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Congenital heart malformations (CHMs) make up between 2 and 3% of annual human births. Bone morphogenetic proteins (BMPs) signalling is required for chamber myocardium development. We examined for possible molecular defects in the bone morphogenetic protein 2 and 4 (BMP2, -4) genes by sequencing analysis of all coding exons, as well as possible transcription or protein expression deregulation by real-time PCR and ELISA, respectively, in 52 heart biopsies with congenital malformations (atrial septal defect (ASD), ventricular septal defect (VSD), tetralogy ofFallot (ToF) and complex cases) compared to 10 non-congenital heart disease (CHD) hearts. No loss of function mutations was found; only synonymous single nucleotide polymorphisms (SNPs) in the BMP2 and BMP4 genes were found. Deregulation of the mRNA expression and co-expression profile of the two genes (BMP2/BMP4) was observed in the affected compared to the normal hearts. BMP2 and -4 protein expression levels were similar in normal and affected hearts. This is the first study assessing the role of BMP-2 and 4 in congenital heart malformations. Our analysis did not reveal molecular defects in the BMP2 and -4 genes that could support a causal relationship with the congenital defects present in our patients. Importantly, sustained mRNA and protein expression of BMP2 and -4 in CHD cases compared to controls indicates possible temporal epigenetic, microRNA or post-transcriptional regulation mechanisms governing the initial stages of cardiac malformation.
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The involvement of growth factors (GFs) in the pathogenesis of lumbar intervertebral disc (ID) herniation and the spontaneous resorption of herniated ID fragments remains only partially elucidated. A simultaneous assessment of the transcript levels of numerous GFs and their association with clinical and epidemiological profiles of human ID herniation would provide valuable insight into the biology and clinical course of the disease. In the present study, we examined simultaneously the transcript levels of vascular endothelial growth factor (VEGF), transforming growth factor ß1 (TGFß1), basic fibroblast growth factor 2 (bFGF2), platelet derived growth factor (PDGF) isoforms and receptors, epidermal growth factor (EGF) and insulin growth factor1 (IGF1) in herniated and control ID specimens and investigated their correlation with the clinicopathological profiles of patients suffering from symptomatic lumbar ID herniation. GF mRNA expression levels were determined by RT-qPCR in 63 surgical specimens from lumbar herniated discs and 10 control ID specimens. Multiple positive correlations were observed between the transcript levels of the GFs examined in the ID herniation group. VEGF mRNA expression was significantly increased in the protruding compared with the extruded discs. Intense and acute pain significantly upregulated the PDGF transcript levels. Significant negative correlations were observed between the patient body mass index and the transcript levels of VEGF and PDGF receptors. Our findings support the hypothesis of the involvement of GFs in the natural history of ID herniation. GFs synergistically act in herniated IDs. Increased VEGF expression possibly induces the neovascularization process in the earliest stages of ID herniation. PDGFC and D play a role in the acute phase of radiculopathy in a metabolic response for tissue healing. A molecular effect, in addition to the biomechanical effect of obesity in the pathogenesis of ID herniation is also implied.
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Péptidos y Proteínas de Señalización Intercelular/genética , Desplazamiento del Disco Intervertebral/genética , Desplazamiento del Disco Intervertebral/patología , Disco Intervertebral/patología , Vértebras Lumbares/patología , Transcriptoma , Adulto , Anciano , Femenino , Humanos , Disco Intervertebral/metabolismo , Desplazamiento del Disco Intervertebral/epidemiología , Vértebras Lumbares/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/genéticaRESUMEN
Thrombocytopenia and thromboembolism(s) may develop in heparin immune-mediated thrombocytopenia (HIT) patients after reexposure to heparin. At the Onassis Cardiac Surgery Center, 530 out of 17,000 patients requiring heart surgery over an 11-year period underwent preoperative HIT assessment by ELISA and a three-point heparin-induced platelet aggregation assay (HIPAG). The screening identified 110 patients with HIT-reactive antibodies, out of which 46 were also thrombocytopenic (true HIT). Cardiac surgery was performed in HIT-positive patients under heparin anticoagulation and iloprost infusion. A control group of 118 HIT-negative patients received heparin but no iloprost during surgery. For the first 20 patients, the dose of iloprost diminishing the HIPAG test to ≤5% was determined prior to surgery by in vitro titration using the patients' own plasma and donor platelets. In parallel, the iloprost "target dose" was also established for each patient intraoperatively, but before heparin administration. Iloprost was infused initially at 3 ng/kg/mL and further adjusted intraoperatively, until ex vivo aggregation reached ≤5%. As a close correlation was observed between the "target dose" identified before surgery and that established intraoperatively, the remaining 90 patients were administered iloprost starting at the presurgery identified "target dose." This process significantly reduced the number of intraoperative HIPAG reassessments needed to determine the iloprost target dose, and reduced surgical time, while maintaining similar primary clinical outcomes to controls. Therefore, infusion of iloprost throughout surgery, under continuous titration, allows cardiac surgery to be undertaken safely using heparin, while avoiding life-threatening iloprost-induced hypotension in patients diagnosed with HIT-reactive antibodies or true HIT.
