Integrin Expression Research Articles

Knockdown of ANO1 decreases TGF-β- and IL-6-induced adhesion and migration of cardiac fibroblasts by inhibiting the expression of integrin and focal adhesion kinase

Ischemic cardiac injury triggers a significant inflammatory response, activating and mobilizing cardiac fibroblasts (CFs), which ultimately contributes to myocardial fibrosis. In this study, we investigated the role of ANO1, a calcium-activated chloride channel (CaCC) protein, in regulating CFs migration and adhesion under inflammatory conditions. Our results demonstrated that ANO1 knockdown significantly attenuated TGF-β- and IL-6-induced adhesion and migration of CFs. This inhibitory effect was mediated through the downregulation of integrin expression and reduced activation of focal adhesion kinase (FAK), key components in cellular adhesion and motility pathways. This study provides new insights into the mechanisms underlying CFs migration and adhesion, highlighting the potential of ANO1 as a therapeutic target for mitigating adverse fibrotic remodeling following myocardial infarction.

Synthetic hydrogel substrate for human induced pluripotent stem cell definitive endoderm differentiation

Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G’ = 1.0–4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation.

Innovative 3D-Printed Titanium Specimens Favor a Modulation of Inflammation in Dental Pulp Stem Cells During Liposome-Triggered Mineralization.

developing customized titanium specimens, with innovative surfaces, is a suitable strategy to overcome implant failure. Additionally, a faster and efficient osteogenic commitment assists tissue regeneration. To investigate the interplay between inflammation and differentiation upon implantation, Dental Pulp Stem Cells (DPSCs) were cultured on 3D-printed titanium owning an internal open cell form, administering osteogenic factors by a liposomal formulation (LipoMix) compared to traditional delivery of differentiation medium (DM). osteogenic differentiation was evaluated by western blot, by measuring β1 integrin expression, and by and real-time RT-PCR, by measuring SP7 and Collagen I gene expression; while.angiogenesis was characterized by measuring VEGF secretion levels. Matrix mineralization was assessed by means of Alizarin Red Staining, cell adhesion and inflammation responses through western blot, enzymatic and ELISA assays evaluating Nrf2 expression, catalase activity and Prostaglandin E2 secretion, respectively. LipoMix enhances cell proliferation and adhesion, as revealed by increased integrin β1 expression. Mineralized matrix deposition, SP7 gene expression, Collagen I release and Alkaline Phosphatase activity appear increased in LipoMix condition. Additionally, the redox-sensitive transcription factor Nrf2 is overexpressed at the earliest experimental times, triggering the catalase activity. data reported confirm that internal topography and post-production treatments on titanium surfaces dynamically and positively condition the DPSC progress towards the osteogenic phenotype, moreover, the combination with LipoMix fastens the positive modulation of inflammation under osteogenic conditions. Therefore, the development of customized surfaces along with the administration of differentiating factors enclosed in a liposomal delivery system, could represent a promising and innovative tool in regenerative dentistry.

Acute contact with profibrotic macrophages mechanically activates fibroblasts via αvβ3 integrin-mediated engagement of Piezo1.

Fibrosis-excessive scarring after injury-causes >40% of disease-related deaths worldwide. In this misguided repair process, activated fibroblasts drive the destruction of organ architecture by accumulating and contracting extracellular matrix. The resulting stiff scar tissue, in turn, enhances fibroblast contraction-bearing the question of how this positive feedback loop begins. We show that direct contact with profibrotic but not proinflammatory macrophages triggers acute fibroblast contractions. The contractile response depends on αvβ3 integrin expression on macrophages and Piezo1 expression on fibroblasts. The touch of macrophages elevates fibroblast cytosolic calcium within seconds, followed by translocation of the transcription cofactors nuclear factor of activated T cells 1 and Yes-associated protein, which drive fibroblast activation within hours. Intriguingly, macrophages induce mechanical stress in fibroblasts on soft matrix that alone suppresses their spontaneous activation. We propose that acute contact with suitable macrophages mechanically kick-starts fibroblast activation in an otherwise nonpermissive soft environment. The molecular components mediating macrophage-fibroblast mechanotransduction are potential targets for antifibrosis strategies.

Pleiotrophin Activates cMet- and mTORC1-Dependent Protein Synthesis through PTPRZ1-The Role of ανβ3 Integrin.

