[Expression of transferrin receptor 1 and ß1-integrins correlates with estrogen receptor status and immune infiltration in breast cancer]. / Ekspressiya transferrinovykh retseptorov 1 i ß1-integrinov korreliruet so statusom retseptorov estrogenov i immunnoi infil'tratsiei pri rake molochnoi zhelezy.

Cancer cells can aberrantly express various markers, including transferrin receptor 1 (CD71) and ß1-integrin molecules. Their role in invasion, migration and metastasis has been demonstrated. Determination of their expression in breast cancer (BC) may be an important point to characterize the clinical course of the tumor and prognosis of the disease. OBJECTIVE: To study of transferrin receptor 1 (CD71) expression by primary breast cancer cells in correlation with tumor cell phenotype. MATERIAL AND METHODS: Determination of BC phenotype: immunohistochemical staining method (immunofluorescence). Antibodies to ER (estrogen receptors), KL-1 (pancytokeratin), CD71 (transferrin receptor), CD29 (ß1-integrins). CD45, CD3, CD4, CD8, CD20 infiltration was also evaluated. ZEISS microscope (AXIOSKOP; Germany), method of G.J. Hammerling et al. Statistical processing: IBM-SPSS Statistics v.21. RESULTS: 63% of BC cases had CD71+ phenotype. CD71-mosaic tumors were observed in 14.4%. ß1-integrin expression was monomorphic in 51.6% of cases and mosaic in 38.7%. 85% of ER-positive tumors were CD71-positive with a monomorphic type of reaction; p=0.014. Among ER-negative tumors, CD71-negative reactions were 2-fold more frequent and the monomorphic type was less frequent. ER-positive tumors were CD29-positive in 73%; p=0.031. 45.5% of ER+ tumors were CD29-monomorphic. Among ER-negative tumors, the frequency of CD29-monomorphic tumors was 55%. Significant infiltration by CD3+ cells was predominant in CD71-positive tumors; p=0.016. In the CD29-monomorphic phenotype, CD45+ infiltration was 31.3%, and in the mosaic phenotype, 67.1%. CONCLUSION: BC aberrantly expresses transferrin receptors, ß1-integrins. CD71 expression is associated with ER expression. ER-positive tumors are often monomorphic for CD71. Prominent CD3+ infiltration was present in CD71+ tumors. Expression of ß1-integrins correlated with ER+ status and weak immune infiltration.

Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5ß1 integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

Advancing Cancer Theranostics Through Integrin αVß3-Targeted Peptidomimetic IAC: From Bench to Bedside.

Introduction: The expression of alpha-five beta-three (αVß3) integrins is upregulated in various malignancies undergoing angiogenesis. The development of integrin antagonists as diagnostic probes makes the αVß3 integrin a suitable candidate for targeting tumor angiogenesis. The goal of this study was to optimize the radiolabeling and evaluate the potential of conjugated integrin antagonist carbamate (IAC), a peptidomimetic, as a theranostic radiopharmaceutical for targeting tumor angiogenesis. Methodology: Radiolabeling of DOTAGA [2,2',2" -{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid]-IAC with [68Ga]Ga, [177Lu]Lu, and [225Ac]Ac was optimized. The binding affinity (Kd) of DOTAGA-IAC for the αVß3 receptor and cancer cell lines was quantified. The biodistribution studies were conducted in healthy Wistar rats. Dosimetry analysis was performed on [177Lu]Lu-DOTAGA-IAC distribution data. A pilot study of [68Ga]Ga-DOTAGA-IAC and [18F]FDG Positron Emission Tomography (PET/CT) imaging was performed in five patients with histopathologically confirmed breast cancer. PET/CT findings were compared between [68Ga]Ga-DOTAGA-IAC and [18F]FDG in these patients. Results: Radiopharmaceuticals were prepared with high radiochemical purity (>99.9%). Kd and Bmax measurements were 15.02 nM and 417 fmol for αVß3 receptor protein: 115.7 nM and 295.3 fmol for C6 glioma cells. Biodistribution studies in rats suggested the excretion via kidneys and partially through the hepatobiliary route. The effective dose of [177Lu]Lu-DOTAGA-IAC was found to be 0.17 mSv/MBq. The dynamic study in patients revealed the optimal imaging time to be 30-35 mins postadministration. Out of the cohort, [68Ga]Ga-DOTAGA-IAC detected the primary lesions in all five patients with a mean standard uptake value (SUVmax) of 3.94 ± 0.58 compared with [18F]FDG (SUVmax 13.8 ± 6.53). Conclusion: The study demonstrates that DOTAGA-IAC exhibits strong binding to αVß3 integrin, positioning it as a promising PET agent for assessing primary and metastatic cancers. The outcomes from the pilot study suggest the potential of [68Ga]Ga-DOTAGA-IAC PET/CT in breast carcinoma diagnosis. While recognizing the theranostic potential of DOTAGA-IAC for αVß3 integrin-expressing tumors, further clinical investigations are warranted to comprehensively assess therapeutic efficacy.