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Anticuerpos/sangre , Fármacos Cardiovasculares/uso terapéutico , Iloprost/uso terapéutico , Trombocitopenia/patología , Tromboembolia/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticoagulantes/efectos adversos , Aneurisma de la Aorta/patología , Aneurisma de la Aorta/cirugía , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Plaquetas/patología , Anuloplastia de la Válvula Cardíaca/métodos , Puente de Arteria Coronaria/métodos , Esquema de Medicación , Monitoreo de Drogas , Femenino , Heparina/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Atención Perioperativa/métodos , Agregación Plaquetaria/efectos de los fármacos , Recuento de Plaquetas , Trombocitopenia/inducido químicamente , Trombocitopenia/inmunología , Tromboembolia/inmunología , Tromboembolia/patología , Resultado del TratamientoRESUMEN
BACKGROUND: Angiogenesis is a prerequisite for tumour development, progression and metastasis; however, its underlying molecular mechanisms in endometrial carcinoma are poorly understood. DESIGN: In this study, the mRNA and protein expression profiles of two key regulators of angiogenesis, vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGFB1), were evaluated by real-time PCR and western blot analysis in 23 endometrial cancer tissue-paired specimens (malignant vs. adjacent normal tissues). We aimed to investigate whether VEGF and TGFB1 serve as markers of the malignant transformation of the endometrium and whether VEGF or TGFB1 expression can constitute a useful prognostic marker of survival in patients with endometrial carcinoma. RESULTS: Tissue-pair analysis revealed VEGF transcriptional up-regulation and TGFB1 mRNA down-regulation as the most frequent transcriptional features. VEGF and TGFB1 mRNA were positively correlated (P < 0·001). VEGF protein levels were higher in endometrioid-type tissue pairs (P = 0·047). TGFB1 protein and mRNA levels were negatively correlated (P = 0·042). TGFB1 protein expression was related to survival only in patients with endometrioid adenocarcinoma (P = 0·045). CONCLUSIONS: Tissue-pair mRNA and protein analysis reveals VEGF transcriptional up-regulation and TGFB1 down-regulation that are correlated with the malignant transformation of the endometrium, while post-transcriptional mechanisms control VEGF and TGFB1 protein. TGFB1 protein demonstrated a prognostic value only in endometrioid adenocarcinoma.
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Carcinoma Endometrioide/genética , Transformación Celular Neoplásica/genética , Neoplasias Endometriales/genética , Regulación Neoplásica de la Expresión Génica/genética , Factor de Crecimiento Transformador beta1/genética , Factor A de Crecimiento Endotelial Vascular/genética , Anciano , Anciano de 80 o más Años , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patología , Transformación Celular Neoplásica/metabolismo , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , ARN Neoplásico , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Idiopathic Pulmonary Fibrosis and Rheumatoid Arthritis associated usual interstitial pneumonia seem to have the same poor outcome as there is not an effective treatment. The aim of the study is to explore the reparative ability of bone marrow mesenchymal stem cells by evaluating the system telomerase/telomeres and propose a novel therapeutic approach. METHODS: BM-MSCs were studied in 6 IPF patients, 7 patients with RA-UIP and 6 healthy controls. We evaluated the telomere length as well as the mRNA expression of both components of telomerase (human telomerase reverse transcriptase, h-TERT and RNA template complementary to the telomeric loss DNA, h-TERC). RESULTS: We found that BM-MSCs from IPF, RA-UIP cases do not present smaller telomere length than the controls (p = 0.170). There was no significant difference regarding the expression of both h-TERT and h-TERC genes between patients and healthy controls (p = 0.107 and p = 0.634 respectively). CONCLUSIONS: We demonstrated same telomere length and telomerase expression in BM-MSCs of both IPF and RA-UIP which could explain similarities in pathogenesis and prognosis. Maintenance of telomere length in these cells could have future implication in cell replacement treatment with stem cells of these devastating lung disorders.