Pleiotrophin (PTN) is a secreted factor that regulates endothelial cell migration through protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) and αvβ3 integrin. Genetic deletion of Ptprz1 results in enhanced endothelial cell proliferation and migration, due to the decreased expression of β3 integrin and the subsequent, enhanced cMet phosphorylation. In the present study, we investigated the effect of PTN and PTPRZ1 on activating the mTORC1 kinase and protein synthesis and identified part of the implicated signaling pathway in endothelial cells. PTN or genetic deletion of Ptprz1 activates protein synthesis in a mTORC1-dependent manner, as shown by the enhanced phosphorylation of the mTORC1-downstream targets ribosomal protein S6 kinase 1 (SK61) and 4E-binding protein 1 (4EBP1) and the upregulation of HIF-1α. The cMet tyrosine kinase inhibitor crizotinib abolishes the stimulatory effects of PTN or PTPRZ1 deletion on mTORC1 activation and protein synthesis, suggesting that mTORC1 activation is downstream of cMet. The mTORC1 inhibitor rapamycin abolishes the stimulatory effect of PTN or PTPRZ1 deletion on endothelial cell migration, suggesting that mTORC1 is involved in the PTN/PTPRZ1-dependent cell migration. The αvβ3 integrin blocking antibody LM609 and the peptide PTN112-136, both known to bind to ανβ3 and inhibit PTN-induced endothelial cell migration, increase cMet phosphorylation and activate mTORC1, suggesting that cMet and mTORC1 activation are required but are not sufficient to stimulate cell migration. Overall, our data highlight novel aspects of the signaling pathway downstream of the PTN/PTPRZ1 axis that regulates endothelial cell functions.

Heterobivalent Dual-Target Peptide for Integrin-αvβ3 and Neuropeptide Y Receptors on Breast Tumor.

Background/Objectives: Heterodimer peptides targeting more than one receptor can be advantageous, as tumors can simultaneously express more than one receptor type. For human breast cancer, a promising biological target is tumor angiogenesis through αvβ3 integrin expression. Another promising target is Neuropeptide Y receptors, considering Y1R is overexpressed in 90% of human breast tumors. This article details the development and preclinical evaluation, both in vitro and in vivo, of a novel heterodimer peptide dual-receptor-targeting probe, [99mTc]HYNIC-cRGDfk-NPY, designed for imaging breast tumors. Methods: Female BALB/c healthy mice were used to perform biodistrubution studies and female SCID mice were subcutaneously injected with MCF-7 and MDA-MB-231 tumor cells. [99mTc]HYNIC-cRGDfk-NPY was intravenously administered to the mice, followed by ex vivo biodistribution studies and small-animal SPECT/CT imaging. Nonspecific tracer uptake in both models was determined by coinjecting an excess of unlabeled HYNIC-cRGDfk-NPY (100 µg) along with the radiolabeled tracer. Results: Imaging and biodistribution data demonstrate good uptake to estrogen receptor-positive (MCF-7) and triple-negative (MDA-MB-231) tumor models. The in vivo tumor uptakes of radiolabeled conjugate were 9.30 ± 3.25% and 4.93 ± 1.01% for MCF-7 and MDA-MB231, respectively. The tumor/muscle ratios were 5.65 ± 0.94 for the MCF-7 model and 7.78 ± 3.20 for MDA-MB231. Conclusions: [99mTc]HYNIC-cRGDfk-NPY demonstrated rapid blood clearance, renal excretion, and in vivo tumor uptake, highlighting its potential as a tumor imaging agent.

ROCK Inhibitor Enhances Neurite Outgrowth In Vitro and Corneal Sensory Nerve Reinnervation In Vivo.

