Binding of Intracellular Myeloperoxidase to αV/ß1 Integrin Serves as a Mechanism of Survival in Epithelial Ovarian Cancer.

We were the first to report that epithelial ovarian cancer (EOC) cells and tissues express myeloperoxidase (MPO) that is known to play a role in immune surveillance and inflammation by myeloid cells. Additionally, we reported that MPO is colocalized with inducible nitric oxide synthase (iNOS), a key pro-oxidant enzyme, and plays a key role in regulating apoptosis in EOC cells. Whereas myeloid cells express MPO in a dimeric form, intriguingly, here we report the unique expression of only the monomeric form of MPO in EOC cells, tissues, and blood of an ovarian cancer patient. Additionally, we have identified a cell membrane receptor, αV/ß1 integrin, that is uniquely expressed by both chemosensitive and chemoresistant EOC cells with significantly higher expression in chemoresistant EOC cells. More importantly, we have demonstrated that monoclonal antibodies against αV/ß1 integrin induced cytotoxicity in EOC cells, but not in normal cells, that is also synergistic with conventional chemotherapies. Cytotoxicity of αV/ß1 antibodies is due to conformational changes in αV/ß1 integrin which prevents monomeric MPO binding to αV/ß1 integrin inhibiting the activation of MPO, leading to increased apoptosis. Since normal epithelial cells and macrophages lack monomeric MPO and αV/ß1 integrin system, targeting this unique MPO-dependent survival mechanism will selectively eliminate EOC cells and will be the target for developing specific ovarian cancer therapies.

αvß1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin-3.

Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote metastasis. To identify EV-bound proteins related to metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that integrins, including αv and ß1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that integrin αv is associated with disease progression in breast cancer patients. Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that integrins αv and ß1 are enriched in the CD63+ subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the galectin family as potential shuttlers of integrin αvß1 into EVs. In particular, knockdown of galectin-3, but not galectin-1, causes a reduction in the levels of cell surface integrins ß1 and αv, and decreases the colocalization of these integrins with CD63. Importantly, knockdown of galectin-3 leads to a decrease of integrin αvß1 export into the EVs concomitant with a decrease in the metastatic potential of breast cancer cells. Moreover, inhibition of the integrin αvß1 complex leads to a reduction in the binding of EVs to fibronectin, suggesting that integrin αvß1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound integrin αvß1 with breast cancer metastasis and provide additional insights into the export of integrin αvß1 into EVs in the context of metastasis.

TGFBI secreted by tumor-associated macrophages promotes glioblastoma stem cell-driven tumor growth via integrin αvß5-Src-Stat3 signaling.

Rationale: In the glioblastoma (GBM) microenvironment, tumor-associated macrophages (TAMs) are prominent components and facilitate tumor growth. The exact molecular mechanisms underlying TAMs' function in promoting glioma stem cells (GSCs) maintenance and tumor growth remain largely unknown. We found a candidate molecule, transforming growth factor beta-induced (TGFBI), that was specifically expressed by TAMs and extremely low in GBM and GSC cells, and meanwhile closely related to glioma WHO grades and patient prognosis. The exact mechanism of TGFBI linking TAM functions to GSC-driven tumor growth was explored. Methods: Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemistry staining (IHC) and public datasets were used to evaluate TGFBI origin and level in GBM. The response of GSCs to recombinant human TGFBI was assessed in vitro and orthotopic xenografts were established to investigate the function and mechanism in vivo. Results: M2-like TAMs infiltration was elevated in high-grade gliomas. TGFBI was preferentially secreted by M2-like TAMs and associated with a poor prognosis for patients with GBM. TGFBI promoted the maintenance of GSCs and GBM malignant growth through integrin αvß5-Src-Stat3 signaling in vitro and in vivo. Of clinical relevance, TGFBI was enriched in the serum and CSF of GBM patients and significantly decreased after tumor resection. Conclusion: TAM-derived TGFBI promotes GSC-driven tumor growth through integrin αvß5-Src-Stat3 signaling. High serum or CSF TGFBI may serve as a potential diagnostic and prognostic bio-index for GBMs.

RGDX1 X2 motif regulates integrin αvß5 binding for pluripotent stem cell adhesion.

