The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.
It is unclear how sublethal hypoxia affects lung development. To investigate the effects of chronic hypoxia on postnatal lung remodeling, we treated neonatal rats with FIO 2 of 0.12 for 10 d and analyzed lung development by morphometry and gene expression by DNA microarray. Our results showed the neonatal rats exposed to hypoxia reduced body weight by 42% and wet lung weight by 32% compared with the neonatal rats exposed to normoxia. In the neonatal rats exposed to hypoxia, the radial alveolar counts were decreased to 5.6 from 7.9 and the mean linear intercepts were increased to 56.5 m from 38.2 m. In DNA microarray analysis, approximately half of probed genes were unknown. Chronic hypoxia significantly regulated expression of genes that are involved in pathogenesis of pulmonary hypertension and postnatal lung remodeling. Chemokine ligand 12, jagged 2 were among those upregulated; c-kit, ephrin A1, and Hif-2␣ were among those downregulated. The altered expression of those genes was correlated with the lung development and remodeling. (Pediatr Res 64: 56-62, 2008)
We assessed the mechanics and morphology of the lung in 165 rats treated neonatally with either room air (RA), O2, RA + steroids, or O2 + steroids. Newborn Sprague-Dawley male rats were randomly assigned to these groups. O2-exposure (0.96-1.0 FiO2) lasted 5 days, and dexamethasone treatment consisted of eight daily S.C. injections of drug or buffer in successive doses of 0.5, 0.4, 0.3, 0.2, 0.1, 0.1, 0.1, and 0.1 mg/kg. At 58 days, right ventricular systolic pressure (RVP) was measured. At 60 days, all rats were sacrificed for obtaining lung weight and DNA, saline pressure-volume (P-V) curves, and morphometry. We weighted right ventricles (RV) and left ventricles + septa (LV). Hyperoxia alone did not, but steroid decreased survival rate to 79.4% (95.3% in RA rats, P < 0.02). Only 21 of 40 (52%) O2 + steroids rats survived, less than in both RA groups (P < 0.001). RV weight, RVP and muscularization of alveolar duct arteries were significantly increased in O2 vs. RA rats. In RA + steroids rats, weight of the LV was decreased but RV, RVP, and lung vasculature were not affected. These effects were additive in the O2 + steroid group. Wet lung weights and DNA were increased for RA + steroid rats over all others. O2 and steroids shifted the P-V curve to the left and O2 + steroids still further. Maximal lung volume increased significantly with RA + steroids and still further in O2 + steroids but not in O2 alone. O2 and steroids significantly increased the mean linear intercept and O2 + steroids even more so.(ABSTRACT TRUNCATED AT 250 WORDS)
The aims of this study were to determine if neonatal hyperoxia exposure causes permanent lung damage and to define the relationship between neonatal lung oxygen toxicity and aging. Sprague-Dawley newborn rats (n = 85) breathed 100% oxygen (O2) or room air (RA) during the first 8 days of life, and then RA. At 2 and 22 months of age we assessed right ventricular (RV) systolic pressure (RVSP), RV weight, saline and air pressure-volume curves, volume density of lung parenchyma and nonparenchyma, parenchymal air space (PAS), mean linear intercept (Lm), number of small arteries/mm2 and the extent of their medial muscularization. Aging in RA did not affect the RVSP, RV weight, the number of small arteries/mm2, or their muscularization. The maximal lung volume/g of dry lung and the elastic recoil pressure between 40 and 90% maximal lung volume decreased. The volume density of lung parenchyma increased but the fraction of the lung parenchyma that was PAS decreased and that of the alveolar septa and Lm increased. The O2-treated rats at 60 days of age had increased RVSP and RV weights with a decrease in the small arteries/mm2. The lung parenchymal volume density and PAS increased and the density of alveolar septa decreased. The Lm increased and the alveoli/mm2 and elastic recoil pressure decreased. The lung damage seen in the O2-treated rats at 60 days persisted and in addition underwent the changes seen in the aging controls. However, the extent of muscularization of the arteries decreased. We conclude that neonatal hyperoxia causes permanent functional and structural changes of the lung but these do not interact with aging; that is, the effects of O2 toxicity and aging are additive but not synergistic.
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