RESUMEN
OBJECTIVE: COVID-19 pneumonia, caused by the virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), was declared a pandemic by the WHO on 11th March 2020. While Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) represents the diagnostic gold standard of infection, computed tomography (CT) has been shown to have an important role in supporting the diagnosis, quantifying the severity, and assessing the efficacy of treatment and its response. Coronary artery calcification (CAC) is a CT finding that estimates atherosclerosis and can be quantified using the coronary artery calcium score (CACS). The purpose of this study is to demonstrate the correlation between coronary artery calcification and mortality rate in COVID-19 patients. PATIENTS AND METHODS: Three hundred seventeen (317) hospitalized patients with SARS-CoV-2 infection were ruled in this retrospective study. All patients underwent a non-ECG-gated chest CT to evaluate lung parenchymal involvement. In the same cohort, we observed the two main coronary arteries (common trunk, circumflex, anterior interventricular and right coronary heart) using a visual score, so patients were divided into four groups based on Ordinal CAC Score (OCS) levels. RESULTS: The multivariate analysis proved that the OCS value was statistically correlated with the mortality rate (p < 0.001). In fact, in the group of patients with an OCS value of 0, the mortality rate was 10.1% (10/99 patients), in the group with OCS between 1 and 4 was 18.9% (21/111), in the OCS group of patients ranged from 5 to 8 was 30.4% (24/79) and in the OCS group between 9 and 12 was 46.4% (13/28). CONCLUSIONS: We suggest that calcific atheromasia of the coronary arteries in patients with COVID-19 can be considered a prognostic marker of clinical outcome.
Asunto(s)
COVID-19 , Enfermedad de la Arteria Coronaria , Calcificación Vascular , Humanos , COVID-19/diagnóstico por imagen , Estudios Retrospectivos , Pronóstico , SARS-CoV-2 , Calcificación Vascular/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagenRESUMEN
[This corrects the article DOI: 10.1155/2016/6481862.].
RESUMEN
The aim of this study was to show the anti-adhesive potential of an antisense oligodeoxynucleotide (ODN) approach when designed to suppress the cellular function of the alphaV integrin subunit in breast cancer cells. The alphaV integrins play major roles in favouring breast cancer spreading. In this study, we inhibited alphaV subunit synthesis in the human breast carcinoma cell line, MDA-MB231, by a partially phosphorothioated antisense oligodeoxynucleotide (5543-ODN). The alphaV antisense 5543-ODN reduced alphaV, but not actin, mRNA transcription and protein expression by 55% and 65% respectively (1 microM, 72 h). Control sense and mismatch reagents were inactive. The antisense, but not the sense and mismatch, 5543-ODN induced dose- and time-dependent inhibition of MDA-MB231 adhesion to serum, vitronectin, fibrinogen and fibronectin substrates but was inactive on adhesion to laminin. Thus, the alphaV integrin was located in adhesion structures, which were disrupted by treatment with the alphaV antisense 5543-ODN. Antisense treated cells also showed evidence of programmed cell death with the appearance of apoptotic bodies. MDA-MB231 cells express a mutant form of the pro-apoptotic factor p53; however, no changes in the expression of p53 were observed by Western blotting. Immunofluorescence did reveal an increased nuclear translocation of p53 suggesting activation of the protein, but such a translocation did not lead to significant changes in either the expression of the cyclin dependent kinase inhibitor, p21(WAF1/CIP1) the cell survival factor Bcl-2 or the pro-apoptotic factor Bax.