The intraepithelial corneal nerves are essential to corneal health. Rho kinase or ROCK inhibitors (RIs) have been reported to play a role in neuron survival after injury. Here we assess integrin and extracellular matrix expression in primary mouse neurons and determine whether treating cells with RI impacts neurite outgrowth in vitro and reinnervation after trephine and debridement injury in mice in vivo. Cocultures of human corneal limbal epithelial cells and E11.5 mouse trigeminal neurons and neurons alone were grown on glass coverslips. High-resolution imaging was performed to localize integrins and laminin on neurons and to determine whether RI impacts neurite outgrowth in vitro and in vivo after both 1.5-mm trephine and 1.5-mm debridement injuries. Several integrin α (α3, α6, αv) chains as well as β4 integrin are expressed on neuron axons and growth cones in cocultures. RI treatment of isolated neurons, cocultures, and in conditioned media increases neurite outgrowth. In vivo, RI positively impacts sensory nerve reinnervation after trephine and debridement injury. These studies are the first to demonstrate expression of β4 integrin on trigeminal sensory neurons and preferential adhesion of neurons to the laminin-enriched matrices found in footprints deposited by human corneal limbal epithelial cells. In addition, we also document for the first time the positive impact of RI on neurite outgrowth in vitro and reinnervation in vivo.

A-213 αEβ7 Integrin (CD103) expression in both early and advanced stage of Cutaneous T cell Lymphoma's (CTCL)

A-213 αEβ7 Integrin (CD103) expression in both early and advanced stage of Cutaneous T cell Lymphoma's (CTCL)

Modulation of αv integrins by lebecetin, a viper venom-derived molecule, in experimental neuroinflammation and demyelination models

Several neurodegenerative diseases, such as multiple sclerosis and Parkinson’s disease, are linked to alterations in myelin content or structure. Transmembrane receptors such as integrins could be involved in these alterations. In the present study, we investigated the role of αv-integrins in experimental models of neuroinflammation and demyelination with the use of lebecetin (LCT), a C-lectin protein purified from Macrovipera lebetina viper venom, as an αv-integrin modulator. In a model of neuroinflammation, LCT inhibited the upregulation of αv, β3, β5, α5, and β1 integrins, as well as the associated release of pro-inflammatory factor IL-6 and chemokine CXCL-10, and decreased the expression of phosphorylated NfκB. The subsequent “indirect culture” between reactive astrocytes and oligodendrocytes showed a down-regulation of αv and β3 integrins versus upregulation of β1 one, accompanied by a reduced expression of myelin basic protein (MBP). Treatment of oligodendrocytes with LCT rectified the changes in integrin and MBP expression. Through Western blot quantification, LCT was shown to upregulate the expression levels of PI3K and p-mTOR while downregulating expression levels of p-AKT in oligodendrocytes, suggesting the neuroprotective and pro-myelinating effects of LCT may be related to the PI3K/mTor/AKT pathway. Concomitantly, we found that LCT promoted remyelination by tracking the increased expression of MBP in the brains of cuprizone-intoxicated mice. These results point to an involvement of integrins in not only neuroinflammation but demyelination as well. Thus, targeting αv integrins could offer potential therapeutic avenues for the treatment of demyelinating diseases.

A Machine Learning-Based Investigation of Integrin Expression Patterns in Cancer and Metastasis.

Integrins, a family of transmembrane receptor proteins, play complex roles in cancer development and metastasis. These roles could be better delineated through machine learning of transcriptomic data to reveal relationships between integrin expression patterns and cancer. We collected publicly available RNA-Seq integrin expression from 8 healthy tissues and their corresponding tumors, along with data from metastatic breast cancer. We then used machine learning methods, including t-SNE visualization and Random Forest classification, to investigate changes in integrin expression patterns. Integrin expression varied across tissues and cancers, and between healthy and cancer samples from the same tissue, enabling the creation of models that classify samples by tissue or disease status. The integrins whose expression was important to these classifiers were identified. For example, ITGA7 was key to classification of breast samples by disease status. Analysis in breast tissue revealed that cancer rewires co-expression for most integrins, but the co-expression relationships of some integrins remain unchanged in healthy and cancer samples. Integrin expression in primary breast tumors differed from their metastases, with liver metastasis notably having reduced expression. Integrin expression patterns vary widely across tissues and are greatly impacted by cancer. Machine learning of these patterns can effectively distinguish samples by tissue or disease status.

αvβ3 integrin-targeted magnetic resonance imaging in a pancreatic cancer mouse model using RGD-modified liposomes encapsulated with Fe-deferoxamine.

