Integrin Α5 Research Articles

Integrin α5 regulates motility of human monocyte-derived Langerhans cells during immune response.

Langerhans cells (LCs) are mainly present in the epidermis and mucosa, and have important roles during skin infection. Migration of LCs to lymph nodes is essential for antigen presentation. However, due to the difficulties in isolating and culturing human LCs, it is not fully understood how LCs move and interact with the extracellular matrix (ECM) through their adhesion molecules such as integrin, during the immune responses. In this study, we aimed to investigate LC motility, cell shape and the role of integrin under inflammatory conditions using monocyte-derived Langerhans cells (moLCs) as a model. As a result, lipopolysaccharide (LPS) stimulation increased adhesion on fibronectin coated substrate and integrin α5 expression in moLCs. Time-lapse imaging of moLCs revealed that stimulation with LPS elongated cell shape, whilst decreasing their motility. Additionally, this decrease in motility was not observed when pre-treated with a neutralising antibody targeting integrin α5. Together, our data suggested that activation of LCs decreases their motility by promoting integrin α5 expression to enhance their affinity to the fibronectin, which may contribute to their migration during inflammation.

Elevated ITGA1 levels in type 2 diabetes: implications for cardiac function impairment

Aims/hypothesisType 2 diabetes mellitus is known to contribute to the development of heart failure with preserved ejection fraction (HFpEF). However, identifying HFpEF in individuals with type 2 diabetes early on is often challenging due to a limited array of biomarkers. This study aims to investigate specific biomarkers associated with the progression of HFpEF in individuals with type 2 diabetes, for the purpose of enabling early detection and more effective management strategies.MethodsBlood samples were collected from individuals with type 2 diabetes, both with and without HFpEF, for proteomic analysis. Plasma integrin α1 (ITGA1) levels were measured and compared between the two groups. Participants were further categorised based on ITGA1 levels and underwent detailed transthoracic echocardiography at baseline and during a median follow-up period of 30 months. Multivariable linear and Cox regression analyses were conducted separately to assess the associations between plasma ITGA1 levels and changes in echocardiography indicators and re-hospitalisation risk. Additionally, proteomic data for the individuals’ left ventricles, from ProteomeXchange database, were analysed to uncover mechanisms underlying the change in ITGA1 levels in HFpEF.ResultsIndividuals with type 2 diabetes and HFpEF showed significantly higher plasma ITGA1 levels than the individuals with type 2 diabetes without HFpEF. These elevated ITGA1 levels were associated with left ventricular remodelling and impaired diastolic function. Furthermore, during a median follow-up of 30 months, multivariable analysis revealed that elevated ITGA1 levels independently correlated with deterioration of both diastolic and systolic cardiac functions. Additionally, higher baseline plasma ITGA1 levels independently predicted re-hospitalisation risk (HR 2.331 [95% CI 1.387, 3.917], p=0.001). Proteomic analysis of left ventricular myocardial tissue provided insights into the impact of increased ITGA1 levels on cardiac fibrosis-related pathways and the contribution made by these changes to the development and progression of HFpEF.Conclusions/interpretationITGA1 serves as a biomarker for monitoring cardiac structural and functional damage, can be used to accurately diagnose the presence of HFpEF, and can be used to predict potential deterioration in cardiac structure and function as well as re-hospitalisation for individuals with type 2 diabetes. Its measurement holds promise for facilitating risk stratification and early intervention to mitigate the adverse cardiovascular effects associated with diabetes.Data availabilityThe proteomic data of left ventricular myocardial tissue from individuals with type 2 diabetes, encompassing both those with and without HFpEF, is available from the ProteomeXchange database at http://proteomecentral.proteomexchange.org.Graphical

Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15.