The arginine-glycine-aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD-containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD-containing peptides are active. In this study, we elucidated the role of RGD-neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1 X2  sequences, such as RGDVF and RGDNY, and that the X1 X2 residues are essential for the adhesion via integrin αvß5 but not αvß3. iPSCs express integrin αvß5 but not αvß3; therefore, iPSC adhesion requires the RGDX1 X2 -containing sequences. The importance of the X1 X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvß5 but not αvß3. Analysis of RGD-neighboring sequences provides important insights into ligand-binding specificity of integrins. Identification of integrin αvß5-binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering.

Irisin ameliorates neuroinflammation and neuronal apoptosis through integrin αVß5/AMPK signaling pathway after intracerebral hemorrhage in mice.

BACKGROUND: Neuroinflammation is a crucial factor in the development of secondary brain injury after intracerebral hemorrhage (ICH). Irisin is a newly identified myokine that confers strong neuroprotective effects in experimental ischemic stroke. However, whether this myokine can exert neuroprotection effects after ICH remains unknown. This study aimed to investigate the impact of irisin treatment on neuroinflammation and neuronal apoptosis and the underlying mechanism involving integrin αVß5/AMPK pathway after ICH. METHODS: Two hundred and eighty-five adult (8-week-old) male C57BL/6 mice were randomly assigned to sham and ICH surgery groups. ICH was induced via intrastriatal injection of autologous blood. Irisin was administered intranasally at 30 min after ICH. To elucidate the underlying mechanism, cilengitide (a selective integrin αVß5 inhibitor) and dorsomorphin (a selective phosphorylated AMPK inhibitor) were administered before irisin treatment. The short- and long-term neurobehavior tests, brain edema, quantitative-PCR, western blotting, Fluoro-Jade C, TUNEL, and immunofluorescence staining were performed to assess the neurofunctional outcome at the level of molecular, cell, histology, and function. RESULTS: Endogenous irisin and its receptor, integrin αVß5, were increased, peaked at 24 h after ICH. irisin post-treatment improved both short- and long-term neurological functions, reduced brain edema after ICH. Interestingly, integrin αVß5 was mainly located in the microglia after ICH, and irisin post-treatment inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization. Moreover, irisin treatment inhibited neutrophil infiltration and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Mechanistically, irisin post-treatment significantly increased the expression of integrin αVß5, p-AMPK and Bcl-2, and decreased the expression of IL-1ß, TNF-α, MPO, and Bax following ICH. The neuroprotective effects of irisin were abolished by both integrin αVß5 inhibitor cilengitide and AMPK inhibitor dorsomorphin. CONCLUSIONS: This study demonstrated that irisin post-treatment ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the integrin αVß5/AMPK signaling pathway after ICH. Thus, irisin post-treatment may provide a promising therapeutic approach for the early management of ICH.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

CCN2-induced lymphangiogenesis is mediated by the integrin αvß5-ERK pathway and regulated by DUSP6.

Lymphangiogenesis is essential for the development of the lymphatic system and is important for physiological processes such as homeostasis, metabolism and immunity. Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated the effects of CCN2 on lymphangiogenesis. In in vivo Matrigel plug assays, exogenous CCN2 increased the number of Podoplanin-positive vessels. Subsequently, we found that CCN2 induced phosphorylation of ERK in primary cultured LECs, which was almost completely inhibited by the blockade of integrin αvß5 and partially decreased by the blockade of integrin αvß3. CCN2 promoted direct binding of ERK to dual-specific phosphatase 6 (DUSP6), which regulated the activation of excess ERK by dephosphorylating ERK. In vitro, CCN2 promoted tube formation in LECs, while suppression of Dusp6 further increased tube formation. In vivo, immunohistochemistry also detected ERK phosphorylation and DUSP6 expression in Podoplanin-positive cells on CCN2-supplemented Matrigel. These results indicated that CCN2 promotes lymphangiogenesis by enhancing integrin αvß5-mediated phosphorylation of ERK and demonstrated that DUSP6 is a negative regulator of excessive lymphangiogenesis by CCN2.

Dual inhibition of αvß6 and αvß1 reduces fibrogenesis in lung tissue explants from patients with IPF.

RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.

Retraction fibers produced by fibronectin-integrin α5ß1 interaction promote motility of brain tumor cells.

Glioblastoma (GBM) is a refractory disease that has a highly infiltrative characteristic. Over the past decade, GBM perivascular niche (PVN) has been described as a route of dissemination. Here, we investigated that trailed membrane structures, namely retraction fibers (RFs), are formed by perivascular extracellular matrix (ECM) proteins. By using the anatomical GBM database, we validated that the ECM-related genes were highly expressed in the cells within the PVN where fibronectin (FN) induced RF formation. By disrupting candidates of FN-binding integrins, integrin α5ß1 was identified as the main regulator of RF formation. De novo RFs were produced at the trailing edge, and focal adhesions were actively localized in RFs, indicating that adhesive force makes RFs remain at the bottom surface. Furthermore, we observed that GBM cells more frequently migrated along the residual RFs formed by preceding cells in microfluidic channels in comparison to those in the channels without RFs, suggesting that the infiltrative characteristics GBM could be attributed to RFs formed by the preceding cells in concert with chemoattractant cues. Altogether, we demonstrated that shedding membrane structures of GBM cells are maintained by FN-integrin α5ß1 interaction and promoted their motility .

ATN-161 Ameliorates Ischemia/Reperfusion-induced Oxidative Stress, Fibro-inflammation, Mitochondrial damage, and Apoptosis-mediated Tight Junction Disruption in bEnd.3 Cells.

We have previously demonstrated the significance of endothelial cell-expressed α5ß1 integrin in ischemic stroke, having shown that α5ß1 integrin endothelial cell-selective knockout mice are significantly resistance to ischemic stroke injury via preservation of the tight junction protein claudin-5 and subsequent stabilization of the blood-brain barrier (BBB). In addition, inhibition of α5ß1 by the small peptide noncompetitive integrin α5 inhibitor, ATN-161, is beneficial in a mouse model of ischemic stroke through reduction of infarct volume, edema, stabilization of the BBB, and reduced inflammation and immune cell infiltration into the brain. In continuation with our previous findings, we have further evaluated the mechanistic role of ATN-161 in vitro and found that oxygen and glucose deprivation and reperfusion (OGD/R)-induced inflammation, oxidative stress, apoptosis, mitochondrial depolarization, and fibrosis attenuate tight junction integrity via induction of α5, NLRP3, p-FAK, and p-AKT signaling in mouse brain endothelial cells. ATN-161 treatment (10 µM) effectively inhibited OGD/R-induced extracellular matrix (ECM) deposition by reducing integrin α5, MMP-9, and fibronectin expression, as well as reducing oxidative stress by reducing mitochondrial superoxide radicals, intracellular ROS, inflammation by reducing NLRP3 inflammasome, tight junction loss by reducing claudin-5 and ZO-1 expression levels, mitochondrial damage by inhibiting mitochondrial depolarization, and apoptosis via regulation of p-FAK and p-AKT levels. Taken together, our results further support therapeutically targeting α5 integrin with ATN-161, a safe, well-tolerated, and clinically validated peptide, in ischemic stroke.

FNDC5/Irisin attenuates diabetic cardiomyopathy in a type 2 diabetes mouse model by activation of integrin αV/ß5-AKT signaling and reduction of oxidative/nitrosative stress.