Asunto(s)
Antígenos CD/genética , Apoptosis , Neoplasias de la Mama/metabolismo , Oligonucleótidos Antisentido/farmacología , Antígenos CD/biosíntesis , Western Blotting , Adhesión Celular/efectos de los fármacos , Densitometría , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Integrina alfaV , Microscopía Fluorescente , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Factores de Tiempo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Tumor progression and metastasis may result in part from the selection of cell clones competent for survival, invasion and growth at secondary sites and characterized by loss of growth inhibitory responses, acquisition of increased adhesiveness and enhanced motility and protease expression. Transforming growth factor-beta1 (TGF-beta1) is produced by osteoblasts (OB) in a latent form and is activated by proteases in a cell-dependent manner. We show here that OB conditioned medium (OB CM) modulates Matrigel invasion of a bone metastatic prostate cancer cell line (PC3) and that this effect is blocked by antibody against TGF-beta1 and by uPA/plasmin inhibitors, suggesting that TGF-beta1 can modulate OB-mediated cell recruitment and that PC3 cells can activate TGF-beta1. TGF-beta1 induces uPA and PAI-1 secretion and promotes binding of uPA at the external plasma membrane with increased membrane-associated plasmin activity. Matrix metalloprotease-9 (MMP-9) is induced both in the medium and in the membrane associated form. Moreover, the balance between proteolytic activity and inhibition is crucial in the metastatic event. Indeed, the increment of PAI-1 could have an important regulatory role on the extracellular proteolysis and might explain the decrease of net PA and gelatinolytic activities measured in the medium. In addition, PAI-1 plays a regulative role localizing matrix degradation in some specific sites, such as areas of cell-to-cell or cell-to-ECM contacts. In conclusion, TGF-beta1 enhances PC3 Matrigel invasion by a uPA/plasmin-dependent mechanism, also involving the MMP-9, and thus may play a central role in malignant prostate tumor progression as a result of stimulating bone matrix invasion.
Asunto(s)
Neoplasias Óseas/metabolismo , Endopeptidasas/biosíntesis , Matriz Extracelular/metabolismo , Osteoblastos/metabolismo , Neoplasias de la Próstata/enzimología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Antineoplásicos/farmacología , Materiales Biocompatibles/farmacología , Western Blotting , Neoplasias Óseas/secundario , Colágeno/farmacología , Medios de Cultivo Condicionados , Progresión de la Enfermedad , Combinación de Medicamentos , Endopeptidasas/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Laminina/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Invasividad Neoplásica , Osteoblastos/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Pruebas de Precipitina , Neoplasias de la Próstata/patología , Inhibidores de Proteasas/farmacología , Proteoglicanos/farmacología , Conejos , Inhibidores Tisulares de Metaloproteinasas/farmacología , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The alphav integrin subunit is highly expressed in osteoclasts where it dimerizes with beta1 and beta3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by alphavbeta3 antibodies or Arg-Gly-Asp-containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that alphav integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine-uracil-guanine (AUG) translational start site of the human/rabbit alphav gene, a procedure which offers the advantage of affecting all the alphav receptors with high efficiency. The alphav antisense ODN caused a dose-dependent, substrate-specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin-dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl-2. Although the expression of the cell death-promoting gene, bax, remained unchanged, a reduction of the bcl-2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of alphav synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of alphav antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.
Asunto(s)
Antígenos CD/biosíntesis , Apoptosis , Integrinas/biosíntesis , Oligodesoxirribonucleótidos Antisentido/farmacología , Osteoclastos/fisiología , Transducción de Señal , Animales , Antígenos CD/genética , Antígenos CD/fisiología , Secuencia de Bases , Sitios de Unión , Adhesión Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Humanos , Integrina alfaV , Integrinas/genética , Integrinas/fisiología , Líquido Intracelular , Datos de Secuencia Molecular , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Conejos , Transducción de Señal/efectos de los fármacosRESUMEN
The involvement of beta1 integrins in osteoclast function has been investigated by utilising an antisense oligodeoxynucleotide (ODN) approach. 18-mer antisense and control phosphorothioate ODNs were made to a conserved internal region of beta1 integrin sequence (nucleotide positions 1634-1651 of the human beta1 fibronectin receptor). These were tested on rabbit osteoclasts for anti-adhesive and resorptive effects mediated by alphaVbeta3 and alpha2beta1, the major integrins of osteoclasts. Antisense, but not control, beta1 ODNs inhibited osteoclast adhesion to collagen-coated glass (by up to 70%), but not to glass coated with vitronectin, fibronectin or fibrinogen. Adhesion to dentine and subsequent resorption were also inhibited (up to 60%) in a sequence-specific manner. The mechanism of action was verified using both a melanoma cell line, DX3, which expresses multiple integrins at high level including alphaVbeta3 and alpha2beta1, and in a rabbit osteoclast marrow culture (BMC) system. Exposure of DX3 cells to antisense ODN for up to 48 hours reduced adhesion to FCS- and collagen-coated glass, and concomitantly inhibited beta1 protein expression assessed by FACS and Western blot analysis; expression of other integrin subunits, alphaV and beta3, was unaffected. Similarly, the beta1 protein levels in the BMC were reduced by > 75% without any effect on actin expression. These data reveal the utility of antisense ODNs in exploring osteoclast biology and further define the functional role of osteoclastic beta1 integrin(s).