In this study we used a spatial transcriptomics approach to identify genes specifically associated with either high or low outflow regions in the trabecular meshwork (TM) that could potentially affect aqueous humor outflow in vivo. High and low outflow regions were identified and isolated from organ cultured human anterior segments perfused with fluorescently-labeled 200 nm FluoSpheres. The NanoString GeoMx Digital Spatial Profiler (DSP) platform was then used to identified genes in the paraffin embedded tissue sections from within those regions. These transcriptome analyses revealed that 16 genes were statistically upregulated in high outflow regions and 57 genes were statistically downregulated in high outflow regions when compared to low outflow regions. Gene ontology enrichment analysis indicated that the top three biological categories of these differentially expressed genes were ECM/cell adhesion, signal transduction, and transcription. The ECM/cell adhesion genes that showed the largest differential expression (Log2FC ±1.5) were ADAM15, BGN, LDB3, and CRKL. ADAM15, which is a metalloproteinase that can bind integrins, was upregulated in high outflow regions, while the proteoglycan BGN and two genes associated with integrin signaling (LDB3, and CRKL) were downregulated. Immunolabeling studies supported the differential expression of ADAM15 and showed that it was specifically upregulated in high outflow regions along the inner wall of Schlemm's canal and in the juxtacanalicular (JCT) region of the TM. In addition to these genes, the studies showed that genes for decorin, a small leucine-rich proteoglycan, and the α8 integrin subunit were enriched in high outflow regions. These studies identify several novel genes that could be involved in segmental outflow, thus demonstrating that digital spatial profiling could be a useful approach for understanding segmental flow through the TM. Furthermore, this study suggests that changes in the expression of genes involved in regulating the activity and/or organization of the ECM and integrins in the TM are likely to be key players in segmental outflow.

Evaluating the Serum Levels of CCL17, CCL22, and CCL28 Chemokines and the Gene Expression of α4β1 and α4β7 Integrins in Patients With Allergic Rhinitis

Allergic rhinitis (AR) is a chronic inflammatory disease involving the nasal mucosa. Leukocytes recruitment to the inflammation sites is controlled by chemokines, cytokines, and adhesion molecules. Retinoic acid (RA), a vitamin A metabolite, plays an essential role in mucosal immunity and the production of inflammatory cytokines and chemokines. This study intended to evaluate the serum levels of RA, CCL17, CCL22, CCL28, and the mRNA expression levels of α4, β1, and β7 integrins in AR patients compared to healthy subjects. Peripheral blood was collected from 37 patients with AR and 30 age- and gender-matched healthy individuals. Serum levels of RA, CCL17, CCL22, and CCL28 were measured by the enzyme-linked immunosorbent assay (ELISA) technique, and the mRNA expression levels for α4, β1, and β7 integrins were assessed using the quantitative real-time PCR method. Our results showed that the serum levels of CCL22 and CCL28 chemokines are significantly higher in the AR group compared to the healthy controls (P<0.01). However, the gene expression of the β1 integrin in the AR group was significantly lower than that of the control group (P<0.001). Besides, there was a positive association between serum RA and CCL17 levels in patients (P<0.0001, r=0.6). In conclusion, increased serum levels of CCL22 and CCL28 chemokines, as well as decreased gene expression of β1 integrin in AR patients, may contribute to the pathogenesis and/or exacerbation of AR.

Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic tissues by transferring various microRNAs and proteins including ITGa5 and NRP1

Mesenchymal stem cells/stromal cells (MSCs)-derived extracellular vesicles (EVs) mediate pro-regenerative effects in damaged ischemic tissues by regulating angiogenesis. MSCs-EVs modulate functions of cells including endogenous mature cells, progenitors and stem cells, resulting in restoration of blood flow. However, the mechanisms underlying such MSC-EV activity still remain poorly understood. The present study analyzes biological effects of bone marrow (BM) MSC-EVs on endothelial cells (ECs) in ischemic tissues both in in vitro and in vivo conditions and elucidates the molecular mechanisms underlying the tissue repair. MSC-EVs were isolated from murine BM-derived MSCs and their morphological, antigenic and molecular composition regarding protein and microRNA levels were evaluated to examine their properties. Global proteomic analysis demonstrated the presence in MSC-EVs of proteins regulating pro-regenerative pathways, including integrin α5 (Itgα5) and neuropilin-1 (NRP1) involved in lymphangiogenesis. MSC-EVs were also enriched in microRNAs regulating angiogenesis, TGF-β signaling and processes guiding cellular adhesion and interactions with extracellular matrix. The functional effects of MSC-EVs on capillary ECs in vitro included the increase of capillary-like tube formation and cytoprotection under normal and inflammatory conditions by inhibiting apoptosis. Notably, MSC-EVs enhanced also capillary-like tube formation of lymphatic ECs, which may be regulated by Itgα5 and NRP1. Moreover, in a mouse model of critical hind limb ischemia, MSC-EVs increased the recovery of blood flow in ischemic muscle tissue, which was accompanied with increased vascular density in vivo. This pro-angiogenic effect was associated with an increase in nitric oxide (NO) production via endothelial NO-synthase activation in ischemic muscles. Interestingly, MSC-EVs enhanced lymphangiogenesis, which has never been reported before. The study provides evidence on pro-angiogenic and novel pro-lymphangiogenic role of MSC-EVs on ECs in ischemic tissue mediated by their protein and miRNA molecular cargos. The results highlight Itgα5 and NRP1 carried by MSC-EVs as potential therapeutic targets to boost lymphangiogenesis.Graphical