Magnetic resonance (MR) imaging is a powerful imaging modality for obtaining anatomical information with high spatial and temporal resolution. In the drug delivery system (DDS) framework, nanoparticles such as liposomes are potential candidates for MR imaging. We validated that RGD peptides are possible targeting molecules for pancreatic cancer with αvβ3 integrin expression. This study aimed to evaluate RGD-modified liposomes loaded with ferrioxamine B for pancreatic cancer imaging. We synthesized four types of RGD-modified liposomes encapsulated with ferrioxamine B (SH-, H-, M-, and L-RGD-liposomes). The binding affinity of RGD-modified liposomes was evaluated in a competitive inhibition study using 125I-echistatin. To investigate the pharmacokinetics of RGD-modified liposomes, a biodistribution study using RGD-liposomes labeled with 111In was carried out in a human pancreatic cancer PANC-1 xenograft mouse model. Finally, MR was performed using ferrioxamine-B-loaded liposomes. RGD-liposomes inhibited the binding of 125I-echistatin to RGD. The biodistribution study revealed that 111In-RGD-liposomes accumulated significantly in the liver and spleen. Among the 111In-RGD-liposomes, 111In-H-RGD-liposomes showed the highest tumor-to-normal tissue ratio. In the MR study, H-RGD-liposomes loaded with ferrioxamine B showed higher tumor-to-muscle signal ratios than RKG-liposomes loaded with ferrioxamine B (control). We successfully synthesized RGD-liposomes that can target αvβ3 integrin.

Interleukin-33-activated basophils promote asthma by regulating Th2 cell entry into lung tissue.

Asthma is characterized by lung eosinophilia, remodeling, and mucus plugging, controlled by adaptive Th2 effector cells secreting IL-4, IL-5, and IL-13. Inhaled house dust mite (HDM) causes the release of barrier epithelial cytokines that activate various innate immune cells like DCs and basophils that can promote Th2 adaptive immunity directly or indirectly. Here, we show that basophils play a crucial role in the development of type 2 immunity and eosinophilic inflammation, mucus production, and bronchial hyperreactivity in response to HDM inhalation in C57Bl/6 mice. Interestingly, conditional depletion of basophils during sensitization did not reduce Th2 priming or asthma inception, whereas depletion during allergen challenge did. During the challenge of sensitized mice, basophil-intrinsic IL-33/ST2 signaling, and not FcεRI engagement, promoted basophil IL-4 production and subsequent Th2 cell recruitment to the lungs via vascular integrin expression. Basophil-intrinsic loss of the ubiquitin modifying molecule Tnfaip3, involved in dampening IL-33 signaling, enhanced key asthma features. Thus, IL-33-activated basophils are gatekeepers that boost allergic airway inflammation by controlling Th2 tissue entry.

Recoverability of zebrafish from decabromodiphenyl ether exposure: The persisted interference with extracellular matrix production and collagen synthesis and the enhancement of arrhythmias

As a widely used brominated flame retardant, the widespread presence of decabromodiphenyl ether (BDE-209) in the natural environment and the toxicity risks it poses are well established, but the recoverability of BDE-209-induced individual injuries remains unknown. Therefore, a 7-day depuration experiment following a 4-day exposure of zebrafish to BDE-209 was conducted to confirm the recoverability and its mode of action. Oxidative stress after depuration was significantly reduced compared with BDE-209 exposure as indicated by the decreased expression level of oxidative stress-related genes and the reduced MDA, Gpx, and GST in zebrafish, indicating a gradual recovery of antioxidant activity. However, BDE-209 inhibition of extracellular matrix (ECM) proteins worsened after depuration. Mechanistically, BDE-209 mediated ECM production and secretion by down-regulating integrin expression. Furthermore, BDE-209 inhibition of collagen synthesis worsened after depuration. Biochemical assays and histopathological observations revealed a same result in zebrafish. Mechanistically, lysine hydroxylation is inhibited thereby affecting collagen synthesis. Interestingly, zebrafish showed arrhythmia after depuration compared to BDE-209 exposure, and abnormal changes in ATPase levels indicated that disturbances in Ca2+ homeostasis contributed to arrhythmia. Collectively, BDE-209-induced interference with ECM production and collagen synthesis persisted after depuration, which will provide new insights for understanding the recovery patterns of individuals under BDE-209 stress.