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Integrina beta1/genética , Oligonucleótidos Antisentido/farmacología , Osteoclastos/efectos de los fármacos , Animales , Secuencia de Bases , Proteínas Sanguíneas , Células de la Médula Ósea/efectos de los fármacos , Calcitonina/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Colágeno , Dentina , Dimerización , Regulación hacia Abajo/efectos de los fármacos , Proteínas de la Matriz Extracelular , Fibrinógeno , Fibronectinas , Regulación de la Expresión Génica/efectos de los fármacos , Vidrio , Humanos , Integrina beta1/fisiología , Integrinas/fisiología , Melanoma/metabolismo , Melanoma/patología , Datos de Secuencia Molecular , Conejos , Receptores de Colágeno , Receptores de Vitronectina/fisiología , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , VitronectinaRESUMEN
Prostate carcinoma (PRCA) cells metastasize to the skeleton with high frequency. Bone stores growth regulatory factors, which are released in active form during bone remodeling. We propose that bone cell-derived growth factors may induce the development of PRCA bone metastasis by recruiting tumor cells and increasing their proliferation in the bone microenvironment. Serum-free conditioned medium harvested from osteoblast cultures (OB CM) stimulated the in vitro chemotaxis of PRCA cells and invasion of a reconstituted basement membrane (Matrigel), suggesting enhanced invasive activity. Preosteoblastic cell CMs were less effective than CMs obtained from mature OB. CMs harvested from differentiated osteoblast cultures capable of matrix mineralization were more active compared to CMs from proliferating osteoblasts. OB CMs stimulated secretion of urokinase (uPA) and matrix metalloproteinase-9 (MMP-9). Inhibition of these matrix-degrading proteases by neutralizing antibodies and/or by inhibitors of their catalytic activity reduced Matrigel invasion. Secretion of uPA and activation of MMP-9 were most prominent by differentiated OB CMs with respect to poorly differentiated cells in vitro. These results are in agreement with several in vivo studies and indicate that factors produced during osteogenesis by bone cells stimulate PRCA cell chemotaxis and matrix proteases expression, thus representing potential targets for alternative therapies deterring the progression of PRCA metastasis to bone.
Asunto(s)
Colagenasas/metabolismo , Proteínas de Neoplasias/metabolismo , Osteoblastos/metabolismo , Neoplasias de la Próstata/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Colágeno , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Combinación de Medicamentos , Humanos , Laminina , Masculino , Metaloproteinasa 9 de la Matriz , Invasividad Neoplásica , Neoplasias de la Próstata/patología , Proteoglicanos , Células Tumorales CultivadasRESUMEN
Prostate cancers (PRCAs) frequently metastasize to bone. We show here that this process is facilitated by osteoblast-mediated tumor cell recruitment. Transforming growth factor-beta1 (TGF-beta1) is produced by osteoblasts in a latent form and is activated by proteases in a cell-dependent manner. This cytokine exhibits pleiotropic effects on cell-extracellular matrix (ECM) interactions and may influence tumor cell invasion and metastasis. Our purpose was to identify the potential molecular mechanisms involved in osteoblast-mediated cell recruitment and to characterize the effect of TGF-beta1 on adhesion, motility and invasiveness of a human prostate cancer cell line with high bone metastatic potential (PC3 cell line) in vitro. Conditioned media from osteoblast cultures (OB CM) enhanced PC3 cell chemotaxis and invasion of reconstituted basement membrane. These effects were blocked by a neutralizing TGF-beta1 polyclonal antibody but not by elution of the OB CM in agarose-heparin columns, suggesting that TGF-beta1, but not EGF-like proteins, contribute to PC3 cell recruitment. In addition, TGF-beta1 directly induced chemotaxis and invasion of PC3 cells in a dose-dependent manner. The TGF-beta1-mediated invasion and motility were accompanied by increased PC3 cell adhesion, spreading and alpha2beta1 and alpha3beta1 integrin expression. These events are involved in the cell adhesion to several components of basement membrane and ECM and in the selective invasion of metastatic tumor cells. Our results suggest that TGF-beta1 can influence cellular recognition of ECM components by prostatic cancer cells and can modulate cell adhesion and invasion leading to increased invasive potential. Given the widespread tissue distribution of TGF-beta1, and the high levels present in the bone, this cytokine may be an important autocrine-paracrine modulator of the bone invasive phenotype in vivo.
