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.

Design and Discovery of a Potent and Selective Inhibitor of Integrin αvß1.

Selective inhibition of the RGD (Arg-Gly-Asp) integrin αvß1 has been recently identified as an attractive therapeutic approach for the treatment of liver fibrosis given its function, target expression, and safety profile. Our identification of a non-RGD small molecule lead followed by focused, systematic changes to the core structure utilizing a crystal structure, in silico modeling, and a tractable synthetic approach resulted in the identification of a potent small molecule exhibiting a remarkable affinity for αvß1 relative to several other integrin isoforms measured. Azabenzimidazolone 25 demonstrated antifibrotic efficacy in an in vivo rat liver fibrosis model and represents a tool compound capable of further exploring the biological consequences of selective αvß1 inhibition.

Integrin αvß1 facilitates ACE2-mediated entry of SARS-CoV-2.

Integrins have been suggested to be involved in SARS-CoV-2 infection, but the underlying mechanisms remain largely unclear. This study aimed to investigate how integrins facilitate the ACE2-mediated cellular entry of SARS-CoV-2. We first tested the susceptibility of a panel of human cell lines to SARS-CoV-2 infection using the spike protein pseudotyped virus assay and examined the expression levels of integrins in these cell lines by qPCR, western blot and flow cytometry. We found that integrin αvß1 was highly enriched in the SARS-CoV-2 susceptible cell lines. Additional studies demonstrated that RGD (403-405)→AAA mutant was defective in binding to integrin αvß1 compared to its wild type counterpart, and anti-αvß1 integrin antibodies significantly inhibited the entry of SARS-CoV-2 into the cells. Further studies using mouse NIH3T3 cells expressing human ACE2, integrin αv, integrin ß1, and/or integrin αvß1 suggest that integrin αvß1 was unable to function as an independent receptor but could significantly facilitate the cellular entry of SASR-CoV-2. Finally, we observed that the Omicron exhibited a significant increase in the ACE2-mediated viral entry. Our findings may enhance our understanding of the pathogenesis of SARS-CoV-2 infection and offer potential therapeutic target for COVID-19.

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.

Role of Vitronectin and Its Receptors in Neuronal Function and Neurodegenerative Diseases.

Vitronectin (VTN), a multifunctional glycoprotein with various physiological functions, exists in plasma and the extracellular matrix. It is known to be involved in the cell attachment, spreading and migration through binding to the integrin receptor, mainly via the RGD sequence. VTN is also widely used in the maintenance and expansion of pluripotent stem cells, but its effects go beyond that. Recent evidence shows more functions of VTN in the nervous system as it participates in neural differentiation, neuronutrition and neurogenesis, as well as in regulating axon size, supporting and guiding neurite extension. Furthermore, VTN was proved to play a key role in protecting the brain as it can reduce the permeability of the blood-brain barrier by interacting with integrin receptors in vascular endothelial cells. Moreover, evidence suggests that VTN is associated with neurodegenerative diseases, such as Alzheimer's disease, but its function has not been fully understood. This review summarizes the functions of VTN and its receptors in neurons and describes the role of VTN in the blood-brain barrier and neurodegenerative diseases.

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.

Molecular determinants of αVß5 localization in flat clathrin lattices - role of αVß5 in cell adhesion and proliferation.

The vitronectin receptor integrin αVß5 can reside in two distinct adhesion structures - focal adhesions (FAs) and flat clathrin lattices (FCLs). Here, we investigate the mechanism that regulates the subcellular distribution of ß5 in keratinocytes and show that ß5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH (also known as LDLRAP1) and Numb, respectively, than for the talin 1 (TLN1); all proteins that bind to the membrane-proximal NPxY motif of the ß5 cytoplasmic domain. Using mass spectrometry, we identified ß5 interactors, including the Rho GEFs p115Rho-GEF and GEF-H1 (also known as ARHGEF1 and ARHGEF2, respectively), and the serine protein kinase MARK2, depletion of which diminishes the clustering of ß5 in FCLs. Replacement of two serine residues (S759 and S762) in the ß5 cytoplasmic domain with phospho-mimetic glutamate residues causes a shift in the localization of ß5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates ß5 localization. Finally, we present evidence that ß5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.

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).

Dual integrin αvß3 and αvß5 blockade attenuates cardiac dysfunction by reducing fibrosis in a rat model of doxorubicin-induced cardiomyopathy.

OBJECTIVE: The present study aimed to evaluate the protective role of cilengitide (CGT), an integrin αvß3 and αvß5 inhibitor, on doxorubicin (DOX)-induced myocardial fibrosis and cardiac dysfunction in a rat model. Methods. Forty male rats were randomly divided into four groups: DOX (n = 12), intraperitoneal (i.p.) injection of DOX 0.8 ∼ 1.0 mg/kg three times a week for up to 6 weeks, then saline i.p. three times a week for another 3 weeks; CGT (n = 8), CGT 10 mg/kg, i.p. three times a week for 9 weeks; DOX + CGT (n = 12), DOX and CGT co-administration as above for 6 weeks, then CGT alone for another 3 weeks; Control (n = 8), saline i.p. three times a week for 9 weeks. Echocardiography, serum procollagen I C-terminal propeptide (PICP) procollagen III N-terminal propeptide (PIIINP) and C telopeptide type I (CTX-I) were evaluated at baseline and 3, 6 and 9 weeks after initial DOX administration for all surviving rats. The heart tissues were then harvested for myocardial hydroxyproline (HYP) evaluation, qRT-PCR, and western blotting. Results. CGT attenuated DOX-induced eccentric remodeling by improving relative wall thickness at the 9th week. CGT also improved systolic function at the 9th week and diastolic function at the 6th and 9th week. CGT reduced myocardial HYP and serum PICP, PIIINP, CTX-I, and the PICP/PIIINP ratio. RT-PCR and western blot showed that CGT blocked the TGF-ß1/SMAD3 pathway and mitigating extracellular matrix turnover. Conclusions. CGT exerted a cardioprotective effect against doxorubicin-induced fibrosis and improved cardiac function.

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.

Myofibroblast TGF-ß Activation Measurement In Vitro.

Myofibroblasts are critical to processes involved in normal wound healing and during pathological fibrosis. They transdifferentiate from fibroblasts, and in doing so become contractile and capable of secreting large amounts of extracellular matrix proteins. Transforming growth factor-beta (TGFß) is a key cytokine involved in wound healing and fibrogenesis. TGFß signaling has long been the subject of experimental therapeutic approaches to inhibit fibrosis in a variety of organ systems. Inhibition of TGFß can reduce myofibroblast transdifferentiation, contractility, and matrix production. Importantly, TGFß is released from cells and sequestered in the extracellular matrix in a latent form that requires activation for biological function. There have been multiple mechanisms of TGFß activation described in a variety of cell types and in cell free systems; however, myofibroblasts have previously been shown to activate TGFß via cell surface integrins, particularly αvß5 integrins. This chapter will provide detailed protocols for accurately measuring activation of TGFß by myofibroblasts in vitro. Levels of active TGFß usually represent a small proportion of the total amount of latent TGFß present in the matrix. Methods to measure active TGFß therefore need to be sensitive and specific to detect the active cytokine only.

Autoregulation of insulin receptor signaling through MFGE8 and the αvß5 integrin.

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvß5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/ß5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvß5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/ß5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvß5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.