Alessandro Ciucci

Increased expression and activity of matrix metalloproteinases characterize embolic cardiac myxomas

Tumor embolism occurs in 30 to 50% of all cases of cardiac myxoma, but the causes are still uncertain. Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix (ECM) and play a crucial role in plaque instability and aortic aneurysm development, in addition to cancer and heart failure. To determine whether MMP activity contributes to tumor embolism, we examined 27 left atrium-sided myxomas, 10 of which showed clinical signs of peripheral embolism. Immunohistochemistry (in all cases) and Western blotting, and in situ and in-gel zymography (in four embolic and six nonembolic consecutive tumors) demonstrated higher expression and activity of MT1-MMP, pro-MMP-2, and pro-MMP-9 in embolic myxomas, whereas pro-MMP-1, MMP-3, and TIMP-1 levels were similar to those of nonembolic tumors. Reverse transcriptase-polymerase chain reaction demonstrated that increased MMP activity was due, at least in part, to increased transcription and that TIMP-2 transcripts increased in embolic myxomas. In vitro, embolic tumor cells retained higher MT1-MMP and pro-MMP-2 levels in basal conditions and after stimulation with interleukin-1beta and interleukin-6. Increased MMP synthesis and release correlated with enhanced ECM degradation products containing glycosaminoglycan chains in embolic myxoma tissue. Our results strongly suggest that MMP overexpression may contribute to an excessive degradation of tumor ECM and increase the risk of embolism in cardiac myxomas.

Biological and molecular properties of a new αvβ3/αvβ5 integrin antagonist

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist.

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Cardiac myxoma cells exhibit embryonic endocardial stem cell features.

The origin of myxoma, the most frequent tumour of the heart, remains uncertain. Previous phenotypic characterizations have shown heterogeneous results and the most recent hypothesis suggests that cardiac myxoma originates from a primitive pluripotential cardiogenic cell. We investigated the expression of actin isoforms in 30 left atrial myxomas by immunohistochemistry and in eight consecutive tumours by RT‐PCR. α‐Smooth muscle actin (α‐SMA) protein and/or transcripts were detected in all cases, whereas α‐cardiac actin was observed in few cases and α‐skeletal actin was always absent. Besides classical features, vessel‐like structures were characterized by cells expressing CD34 and, less frequently, α‐SMA. Confocal microscopy showed focal co‐expression of CD34 and α‐SMA in myxoma cells, suggesting a gradual loss of stem endothelial markers and the acquisition of myocytic antigens. In order to confirm this hypothesis, early cardiac differentiation markers were also investigated. RT‐PCR documented the presence of transcripts for Sox9 (100%), Notch1 (87.5%), NFATc1 (37.5%), Smad6, metalloproteinases 1 and 2 alone or in variable combinations and the absence of ErbB3 and WT1. Myxoma cells maintained phenotypic heterogeneity in vitro, including the expression of α‐SMA and the presence of stress fibres. These findings document in cardiac myxoma cells phenotypic markers of the embryonic endothelial‐to‐mesenchymal transformation that precedes terminal differentiation of endocardial cushions, supporting the hypothesis that cardiac myxoma cells may derive from adult developmental remnants. Copyright

Cellular retinol binding protein-1 expression in endometrial hyperplasia and carcinoma: diagnostic and possible therapeutic implications

Cellular retinol binding protein-1 (CRBP-1) contributes to the maintenance of the differentiative state of endometrial glandular cells through the regulation of bioavailability of retinol and derivatives, but its role in endometrial oncogenetic process remains unclear. Antibodies to CRBP-1, estrogen and progesterone receptors (ER and PR) were applied to paraffin sections of proliferative (n=10) and secretory endometrium (n=9), and to endometrial polyps (n=6), simple (n=7), complex (n=3) and atypical endometrial hyperplasias (n=9) as well as to 47 endometrioid carcinomas of different histological grade (G) (G1, n=18; G2, n=19; G3, n=10). Four serous and two clear cell carcinomas were also examined. In glandular cells, CRBP-1 positivity was mainly cytoplasmic and rarely in the nuclei. CRBP-1 immunodetection was weakly positive in proliferative and low and focal in secretory endometrium and higher in atypical as compared to simple and complex hyperplasias. CRBP-1 expression in G1 endometrioid carcinomas was similar to that in atypical hyperplasias. In the latter, the highest CRBP-1 expression was observed in areas of squamous differentiation. Semiquantitative evaluation revealed a significant decrease of cytoplasmic CRBP-1 immunoreactivity with the increase of tumor grade. Among G3 endometrioid carcinomas, 60% were CRBP-1 negative, whereas the remaining cases showed a very low and focal positivity. Serous carcinomas were also CRBP-1 negative. When areas of different grading were present within the same tumor, less differentiated areas retained a lower CRBP-1 immunoreaction. The progressive decrease of CRBP-1 paralleled that of ER and PR immunodetection. RT-PCR in eight endometrioid carcinomas suggested a decrease of CRBP-1 with the increase of tumor grade also at transcriptional level. Our results indicate that CRBP-1 immunodetection may constitute an additional tool for histological grading of endometrial carcinoma. The CRBP-1 loss during the progression of endometrial cancer suggests a new potential target for pharmacological strategies aimed to counteract its progression by increased intracellular retinol bioavailability.

Modulation of Clusterin Isoforms Is Associated With All-Trans Retinoic Acid–Induced Proliferative Arrest and Apoptosis of Intimal Smooth Muscle Cells

Objectives— Clusterin is a heterodimeric glycoprotein which is implicated in several biological processes. The nuclear (n-CLU) and cytoplasmic secreted (s-CLU) isoforms have recently been described, but their role is still unclear. The aim of this study is to investigate the expression of clusterin and its isoforms during proliferative arrest and apoptosis of vascular smooth muscle cells (SMCs). Methods and Results— Clusterin expression was evaluated by immunohistochemistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-CLU and n-CLU, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all-trans retinoic acid-induced proliferative arrest and increased &agr;-smooth muscle actin expression did associate to s-CLU and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-CLU to n-CLU isoform. Conclusions— Clusterin overexpression characterized neointimal SMCs; s-CLU expression decreased in IT cells during all-trans retinoic acid–induced proliferative arrest and redifferentiation, whereas n-CLU overexpression was characteristic of apoptosis.

Biological and molecular properties of a new v 3/ v 5 integrin antagonist

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Biological and molecular properties of a new alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Biological and molecular properties of a new αvβ 3/αvβ5 integrin antagonist

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.