Vaginal pH value can affect the susceptibility to human papillomavirus infection

BackgroundCervical cancer is the fourth most common cancer among women, with persistent high-risk human papillomavirus (HPV) infection being responsible for its progression. In healthy, pre-menopausal women, the vaginal pH value is maintained at 3.8–4.5, but various factors can affect it. Previous studies have suggested the relationship between vaginal pH value and HPV infection. In this study, we aimed to explore the relationship between vaginal pH and susceptibility of HPV infection.MethodsIn our study, we retrospectively collected medical information from women who underwent leukorrhea examination at our hospital. We excluded women with infectious diseases or cancer, those who were pregnant or within 6 months post-delivery, and those without HPV test results within 6 months. The association between percentage of HPV infection and vaginal pH value was analyzed. Furthermore, we prepared HPV pseudovirus (PsVs) by co-transfecting structure plasmids and report plasmids in 293FT cells. In vitro, we changed the pH value of cell culture medium to investigate its influence on HPV PsVs infection. In vivo, we changed mouse’s vaginal pH value to investigate its influence on HPV PsVs infection.ResultsOur retrospective study included 3115 women aged 20–78, including 2531 women with HPV negative and 584 women with HPV positive. The percentages of both HPV infection and high-risk HPV infection were higher in women with a vaginal pH value ≥5.0 compared to those with a pH value < 5.0. In vitro, HPV PsVs infection rate was higher in cell culture medium of higher pH value, dominantly due to the influence of pH value on the stage of HPV PsVs adhering to cell surface. Neither of the cell surface HPV receptors Syndecan-1 nor integrin α6 was found to be changed obviously in different pH values. In vivo, more HPV PsVs were adhered to the mouse’s vaginal epithelial cells with the increase of the vaginal pH value.ConclusionsOur study suggests a possible association between vaginal pH value and HPV infection. The pH value can influence the susceptibility of HPV PsVs infection by affecting the adhering of HPV PsVs to cells in vivo and in vitro. Additionally, the cell surface HPV receptors Syndecan-1 and Integrin α6 do not seem to be affected by pH value, and the specific mechanism needs to be further explored.

Abstract 16: Integrin α9β1 and VCAM-1 Interaction Promotes Neutrophil Hyperactivation and Cognitive Decline Following Subarachnoid Hemorrhage

Background: Integrin α9β1 is a transmembrane glycoprotein that is highly expressed on neutrophils and mediates transendothelial migration via vascular cell adhesion molecule-1 (VCAM-1). Neutrophils are major contributors to cerebral thrombosis and oxidative stress following subarachnoid hemorrhage (SAH); however, the mechanism by which neutrophil integrin α9 contributes to SAH pathogenesis is unclear. Here, we evaluated the mechanistic role of neutrophil integrin α9 in the post-SAH sensorimotor and cognitive recovery. Methods: SAH was induced by filament perforation in neutrophil-specific α9 -/- (α9 fl/fl Mrp8Cre +/- ) mice and littermate controls. Cognitive recovery (Y-maze, Barnes maze, and novel object recognition test) and sensorimotor recovery (modified neurological severity score, corner, and cylinder test) were performed for up to 4-weeks. Unbiased RNA sequencing (RNA-seq) was used to evaluate the potential mechanisms by which neutrophil integrin α9-VCAM-1 interactions promote SAH. Computational modeling, docking, virtual screening, and cell-free assays were used to identify small-molecule inhibitors of the integrin α9-VCAM-1 interactions. Results: Experimental SAH resulted in increased levels of neutrophil integrin α9 which was concomitant with neutrophil hyperactivation. Post-SAH neutrophil-specific α9 -/- mice exhibited improved sensorimotor and cognitive recovery, along with reduced plasma elastase and MPO levels and brain neutrophil infiltration. Mechanistically, integrin α9/VCAM-1 interactions promote neutrophil hyperactivation, extracellular signal-regulated kinase (ERK) phosphorylation, and endothelial cell apoptosis. RNA-seq data revealed that α9/VCAM-1 interactions promotes gene expression related to neutrophil inflammation, exocytosis, NF-κB signaling, and chemotaxis. Virtual screening and cell-free assay identified macitentan as a potent inhibitor of integrin α9-VCAM-1 interactions. In vitro, macitentan treatment decreased neutrophil adhesion to VCAM-1 and macitentan treatment at a dose of 5 mg/kg resulted in improved SAH outcomes. Conclusion: The results of this study describe a previously unrecognized pathway involving integrin α9 and VCAM-1 in the pathogenesis of SAH.

