Genetic and epigenetic changes in the eutopic endometrium of women with endometriosis: association with decreased endometrial αvß3 integrin expression.

About 40% of women with infertility and 70% of women with pelvic pain suffer from endometriosis. The pregnancy rate in women undergoing IVF with low endometrial integrin αvß3 (LEI) expression is significantly lower compared to the women with high endometrial integrin αvß3 (HEI). Mid-secretory eutopic endometrial biopsies were obtained from healthy controls (C; n=3), and women with HEI (n=4) and LEI (n=4) and endometriosis. Changes in gene expression were assessed using human gene arrays and DNA methylation data were derived using 385 K Two-Array Promoter Arrays. Transcriptional analysis revealed that LEI and C groups clustered separately with 396 differentially expressed genes (DEGs) (P<0.01: 275 up and 121 down) demonstrating that transcriptional and epigenetic changes are distinct in the LEI eutopic endometrium compared to the C and HEI group. In contrast, HEI vs C and HEI vs LEI comparisons only identified 83 and 45 DEGs, respectively. The methylation promoter array identified 1304 differentially methylated regions in the LEI vs C comparison. The overlap of gene and methylation array data identified 14 epigenetically dysregulated genes and quantitative RT-PCR analysis validated the transcriptomic findings. The analysis also revealed that aryl hydrocarbon receptor (AHR) was hypomethylated and significantly overexpressed in LEI samples compared to C. Further analysis validated that AHR transcript and protein expression are significantly (P<0.05) increased in LEI women compared to C. The increase in AHR, together with the altered methylation status of the 14 additional genes, may provide a diagnostic tool to identify the subset of women who have endometriosis-associated infertility.

Imaging Fibrogenesis in a Diet-Induced Model of Nonalcoholic Steatohepatitis (NASH).

Purpose: Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin α v ß 3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin α v ß 3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures: Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin α v ß 3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results: The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin α v ß 3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions: The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin α v ß 3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin α v ß 3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.

Application of Triarylboron Substituted with Cyclic Arginine-Glycine-Aspartic Acid Motifs as a Multivalent Two-Photon Fluorescent Probe for Tumor Imaging in Vivo.

Detection of cancer in its early stages is difficult, and this is a major issue that impairs the timely diagnosis and treatment of tumors. Integrin αVß3 is expressed on tumoral endothelial cells, as well as other tumor cells. By functionalizing the triarylboron (TAB) compound with multiple cyclic arginine-glycine-aspartic acid (cRGD) motifs, which specifically bind to integrin αVß3, a multivalent two-photon fluorescent probe TAB-3-cRGD was designed and chemically synthesized. Through cell imaging experiments, we showed that TAB-3-cRGD can selectively bind to integrin αVß3 on the cell surface and can effectively distinguish normal cells from tumor cells overexpressing integrin αVß3. Using a mouse model, we also showed that TAB-3-cRGD could target the tumor site in vivo, offering a promising tool for cancer detection.

Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic-to-pulmonary shunt.

Recent studies indicated that osteopontin (OPN) was involved in the genesis and progression of pulmonary arterial hypertension (PAH); however, its role in congenital heart disease-associated PAH (CHD/PAH) remains unknown. Our results showed that OPN was increased in lungs and plasma of patients with Eisenmenger syndrome; moreover, OPN and αVß3-integrin expression levels were augmented in rat lungs exposed to systemic-to-pulmonary shunt. Cell culture assay demonstrated that distal pulmonary arterial smooth muscle cells (PASMCs) from rat lungs suffering from volume and pressure overload exhibited enhanced proliferation compared with those from healthy rats. Mechanical stretch (20% at 1 Hz) increased OPN expression and activated ERK1/2 and protein kinase B (Akt) signal pathway in distal PASMCs from healthy rats. Interestingly, OPN enhanced the proliferation and migration of PASMCs while blocking αVß3-integrin with neutralizing antibody LM609 or Arg-Gly-Asp peptidomimetic antagonist cyclo(Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (XJ735), rectified the proliferative and migratory effects of OPN, which were partially mediated via ERK1/2 and Akt signaling pathways. Furthermore, surgical correction of systemic-to-pulmonary shunt, particularly XJ735 supplementation after surgical correction of systemic-to-pulmonary shunt, significantly alleviated the pulmonary hypertensive status in terms of pulmonary hemodynamic indices, pulmonary vasculopathy, and right ventricular hypertrophy. In summary, OPN alteration in lungs exposed to systemic-to-pulmonary shunt exerts a deteriorative role in pulmonary vascular remodeling through modulating the proliferation and migration of PASMCs, at least in part, via ανß3-ERK1/2 and ανß3-Akt signaling pathways. Antagonizing OPN receptor ανß3-integrin accelerated the regression of pulmonary vasculopathy after surgical correction of systemic-to-pulmonary shunt, indicating a potential therapeutic strategy for patients with CHD/PAH.-Meng, L., Liu, X., Teng, X., Gu, H., Yuan, W., Meng, J., Li, J., Zheng, Z., Wei, Y., Hu, S. Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic-to-pulmonary shunt.