Irisin, the cleaved form of the fibronectin type III domain containing 5 (FNDC5) protein, is involved in metabolism and inflammation. Recent findings indicated that irisin participated in cardiovascular physiology and pathology. In this study, we investigated the effects of FNDC5/irisin on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice. Downregulation of myocardial FNDC5/irisin protein expression and plasma irisin levels was observed in db/db mice compared to db/+ controls. Moreover, echocardiography revealed that db/db mice exhibited normal cardiac systolic function and impaired diastolic function. Adverse structural remodeling, including cardiomyocyte apoptosis, myocardial fibrosis, and cardiac hypertrophy were observed in the hearts of db/db mice. Sixteen-week-old db/db mice were intramyocardially injected with adenovirus encoding FNDC5 or treated with recombinant human irisin via a peritoneal implant osmotic pump for 4 weeks. Both overexpression of myocardial FNDC5 and exogenous irisin administration attenuated diastolic dysfunction and cardiac structural remodeling in db/db mice. Results from in vitro studies revealed that FNDC5/irisin protein expression was decreased in high glucose (HG)/high fat (HF)-treated cardiomyocytes. Increased levels of inducible nitric oxide synthase (iNOS), NADPH oxidase 2 (NOX2), 3-nitrotyrosine (3-NT), reactive oxygen species (ROS), and peroxynitrite (ONOO-) in HG/HF-treated H9C2 cells provided evidence of oxidative/nitrosative stress, which was alleviated by treatment with FNDC5/irisin. Moreover, the mitochondria membrane potential (ΔΨm) was decreased and cytochrome C was released from mitochondria with increased levels of cleaved caspase-3 in HG/HF-treated H9C2 cells, indicating the presence of mitochondria-dependent apoptosis, which was partially reversed by FNDC5/irisin treatment. Mechanistic studies showed that activation of integrin αVß5-AKT signaling and attenuation of oxidative/nitrosative stress were responsible for the cardioprotective effects of FNDC5/irisin. Therefore, FNDC5/irisin mediates cardioprotection in DCM by inhibiting myocardial apoptosis, myocardial fibrosis, and cardiac hypertrophy. These findings implicate that FNDC5/irisin as a potential therapeutic intervention for DCM, especially in type 2 diabetes mellitus (T2DM).

An Integrin-αvß6/α5ß1-Bitargeted Probe for the SPECT Imaging of Pancreatic Adenocarcinoma in Preclinical and Primary Clinical Studies.

Pancreatic adenocarcinoma (PA) is one of the deadliest human malignancies. However, early detection, prediction of surgical resectability, and prognosis of PA are challenging with current conventional imaging technologies in the clinic. Molecular imaging technologies combined with novel imaging probes could be useful for early detection and accurate staging of PA. Integrin αvß6 and α5ß1 are found to be overexpressed in PA. In this study, integrin αvß6/α5ß1-bitargeted probes 99mTc-HYNIC-isoDGR (99mTc-isoDGR) and 99mTc-HYNIC-PEG4-PisoDGR2 (99mTc-3PisoDGR2) were prepared and evaluated in the BxPC-3 human pancreatic tumor model. Both subcutaneous and in situ BxPC-3 tumors could be clearly visualized by 99mTc-isoDGR nanoScan SPECT/CT imaging with a high ratio of tumor to background. The blocking study with excess nonradioactive peptide showed a significantly reduced tumor uptake, which confirmed the specificity of 99mTc-isoDGR. Biodistribution results confirmed the imaging results. The dimer tracer 99mTc-3PisoDGR2 significantly enhanced tumor uptake compared with 99mTc-isoDGR, and the spontaneous PA lesion in the mouse model could be clearly visualized by 99mTc-3PisoDGR2. The primary clinical study also verified the ability of 99mTc-3PisoDGR2 for detection of PA. Therefore, SPECT/CT imaging using the integrin αvß6/α5ß1-bitargeted 99mTc-3PisoDGR2 provided a potential approach for the noninvasive detection of PA.

Convallatoxin Promotes M2 Macrophage Polarization to Attenuate Atherosclerosis Through PPARγ-Integrin αvß5 Signaling Pathway.

