RESUMEN
Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.
Asunto(s)
Fibrosis Quística/patología , Endotelio Vascular/patología , Pulmón/patología , Arteria Pulmonar/patología , Sustitución de Aminoácidos , Biomarcadores/metabolismo , Adhesión Celular , Línea Celular Transformada , Proliferación Celular , Separación Celular , Supervivencia Celular , Células Cultivadas , Colagenasas/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/cirugía , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Impedancia Eléctrica , Endotelio Vascular/metabolismo , Humanos , Inmunofenotipificación , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/cirugía , Mutación , Neumonectomía , Arteria Pulmonar/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced transendothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a ß2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Endoteliales/patología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Proliferación Celular/fisiología , AMP Cíclico/metabolismo , Fibrosis Quística/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Homeostasis/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Insulina/farmacología , Interleucina-8/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxidos de Nitrógeno/metabolismo , Fosforilación , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arrestina beta 2/metabolismoRESUMEN
Colorectal cancer (CRC) is one of the most common malignancies worldwide and the lung is one of the most frequent sites for CRC metastasis. The geriatric population is increasing, but clinical decision making is often influenced by the effect of aging. For this reason, the elderly population does not often receive potentially curative cancer treatments as offered to younger ones. From January 2000 to March 2016, 21 elderly patients (older than 75 years) underwent pulmonary resections for colorectal cancer pulmonary metastases. A postoperative morbidity rate of 23.8 % and a 30-day mortality rate of 4.8 % were reported. A cumulative overall survival of 34.19 ± 23.51 months (95 % CI 23.71-50.28) and a disease-free interval of 24.62 ± 23.79 months (95 % CI 6.44-39.56) were observed. By considering only R0 surgically resected patients, the 1-, 3- and 5-year OS were 94.1, 59.5 and 21.2 % with a mean overall survival and disease-free interval of 51.10 ± 7.82 and 42.75 ± 9.35, respectively. Concerning risk factors, an important correlation between the number of pulmonary metastases, surgical radicality and overall survival was reported (p = 0.030 and p = 0.005, respectively). In summary, according to our series, pulmonary metastasectomy in selected elderly CRC oligometastatic patients seems to be safe and effective.