αvß3 Integrin Mediates Radioresistance of Prostate Cancer Cells through Regulation of Survivin.

The αvß3 integrin is involved in various physiologic and pathologic processes such as wound healing, angiogenesis, tumor growth, and metastasis. The impact of αvß3 integrin on the radiosensitivity of prostate cancer cells and the molecular mechanism controlling cell survival in response to ionizing radiation (IR) was investigated. Both LNCaP cells stably transfected with αvß3 integrin and PC-3 cells that contain endogenous ß3 integrin were used. This study demonstrated that αvß3 integrin increases survival of αvß3-LNCaP cells upon IR while small hairpin RNA (shRNA)-mediated knockdown of αvß3 integrin in PC-3 cells sensitizes to radiation. Expression of αvß3 integrin in LNCaP cells also enhances anchorage-independent cell growth while knockdown of αvß3 integrin in PC-3 cells inhibits anchorage-independent cell growth. The αvß3 antagonist, cRGD, significantly increases radiosensitivity in both αvß3-LNCaP and PC-3 cells. Moreover, αvß3 integrin prevents radiation-induced downregulation of survivin. Inhibition of survivin expression by siRNA or shRNA enhances IR-induced inhibition of anchorage-independent cell growth. Overexpression of wild-type survivin in PC-3 cells treated with αvß3 integrin shRNA increases survival of cells upon IR. These findings reveal that αvß3 integrin promotes radioresistance and regulates survivin levels in response to IR. IMPLICATIONS: Future translational research on targeting αvß3 integrin and survivin may reveal novel approaches as an adjunct to radiotherapy for patients with prostate cancer.

Biodistribution and Internal Radiation Dosimetry of 99mTc-IDA-D-[c(RGDfK)]2 (BIK-505), a Novel SPECT Radiotracer for the Imaging of Integrin αvß3 Expression.

BACKGROUND: Integrin αvß3 is a molecular marker for the estimation of tumor angiogenesis. 99mTc-IDA-D-[c(RGDfK)]2 (also known as BIK-505) is a recently developed radiotracer for single-photon emission computed tomography, with good affinity for integrin αvß3. In this study, the authors investigated the whole-body distribution and internal radiation dosimetry of 99mTc-IDA-D-[c(RGDfK)]2 in elderly human participants. MATERIALS AND METHODS: Six healthy volunteers underwent whole-body simultaneous anterior and posterior scans, preceded by transmission scans using cobalt-57 flood source, with a dual head gamma camera system, at 0, 1, 2, 4, 8, and 24 h postinjection of 99mTc-IDA-D-[c(RGDfK)]2 (injected radioactivity [mean ± SD] = 388.7 ± 29.3 MBq). Anterior and posterior images were geometrically averaged and attenuation corrected to delineate the regions of interest in the liver, gallbladder, kidneys, urinary bladder, spleen, brain, and large intestine. Radiation dose for each organ and the effective doses (EDs) were estimated using OLINDA/EXM 1.1 software. RESULTS: High radiation doses of renal and biliary excretion tracks such as the urinary bladder wall, upper large intestine, kidneys, liver, and gallbladder wall (19.15 ± 6.84, 19.28 ± 4.78, 15.67 ± 0.90, 9.13 ± 1.71, and 9.09 ± 2.03 µGy/MBq, respectively) were observed. The ED and effective dose equivalent were 5.08 ± 0.53 and 7.11 ± 0.58 µSv/MBq, respectively. CONCLUSIONS: Dosimetry results were comparable to other radiolabeled peptides and were considered safe and efficient for clinical usage.

A novel αVß3 ligand-modified HPMA copolymers for anticancer drug delivery.