RESUMEN
The objective of this study was to investigate the hypothesis that the altered epigenetic mechanisms that regulate IGF2 imprinting in placentas from fetal growth restricted (FGR) pregnancies affect IGF2 expression leading to impaired fetal growth. We investigated gene transcription, genotyping and the methylation patterns of IGF2 from 31 and 17 placentas from FGR-complicated and normal pregnancies, respectively. A statistically significant decrease in IGF2 mRNA levels was observed in the placentas from the FGR pregnancies. Loss of imprinting (LOI) was only detected in the abnormal placentas. The evaluation of the percentage of the methylated reference (PMR) of two different potentially differentially methylated regions (DMR) demonstrated significant PMR values in both sites for the normal and FGR pregnancies with no significant differences. Our results suggest the involvement of the IGF2 imprinted gene in placental function and fetal growth and the possible association of epigenetic alterations with the pathophysiology of fetal growth restriction.
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Retardo del Crecimiento Fetal/genética , Factor II del Crecimiento Similar a la Insulina/genética , Placenta/metabolismo , Metilación de ADN/genética , Femenino , Impresión Genómica/genética , Genotipo , Humanos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Intervertebral disc (IVD) degeneration suggests a complex process influenced by genetics, lifestyle and biomechanics, which accounts for the development of low back pain (LBP) and lumbar radiculopathy, a major cause of musculoskeletal disability in humans. The family of Akt/PKB kinases is a principal mediator in the signal transduction pathways, which contribute to transcriptional regulation, cell growth, proliferation, apoptosis, and survival ability. The purpose of this study was to evaluate the transcriptional profile of the AKT family genes in human herniated discs and the involvement of the PI3K-Akt signaling pathway in human IVD degeneration. Real-time PCR analysis was used to assess the mRNA expression pattern of the three Akt/PKB isoforms in 63 herniated and 10 control disc specimens. Our results showed a significant positive correlation between AKT1 and AKT3 mRNA in herniated discs suggesting a synergistic action between these isoforms in disc herniation. Interestingly, AKT2 mRNA was up-regulated in patients with acute pain during the first 12 months, indicating that AKT2 transcriptional activation may be associated with acute rather than chronic inflammation and phagocytosis. Finally, Akt1/PKB transcription presented a stepwise activation as disc herniation deteriorated. Our findings provide evidence on the transcriptional activation of the Akt/PKB pathway indicating that it is involved in lumbar disc degeneration. There is need for further studies to elucidate the exact role and down-stream signaling action of Akt/PKB isoforms in the pathogenesis of lumbar disc herniation.
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Desplazamiento del Disco Intervertebral/enzimología , Desplazamiento del Disco Intervertebral/genética , Vértebras Lumbares/enzimología , Proteínas Proto-Oncogénicas c-akt/genética , Adulto , Anciano , Femenino , Regulación Enzimológica de la Expresión Génica , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Humanos , Desplazamiento del Disco Intervertebral/epidemiología , Isoenzimas/biosíntesis , Isoenzimas/genética , Vértebras Lumbares/patología , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Transducción de Señal/genética , Activación Transcripcional/genética , Adulto JovenRESUMEN
CONTEXT: Yin Yang-1 (YY-1) is implicated in the pathogenesis of lung cancer which can be complicated with idiopathic pulmonary fibrosis (IPF). OBJECTIVE: The aim of the study was to investigate whether YY-1 is involved in the pathogenesis of IPF and whether represents a common pathogenetic pathway which could explain the coexistence of these disorders. MATERIALS AND METHODS: Lung tissue from 52 patients (37 with IPF and 15 controls) and bronchoalveolar lavage fluid (BALF) from 34 patients (25 with IPF and 9 controls) were studied and YY-1 mRNA expression was evaluated by real-time PCR. RESULTS: YY-1 was expressed in 8% (3/37) of IPF patients and in 6% (1/15) of healthy controls in tissue samples. In addition, 12% (3/25) of IPF patients and 33% (3/9) of healthy controls have expressed YY-1 gene in BALF samples. However, no statistical significant difference in mRNA expression between patients and controls has been detected in both tissue and BAL fluid samples. DISCUSSION AND CONCLUSION: Our results do not support the hypothesis of YY-1 involvement in IPF. However, similar expression of YY-1 gene in two biological samples cannot exclude a possible role of this polymorphic gene in the pathway of IPF. Further studies in a larger scale of patients are needed.