Abstract TP314: The α5 Integrin Inhibitor ATN161 Mitigates SARS-CoV-2- Induced Brain Endothelial Barrier Disruption and Neuroinflammation α5 Integrin Inhibitor ATN 161

Introduction: Although SARS-CoV-2 infection can cause a wide range of mild to severe symptoms, the pathophysiology of acute and long-term neurological manifestations remains elusive. The arginine-glycine-aspartic acid motif of the viral spike protein may use to bind integrins receptors in the CNS, which play an important role in cerebrovascular integrity. Here we investigate the role of integrin α5β1 in mediating brain endothelial damage and inflammation during SARS-CoV-2 infection. Method: Mouse brain microvascular endothelial cells (bEnd.3) were treated with SARS-CoV-2 (Isolate USA-WA1/2020) or delta variant spike protein for 24h then later exposed to hypoxia for 6h (to model the effects of in vivo pulmonary infection). Cells were pretreated with ATN-16, 1h before SARS-CoV-2 and hypoxia challenge. Further, BALB/c mice were inoculated intranasally with 2x10 4 -PFU of the MA-10 strain of SARS-CoV-2 and treated with 1mg/kg of ATN-161, an α5β1 integrin inhibitor, retro-orbitally. The brains were collected 3 days post-infection for neuropathological evaluation. Results: SARS-CoV-2 and delta variant spike protein inoculations induced integrin α5 and decreased claudin-5 expression in bEnd.3 cells in a dose-dependent manner. SARS-CoV-2 spike protein challenge at 0.5 μg for 24h followed by hypoxia for 6h resulted in increased α5 and decreased claudin-5 expression in either hypoxia or SARS-CoV-2+hypoxia combination. ATN-161 (10μM) pretreatment inhibited SARS-CoV-2+hypoxia-induced α5 upregulation and restored claudin-5 loss in bEnd.3 cells. The in vivo studies showed a significant increase in the pro-inflammatory response as measured by cytokine IL-6 expression and a decrease in barrier integrity by tight junction claudin-5 expression in the brains of MA10-infected mice compared to mock controls. ATN-161 treatment decreased IL-6 expression and increased claudin 5 expression in infected mice. Conclusion: Therefore, we propose that targeting integrin α5β1 through inhibitors such as ATN-161 offers a promising therapeutic strategy for attenuating SARS-CoV-2 and its immunological impact on brain vasculature. This approach may be pivotal in reducing both acute and chronic neurological morbidities associated with COVID-19.

Imaging Very Late Antigen-4 on MOLT4 Leukemia Tumors with Cysteine Site-Specific 89Zr-Labeled Natalizumab Immuno-Positron Emission Tomography.

Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.

Live imaging of Fibronectin 1a-mNeonGreen and Fibronectin 1b-mCherry knock-in alleles during early zebrafish development