The integrin αVß3 receptor emerged as one of the most promising targets owing to its high expression on the surface of various malignant tumour cells and tumour angiogenesis endothelial cells, but with little expression in mature endothelial cells and the majority of normal cells. Here, we report a new targeting ligand FQSIYPpIK (FQS) with high affinity to integrin αVß3 receptor. To take the advantage of the particular interaction between FQS and integrin αVß3 receptor, FQS was linked to N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. A model drug doxorubicin (DOX) was simultaneously conjugated to the same HPMA copolymers via pH-sensitive hydrazone linkages (FQS-HPMA-DOX). In in vitro study, FQS-HPMA-DOX could be internalised into αVß3 receptor-overexpressed B16F10 cells via a highly specific ligand - receptor pathway (5.0 times and 4.5 times higher cellular internalisation than HPMA-DOX and a scrambled peptide (s)-FQS (sequence: SYFIPKQIp)-modified copolymers ((s)-FQS-HPMA-DOX)). It is worth noting that compared with the classical αVß3 ligand cRGDfK-modified HPMA copolymers (cRGDfK-HPMA-DOX), FQS-HPMA-DOX also showed superior targeting efficiency. In in vivo study in the B16F10 melanoma bearing mice model showed the antitumour efficiency of FQS-HPMA-DOX (83.9%) were significantly higher than HPMA-DOX (44.9%) and cRGDfK-HPMA-DOX (77.5%). These results suggest that FQS peptide can act as an effective targeting ligand for the delivery of therapeutic agents.

Triazole RGD antagonist reverts TGFß1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor ß1 (TGFß1) play a central role. When exposed to TGFß1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFß1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvß3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFß1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFß1, and reduces both ALK5/TGFß1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Tat PTD-Endostatin-RGD: A novel protein with anti-angiogenesis effect in retina via eye drops.

BACKGROUND: Diabetic retinopathy is a leading cause of blindness. The objective was to design a novel fusion protein, Tat PTD-Endostatin-RGD, to treat retinal neovascularization via eye drops instead of traditional intravitreal injection trepapeutical methods. METHOD: The anti-angiogenesis ability was evaluated in vitro by chick embryo chorioallantoic membrane assay, wound healing assay and tube formation assay. Corneal barrier and blood-retina barrier were constructed in vitro to investigate the penetration ability of Tat PTD-Endostatin-RGD. Western blot was used to detect the integrin αvß3 expression level in rat retina microvascular endothelial cells which was stimulated by S-nitroso-N-acetylpenicillamine. The binding affinity of Tat PTD-Endostatin-RGD to integrin αvß3 was investigated by evaluating the penetration ability on blood-retina barriers treated with S-nitroso-N-acetylpenicillamine. The pharmacodynamics and efficacy analysis were further carried out in the oxygen-induced retinopathy model in vivo. In addition, the pharmacokinetic profile via eye drops was studied on a C57BL/6 mice model. RESULT: Tat PTD-Endostatin-RGD showed high anti-angiogenesis activity and high ability to penetrate these two barriers in vitro. The Western blot results indicated S-nitroso-N-acetylpenicillamine upregulated the expression level of integrin αvß3 in a dose-dependent manner. Tat PTD-Endostatin-RGD showed a high affinity to rat retina microvascular endothelial cells treated with S-nitroso-N-acetylpenicillamine. The results showed that Tat PTD-Endostatin-RGD could inhibit abnormal angiogenesis in retina via eye drops. CONCLUSION: Tat PTD-Endostatin-RGD showed high penetration ability through ocular barriers, bound specifically to integrin αvß3 and effectively inhibited the abnormal angiogenesis. GENERAL SIGNIFICANCE: Tat PTD-Endostatin-RGD represents a potent novel drug applied via eye drops for fundus oculi neovascularization diseases.

The disintegrin tzabcanin inhibits adhesion and migration in melanoma and lung cancer cells.