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Fibrosis Pulmonar Idiopática/metabolismo , Factor de Transcripción YY1/metabolismo , Líquido del Lavado Bronquioalveolar , Estudios de Casos y Controles , Regulación de la Expresión Génica , Humanos , Factor de Transcripción YY1/genéticaRESUMEN
The involvement of matrix metalloproteinases (MMPs) in both the pathogenesis of intervertebral disc (ID) herniation and the spontaneous regression of herniated ID fragments remains only partially elucidated. The purpose of the present study was to simultaneously examine the transcript levels of a large number of MMPs (-1, -3, -8, -9, -13 and -14) and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs) and to investigate their correlation with the clinicopathologic profile of patients suffering from symptomatic lumbar ID herniation. mRNA expression levels were determined by means of the real-time polymerase chain reaction in 63 herniated and 10 control ID specimens. Our results showed multiple positive correlations among all MMPs and ADAMTS-4 mRNA in herniated samples, indicating their possible synergistic effect in ID herniation. MMP-9 and -13 mRNA levels were significantly elevated in patients with chronic pain, presumably as a consequence of neovascularization and chronic inflammation. Smoking habits were found to have a negative dose-dependent effect on the transcript levels of MMP-3 and MMP-13 and a positive correlation with pain intensity, suggesting an unfavorable role for smoking in the regression process of herniated disc fragments. Our findings provide evidence of the molecular portrait of MMPs and ADAMTS-4 in lumbar ID herniation, as well as of its association with the clinicopathological profile of the patients included in this study, reinforcing the hypothesis of MMPs involvement in the natural history of ID herniation. However, further studies are necessary to elucidate the exact role of MMPs in the resorption process of herniated lumbar discs.
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Proteínas ADAM/genética , Desplazamiento del Disco Intervertebral/enzimología , Desplazamiento del Disco Intervertebral/genética , Disco Intervertebral/enzimología , Metaloproteinasas de la Matriz/genética , Procolágeno N-Endopeptidasa/genética , Proteínas ADAM/fisiología , Proteína ADAMTS4 , Adulto , Anciano , Comorbilidad/tendencias , Femenino , Predisposición Genética a la Enfermedad , Humanos , Disco Intervertebral/patología , Desplazamiento del Disco Intervertebral/epidemiología , Masculino , Metaloproteinasas de la Matriz/biosíntesis , Metaloproteinasas de la Matriz/fisiología , Persona de Mediana Edad , Procolágeno N-Endopeptidasa/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The events that result in the establishment and progression of aortic aneurysms are complex and multifactorial. However, degradation of the extracellular matrix (ECM) of aortic tunica media appears to be a consistent histopathological and biochemical feature. An increased local expression of matrix metalloproteinases (MMPs) as well as an imbalance between MMP expression and the expression of their natural tissue inhibitors (TIMPs) have been demonstrated in dilated aortic wall. We hypothesized that a distinct MMP and TIMP expression pattern underlies the development of ascending aorta dilation. To test our hypothesis, expression levels of 10 MMPs and 4 TIMPs were assessed by real-time PCR in dilated and normal aortic tissue derived from patients that underwent elective surgical repair of ascending aorta aneurysm (AAA) and coronary artery by-pass grafting, respectively. We found no statistically significant up- or down-regulation of any individual MMP. Surprisingly, the tissue inhibitor of metalloproteinases (TIMP)-3 was significantly more expressed in dilated aortic tissue compared to control tissue, thereby reflecting an effort to counteract MMP activity. Finally, when we evaluated the MMP and TIMP co-expression pattern in normal and dilated aortic tissue, we observed that in aortic aneurysms activation of the MMP system was characterised by the co-expression of more than one proteinase and the down-regulation of TIMP-1 and -2. The latter observation is the key regulatory point that leads to ECM degradation and, subsequently, to AAA formation.
