytic trafficking. It has been found that the recycling ability of integrin β1 through Rab11-controlled long loop pathways is directly associated with cancer invasion. Previous studies showed that gain-of-function mutant p53 regulates the Rab-coupling protein [RCP]-mediated integrin β1 recycling by inactivating tumor suppressor TAp63. So, we were interested to investigate the involvement of miR-205 in this process. In the current study first, we evaluated that the lower expression of miR-205 in MDA-MB-231 cell line is associated with high motility and invasiveness. Further investigation corroborated that miR-205 directly targets RCP resulting in attenuated RCP-mediated integrin β1 recycling. Overexpression of TAp63 validates our in vitro findings. To appraise the anti-metastatic role of miR-205, we developed two in vivo experimental models- xenograft-chick embryo and xenograft-immunosuppressed BALB/c mice. Our in vivo results support the negative effect of miR-205 on metastasis. Therefore, these findings advocate the tumor suppressor activity of miR-205 in breast cancer cells and suggest that in the future development of miR-205-targeting RNAi therapeutics could be a smart alternative approach to prevent the metastatic fate of the disease.

Abstract 14438: Loss of the ROR2 Tyrosine Kinase Receptor is Associated With Dysfunctional Angiogenesis in Pulmonary Hypertension via Inappropriate Focal Adhesion Activation and Disruption of the Endothelial Barrier

Background: Dysfunctional angiogenesis is a hallmark of pulmonary arterial hypertension (PAH), but its mechanisms are unclear. We recently demonstrated a key role for Wnt7a in lung angiogenesis. Wnt7a acts through ROR2, a Wnt pathway tyrosine kinase receptor. We elucidate how ROR2 controls angiogenic functions in pulmonary microvascular endothelial cells (PMVECs). Hypothesis: Loss of ROR2 activity is associated with PAH. Methods: Healthy and PAH PMVECs were obtained from explanted lungs. PMVECs were transfected with ROR2 siRNA/constructs, followed by functional and molecular studies. Focal adhesion (FA) activation and force generation was measured using FRET-based total internal reflection microscopy (TIRF). Mice with endothelial-specific conditional ROR2 KO (ROR2 ECKO) were phenotyped in normoxia and hypoxia. Results: ROR2 expression was found predominantly in the endothelium of healthy lung vessels but significantly reduced within PAH lesions. ROR2 knockdown (siROR2) reduced vascular sprouting comparable to PAH PMVECs. Interestingly, siROR2 and ROR2-deficient PAH PMVECs displayed increased fibronectin adhesion and hyperpermeability, which led us to look at focal adhesions (FA) and cell-cell junctions. Confocal and TIRF microscopy of siROR2 and ROR2-deficient PAH PMVECs revealed an increase in the FA number and force that inversely correlated with reduced VE-cadherin at cell junctions. However, transfection of a ROR2 construct in PAH PMVECs resulted in FAs activity normalization and junctional integrity accompanied by recovery of adhesion and permeability. Proteomic studies demonstrated that ROR2 interacts with Integrin b1 in focal adhesions, and its loss results in Integrin b1 activation, FA clustering, and high mechanical forces. In animal studies, we found that, compared to controls, ROR2 ECKO developed more severe pulmonary hypertension, right ventricular remodeling, microvascular reduction, and muscularization in hypoxia, which correlated with higher levels of integrin b1 activation and reduced VE-cadherin expression. Conclusions: ROR2 promotes PMVEC adhesion and barrier formation, and its loss is associated with dysfunctional angiogenesis. Restoring ROR2 could be a novel therapeutic strategy in PAH.

The role of CEACAMs versus integrins in Helicobacter pylori CagA translocation: a systematic review

The delivery of Helicobacter pylori CagA into host cells was long believed to occur through the integrin cell surface receptors. However, the role of CEACAM receptors has recently been highlighted, instead. Here, we have categorized the existing experimental evidence according to whether deletion, upregulation, downregulation, or inhibition of the target ligands (T4SS or HopQ) or receptors (integrins or CEACAMs), result in alterations in CagA phosphorylation, cell elongation, or IL-8 production. According to our analysis, the statistics favor the essence of most of the T4SS constituents and the involvement of HopQ adhesin in all three functions. Concerning the integrin family, the collected data is controversial, but yielding towards it being dispensable or involved in CagA translocation. Yet, regarding cell elongation, more events are showing β1 integrin being involved, than αvβ4 being inhibitory. Concerning IL-8 secretion, again there are more events showing α5, β1 and β6 integrins to be involved, than those showing inhibitory roles for β1, β4 and β6 integrins. Finally, CEACAM 1, 3, and 5 are identified as mostly essential or involved in CagA phosphorylation, whereasCEACAM 4, 7, and 8 are found dispensable and CEACAM6 is under debate. Conversely, CEACAM1, 5 and 6 appear mostly dispensable for cell elongation. Noteworthy is the choice of cell type, bacterial strain, multiplicity and duration of infection, as well as the sensitivity of the detection methods, all of which can affect the variably obtained results.