Integrins play an essential role in cancer survival and invasion, and they have been major targets in drug development and design. Disintegrins are small (4-16kDa) viperid snake venom proteins that exhibit a canonical integrin-binding site (often RGD). These non-enzymatic proteins inhibit integrin-mediated cell-cell and cell-extracellular matrix interactions, making them potential candidates as therapeutics in cancer and numerous other human disorders. The present study examined the cytotoxic, anti-adhesion, and anti-migration effects of a recently characterized disintegrin, tzabcanin, towards melanoma (A-375) and lung (A-549) cancer cell lines. Tzabcanin inhibits adhesion of both cells lines to vitronectin and exhibited very weak cytotoxicity towards A-375 cells; however, it had no effect on cell viability of A-549 cells. Further, tzabcanin significantly inhibited migration of both cell lines in cell scratch/wound healing assays. Flow cytometric analysis indicates that both A-375 and A-549 cell lines express integrin αvß3, a critical integrin in tumor motility and invasion, and a major receptor of the extracellular matrix protein vitronectin. Flow cytometric analysis also identified αvß3 as a binding site of tzabcanin. These results suggest that tzabcanin may have utility in the development of anticancer therapies, or may be used as a biomarker to detect neoplasms that over-express integrin αvß3.

Integrin αvß3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418).

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.

PKCδ regulates integrin αVß3 expression and transformed growth of K-ras dependent lung cancer cells.

We have previously shown that Protein Kinase C delta (PKCδ) functions as a tumor promoter in non-small cell lung cancer (NSCLC), specifically in the context of K-ras addiction. Here we define a novel PKCδ -> integrin αVß3 ->Extracellular signal-Regulated Kinase (ERK) pathway that regulates the transformed growth of K-ras dependent NSCLC cells. To explore how PKCδ regulates tumorigenesis, we performed mRNA expression analysis in four KRAS mutant NSCLC cell lines that stably express scrambled shRNA or a PKCδ targeted shRNA. Analysis of PKCδ-dependent mRNA expression identified 3183 regulated genes, 210 of which were specifically regulated in K-ras dependent cells. Genes that regulate extracellular matrix and focal adhesion pathways were most highly represented in this later group. In particular, expression of the integrin pair, αVß3, was specifically reduced in K-ras dependent cells with depletion of PKCδ, and correlated with reduced ERK activation and reduced transformed growth as assayed by clonogenic survival. Re-expression of PKCδ restored ITGAV and ITGB3 mRNA expression, ERK activation and transformed growth, and this could be blocked by pretreatment with a αVß3 function-blocking antibody, demonstrating a requirement for integrin αVß3 downstream of PKCδ. Similarly, expression of integrin αV restored ERK activation and transformed growth in PKCδ depleted cells, and this could also be inhibited by pretreatment with PD98059.Our studies demonstrate an essential role for αVß3 and ERK signalingdownstream of PKCδ in regulating the survival of K-ras dependent NSCLC cells, and identify PKCδ as a novel therapeutic target for the subset of NSCLC patients with K-ras dependent tumors.

ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells.

Cancer cells contain a small population of cancer stem cells or cancer initiating cells, which can be enriched in the side population (SP) after fluorescence activated cell sorting. To examine the members of the ADAM, ADAMTS and MMP gene families related to phenotypes of the SP and the main population (MP), we screened the expression of all the members in the propagated SP and MP of A549 lung adenocarcinoma cells, and found that the relative expression ratio of ADAM23 in the MP to the SP is most highly increased, but none of them are increased in the SP. A similar result on the ADAM23 expression was obtained with another cell line, Calu-3 cells. Overexpression of ADAM23 inhibited colony formation, cell adhesion and migration, and knockdown of ADAM23 by shRNA showed the reverse effects. ADAM23-mediated suppression of colony formation, cell adhesion and migration was greatly reduced by treatment with neutralizing anti-ADAM23 antibody, anti-αvß3 integrin antibody and/or ADAM23 disintegrin peptide. Expression of cancer stem cell-related genes, including AKRC1/2, TM4SF1 and NR0B1, was increased by knockdown of ADAM23. In addition, lung metastasis of A549 transfectants with different levels of ADAM23 expression was negatively regulated by the ADAM23 expression levels. Our data provide evidence that ADAM23 plays a role in suppression of cancer cell progression through interaction with αvß3 integrin, and suggest that downregulation of ADAM23 in SP cells may contribute toward providing a cancer stem cell phenotype by facilitating the activity of integrin αvß3.

PET-Based Human Dosimetry of the Dimeric αvß3 Integrin Ligand 68Ga-DOTA-E-[c(RGDfK)]2, a Potential Tracer for Imaging Tumor Angiogenesis.