Integrin profle of circulating tumour cells in breast cancer patients

Background. Integrins, as adhesion molecules, play a key role in the interaction of cells with the basal membrane and intercellular matrix. Numerous studies demonstrate evidence of increased expression of integrins on tumor cells in different types of cancer. Thus, β3 and αV integrins are associated with stem-like features of tumor cells, and β4 integrin as α6β4 heterodimer provides anchorage-independent survival of malignant mammary epithelial cells. However, all the described functions of integrins have been investigated exclusively on primary tumor cells. The functional significance and expression pattern of integrins on circulating tumor cells (CTCs) remains unclear. The aim of the study was to evaluate the β3, β4 and αvβ5 integrin expression on CTCs and its association with molecular subtype, stage and lymph node metastasis in breast cancer patients Material and Methods. The study included 22 patients with T1–4N0–3M0 invasive ductal breast carcinoma. Venous blood was taken from patients without neoadjuvant chemotherapy (group 1) and after neoadjuvant chemotherapy (group 2) in the volume of 12 ml into vacuum tubes with EDTA. The expression of CTC integrins including stemness features CD44/CD24, CD133 and ALDH1, and epithelial-mesenchymal transition (EMT) (N-cadherin) was evaluated by flow cytometry. Results. CTCs with β3+β4-αvβ5- and β3-β4+αvβ5+ phenotypes and stemness properties were associated with larger tumor size (T4) in breast cancer patients. The β3 integrin expression was associated with more aggressive molecular subtypes of breast cancer. Administration of neoadjuvant chemotherapy did not affect the expression pattern of β3, β4 and αvβ5 integrins in CTCs. Conclusion. In breast cancer, most CTCs expressed β3, β4 and αvβ5 integrins despite the lack of attachment to the basal membrane and intercellular matrix. The expression of the above integrins on CTCs was associated with breast cancer molecular subtype, stage and lymph node metastasis, and therefore its evaluation can be considered as one of the objectives of liquid biopsy study.

The Role of αvβ3 Integrin in Lamina Cribrosa Cell Mechanotransduction in Glaucoma.

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVβ3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVβ3 integrin expression using cilengitide (a known potent αVβ3 and αVβ5 inhibitor) and β3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVβ3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVβ3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVβ3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVβ3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVβ3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVβ3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVβ3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVβ3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma.

Abstract P455: Integrins play a role in stress relaxation of perivascular adipose tissue

Perivascular adipose tissue (PVAT) is well known for being anti-contractile (reducing potency, maximum) in healthy tissues. We discovered a new function of PVAT, the ability to stress relax in response to a stretch. This is of note because stress relaxation has been attributed largely to smooth muscle, of which PVAT has none that is organized in a functional layer. Here we test the hypothesis that stress relaxation depends on the interactions of the cell membrane delimited heterodimer integrins with collagen. Our model is the thoracic aorta of the male Dahl SS rat on a normal diet. The PVAT and aorta were physically separated for most assays. Results from snRNA seq in PVAT, histochemistry and isometric contractility were used. Collagen was evident in PVAT as stained by Masson Trichrome. snRNA seq of whole PVAT determined expression of many collagen isoforms and integrins, with mRNA for both the α1 and β1 integrin being most highly expressed. Isolated aortic rings and separated PVAT were challenged with 2 or 4 grams of passive tension. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1β1 distintegrin obtustatin (5 uM) or general inhibitor RGD peptide (1 mM). RGD peptide but not obtustatin reduced the ability of the isolated PVAT ring to maintain applied tone at both the 2 and 4 gram challenge (~15% gain in relaxation/loss of tone, figure ). By contrast, stress relaxation in the aorta itself was not reduced by either inhibitor. In addition to a1b1 and collagen interaction, cell-cell communication inference identified integrins av and a5, two major RGD motif containing isoforms, as potential partners of collagens with fibroblast-like cells most likely involved. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so at least in part through integrin-collagen interactions that may not include α1β1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

Hepatocyte growth factor promotes melanoma metastasis through ubiquitin-specific peptidase 22-mediated integrins upregulation