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Aneurisma de la Aorta Torácica/genética , Inhibidores Tisulares de Metaloproteinasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Aneurisma de la Aorta Torácica/enzimología , Aneurisma de la Aorta Torácica/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Metaloproteinasas de la Matriz/biosíntesis , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Persona de Mediana Edad , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Transcripción GenéticaRESUMEN
PURPOSE OF THE STUDY: Several studies in patients with lung cancer have shown that epidermal growth factor receptor regulates various tumorigenic processes through the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin and Ras/Raf/Mek/Erk (mitogen-activated protein kinase (MAPK)) signalling pathways. The aim of our study is to evaluate whether these pathways are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and to seek indirect evidence of a common pathogenetic pathway with lung cancer. m-RNA expression of oncogenes participating in these two signaling pathways, as well as the combined m-RNA expression of the suppressor genes R-kip and p53 in lung tissue of patients with IPF were evaluated. BASIC PROCEDURES: The study population was composed by two distinct groups. Patients with IPF (n = 25) and control subjects who underwent thoracic surgery for reasons other than interstitial lung disease (n = 10). Expression analysis of the aforementioned oncogenes and suppressor genes was performed using real-time reverse transcription polymerase chain reaction. MAIN FINDINGS: We found no difference in the overall m- RNA expression between controls and IPF in both investigated pathways. However, Braf has been overexpressed in IPF samples (P = 0.01) in contrast with K-ras that has been found downregulated (P < 0.001) in comparison with controls. PRINCIPAL CONCLUSIONS: These findings cannot exclude the hypothesis of involvement of Akt and MAPK signalling pathways in pathogenesis of IPF. However, further investigation is needed in order to verify these data.
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Fibrosis Pulmonar Idiopática/enzimología , Fibrosis Pulmonar Idiopática/patología , Pulmón/enzimología , Pulmón/patología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Análisis Mutacional de ADN , Demografía , Femenino , Regulación de la Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/fisiopatología , Pulmón/fisiopatología , Sistema de Señalización de MAP Quinasas/genética , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Espirometría , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
BACKGROUND: Experimental data have provided evidence that progenitor cells of bone marrow (BM) origin may play a role in the fibrogenic process of the lung. OBJECTIVE: To probe the possible involvement of BM mesenchymal stem cells (MSCs) in the pathophysiology of Idiopathic Pulmonary Fibrosis (IPF) by investigating the molecular profile of these cells. DESIGN: BM MSCs were studied in 10 IPF patients and 10 healthy controls. MSCs were identified by their immunophenotypic characteristics and their potential to differentiate towards adipocytes/osteocytes/chondrocytes. We evaluated the mRNA expression of genes involved in the lung injury of IPF, namely the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor beta-1 (TGF-beta1) and the axis stromal-cell-derived factor-1 (SDF-1)/CXCR4 in BM MSCs using quantitative RT-PCR. RESULTS: The BM MSCs of IPF patients displayed normal immunophenotypic characteristics and differentiation potential. No statistically significant difference was found between patients and controls in VEGF and FGF mRNA expression. TGF-beta1 was not expressed in either patients or controls. A significant increase in SDF-1-TR1 and CXCR4 mRNA expression was detected in IPF patients (1.6 x 10(25) +/- 1.2 x 10(25) and 3.1 x 10(7) +/- 3.1 x 10(7), respectively) compared to controls (0.32 x 10(25) +/- 0.07 x 10(25) and 1.67 x 10(7) +/- 0.30 x 10(7), respectively) (p = 0.001 and p = 0.001, respectively) whereas SDF-1 levels in MSC supernatants were similar in patients and controls. CONCLUSIONS: The increased CXCR4 expression by patient MSCs suggests that the BM is probably implicated in the pathophysiology of IPF by mobilizing MSCs in response to or preceding lung injury. The potential role of BM MSCs in IPF is another interesting field for further investigation.