UNLABELLED: Peptides containing the Arg-Gly-Asp (RGD) sequence have high affinity for αvß3 integrin receptors overexpressed in tumor cells. The objective of this research was to determine the biodistribution and estimate the radiation dose from (68)Ga-DOTA-E-[c(RGDfK)]2 using whole-body PET scans in humans. METHODS: Five healthy volunteers (2 women, 3 men; mean age ± SD, 37.2 ± 15.6 y; range, 28-65 y; mean weight, 79.2 ± 21.0 kg; range, 64-115 kg) were included. After intravenous injection of the tracer (198.3 ± 3.3 MBq), 3 successive whole-body (vertex to mid thigh) PET/CT scans at 3 time points (30, 60, and 120 min) were obtained on a 16-slice PET/CT scanner. The subjects did not void the bladder until the entire series of images was completed. Low-dose CT without contrast agent was used for anatomic localization and attenuation correction. OLINDA/EXM software was applied to calculate human radiation doses using the reference adult model. RESULTS: The highest uptake was in the urinary bladder, followed by the liver, kidneys, and spleen, in descending order. The critical organ was the urinary bladder wall. The mean effective doses (all subjects, men and women) were 34.1 ± 4.9, 31.0 ± 2.4, and 20.9 ± 5.2 µSv/MBq for the no-voiding, 2.5-h-voiding, and 1-h-voiding models, respectively. CONCLUSION: Of particular interest in this research was the visualization of the choroid plexus and ventricular system, which seems to be a characteristic of RGD-dimeric peptides. Measured absorbed doses and effective doses are comparable to other previously reported RGD-based radiopharmaceuticals labeled with (68)Ga and (18)F. Therefore, (68)Ga-DOTA-E-[c(RGDfK)]2 can safely be used for imaging integrin αVß3 expression.

Complementary, Selective PET Imaging of Integrin Subtypes α5ß1 and αvß3 Using 68Ga-Aquibeprin and 68Ga-Avebetrin.

UNLABELLED: Despite in vivo mapping of integrin αvß3 expression being thoroughly investigated in recent years, its clinical value is still not well defined. For imaging of angiogenesis, the integrin subtype α5ß1 appears to be a promising target, for which purpose we designed the PET radiopharmaceutical (68)Ga-aquibeprin. METHODS: (68)Ga-aquibeprin was obtained by click-chemistry (CuAAC) trimerization of a α5ß1 integrin-binding pseudopeptide on the triazacyclononane-triphosphinate (TRAP) chelator, followed by automated (68)Ga labeling. Integrin α5ß1 and αvß3 affinities were determined in enzyme linked immune sorbent assay on immobilized integrins, using fibronectin and vitronectin, respectively, as competitors. M21 (human melanoma)-bearing severe combined immunodeficient mice were used for biodistribution, PET imaging, and determination of in vivo metabolization. The expression of α5 and ß3 subunits was determined by immunohistochemistry on paraffin sections of M21 tumors. RESULTS: (68)Ga-aquibeprin shows high selectivity for integrin α5ß1 (50% inhibition concentration [IC50] = 0.088 nM) over αvß3 (IC50 = 620 nM) and a pronounced hydrophilicity (log D = -4.2). Severe combined immunodeficient mice xenografted with M21 human melanoma were found suitable for in vivo evaluation, as M21 immunohistochemistry showed not only an endothelial and strong cytoplasmatic expression of the ß3 integrin subunit but also an intense expression of the α5 integrin subunit particularly in the endothelial cells of intratumoral small vessels. Ex vivo biodistribution (90 min after injection) showed high uptake in M21 tumor (2.42 ± 0.21 percentage injected dose per gram), fast renal excretion, and low background; tumor-to-blood and tumor-to-muscle ratios were 10.6 ± 2.5 and 20.9 ± 2.4, respectively. (68)Ga-aquibeprin is stable in vivo; no metabolites were detected in mouse urine, blood serum, kidney, and liver homogenates 30 min after injection. PET imaging was performed for (68)Ga-aquibeprin and the previously described, structurally related c(RGDfK) trimer (68)Ga-avebetrin, which shows an inverse selectivity for integrin αvß3 (IC50 = 0.22 nM) over α5ß1 (IC50 = 39 nM). In vivo target specificity was proven by cross-competition studies; tumor uptake of either tracer was not affected by the coadministration of 40 nmol (∼5 mg/kg) of the respective other compound. CONCLUSION: (68)Ga-aquibeprin and (68)Ga-avebetrin are recommendable for complementary mapping of integrins α5ß1 and αvß3 by PET, allowing for future studies on the role of these integrins in angiogenesis, tumor progression, metastasis, and myocardial infarct healing.