Potential of αvß6 and αvß1 integrin inhibition for treatment of idiopathic pulmonary fibrosis.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease of unknown cause with a dismal prognosis. Nintedanib and Pirfenidone are approved worldwide for the treatment of IPF, but they only slow the rate of functional decline and disease progression. Therefore, there is an urgent need for more efficacious and better tolerated drugs. AREAS COVERED: αvß6 and αvß1 are two integrins overexpressed in fibrotic tissue, which play a critical role in the development of lung fibrosis. They act by converting transforming growth factor (TGF)-ß, one of the most important profibrotic cytokine, in its active form. Here, we summarize and critically discuss the potential of a dual αvß6/αvß1 integrin inhibitor for the treatment of IPF. EXPERT OPINION: Bexotegrast, a dual αvß6/αvß1 integrin inhibitor, has the potential to slow or even halt disease progression in IPF. Indeed, the strong pre-clinical rationale and promising early phase clinical trial data have raised expectations.

Integrins as a bridge between bacteria and cells: key targets for therapeutic wound healing.

Integrins are heterodimers composed of α and ß subunits that are bonded through non-covalent interactions. Integrins mediate the dynamic connection between extracellular adhesion molecules and the intracellular actin cytoskeleton. Integrins are present in various tissues and organs where these heterodimers participate in diverse physiological and pathological responses at the molecular level in living organisms. Wound healing is a crucial process in the recovery from traumatic diseases and comprises three overlapping phases: inflammation, proliferation and remodeling. Integrins are regulated during the entire wound healing process to enhance processes such as inflammation, angiogenesis and re-epithelialization. Prolonged inflammation may result in failure of wound healing, leading to conditions such as chronic wounds. Bacterial colonization of a wound is one of the primary causes of chronic wounds. Integrins facilitate the infectious effects of bacteria on the host organism, leading to chronic inflammation, bacterial colonization, and ultimately, the failure of wound healing. The present study investigated the role of integrins as bridges for bacteria-cell interactions during wound healing, evaluated the role of integrins as nodes for bacterial inhibition during chronic wound formation, and discussed the challenges and prospects of using integrins as therapeutic targets in wound healing.

Dental pulp mesenchymal stem cells-response to fibrin hydrogel reveals ITGA2 and MMPs expression.

Regenerative endodontic procedures (REP) aim at reestablishing tooth vitality by replacing the irreversibly damaged dental pulp removed by the dental practitioner with a new functional one. The current treatment of advanced caries relies on the replacement of the inflamed or necrosed dental pulp with an inert filling material. This leads to a functional but non-vital tooth, which lacks the ability to sense dental tissue damage, and to protect from further bacterial attack. Therapeutic strategies inspired by tissue engineering called REP propose to regenerate a fully functional dental pulp directly in the canal space. Promising results were obtained using dental pulp mesenchymal stem cells (DP-MSCs) in combination with bio-inspired artificial and temporary 3D hydrogels made of extracellular matrix molecules such as collagen and fibrin biomacromolecules. However, the uncontrolled mechanisms of DP regeneration from DP-MSCs in 3D biomacromolecules fail to regenerate a fully functional DP and can induce fibrotic scarring or mineralized tissue formation to a non-negligible extent. The lack of knowledge regarding the early molecular mechanisms initiated by DP-MSCs seeded in ECM-made hydrogels is a scientific lock for REP. In this study, we investigated the early DP-MSC-response in a 3D fibrin hydrogel. DP-MSCs isolated from human third molars were cultured for 24 h in the fibrin hydrogel. The differential transcript levels of extracellular and cell surface genes were screened with 84-gene PCR array. Out of the 84 genes screened, 9 were found to be overexpressed, including those coding for the integrin alpha 2 subunit, the collagenase MMP1 and stromelysins MMP3, MMP10 and MMP12. Over-expression of ITGA2 was confirmed by RT-qPCR. The expression of alpha 2 integrin subunit protein was assessed over time by immunoblot and immunofluorescence staining. The increase in the transcript level of MMP1, MMP3, MM10 and MMP12 was confirmed by RT-qPCR. The overexpression of MMP1 and 3 at the protein level was assessed by immunoblot. MMP3 expression by DP-MSCs was observed by immunofluorescence staining. This work demonstrates overexpression of ITGA2 and of MMP1, 3, 10 and 12 by DP-MSCs cultured in a fibrin hydrogel. The main preliminary extracellular and cell surface response of the DP-MSCs to fibrin hydrogel seems to rely on a ITGA2/MMP3 axis. Further investigations are needed to precisely decipher the role of this axis in dental pulp tissue building. Nevertheless, this work identifies extracellular and cell surface molecules that could be potential checkpoints to be targeted to guide proper dental pulp tissue regeneration.