Hepatocyte growth factor (HGF) plays a critical role in promoting tumor migration, invasion, and metastasis, partly by upregulating integrins. The molecular mechanisms behind how HGF facilitates integrin-mediated tumorigenesis are not fully understood. In this study, we demonstrate that the ubiquitin-specific peptidase 22 (USP22) is essential for HGF-induced melanoma metastasis. HGF treatment dramatically increased the expression of both USP22 and multiple integrin family members in particular ITGAV, ITGB3, and ITGA1. An unbiased analysis of the TCGA database reveals integrins as common downstream targets of both USP22 and HGF across multiple human cancer types. Notably, CRISPR-mediated deletion of USP22 completely eliminates HGF-induced integrin expression in melanoma cells. At the molecular level, USP22 acts as a bona fide deubiquitinase for Sp1, a transcription factor for the ITGAV, ITGB3, and ITGA1 genes. USP22 interacts with and inhibits Sp1 ubiquitination, protecting against Sp1 proteasomal degradation. Supporting this, immunohistology analysis detects a positive correlation among USP22, Sp1, and integrin αv in human melanoma tissues. This study identifies the death from the signature gene USP22 as a critical positive regulator for HGF-induced integrin expression by deubiquitinating the Sp1 transcription factor during melanoma metastasis.

A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis.

Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVβ3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4-αVβ3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of β3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown β3 Integrin upregulation. However, it is not known if astrocytes upregulate β3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase β3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated β3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, β3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.

Adhesion of retinal cells to gold surfaces by biomimetic molecules.

Neural cell-electrode coupling is crucial for effective neural and retinal prostheses. Enhancing this coupling can be achieved through surface modification and geometrical design to increase neuron-electrode proximity. In the current research, we focused on designing and studying various biomolecules as a method to elicit neural cell-electrode adhesion via cell-specific integrin mechanisms. We designed extracellular matrix biomimetic molecules with different head sequences (RGD or YIGSR), structures (linear or cyclic), and spacer lengths (short or long). These molecules, anchored by a thiol (SH) group, were deposited onto gold surfaces at various concentrations. We assessed the modifications using contact angle measurements, fluorescence imaging, and X-ray Photoelectron Spectroscopy (XPS). We then analyzed the adhesion of retinal cells and HEK293 cells to the modified surfaces by measuring cell density, surface area, and focal adhesion spots, and examined changes in adhesion-related gene and integrin expression. Results showed that YIGSR biomolecules significantly enhanced retinal cell adhesion, regardless of spacer length. For HEK293 cells, RGD biomolecules were more effective, especially with cyclic RGD and long spacers. Both cell types showed increased expression of specific adhesion integrins and proteins like vinculin and PTK2; these results were in agreement with the adhesion studies, confirming the cell-specific interactions with modified surfaces. This study highlights the importance of tailored biomolecules for improving neural cell adhesion to electrodes. By customizing biomolecules to foster specific and effective interactions with adhesion integrins, our study provides valuable insights for enhancing the integration and functionality of retinal prostheses and other neural implants.

Urolithin A Ameliorates the TGF Beta-Dependent Impairment of Podocytes Exposed to High Glucose.

Increased activity of transforming growth factor-beta (TGF-β) is a key factor mediating kidney impairment in diabetes. Glomerular podocytes, the crucial component of the renal filter, are a direct target of TGF-β action, resulting in irreversible cell loss and progression of chronic kidney disease (CKD). Urolithin A (UA) is a member of the family of polyphenol metabolites produced by gut microbiota from ellagitannins and ellagic acid-rich foods. The broad spectrum of biological activities of UA makes it a promising candidate for the treatment of podocyte disorders. In this in vitro study, we investigated whether UA influences the changes exerted in podocytes by TGF-β and high glucose. Following a 7-day incubation in normal (NG, 5.5 mM) or high (HG, 25 mM) glucose, the cells were treated with UA and/or TGF-β1 for 24 h. HG and TGF-β1, each independent and in concert reduced expression of nephrin, increased podocyte motility, and up-regulated expression of b3 integrin and fibronectin. These typical-for-epithelial-to-mesenchymal transition (EMT) effects were inhibited by UA in both HG and NG conditions. UA also reduced the typically elevated HG expression of TGF-β receptors and activation of the TGF-β signal transducer Smad2. Our results indicate that in podocytes cultured in conditions mimicking the diabetic milieu, UA inhibits and reverses changes underlying podocytopenia in diabetic kidneys. Hence, UA should be considered as a potential therapeutic agent in podocytopathies.