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Factores de Crecimiento de Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adulto , Anciano , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Humanos , Fibrosis Pulmonar Idiopática/patología , Masculino , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Fenotipo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
CONTEXT AND OBJECTIVE: It has been suggested that stromal cell-derived factor-1alpha ((SDF-1alpha) or CXCL12, both transcripts, TR1 and TR2) and its cognate receptor CXCR4 may regulate cancer metastasis. We have investigated the role of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2) and the biological axis of CXCL12-CXCR4, in patients with malignant pleural effusions (PEs). MATERIAL AND METHODS: Twenty five patients, seven with transudative PEs due to heart failure and 18 with exudative malignant PEs (7 with small cell lung cancer (SCLC) and 11 with nonsmall cell lung cancer (NSCLC)) were included in the study. Expression analysis of the mediators was performed in pleural fluid pellet using real-time reverse transcription-PCR. Protein expression has been evaluated by western blot analysis. RESULTS: SDF-TR1 (P = 0.02) but not SDF-TR2 (P = 0.23) or CXCR4 levels (P = 0.23) were higher in malignant PEs than in transudates. SDF-TR1 (P = 0.04) and SDF- TR2 levels (P = 0.04) but not CXCR4 levels (P = 0.123) were higher in SCLC PEs than in heart failure PEs. SDF-TR1 (P = 0.03) but not SDF-TR2 levels (P = 0.6) and CXCR4 levels (P = 0.4) were higher in NSCLC PEs than in transudates. Ang-1 has not been expressed in PEs, whereas no significant difference has been detected in VEGF and Ang-2 expression between malignant PEs and transudates. However, protein expression showed increased VEGF and SDF expression in malignant PEs. CONCLUSIONS: These results suggest that elevated SDF-1alpha/CXCL12 levels would be suggestive of a link to metastasis and may participate in pleural trafficking in lung cancer.
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Quimiocina CXCL12/metabolismo , Regulación de la Expresión Génica , Neoplasias Pulmonares/enzimología , Derrame Pleural/metabolismo , Carcinoma Pulmonar de Células Pequeñas/enzimología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Pulmón/patología , Masculino , Persona de Mediana Edad , Metástasis de la NeoplasiaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is associated with aberrant repair, persistence of collagen deposition, and the development of vascular remodeling. However, the role of angiogenesis in the pathogenesis of IPF is still undetermined. The aim of this study was to evaluate the combined mRNA expression of vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF1) epidermal growth factor (EGF), and its receptor (EGFR) in lung tissue obtained from IPF patients. We have also investigated the expression of chemokine CXCL12/stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, to identify alterations that maybe implicated in the pathogenesis of IPF. The subjects studied consisted of two distinct groups: patients with IPF (n = 25) and subjects (control) undergoing thoracic surgery for reasons other than interstitial lung disease (n = 10). Expression analysis of the aforementioned growth factors and biological axis CXCL12/CXR4 analysis were performed using real-time RT-PCR. IGF-1, EGF, and FGF2 mRNA levels are significantly decreased in the patients compared to the controls (p = 0.028, p = 0.023 and p = 0.009, respectively). SDF1-TR1 and SDF1-TR2 transcript levels were significantly lower in patients compared to controls (p = 0.017 and p = 0.001). Significant coexpression of VEGF mRNA with IGF mRNA was observed in the group of the patients (p = 0.017). An additional coexpression of VEGF mRNA with SDF1-TR1 mRNA was demonstrated(p = 0.030). Our results show a downregulation in angiogenetic mechanisms in IPF. However, our results should be further verified by measuring other angiogenetic pathways in more samples.
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Proteínas Angiogénicas/genética , Quimiocina CXCL12/genética , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Receptores CXCR4/genética , Proteínas Angiogénicas/análisis , Proteínas Angiogénicas/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Quimiocina CXCL12/análisis , Quimiocina CXCL12/metabolismo , Regulación hacia Abajo/fisiología , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Fibrosis Pulmonar Idiopática/inmunología , Fibrosis Pulmonar Idiopática/fisiopatología , Factor I del Crecimiento Similar a la Insulina/genética , Pulmón/inmunología , Pulmón/fisiopatología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/fisiopatología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores CXCR4/análisis , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: The placental anticoagulant protein Annexin A5 (ANXA5) is highly expressed on the apical surfaces of syncytiotrophoblasts and plays an important role in maintaining blood fluidity in the placental circulation. We investigated the mRNA and protein expression of ANXA5 in placentas from pregnancies complicated by fetal growth restriction (FGR) compared with uncomplicated pregnancies. MATERIALS AND METHODS: Placental tissue was collected from 18 pregnancies complicated by FGR and 16 pregnancies with a normal outcome. ANXA5 mRNA expression was quantified by Real-Time PCR (RT-PCR), and protein concentrations were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: A decreased ANXA5 mRNA expression was observed in placenta samples from FGR-affected pregnancies compared to those from uncomplicated pregnancies. However, similar ANXA5 protein levels were measured in both specimen groups. No correlation was observed between ANXA5 mRNA and protein levels. CONCLUSIONS: Transcriptional ANXA5 down-regulation was demonstrated in FGR-affected pregnancies, although protein levels were similar in FGR-related placentas and controls. We can speculate that either recruitment of the protein from the bloodstream or increased apoptosis or post-transcriptional modifications occur, which affect ANXA5 protein levels in FGR-related placentas. Further studies are required to reveal the role of ANXA5 in FGR pathology.