Augmenting fibronectin levels in injured adult CNS promotes axon regeneration in vivo.

In an attempt to repair injured central nervous system (CNS) nerves/tracts, immune cells are recruited into the injury site, but endogenous response in adult mammals is insufficient for promoting regeneration of severed axons. Here, we found that a portion of retinal ganglion cell (RGC) CNS projection neurons that survive after optic nerve crush (ONC) injury are enriched for and upregulate fibronectin (Fn)-interacting integrins Itga5 and ItgaV, and that Fn promotes long-term survival and long-distance axon regeneration of a portion of axotomized adult RGCs in culture. We then show that, Fn is developmentally downregulated in the axonal tracts of optic nerve and spinal cord, but injury-activated macrophages/microglia upregulate Fn while axon regeneration-promoting zymosan augments their recruitment (and thereby increases Fn levels) in the injured optic nerve. Finally, we found that Fn's RGD motif, established to interact with Itga5 and ItgaV, promotes long-term survival and long-distance axon regeneration of adult RGCs after ONC in vivo, with some axons reaching the optic chiasm when co-treated with Rpl7a gene therapy. Thus, experimentally augmenting Fn levels in the injured CNS is a promising approach for therapeutic neuroprotection and axon regeneration of at least a portion of neurons.

A synthetic bioactive peptide of the C-terminal fragment of adhesion protein Fibulin7 attenuates the inflammatory functions of innate immune cells in LPS-induced systemic inflammation.

OBJECTIVE: Systemic inflammation is associated with improper localization of hyperactive neutrophils and monocytes in visceral organs. Previously, a C-terminal fragment of adhesion protein Fibulin7 (Fbln7-C) was shown to regulate innate immune functionality during inflammation. Recently, a shorter bioactive peptide of Fbln7-C, FC-10, via integrin binding was shown to reduce ocular angiogenesis. However, the role of FC-10 in regulating the neutrophils and monocyte functionality during systemic inflammatory conditions is unknown. The study sought to explore the role of FC-10 peptide on the functionality of innate immune cells during inflammation and endotoxemic mice. METHODS: Neutrophils and monocytes were isolated from healthy donors and septic patient clinical samples and Cell adhesion assay was performed using a UV spectrophotometer. Gene expression studies were performed using qPCR. Protein level expression was measured using ELISA and flow cytometry. ROS assay, and activation markers analysis in vitro, and in vivo were done using flow cytometry. TREATMENT: Cells were stimulated with LPS (100 ng/mL) and studied in the presence of peptides (10 µg, and 20 µg/mL) in vitro. In an in vivo study, mice were administered with LPS (36.8 mg/kg bw) and peptide (20 µg). RESULTS: This study demonstrates that human neutrophils and monocytes adhere to FC-10 via integrin ß1, inhibit spreading, ROS, surface activation markers (CD44, CD69), phosphorylated Src kinase, pro-inflammatory genes, and protein expression, compared to scrambled peptide in cells isolated from healthy donors and clinical sample. In line with the in vitro data, FC-10 (20 µg) administration significantly decreases innate cell infiltration at inflammatory sites, improves survival in endotoxemia animals & reduces the inflammatory properties of neutrophils and monocytes isolated from septic patients. CONCLUSION: FC-10 peptide can regulate neutrophils and monocyte functions and has potential to be used as an immunomodulatory therapeutic in inflammatory diseases.

Deletion of Talin1 in Myeloid Cells Facilitates Atherosclerosis in Mice.

