The combined effects of hypoxia and interleukin 1, lipopolysaccharide, or tumor necrosis factor a on the expression of genes encoding endothelial constitutive and inducible nitric oxide synthases, endothelin 1, interleukin 6, and interleukin 8 were investigated in human primary pulmonary endothelial cells and whole pulmonary artery organoid cultures. Hypoxia decreased the expression of constitutive endothelial nitric oxide synthase (NOS-3) mRNA and NOS-3 protein as compared with normoxic conditions. The inhibition of expression of NOS-3 corresponded with a reduced production of NO. A combination of hypoxia with bacterial lipopolysaccharide, interleukin 113, or tumor necrosis factor a augmented both effects. In contrast, the combination of hypoxia and the inflammatory mediators superinduced the expression of endothelin 1, interleukin 6, and interleukin 8. Here, we have shown that inflammatory mediators aggravate the effect of hypoxia on the down-regulation of NOS-3 and increase the expression of proinflammatory cytokines in human pulmonary endothelial cells and whole pulmonary artery organoid cultures.The pathogenesis of pulmonary hypertension is unknown. However, both experimental and clinical evidence indicate that hypoxia or inflammation dramatically aggravates the extent of pulmonary hypertension, e.g., during acute bacterial infection in patients with chronic obstructive pulmonary disease. Experimental evidence suggests endothelial nitric oxide production as the main regulator ofvascular tone in pulmonary arteries. Nitric oxide (NO) is mainly produced by three isoforms of NO synthases (NOS 1-3), two of which are constitutively present in neuronal or endothelial cells (NOS-1 or NOS-3, respectively) (1). In human umbilical vein endothelial cells, hypoxia was found to be responsible for both a decrease of human NOS-3 and an increase of human endothelin 1 (ET-1) mRNA (2). Conversely, exposure of rat lungs to hypoxia for more than 1 week caused an increase of both NOS-3 mRNA and protein leading to a doubled production of NO (3). In short-term cultures using human aortic endothelial cells, inflammatory mediators have been demonstrated both to increase the mRNA of the inducible isoform of nitric oxide synthases (iNOS or NOS-2) and to decrease the amount of NOS-3 mRNA, which may imply a major influence of NOS-2 on the regulation of vascular tone during inflammatory states (4). NO has been shown to down-regulate the expression of vasoconstrictors and growth factors such as ET-1, interleukin 1 (IL-1), and platelet-derived growth factor in human umbilical vein endothelial cells (5). Similarly, proinflammatory cytokines were shown to induce the synthesis of NO, suggesting a crucial role of inflammatory mediators in the regulation of arterial tissues (6). Analogously, hypoxia stimulated the induction of major vascular growth factors such as ET-1 (5) and platelet-derived growth factor B (PDGF-B) (8). To evaluate the influence of inflammation and/or hypoxia on the regulation of NO biosynthesis, we characterized the...
The formation and development of nucleoli and their connections with the nucleolar chromosomes were studied in human spermatocytes using electron microscopy, silver staining of nucleolus organizer regions (NORs), high resolution autoradiography and in situ hybridization in order to localize rRNA genes and their transcription in the different stages of meiotic prophase I. At leptotene, new nucleoli were formed, consisting of a fibrillar centre surrounded by a cap of dense fibrillar component. Following [3H]uridine uptake, label was found only over the dense fibrillar component. In situ hybridization revealed rDNA mainly in the dense fibrillar component and in the chromatin. During zygotene, nucleoli increased in size. The fibrillar centre was connected with the secondary constriction region of the nucleolar bivalent and was partially surrounded by dense fibrillar component. This shell of dense fibrillar component merged into a fibrillo-granular mesh that extended away from the fibrillar centre. Autoradiography following [3H]uridine uptake again showed the label overlaying the dense fibrillar component and the proximal part of the fibrillo-granular strands. With in situ hybridization in both the light and electron microscope, signal was mainly found in the dense fibrillar component. A small quantity of label was observed in the peripheral region of the fibrillar centre and in the adjacent chromatin. From early to late pachytene segregation of nucleolar components occurred, with a reduction in the dense fibrillar component that formed a narrow rim around the fibrillar centre with small extensions along the granular component. [3H]uridine incorporation progressively decreased. In situ hybridization showed signal located mainly in the dense fibrillar component and in the chromatin corresponding to the condensed short arm of the nucleolar bivalent. Our results indicate that the majority of rDNA is located and transcribed in the dense fibrillar component; only a small amount is present in the peripheral part of the fibrillar centre and may be transcribed there. Moreover, from leptotene to zygotene, rDNA unravels from the nucleolar chromosome into the nucleolar dense fibrillar component. From zygotene to late pachytene a progressive return to the condensed acrocentric short arm is observed.
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