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Anexina A5/metabolismo , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Placenta/metabolismo , ARN Mensajero/metabolismo , Animales , Anexina A5/genética , Apoptosis/genética , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Femenino , Mamíferos/genética , Mamíferos/metabolismo , Procedimientos Quirúrgicos Obstétricos , Circulación Placentaria/genética , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/metabolismoRESUMEN
OBJECTIVE: We speculated that distinct angiogenic profiles are involved in idiopathic interstitial pneumonias (IIPs) in comparison with interstitial pneumonias associated with collagen vascular disease (CVD-IPs). This hypothesis was investigated by measuring the expression of a cardinal biologic axis, the vascular endothelial growth factor (VEGF)-stromal derived growth factor [SDF-1alpha, transcripts 1 and 2 (TR1 and TR2)] and receptor, CXCR4 and the angiogenetic receptors CXCR2 and CXCR3 in bronchoalveolar lavage fluid (BALF) in both conditions. METHODS: We studied prospectively 25 patients with fibrotic IIPs (f-IIPs) [20 with idiopathic pulmonary fibrosis (IPF) and 5 with idiopathic non-specific interstitial pneumonia (NSIP)] and 16 patients with CVD-IPs. mRNA expression was measured by Real-Time RT-PCR and protein was evaluated by Western Blotting. RESULTS: A significantly greater value has been detected in SDF-1alpha-TR1 mRNA expression levels of CVD-IPs (p=0.05) in comparison with IPF group. A similar trend has been also detected in protein expression in favor of CVD-IP group. In addition, VEGF mRNA levels have been found significantly increased in CVD-IPs in comparison with the NSIP group (p=0.05). No significant difference has been found in SDF-1alpha-TR2-CXCR4 mRNA and CXCR2-CXCR3 between the two groups. CONCLUSION: These results showed increased expression of SDF-1alpha in CVD-IPs, suggesting different angiogenic procedures. Further studies are needed in order to better explore the angiogenetic pathway in these disorders.
Asunto(s)
Quimiocina CXCL12/genética , Fibrosis Pulmonar Idiopática/genética , Enfermedades Pulmonares Intersticiales/genética , Regulación hacia Arriba , Anciano , Western Blotting , Líquido del Lavado Bronquioalveolar/química , Enfermedades del Colágeno/genética , Enfermedades del Colágeno/fisiopatología , Femenino , Humanos , Fibrosis Pulmonar Idiopática/fisiopatología , Enfermedades Pulmonares Intersticiales/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , ARN Mensajero/metabolismo , Receptores CXCR3/genética , Receptores CXCR4/genética , Receptores de Interleucina-8B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades Vasculares/genética , Enfermedades Vasculares/fisiopatología , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Ras genes, a class of nucleotide-binding proteins that regulate normal and transformed cell growth, have been scarcely investigated in human brain tumours. We evaluated the mutational, mRNA and protein expression profile of the ras genes in 21 glioblastomas multiforme (grade IV), four fibrillary astrocytoma (grade II), four anaplastic astrocytoma (grade III) and 15 normal specimens. K-, H- and N-ras transcript levels were determined by real-time RT-PCR and mutational status by PCR-restriction fragment length polymorphism (RFLP) and direct sequencing. p21 protein was evaluated by Western blot analysis. Two K-ras mutations were found in codons 16 and 26 in one pathological and one normal sample, respectively. Glioblastoma multiforme cases exhibited significantly lower K- and H-ras mRNA levels compared to controls (P < 10(-4)). K- and H-ras mRNA down-regulation was not associated with patient outcome or survival. K-ras was positively correlated with H-ras in glioblastomas (P = 0.005), but not in normal specimens. p21 protein was absent in all samples. Our findings provide evidence of K- and H-ras involvement in brain malignant transformation through transcriptional down-regulation, while N-ras seems to contribute less to brain carcinogenesis.
