RESUMEN
Pulmonary arterial hypertension (PAH) is a fatal disease associated with severe remodeling of the large and small pulmonary arteries. Increased accumulation of inflammatory cells and apoptosis-resistant cells are contributing factors. Proliferative apoptosis-resistant cells expressing CD133 are increased in the circulation of PAH patients. Circulating cells can contribute to tissue repair via cell fusion and heterokaryon formation. We therefore hypothesized that in the presence of increased leukocytes and CD133-positive (CD133(pos)) cells in PAH lung tissue, cell fusion and resulting genomic instability could account for abnormal cell proliferation and the genesis of vascular lesions. We performed analyses of CD45/CD133 localization, cell fusion, and proliferation during late-stage PAH in human lung tissue from control subjects and subjects with idiopathic (IPAH) and familial (FPAH) PAH. Localization, proliferation, and quantitation of cell populations in individual patients were performed by immunolocalization. The occurrence of cellular fusion in vascular lesions was analyzed in lung tissue by fluorescence in situ hybridization. We found the accumulation of CD45(pos) leukocytic cells in the tissue parenchyma and perivascular regions in PAH patients and less frequently observed myeloid cells (CD45/CD11b). CD133(pos) cells were detected in occlusive lesions and perivascular areas in those with PAH and were more numerous in those with IPAH lesions than in FPAH lesions. Cells coexpressing CD133 and smooth muscle alpha-actin were occasionally observed in occlusive lesions and perivascular areas. Proliferating cells were more prominent in IPAH lesions and colocalized with CD45 or CD133. We found no evidence of increased ploidy to suggest cell fusion. Taken together, these data suggest that abnormal lesion formation in PAH occurs in the absence of cell fusion.
Asunto(s)
Apoptosis , Hipertensión Pulmonar/patología , Pulmón/patología , Células Mieloides/patología , Arteria Pulmonar/patología , Antígeno AC133 , Adulto , Antígenos CD/metabolismo , Fusión Celular , Proliferación Celular , Niño , Femenino , Células Gigantes/metabolismo , Células Gigantes/patología , Glicoproteínas/metabolismo , Humanos , Hipertensión Pulmonar/metabolismo , Antígenos Comunes de Leucocito/biosíntesis , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Células Mieloides/metabolismo , Péptidos/metabolismo , Arteria Pulmonar/metabolismoRESUMEN
Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.
Asunto(s)
Células Endoteliales/patología , Hiperoxia/patología , Pulmón/patología , Células Madre/patología , Animales , Animales Recién Nacidos , Vasos Sanguíneos/patología , Displasia Broncopulmonar/patología , Displasia Broncopulmonar/fisiopatología , Modelos Animales de Enfermedad , Humanos , Hiperoxia/fisiopatología , Técnicas In Vitro , Recién Nacido , Antígenos Comunes de Leucocito/metabolismo , Pulmón/irrigación sanguínea , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Microcirculación , Células Madre/metabolismo , Factores de Tiempo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.
Asunto(s)
Galectina 1/deficiencia , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Hipoxia/complicaciones , Hipoxia/fisiopatología , Animales , Secuencia de Bases , Enfermedad Crónica , Cartilla de ADN/genética , Matriz Extracelular/metabolismo , Galectina 1/genética , Galectina 1/fisiología , Técnicas In Vitro , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Ratones Noqueados , Microcirculación/metabolismo , Microcirculación/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ovinos , Resistencia Vascular/fisiologíaRESUMEN
BACKGROUND: The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice. METHOD: Mice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed. RESULTS: Hypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure [RVsP] 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia. CONCLUSION: In mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice.
Asunto(s)
Hipertensión Pulmonar/enzimología , Hipoxia/enzimología , Óxido Nítrico Sintasa/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Presión Sanguínea/fisiología , Western Blotting , Hematócrito , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Hipoxia/complicaciones , Hipoxia/patología , Inmunohistoquímica , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Función Ventricular DerechaRESUMEN
Vascular remodeling due to pulmonary arterial smooth muscle cell (PASMC) proliferation is central to the development of pulmonary hypertension. Cell proliferation requires the coordinated interaction of cyclins and cyclin-dependent kinases (cdk) to drive cells through the cell cycle. Cdk inhibitors can bind cyclin-cdk complexes and cause G(1) arrest. To determine the importance of the cdk inhibitor p27(Kip1) in PASMC proliferation we studied [(3)H]thymidine incorporation, changes in cell cycle, cell proliferation, and protein expression of p27(Kip1) following serum stimulation in early passage rat PASMC. p27(Kip1) expression decreased to 40% of baseline after serum stimulation, which was associated with an increase in both [(3)H]thymidine incorporation and the percent of cells in S phase. p27(Kip1) binding to cyclin E decreased at 24 h, and this correlated with an increase in phosphorylation of retinoblastoma both in vivo and in vitro. Overexpression of p27(Kip1) decreased [(3)H]thymidine incorporation and reduced cell counts at 5 d compared with controls. PASMC obtained from p27(Kip1-/-) mice showed a 2-fold increase in [(3)H]thymidine incorporation (at 24 h) and cell proliferation compared with p27(Kip1+/+) PASMC when cultured in 10% fetal bovine serum (FBS). These results suggest an important role for p27(Kip1) in regulating PASMC mitogenesis and proliferation.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Músculo Liso Vascular/citología , Arteria Pulmonar/citología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Sanguíneas/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , ADN/biosíntesis , Citometría de Flujo , Expresión Génica/fisiología , Hipertensión/metabolismo , Masculino , Músculo Liso Vascular/enzimología , Mutagénesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Arteria Pulmonar/enzimología , Ratas , Ratas Sprague-Dawley , Timidina/farmacocinética , TritioRESUMEN
The relationship between the number of species and the area sampled is one of the oldest and best-documented patterns in community ecology. Several theoretical models and field data from a wide range of plant and animal taxa suggest that the slope, z, of a graph of the logarithm of species richness against the logarithm of area is roughly constant, with z approximately 0.25. We collected replicated and randomized plant data at 11 spatial scales from 0.01 to 10(8) square meters in Great Britain which show that the slope of the log-log plot is not constant, but varies systematically with spatial scale, and from habitat to habitat at the same spatial scale. Values of z were low (0.1 to 0.2) at small scales (<100 square meters), high (0.4 to 0.5) at intermediate scales (1 hectare to 10 square kilometers), and low again (0.1 to 0.2) for the largest scale transitions (e.g., East Berks to all of Berkshire). Instead of one process determining changes in species richness across a wide range of scales, different processes might determine plant biodiversity at different spatial scales.