INTRODUCTION: As the primary immune cells, macrophages play a key role in atherosclerotic progression. M2 macrophage polarization has been reported to promote tissue repair and attenuate plaque formation upon the expression of anti-inflammatory factors. Convallatoxin (CNT) is a natural cardiac glycoside with anti-inflammatory pharmacological properties. However, whether CNT protects against atherosclerosis (AS) and underlying mechanisms is unknown. This work was designed to explore the potential effects of CNT on atherosclerosis. METHODS: In this study, Apolipoprotein E deficiency (ApoE-/-) mice fed with high-fat diet were established, and CNT (50 or 100 µg/kg) were intragastrically administrated for 12 weeks every day. In vitro, RAW264.7 macrophages stimulated with ox-LDL were treated with CNT (50 or 100 nM) for 24 h. The specific PPARγ antagonist, GW9662, was used to block the PPARγ signaling pathway in vitro. Then, the atherosclerotic lesions, macrophage polarization markers, inflammatory cytokines and PPARγ signaling pathway were examined in further examinations. RESULTS: Our results showed that the atherosclerotic lesions were reduced by CNT, as demonstrated by the downregulation of serum lipid level and aortic plaque area in AS mice. Furthermore, we found that CNT treatment promoted the expression of M2 macrophage markers (Arg1, Mrc1, Retnla and Chi3l3), and decreased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), accompanied by the increase of anti-inflammatory factor (IL-10) in aortic vessels of AS mice. In ox-LDL-induced RAW264.7 cells, CNT administration also facilitated macrophages polarizing towards M2 subtype and inhibited inflammatory responses. Furthermore, both the in vivo and in vitro experiments showed CNT could increase the expression of PPARγ, Integrin αv and Integrin ß5, and GW9662 could block CNT-induced M2 macrophage polarization. CONCLUSION: Taken together, these data suggest that CNT may promote M2 macrophage polarization to exert an anti-atherosclerotic effect, partially through activating PPARγ-Integrin αvß5 signaling pathway.

Vitronectin-activated αvß3 and αvß5 integrin signalling specifies haematopoietic fate in human pluripotent stem cells.

OBJECTIVES: Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder-free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs. MATERIALS AND METHODS: A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN-mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells. RESULTS: We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial-haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvß3 and αvß5 integrins were required for VTN-promoted haematopoietic differentiation. Blocking αvß3 and αvß5 integrins by the integrin antagonists impaired the development of HE, but not endothelial-to-haematopoietic transition (EHT). Finally, both αvß3 and αvß5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, ß3 or ß5. CONCLUSION: The established VTN-based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development.

Vitronectin regulates the axon specification of mouse cerebellar granule cell precursors via αvß5 integrin in the differentiation stage.

Vitronectin, an extracellular matrix protein, controls the differentiation of cerebellar granule cell precursors (CGCPs) via αvß5 integrin, particularly in the initial stage of differentiation to granule cells. In this study, we determined whether vitronectin regulates axon specification in this initial differentiation stage of CGCPs. First, we analyzed whether vitronectin deficiency, ß5 integrin knockdown (KD), and ß5 integrin overexpression affect axon specification of primary cultured CGCPs. Vitronectin deficiency and ß5 integrin KD inhibited axon formation, while vitronectin administrated- and ß5 integrin overexpressed-neurons formed multiple axons. Moreover, KD of ß5 integrin suppressed vitronectin-induced multiple axon formation. These findings indicate that vitronectin contributes to regulating axon specification via αvß5 integrin in CGCPs. Next, we determined the signaling pathway involved in regulating vitronectin-induced axon specification. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited vitronectin-induced multiple axon specification, and lithium chloride, an inhibitor of glyocogen synthase kinase 3 beta (GSK3ß), attenuated the inhibitory effect of vitronectin-KO and ß5 integrin KD on the specification of CGCPs. In addition, vitronectin induced the phosphorylation of protein kinase B (Akt) and GSK3ß in neuroblastoma Neuro2a cells. Taken together, our results indicate that vitronectin plays an important factor in axon formation process in CGCPs via a ß5 integrin/PI3K/GSK3ß pathway.

Combination therapy of cold atmospheric plasma (CAP) with temozolomide in the treatment of U87MG glioblastoma cells.

Cold atmospheric plasma (CAP) technology, a relatively novel technique mainly investigated as a stand-alone cancer treatment method in vivo and in vitro, is being proposed for application in conjunction with chemotherapy. In this study, we explore whether CAP, an ionized gas produced in laboratory settings and that operates at near room temperature, can enhance Temozolomide (TMZ) cytotoxicity on a glioblastoma cell line (U87MG). Temozolomide is the first line of treatment for glioblastoma, one of the most aggressive brain tumors that remains incurable despite advancements with treatment modalities. The cellular response to a single CAP treatment followed by three treatments with TMZ was monitored with a cell viability assay. According to the cell viability results, CAP treatment successfully augmented the effect of a cytotoxic TMZ dose (50 µM) and further restored the effect of a non-cytotoxic TMZ dose (10 µM). Application of CAP in conjunction TMZ increased DNA damage measured by the phosphorylation of H2AX and induced G2/M cell cycle arrest. These findings were supported by additional data indicating reduced cell migration and increased αvß3 and αvß5 cell surface integrin expression as a result of combined CAP-TMZ treatment. The data presented in this study serve as evidence that CAP technology can be a suitable candidate for combination therapy with existing chemotherapeutic drugs. CAP can also be investigated in future studies for sensitizing glioblastoma cells to TMZ and other drugs available in the market.