Asunto(s)
Neoplasias Encefálicas/genética , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Genes ras , Glioma/genética , Adulto , Anciano , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/mortalidad , Western Blotting/métodos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Estudios de Casos y Controles , Codón , Femenino , Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioma/metabolismo , Glioma/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Proteína Oncogénica p21(ras)/análisis , Proteína Oncogénica p21(ras)/metabolismo , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Estadísticas no Paramétricas , Tasa de SupervivenciaRESUMEN
BACKGROUND: We have previously shown a different local and systemic angiogenic profile of CXC chemokines in Idiopathic Pulmonary Fibrosis (IPF) patients compared to sarcoidosis. In particular, sarcoidosis showed an angiostatic microenvironment, as compared with the angiogenic cytokine milieu seen in IPF. Purpose of the Study. Our aim was to further investigate the aforementioned finding by measuring the expression of different chemokines in granulomatous and fibrotic diseases. We estimated the levels of vascular endothelial growth factor (VEGF) and its high-affinity receptor, Flt-1 (fms-like tyrosine kinase 1), in bronchoalveolar lavage fluid (BALF) of patients with IPF and pulmonary sarcoidosis. We have also investigated the mRNA expression of angiogenetic chemokines' receptors such as CXCR2 and CXCR3 and the biological axis of stromal derived factor-1 alpha (SDF-1 alpha or CXCL12 alpha/CXCL12 beta) and receptor, CXCR4. METHODS: We studied prospectively three groups of patients: (i) one group of 18 patients with IPF, (ii) one group of 16 patients with sarcoidosis, and (iii) 10 normal subjects. RESULTS: A statistically significant increase has been detected in VEGF mRNA expression in IPF in comparison with pulmonary sarcoidosis (P = .03). In addition, a significant increase has been measured in CXCL12 alpha in sarcoidosis in comparison to IPF (P = .02). Moreover, a statistically significant decrease has been found in Flt-1 protein levels in pulmonary sarcoidosis in comparison with IPF (P = .03). A significant increase in VEGF (P = .03) and CXCR4 (P = .03) mRNA levels has been also detected in sarcoidosis' patients when compared with healthy controls. CONCLUSIONS: Our data suggest that increased expression of Flt-1 and downregulation of CXCL12 alpha in IPF may further support the hypothesis of a different angiogenetic profile between fibrotic and granulomatous diseases. However, further studies are needed in order to better investigate these enigmatic diseases.
Asunto(s)
Fibrosis Pulmonar Idiopática/inmunología , ARN Mensajero/análisis , Sarcoidosis Pulmonar/inmunología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Líquido del Lavado Bronquioalveolar/química , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Femenino , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sarcoidosis Pulmonar/genética , Sarcoidosis Pulmonar/fisiopatología , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
A better understanding of the expression profile of a group of angiogenic markers in nasal polyps (NPs) would contribute considerably to the investigation of the formation of NPs. The aim of this study was to evaluate the combined mRNA expression of vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), transforming growth factor beta1 (TGFbeta1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF1) in NPs obtained from 21 patients undergoing nasal polypectomy. Nasal mucosae were obtained from the adjacent inferior turbinates (AIT) and middle turbinates (AMT) of the patients, as well as from 11 control subjects undergoing nasal corrective surgery. Analysis was performed using real-time RT-PCR. VEGFA, TGFbeta1 and IGF1 exhibited significant over-expression in the NPs compared to the control turbinates, EGF did not exhibit significant expression, and FGF2 presented constant over-expression in the NPs compared to both the adjacent and control turbinates. Since its mRNA levels were positively correlated with all the corresponding levels of the rest of the growth factors studied, TGFbeta1 seems to be a key cytokine in interactions between NP cells and the leading molecule of the epithelial differentiation and tissue remodelling present in the disease. Many correlations between the transcript levels of the other growth factors arose in the NP group as well, supporting a co-regulation of these genes in nasal polyposis. Our conclusions were that that VEGFA and TGFbeta1 participate significantly in the formation of NPs, whereas FGF2 and IGF1 are implicated in nasal polyposis to a lesser, but still significant, extent. EGF does not seem to be actively involved in the NP evolution process.