Within the developing embryo, cells assemble and remodel their surrounding extracellular matrix during morphogenesis. Fibronectin is an extracellular matrix glycoprotein and is a ligand for several members of the Integrin adhesion receptor family. Here, we compare the expression pattern and loss of function phenotypes of the two zebrafish fibronectin paralogs fn1a and fn1b. We engineered two fluorescently tagged knock-in alleles to facilitate live in vivo imaging of the Fibronectin matrix. Genetic complementation experiments indicate that the knock-in alleles are fully functional. Fn1a-mNeonGreen and Fn1b-mCherry are co-localized in ECM fibers on the surface of the paraxial mesoderm and myotendinous junction. In 5-days old zebrafish larvae, Fn1a-mNeonGreen predominantly localizes to the branchial arches, heart ventricle, olfactory placode and within the otic capsule while Fn1b-mCherry is deposited at the pericardium, proximal convoluted tubule, posterior hindgut and at the ventral mesoderm/cardinal vein. We examined Fn1a-mNeonGreen and Fn1b-mCherry in maternal zygotic integrin α5 mutants and integrin β1a; β1b double mutants and find distinct requirements for these Integrins in assembling the two Fibronectins into ECM fibers in different tissues. Rescue experiments via mRNA injection indicate that the two fibronectins are not fully inter-changeable. Lastly, we examined cross-regulation between the two Fibronectins and find fn1a is necessary for normal Fn1b fibrillogenesis in the presomitic mesoderm, but fn1b is dispensable for the normal pattern of Fn1a deposition.

Rejuvenating Hyaline Cartilaginous Phenotype of Dedifferentiated Chondrocytes in Collagen II Scaffolds: A Mechanism Study Using Chondrocyte Membrane Nanoaggregates as Antagonists.

Joint cartilage lesions affect the global population in the current aging society. Maintenance and rejuvenation of articular cartilage with hyaline phenotype remains a challenge as the underlying mechanism has not been completely understood. Here, we have designed and performed a mechanism study using scaffolds made of type II collagen (Col2) as the 3D cell cultural platforms, on some of which nanoaggregates comprising extracts of chondrocyte membrane (CCM) were coated as the antagonist of Col2. Dedifferentiated chondrocytes were, respectively, seeded into these Col2 based scaffolds with (antCol2S) or without (Col2S) CCM coating. After 6 weeks, in Col2S, the chondrocytes were rejuvenated to regain hyaline phenotype, whereas this redifferentiation effect was attenuated in antCol2S. Transcriptomic and proteomic profiling indicated that the Wnt/β-catenin signaling pathway, which is an opponent to maintenance of the hyaline cartilaginous phenotype, was inhibited in Col2S, but it was contrarily upregulated in antCol2S due to the antagonism and shielding against Col2 by the CCM coating. Specifically, in antCol2S, since the coated CCM nanoaggregates contain the same components as those present on the surface of the seeded chondrocytes, the corresponding ligand sites on Col2 had been preoccupied and saturated by CCM coating before exposure to the seeded cells. The results indicated that the ligation between Col2 ligands and integrin α5 receptors on the surface of the seeded chondrocytes in antCol2S was antagonized by the CCM coating, which facilitates the Wnt/β-catenin signaling toward the loss of hyaline cartilaginous phenotype. This finding reveals the contribution of Col2 for maintenance and rejuvenation of the hyaline cartilaginous phenotype in chondrocytes.

ITGA7 loss drives the differentiation of adipose-derived mesenchymal stem cells to cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major cellular component of the tumor (pre-)metastatic niche and play an essential role in omental dissemination of ovarian cancer. The omentum is rich in adipose, and adipose-derived mesenchymal stem cells (ADSCs) have been identified as a source of CAFs. However, the molecular events driving the phenotype shift of ADSCs remain largely unexplored. In this research, we focus on integrins, transmembrane receptors that have been widely involved in cellular plasticity. We found that integrin α7(ITGA7) was the only member of the integrin family that positively correlated with both overall survival and progression-free survival in ovarian cancer through GEPIA2. The immunohistochemistry signal of ITGA7 was apparent in the tumor stroma, and a lower omental ITGA7 level was associated with metastasis. Primary ADSCs were isolated from the omentum of patients with ovarian cancer and identified by cellular morphology, biomarkers, and multilineage differentiation. The conditional medium of ovarian cancer cells induced ITGA7 expression decrease and phenotypic changes in ADSCs. Downregulation of ITGA7 in primary omental ADSCs led to decrease in stemness properties and emerge of characteristic morphology and biomarkers of CAFs. Moreover, the conditioned medium of ADSCs with ITGA7 depletion exhibited enhanced abilities to improve the migration and invasion of ovarian cancer cells in vitro. Overall, these findings indicate that loss of ITGA7 may induce the differentiation of ADSCs to CAFs that contribute to a tumor-supportive niche.

KRT17 From Keratinocytes With High Glucose Stimulation Inhibit Dermal Fibroblasts Migration Through Integrin α11.