Asunto(s)
Proteínas de la Matriz Extracelular/fisiología , Osteoblastos/fisiología , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta/fisiología , Animales , Adhesión Celular , Movimiento Celular , Quimiotaxis , Medios de Cultivo Condicionados , Humanos , Integrinas/análisis , Masculino , Invasividad Neoplásica , ConejosRESUMEN
We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase-2 (MMP-2) secreted by human GCT23 giant cell tumour cells. Activation of MMP-2 is RGD sequence dependent, possibly involves anti-alphaVbeta3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP-2, but subsequent addition of soluble GRGDSP induced rounding and MMP-2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase-2 (TIMP-2) and carboxyl terminal MMP-2 consistent with a role for membrane type (MT)-MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT-MMP-1, MMP-1, MMP-2, TIMP-1 or TIMP-2. GRGDSP and cytochalasin B enhanced levels of membrane-associated pro- and active form MMP-1 and MMP-2 but not MT-MMP-1, stimulated cell surface MMP-1 staining and induced that of MT-MMP-1, MMP-2 and TIMP-2. This was consistent with the possible relocation of constitutive MT-MMP-1 to the cell surface as a prerequisite for subsequent cell surface MMP-2/TIMP-2/MT-MMP-1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP-1/MMP-2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology.
Asunto(s)
Carcinoma de Células Gigantes/enzimología , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Oligopéptidos/genética , Oligopéptidos/farmacología , Sialoglicoproteínas/farmacología , Carcinoma de Células Gigantes/genética , Carcinoma de Células Gigantes/patología , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sialoproteína de Unión a Integrina , Metaloproteinasa 2 de la Matriz , Osteopontina , Células Tumorales CultivadasRESUMEN
The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the alpha vbeta3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.
Asunto(s)
Genes src/fisiología , Factor Estimulante de Colonias de Macrófagos/farmacología , Osteoclastos/citología , Osteoclastos/fisiología , Receptores de Vitronectina/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Animales , Células Cultivadas , Genes src/efectos de los fármacos , Humanos , Osteoclastos/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Conejos , Receptores de Vitronectina/efectos de los fármacos , Proteínas Recombinantes/farmacologíaRESUMEN
Prostate cancer (PRCA) cells metastasize to bone with high frequency, inducing typical osteosclerotic lesions. To establish if local stimuli on the bone tissue may derive from metastatic colonies of prostatic origin, we evaluated the biologic activities secreted by human prostatic epithelium and effective on osteoblast-like cells in vitro. Supernatant from short-term tissue cultures of human prostatic tissue samples obtained from PRCA (35 cases) and benign prostatic hyperplasia (BPH, 12 cases) patients were applied to three models of cells with osteoblastic phenotype: two normal [rabbit osteoblasts (OB) and rat periosteal cells (PO)] and one transformed (human osteosarcoma cell line, MG63). Proliferative activity was monitored through enzymatic reduction of tetrazolium salts and expressed as relative mitogenic activities (RMA). Analysis of proliferation and alkaline phosphatase (ALP) activity, a marker of osteoblast function, demonstrates that conditioned media (CM) from PRCA cultures stimulate both growth and activity of osteoblast-like cells to a greater extent compared to CM from BPH. Furthermore, cell growth and activity of osteoblast-like cells are progressively increased by CM derived from patients with stage B (tumor confined within the prostate capsule), stage C (locally invasive tumor), and stage D (invasive tumor with distant metastasis) disease. One of the mechanisms potentially underlying the CM-stimulated effects on bone cells is associated with the urokinase (uPA) enzyme route, whose release progressively increases with the stage of disease. However, antibodies against uPA and p-aminobenzamidine (a low molecular weight urokinase inhibitor) treatment, which both inhibit the proliferative and differentiative effects induced by exogenous urokinase, partially slow down the effects of CM from PRCA tissue cultures, suggesting that additional factors are secreted by prostatic tumor cells in vitro. In conclusion, we show that the mitogenic and differentiative activities for osteoblasts produced by prostatic tumor cells in short-term tissue cultures are related to PRCA stage and may predict the behavior of skeletal metastases in single cases of tumor. In addition, the culture methods used may represent a valid model to study prostatic and bone cellular interactions, which may indicate new therapeutic approaches in metastatic prostate tumors.