Selective Targeting of αvß5 Integrin in HepG2 Cell Line by RGDechi15D Peptide.

Recently, the research community has become increasingly concerned with the receptor αvß5, a member of the well-known integrin family. Different ongoing studies have evidenced that αvß5 integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. Remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. In this scenario, the availability of a selective αvß5 antagonist without 'off-target' protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. We recently reported a cyclic peptide, RGDechi15D, obtained by structure-activity studies. To our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvß5 integrin and not cross react with αvß3. Here we demonstrated the ability of the peptide to diminish both adhesion and invasion of HepG2 cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the PI3K pathway. The peptide, also decreases the formation of new vessels in endothelial cells. Taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma.

Tenascin-C Induces Phenotypic Changes in Fibroblasts to Myofibroblasts with High Contractility through the Integrin αvß1/Transforming Growth Factor ß/SMAD Signaling Axis in Human Breast Cancer.

Tenascin-C (TNC) is strongly expressed by fibroblasts and cancer cells in breast cancer. To assess the effects of TNC on stromal formation, we examined phenotypic changes in human mammary fibroblasts treated with TNC. The addition of TNC significantly up-regulated α-smooth muscle actin (α-SMA) and calponin. TNC increased the number of α-SMA- and/or calponin-positive cells with well-developed stress fibers in immunofluorescence, which enhanced contractile ability in collagen gel contraction. The treatment with TNC also significantly up-regulated its own synthesis. Double immunofluorescence of human breast cancer tissues showed α-SMA- and/or calponin-positive myofibroblasts in the TNC-deposited stroma. Among several receptors for TNC, the protein levels of the αv and ß1 integrin subunits were significantly increased after the treatment. Immunofluorescence showed the augmented colocalization of αv and ß1 at focal adhesions. Immunoprecipitation using an anti-αv antibody revealed a significant increase in coprecipitated ß1 with TNC in lysates. The knockdown of αv and ß1 suppressed the up-regulation of α-SMA and calponin. The addition of TNC induced the phosphorylation of SMAD2/3, whereas SB-505124 and SIS3 blocked myofibroblast differentiation. Therefore, TNC enhances its own synthesis by forming a positive feedback loop and increases integrin αvß1 heterodimer levels to activate transforming growth factor-ß signaling, which is followed by a change to highly contractile myofibroblasts. TNC may essentially contribute to the stiffer stromal formation characteristic of breast cancer tissues.

GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis.

Mucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that Rhizopus delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin ß1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin ß1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands.IMPORTANCE Mucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin ß1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

MERTK-Dependent Ensheathment of Photoreceptor Outer Segments by Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Maintenance of a healthy photoreceptor-retinal pigment epithelium (RPE) interface is essential for vision. At the center of this interface, apical membrane protrusions stemming from the RPE ensheath photoreceptor outer segments (POS), and are possibly involved in the recycling of POS through phagocytosis. The molecules that regulate POS ensheathment and its relationship to phagocytosis remain to be deciphered. By means of ultrastructural analysis, we revealed that Mer receptor tyrosine kinase (MERTK) ligands, GAS6 and PROS1, rather than αVß5 integrin receptor ligands, triggered POS ensheathment by human embryonic stem cell (hESC)-derived RPE. Furthermore, we found that ensheathment is required for POS fragmentation before internalization. Consistently, POS ensheathment, fragmentation, and internalization were abolished in MERTK mutant RPE, and rescue of MERTK expression in retinitis pigmentosa (RP38) patient RPE counteracted these defects. Our results suggest that loss of ensheathment due to MERTK dysfunction might contribute to vision impairment in RP38 patients.