The thyroid hormone-αvß3 integrin axis in ovarian cancer: regulation of gene transcription and MAPK-dependent proliferation.

Ovarian carcinoma is the fifth common cause of cancer death in women, despite advanced therapeutic approaches. αvß3 integrin, a plasma membrane receptor, binds thyroid hormones (L-thyroxine, T4; 3,5,3'-triiodo-L-thyronine, T3) and is overexpressed in ovarian cancer. We have demonstrated selective binding of fluorescently labeled hormones to αvß3-positive ovarian cancer cells but not to integrin-negative cells. Physiologically relevant T3 (1 nM) and T4 (100 nM) concentrations in OVCAR-3 (high αvß3) and A2780 (low αvß3) cells promoted αv and ß3 transcription in association with basal integrin levels. This transcription was effectively blocked by RGD (Arg-Gly-Asp) peptide and neutralizing αvß3 antibodies, excluding T3-induced ß3 messenger RNA, suggesting subspecialization of T3 and T4 binding to the integrin receptor pocket. We have provided support for extracellular regulated kinase (ERK)-mediated transcriptional regulation of the αv monomer by T3 and of ß3 monomer by both hormones and documented a rapid (30-120 min) and dose-dependent (0.1-1000 nM) ERK activation. OVCAR-3 cells and αvß3-deficient HEK293 cells treated with αvß3 blockers confirmed the requirement for an intact thyroid hormone-integrin interaction in ERK activation. In addition, novel data indicated that T4, but not T3, controls integrin's outside-in signaling by phosphorylating tyrosine 759 in the ß3 subunit. Both hormones induced cell proliferation (cell counts), survival (Annexin-PI), viability (WST-1) and significantly reduced the expression of genes that inhibit cell cycle (p21, p16), promote mitochondrial apoptosis (Nix, PUMA) and tumor suppression (GDF-15, IGFBP-6), particularly in cells with high integrin expression. At last, we have confirmed that hypothyroid environment attenuated ovarian cancer growth using a novel experimental platform that exploited paired euthyroid and severe hypothyroid serum samples from human subjects. To conclude, our data define a critical role for thyroid hormones as potent αvß3-ligands, driving ovarian cancer cell proliferation and suggest that disruption of this axis may present a novel treatment strategy in this aggressive disease.

Tenascin-C may accelerate cardiac fibrosis by activating macrophages via the integrin αVß3/nuclear factor-κB/interleukin-6 axis.

Tenascin-C (TN-C) is an extracellular matrix protein not detected in normal adult heart, but expressed in several heart diseases closely associated with inflammation. Accumulating data suggest that TN-C may play a significant role in progression of ventricular remodeling. In this study, we aimed to elucidate the role of TN-C in hypertensive cardiac fibrosis and underlying molecular mechanisms. Angiotensin II was administered to wild-type and TN-C knockout mice for 4 weeks. In wild-type mice, the treatment induced increase of collagen fibers and accumulation of macrophages in perivascular areas associated with deposition of TN-C and upregulated the expression levels of interleukin-6 and monocyte chemoattractant protein-1 as compared with wild-type/control mice. These changes were significantly reduced in TN-C knockout/angiotensin II mice. In vitro, TN-C accelerated macrophage migration and induced accumulation of integrin αVß3 in focal adhesions, with phosphorylation of focal adhesion kinase (FAK) and Src. TN-C treatment also induced nuclear translocation of phospho-NF-κB and upregulated interleukin-6 expression of macrophages in an NF-κB-dependent manner; this being suppressed by inhibitors for integrin αVß3 and Src. Furthermore, interleukin-6 upregulated expression of collagen I by cardiac fibroblasts. TN-C may enhance inflammatory responses by accelerating macrophage migration and synthesis of proinflammatory/profibrotic cytokines via integrin αVß3/FAK-Src/NF-κB, resulting in increased fibrosis.

Inhibition of osteoclast activation by phloretin through disturbing αvß3 integrin-c-Src pathway.