To investigate the effects of overexpressed keratin 17 (KRT17) on the biology of human dermal fibroblasts (HDFs) and to explore the mechanism of KRT17 in diabetic wound healing. KRT17 expression was tested in diabetic keratinocytes, animal models, and patient skin tissues (Huazhong University of Science and Technology Ethics Committee, [2022] No. 3110). Subsequently, HDFs were stimulated with different concentrations of KRT17 in vitro. Changes in the proliferation and migration of HDFs were observed. Then, identification of KRT17-induced changes in dermal fibroblast of RNA sequencing-based transcriptome analysis was performed. KRT17 expression was upregulated under pathological conditions. In vitro stimulation of HDFs with different concentrations of KRT17 inhibited cell migration. RNA-seq data showed that enriched GO terms were extracellular matrix components and their regulation. KEGG analysis revealed that the highest number of enriched genes was PI3K-Akt, in which integrin alpha-11 (ITGA11) mRNA, a key molecule that regulates cell migration, was significantly downregulated. Decreased ITGA11 expression was observed after stimulation of HDFs with KRT17 in vitro. Increased expression of KRT17 in diabetic pathological surroundings inhibits fibroblast migration by downregulating the expression of ITGA11. Thus, KRT17 may be a molecular target for the treatment of diabetic wounds.

Hypoxia-driven TNS4 fosters HNSCC tumorigenesis by stabilizing integrin α5β1 complex and triggering FAK-mediated Akt and TGFβ signaling pathways.

Head and neck squamous cell carcinoma (HNSCC) remains a formidable clinical challenge due to its high recurrence rate and limited targeted therapeutic options. This study aims to elucidate the role of tensin 4 (TNS4) in the pathogenesis of HNSCC across clinical, cellular, and animal levels. We found a significant upregulation of TNS4 expression in HNSCC tissues compared to normal controls. Elevated levels of TNS4 were associated with adverse clinical outcomes, including diminished overall survival. Functional assays revealed that TNS4 knockdown attenuated, and its overexpression augmented, the oncogenic capabilities of HNSCC cells both in vitro and in vivo. Mechanistic studies revealed that TNS4 overexpression promotes the interaction between integrin α5 and integrin β1, thereby activating focal adhesion kinase (FAK). This TNS4-mediated FAK activation simultaneously enhanced the PI3K/Akt signaling pathway and facilitated the interaction between TGFβRI and TGFβRII, leading to the activation of the TGFβ signaling pathway. Both of these activated pathways contributed to HNSCC tumorigenesis. Additionally, we found that hypoxia-inducible factor 1α (HIF-1α) transcriptionally regulated TNS4 expression. In conclusion, our findings provide the basis for innovative TNS4-targeted therapeutic strategies, which could potentially improve prognosis and survival rates for patients with HNSCC.

Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis.

Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. Our findings define integrin α2β1 as an early marker of OC differentiation.

ITGA1 Promotes Glioma Cell Proliferation and Affects Immune Cell Infiltration in Low-Grade Glioma.

Background: Low-grade glioma (LGG) is a commonly occurring type of central nervous system cancer. Integrin α1 (ITGA1), a family member of integrins, is implied in the malignant development of cancers, but the fundamental role of ITGA1 has not been illustrated yet in glioma. This study aimed to evaluate the prognostic value of ITGA1. Methods: Correlations between ITGA1 and relevant clinical features were analyzed in the LGG datasets based on Chinese Glioma Genome Atlas (CGGA) and Tumor Genome Atlas (TCGA). Glioma clinical samples and glioma cell lines were identified at the level of mRNA and protein level by Western blot. Cox regression were developed to assess the involvement of ITGA1 expression in predicting survival in LGG patients. Application of GSEA enrichment analysis to reveal ITGA1-mediated biological functions in LGG. Using TIMER 2.0 to analyze correlations between immune cell infiltration. In addition, ITGA1 high expression was analyzed for correlation with immune checkpoint-related genes and cumulative survival time. Results: ITGA1 was significantly more expressed in LGG than in normal samples. Cox regression indicated that ITGA1 was a risk factor independently for prognosis in LGG patients. GSEA enrichment analysis indicated that ITGA1 was engaged in several immunomodulatory processes. ITGA1 expression was shown to be highly correlated with the immune score, stromal score, and estimate score in LGG. ITGA1 was significantly related to the immune checkpoint-associated gene expression. In vivo experiments showed that overexpression of ITGA1 promoted glioma cell invasion. Conclusion: High ITGA1 expression is correlated with immune infiltration of the low-grade tumor, leading to poor prognoses in LGG patients.