BACKGROUND: Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied. Our study investigated how talin1 in myeloid cells regulates the progression of atherosclerosis. METHODS: On an Apoe-/- background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. The atherosclerosis development in the aorta and monocyte recruitment into atherosclerotic lesions were analyzed. RESULTS: Myeloid talin1 deletion facilitated the formation of atherosclerotic lesions and macrophage deposition in lesions. Talin1 deletion abolished integrin ß2-mediated adhesion of monocytes but did not impair integrin α4ß1-dependent cell adhesion in a flow adhesion assay. Strikingly, talin1 deletion did not prevent Mn2+- or chemokine-induced activation of integrin α4ß1 to the high-affinity state for ligands. In an in vivo competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4ß1 but was not affected by talin1 deletion or antibodies to integrin ß2. Furthermore, quantitative polymerase chain reaction and ELISA (enzyme-linked immunosorbent assay) analysis showed that macrophages produced cytokines to promote inflammation and the proliferation of smooth muscle cells. Ligand binding to integrin ß3 inhibited cytokine generation in macrophages, although talin1 deletion abolished the negative effects of integrin ß3. CONCLUSIONS: Integrin α4ß1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4ß1 activation to the high-affinity state and integrin α4ß1-mediated monocyte recruitment. Yet, talin1 is required for integrin ß3 to inhibit the production of inflammatory cytokines in macrophages. Thus, intact monocyte recruitment and elevated inflammatory responses cause enhanced atherosclerosis in talin1-deficient mice. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.

Integrins play a role in stress relaxation of perivascular adipose tissue.

Perivascular adipose tissue (PVAT) is known for being anti-contractile in healthy tissues. We discovered a new function of PVAT, the ability to stress relax and maintain a tone in response to a stretch. This is of note because stress relaxation has been attributed to smooth muscle, of which PVAT has none that is organized in a functional layer. We test the hypothesis the interactions of integrins with collagen play a role in stress relaxation. Our model is the thoracic aorta of the male Dahl SS rat. The PVAT and aorta were physically separated for most assays. Results from single nuclei RNA sequencing (snRNAseq) experiments, histochemistry and isometric contractility were also used. Masson Trichrome staining made evident the expression of collagen in PVAT. From snRNA seq experiments of the PVAT, mRNA for multiple collagen and integrin isoforms were detected: the α1 and ß1 integrin were most highly expressed. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1ß1 distintegrin obtustatin or general integrin inhibitor RGD peptide. RGD peptide but not obtustatin increased the stress relaxation. Cell-cell communication inference identified integrins αv and α5, two major RGD motif containing isoforms, as potential signaling partners of collagens. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so in part through integrin-collagen interactions that may not include α1ß1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4ß1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

Characterization and preparation of food-derived peptides on improving osteoporosis: A review.

Osteoporosis is a systemic bone disease characterized by reduced bone mass and deterioration of the microstructure of bone tissue, leading to an increased risk of fragility fractures and affecting human health worldwide. Food-derived peptides are widely used in functional foods due to their low toxicity, ease of digestion and absorption, and potential to improve osteoporosis. This review summarized and discussed methods of diagnosing osteoporosis, treatment approaches, specific peptides as alternatives to conventional drugs, and the laboratory preparation and identification methods of peptides. It was found that peptides interacting with RGD (arginine-glycine-aspartic acid)-binding active sites in integrin could alleviate osteoporosis, analyzed the interaction sites between these osteogenic peptides and integrin, and further discussed their effects on improving osteoporosis. These may provide new insights for rapid screening of osteogenic peptides, and provide a theoretical basis for their application in bone materials and functional foods.

A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication.

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

The Role of αvß3 Integrin in Cancer Therapy Resistance.

A relevant challenge for the treatment of patients with neoplasia is the development of resistance to chemo-, immune-, and radiotherapies. Although the causes of therapy resistance are poorly understood, evidence suggests it relies on compensatory mechanisms that cells develop to replace specific intracellular signaling that should be inactive after pharmacological inhibition. One such mechanism involves integrins, membrane receptors that connect cells to the extracellular matrix and have a crucial role in cell migration. The blockage of one specific type of integrin is frequently compensated by the overexpression of another integrin dimer, generally supporting cell adhesion and migration. In particular, integrin αvß3 is a key receptor involved in tumor resistance to treatments with tyrosine kinase inhibitors, immune checkpoint inhibitors, and radiotherapy; however, the specific inhibition of the αvß3 integrin is not enough to avoid tumor relapse. Here, we review the role of integrin αvß3 in tumor resistance to therapy and the mechanisms that have been proposed thus far. Despite our focus on the αvß3 integrin, it is important to note that other integrins have also been implicated in drug resistance and that the collaborative action between these receptors should not be neglected.