This study was to explore the sequential signaling of disorganization of the actin cytoskeletal architecture by phloretin. RAW 264.7 macrophages were incubated with 1-20 µM phloretin for 5 days in the presence of RANKL. C57BL/6 mice were ovariectomized (OVX) and orally treated with 10 mg/kg phloretin once a day for 8 weeks. Phloretin allayed RANKL stimulated formation of actin podosomes with the concomitant retardation of the vinculin activation. Oral administration of phloretin suppressed the induction of femoral gelsolin and vinculin in OVX mice. The RANK-RANKL interaction resulted in the αvß3 integrin induction, which was demoted by phloretin. The RANKL induction of actin rings and vacuolar-type H(+)-ATPase entailed Pyk2 phosphorylation and c-Src and c-Cbl induction, all of which were blunted by phloretin. Similar inhibition was also observed in phloretin-exposed OVX mouse femoral bone tissues with decreased trabecular collagen formation. Phloretin suppressed the paxillin induction in RANKL-activated osteoclasts and in OVX epiphyseal bone tissues. Also, phloretin attenuated the Syk phosphorylation and phospholipase Cγ induction by RANKL in osteoclasts. These results suggest that phloretin was an inhibitor of actin podosomes and sealing zone, disrupting αvß3 integrin-c-Src-Pyk2/Syk signaling pathway for the regulation of actin cytoskeletal organization in osteoclasts.

Integrins αvß3 and αvß5 as prognostic, diagnostic, and therapeutic targets in gastric cancer.

BACKGROUND: We investigated the expression of two αv integrins, αvß3 and αvß5, in gastric cancer (GC) by testing the following hypotheses: that these molecules are expressed in GC; that they are implicated in GC biology; that they help to distinguish between the two major histological subtypes of GC, according to Laurén; and that they are prognostically relevant. METHODS: Formalin-fixed and paraffin-embedded tissue samples from 482 GC samples were stained immunohistochemically using rabbit monoclonal antibodies directed against αvß3 (EM22703) and αvß5 (EM09902). Immunostaining of tumor, stroma, and endothelial cells was evaluated separately by the quantity and intensity, generating an immunoreactivity score. The immunoreactivity score of both antibodies was correlated with clinicopathology data and patient survival. RESULTS: Each integrin was expressed in at least one tumor component in all GCs. Both were expressed significantly more often in the intestinal phenotype according to Laurén. Moreover, patients who grouped as "positive" for expression of αvß3 on endothelial cells, and patients with an intestinal type GC, grouped as "negative" for expression of αvß5 on stroma cells, had significantly longer survival. The expression of αvß5 on stroma cells was confirmed to be an independent prognostic factor of intestinal-type GC. CONCLUSION: The expression of αvß3 and αvß5 in at least one tumor component in all GC samples is an interesting new result that should form a basis for further investigations; for example, regarding selective integrin antagonists and the value of αvß3 and αvß5 as putative prognostic biomarkers. Moreover, both markers might be helpful in the routine classification of GC subtypes.

(99m)Tc-3P-RGD2 micro-single-photon emission computed tomography/computed tomography provides a rational basis for integrin αvß3-targeted therapy.

PURPOSE: This study was to demonstrate the utility of (99m)Tc-3P-RGD2 micro-single-photon emission computed tomography/computed tomography (SPECT/CT) for the integrin αvß3 expression quantification in NCI-H446 and A549 lung cancer xenografts. MATERIALS AND METHODS: (99m)Tc-3P-RGD2 was prepared with high radiochemical purity (97%±2%) and showing high in vitro stability. The in vitro affinities of (99m)Tc-3P-RGD2 to NCI-H446 and A549 tumor cells were analyzed with γ-counter, while the in vivo uptakes in NCI-H446 and A549 xenografts were evaluated with micro-SPECT/CT. The region of interest was drawn over the tumor site and contralateral muscle on the SPECT/CT image, and the tumor to nontumor (T/NT) ratio was calculated to estimate αvß3 expression and tumor uptake. The expressions of integrin αvß3 in vitro and in vivo were analyzed using a flow cytometer and immunofluorescence. RESULTS: Micro-SPECT/CT demonstrated focal uptake in the tumors. T/NT ratio in NCI-H446 xenografts was significantly higher compared with the A549 tumor model, as 5.92±0.82 and 3.62±0.91, respectively, with p<0.05. In addition, integrin αvß3 expression in NCI-H446 cells was significantly higher compared with the A549 cells, which was consistent with the imaging data. A linear relationship was observed between (99m)Tc-3P-RGD2 uptake and αvß3 expression (R(2)=0.7667, p<0.001). CONCLUSION: (99m)Tc-3P-RGD2 SPECT/CT could be used to quantify integrin αvß3 expression within tumors, providing a rational basis for integrin αvß3-targeted cancer therapy.