Targeting Reprogrammed Cancer-Associated Fibroblasts with Engineered Mesenchymal Stem Cell Extracellular Vesicles for Pancreatic Cancer Treatment.

Background: As one of the most aggressive and lethal cancers, pancreatic cancer is highly associated with cancer-associated fibroblasts (CAFs) that influence the development and progression of cancer. Targeted reprogramming of CAFs may be a promising strategy for pancreatic cancer. This study aims to construct engineered extracellular vesicles (EVs) with surface modification of integrin α5 (ITGA5)-targeting peptide and high internal expression of miR-148a-3p by endogenous modification for targeted reprogramming of pancreatic CAFs. Methods: Bone marrow mesenchymal stem cells (BMSCs) and pancreatic CAFs were cocultured to examine the effect of BMSC-derived EVs on the expression levels of CAF markers. miR-148a-3p was identified as a functional molecule. The mechanism of miR-148a-3p was elucidated using the dual-luciferase reporter assay. BMSCs were infected with TERT-encoding and miR-148a-3p-encoding lentiviruses. Subsequently, BMSCs were modified with ITGA5-specific targeting peptide. The supernatant was ultracentrifuged to obtain the engineered EVs (ITGA5-EVs-148a), which were used to reprogram CAFs. Results: BMSCs modulated CAF marker expressions through EVs. miR-148a-3p was up-regulated in BMSCs. The expression of miR-148a-3p in pancreatic CAFs was down-regulated when compared with that in normal fibroblasts (NFs). Mechanistically, ITGA5-EVs-148a effectively suppressed the proliferation and migration of pancreatic CAFs by targeting ITGA5 through the TGF-β/SMAD pathway. ITGA5-EVs-148a was associated with enhanced cellular uptake and exhibited enhanced invitro and invivo targeting ability. Moreover, ITGA5-EVs-148a exerted strong reconfiguration effects in inactivating CAFs and reversing tumor-promoting effects in 3D heterospheroid and xenograft pancreatic cancer models. Conclusions: This targeted CAF reprogramming strategy with genetically engineered ITGA5-EVs-148a holds great promise as a precision therapeutics in clinical settings.

SARS-CoV-2-induced brain endothelial barrier disruption and neuroinflammation are ameliorated by α5 integrin inhibitor ATN 161

SARS-CoV-2-induced brain endothelial barrier disruption and neuroinflammation are ameliorated by α5 integrin inhibitor ATN 161

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

Oleuropein Stimulates Migration of Human Trophoblast Cells and Expression of Invasion-Associated Markers.

Successful pregnancy establishment requires highly synchronized cross talk between the invasive trophoblast cells and the receptive maternal endometrium. Any disturbances in this tightly regulated process may lead to pregnancy complications. Local factors such as nutrients, hormones, cytokines and reactive oxygen species modulate the invasion of extravillous trophoblasts through critical signaling cascades. Epidemiological studies strongly indicate that a Mediterranean diet can significantly impact molecular pathways during placentation. Therefore, the aim of the current study was to examine whether oleuropein (OLE), one of the main compounds of the Mediterranean diet, may influence trophoblast cell adhesion and migration, as well as the expression of invasion-associated molecular markers and inflammatory pathways fostering these processes. HTR-8/SVneo cells were incubated with OLE at selected concentrations of 10 and 100 µM for 24 h. Results showed that OLE did not affect trophoblast cell viability, proliferation and adhesion after 24 h in in vitro treatment. The mRNA expression of integrin subunits α1, α5 and β1, as well as matrix-degrading enzymes MMP-2 and -9, was significantly increased after treatment with 10 µM OLE. Furthermore, OLE at a concentration of 10 µM significantly increased the protein expression of integrin subunits α1 and β1. Also, OLE inhibited the activation of JNK and reduced the protein expression of COX-2. Finally, a lower concentration of OLE 10 µM significantly stimulated migration of HTR-8/SVneo cells. In conclusion, the obtained results demonstrate the effects of OLE on the function of trophoblast cells by promoting cell migration and stimulating the expression of invasion markers. As suggested from results, these effects may be mediated via inhibition of the JNK signaling pathway.