Interactions of Cardiac Proteins with Plasma-Synthesized Polypyrrole (PSPy) to Improve Adult Cardiomyocytes Culture.

Plasma-Synthesized Polypyrrole (PSPy) has been reported as a biomaterial suitable for cell growth in vitro and in vivo. An experimental duplicate was carried out that showed the growth of cardiomyocytes with PSPy, following a protocol previously reported by the working group. The cardiomyocytes cultured with the biomaterial retained their native morphological characteristics, a fundamental key to improving cardiac cell therapy procedures. Such observations motivated us to investigate the molecular characteristics of the biomaterial and the type of interactions that could be occurring (mainly electrostatic, hydrogen bonds, and non-polar). Additionally, PSPy has been studied to establish the probable mechanisms of action of the biomaterial, in particular, its action on a group of cell membrane proteins, integrins, which we know participate in the adhesion of cells to the extracellular matrix, in adhesion between cells and as bidirectional signal transducer mechanisms. In this work, we carried out studies of the interactions established between cardiac integrins α2ß1 and α5ß1 with different PSPy models by molecular docking studies and binding free energies (ΔGb) calculations. The models based on a previously reported PSPy molecule have three variable terminal chemical groups, with the purpose of exploring the differences in the type of interaction that will be established by modifying the position of an amino (-NH2), a hydroxyl (-OH), and a nitrile (C≡N) in (fixed) groups, as well as the length of the terminal chains (a long/short -NH2). A model with short chains for the -OH and -NH2 (lateral) group was the model with the best interactions with cardiac integrins. We experimentally verified the direct interaction of cardiomyocytes with the PSPy biomaterial observed in rat primary cultures, allowing us to validate the favorable interactions predicted by the computational analysis.

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion.

BACKGROUND: The aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell-matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis. METHODS: We used Blg-Cre; Brca1F/F; Trp53F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures. RESULTS: We show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers. CONCLUSIONS: Our data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1F/F; Trp53F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.

Binding requirements for latent transforming growth factor Beta2 activation.

Although the mechanism for activation of latent TGFß1 and TGFß3 is understood to involve the binding of the TGFß propeptide (LAP) to both an integrin and an insoluble substrate, the activation of latent TGFß2 has been unclear because the TGFß2 LAP does not have the classical integrin binding sequence found in the other two TGFß isoform LAPs. To assess the potential requirement for covalent linkage with a matrix or cell surface protein for the activation of latent TGFß2, we generated mice in which the TGFß2 Cys residue predicted to be involved in binding was mutated to Ser (Tgfb2C24S). We reasoned that, if covalent interaction with a second molecule is required for latent TGFß2 activation, mutant mice should display a Tgfb2 null (Tgfb2-/-)-like phenotype. Tgfb2C24S mice closely phenocopy Tgfb2-/- mice with death in utero between E18 and P1 and with congenital heart and kidney defects similar to those described for Tgfb2-/- mice. The mutant latent TGFß2 is secreted at levels similar to WT, yet TGFß signaling monitored as nuclear pSmad2 is suppressed. We conclude that, like latent TGFß1, latent TGFß2 activation requires binding to an immobilized matrix or plasma membrane molecule.

The protective roles of integrin α4ß7 and Amphiregulin-expressing innate lymphoid cells in lupus nephritis.

Type 2 innate lymphoid cells (ILC2s) have emerged as key regulators of the immune response in renal inflammatory diseases such as lupus nephritis. However, the mechanisms underlying ILC2 adhesion and migration in the kidney remain poorly understood. Here, we revealed the critical role of integrin α4ß7 in mediating renal ILC2 adhesion and function. We found that integrin α4ß7 enables the retention of ILC2s in the kidney by binding to VCAM-1, E-cadherin, or fibronectin on structural cells. Moreover, integrin α4ß7 knockdown reduced the production of the reparative cytokine amphiregulin (Areg) by ILC2s. In lupus nephritis, TLR7/9 signaling within the kidney microenvironment downregulates integrin α4ß7 expression, leading to decreased Areg production and promoting the egress of ILC2s. Notably, IL-33 treatment upregulated integrin α4ß7 and Areg expression in ILC2s, thereby enhancing survival and reducing inflammation in lupus nephritis. Together, these findings highlight the potential of targeting ILC2 adhesion as a therapeutic strategy for autoimmune kidney diseases.

Platelet Spreading and Clot Retraction are Regulated by two Distinct αIIbß3 Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αIIbß3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbß3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbß3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbß3 outside-in signaling and should not be used interchangeably as readout for αIIbß3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbß3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.