Ikechukwu I. Ekekezie

T1α/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

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.

Independent and Combined Effects of Prolonged Inhaled Nitric Oxide and Oxygen on Lung Inflammation in Newborn Piglets

Clinical use of nitric oxide (NO) is usually in conjunction with high oxygen concentrations, the effects of which may include lung neutrophil accumulation, apoptosis and upregulation of antioxidant enzyme activity. To define the effects of NO on neutrophils from young piglets and its relationship to lung neutrophil dynamics during hyperoxia we exposed thirty piglets to room air (RA), RA+NO (50 ppm NO), O2 (FiO2≥0.96) or O2+NO for 5 days. Ten additional animals breathed RA+NO or O2+NO, then recovered in RA for 3 days before sacrifice. Neutrophil CD18 and intracellular oxidant production were measured by flow cytometry. Lung apoptosis were assessed by TUNEL assay. Lung myeloperoxidase, SOD and catalase were measured biochemically. When compared to RA group, there was significant reduction in neutrophil CD18 and intracellular oxidant production in the RA+NO group, but lung MPO was unchanged. The O2 and O2+NO groups did not differ in CD18 expression or in intracellular oxidant production, but had significant increase in lung myeloperoxidase compared to the RA group. Apoptosis increased significantly only in the O2+NO group. The O2 group showed significantly increased lung SOD and catalase activity compared to the RA group, whereas the RA+NO and O2+NO groups did not. We conclude that inhaled NO at 50 ppm decreases neutrophil CD18 expression as well as intracellular oxidant production. However, this effect does not impact lung neutrophil accumulation during concurrent hyperoxia. The combination of NO and O2 exposure produces an increase in lung apoptosis. Finally, NO may prevent upregulation of SOD and catalase activity during hyperoxia, potentially increasing injury.

Endostatin and vascular endothelial cell growth factor (VEGF) in piglet lungs: Effect of inhaled nitric oxide and hyperoxia

Pulmonary hyperoxic injury manifests as widespread alveolar-epithelial and microvascular endothelial cell necrosis, resolution of which requires angiogenesis. We investigated the hypothesis that inhaled nitric oxide (iNO) and hyperoxia each decreases lung vascular endothelial growth factor (VEGF) expression but increases endostatin and that concurrent administration of both gases will show a greater effect. Piglets were randomized to breathe for 5 d room air (RA); RA + NO (RA + 50 ppm NO), O2 (hyperoxia, FIO2 >0.96), O2 + NO, or O2 + NO + REC (O2 + NO plus recovery in 50% O2 for 72 h. After the piglets were killed, we measured lung capillary leak, VEGF mRNA, VEGF, and endostatin protein in homogenates, plasma, and lavage. VEGF mRNA decreased significantly with O2 and O2 + NO compared with breathing RA (p ≤ 0.05). VEGF protein declined in the experimental groups with a significant reduction in the recovery group compared with the RA group (p ≤ 0.05). Similar but more dramatic, endostatin declined in all groups relative to the RA group (p < 0.001). Lavage fluid VEGF protein and lung capillary leak rose significantly with O2 and O2 + NO compared with RA, but endostatin was unchanged. At 72 h of recovery from hyperoxia, VEGF mRNA and lavage fluid VEGF but not lung VEGF protein had normalized. Hyperoxia and iNO suppresses lung endostatin expression, but iNO unlike hyperoxia alone does not alter lung VEGF production. Hyperoxia paradoxically raises lavageable VEGF levels. This latter effect and that on VEGF mRNA level but not protein is abrogated by recovery in reduced FIO2 for 72 h.

Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.

Chronic hypoxia and rat lung development: analysis by morphometry and directed microarray.

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 FIO2 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 μm from 38.2 μm. 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-2α were among those downregulated. The altered expression of those genes was correlated with the lung development and remodeling.

High-dose inhaled nitric oxide and hyperoxia increases lung collagen accumulation in piglets

Nitric oxide (NO), a pro-oxidant gas, is used with hyperoxia (O2) to treat neonatal pulmonary hypertension and recently bronchopulmonary dysplasia, but great concerns remain regarding NO’s potential toxicity. Based on reports that exposure to oxidant gases results in pulmonary extracellular matrix injury associated with elevated lavage fluid levels of extracellular matrix components, we hypothesized that inhaled NO with or without hyperoxia will have the same effect. We measured alveolar septal width, lung collagen content, lavage fluid hydroxyproline, hyaluronan and laminin levels in neonatal piglets after 5 days’ exposure to room air (RA), RA + 50 ppm NO (RA + NO), O2 (FiO2 > 0.96) or O2 + NO. Matrix metalloproteinase (MMP) activity and MMP-2 mRNA were also measured. In recovery experiments, we measured lung collagen content in piglets exposed to RA + NO or O2 + NO and then allowed to recover for 3 days. The results show that lung collagen increased 4-fold in the RA + NO piglets, the O2 and O2 + NO groups had only a 2-fold elevation relative to RA controls. Unlike the RA + NO piglets, the O2 and O2 + NO groups had more than 20-fold elevation in lung lavage fluid hydroxyproline compared to the RA group. O2 and O2 + NO also had increased lung MMP activity, extravascular water, and lavage fluid proteins. MMP-2 mRNA levels were unchanged. After 3 days’ recovery in room air, the RA + NO groups’ lung collagen had declined from 4-fold to 2-fold above the RA group values. The O2 + NO group did not decline. Alveolar septal width increased significantly only in the O2 and O2 + NO groups. We conclude that 5 days’ exposure to NO does not result in pulmonary matrix degradation but instead significantly increases lung collagen content. This effect appears potentially reversible. In contrast, hyperoxia exposure with or without NO results in pulmonary matrix degradation and increased lung collagen content. The observation that NO increased lung collagen content represents a new finding and suggests NO could potentially induce pulmonary fibrosis.

Monocyte chemoattractant protein-1 and its receptor CCR-2 in piglet lungs exposed to inhaled nitric oxide and hyperoxia.

Monocyte chemoattractant protein-1 (MCP-1), acting through its C-C chemokine receptor 2 (CCR-2), has important roles in inflammation, angiogenesis, and wound repair. The individual and combined effects of inhaled nitric oxide (NO) and hyperoxia on lung MCP-1 and CCR-2 in relation to lung leukocyte dynamics are unknown. Because MCP-1 gene is up-regulated by oxidants, we hypothesized that inhaled NO with hyperoxia will increase MCP-1 production and CCR-2 expression more than either gas alone. We randomly assigned young piglets to breathe room air (RA), RA+50 ppm NO (RA+NO), O2, or O2+NO for 1 or 5 d before sacrifice. Lungs were lavaged and tissues preserved for hybridization studies, Western blotting, histology, and immunohistochemistry. The results show that lung MCP-1 production and alveolar macrophage count were significantly elevated in the 5-d O2 and O2+NO groups relative to the RA group (p ≤ 0.05). In contrast, lung CCR-2 abundance was diminished in the O2 group (p ≤ 0.05), but not in the O2+NO group, compared with the RA group. No difference was detected in any variable studied at 24 h. CCR-2 distribution was similar in all groups with staining of alveolar septa, macrophages, vascular endothelium, and the luminal epithelial surface of airways. We conclude that although hyperoxia increases MCP-1 in young piglet lungs, it also decreases CCR-2 abundance, which may limit participation of MCP-1 in alveolar macrophage recruitment. Inhaled NO, unlike hyperoxia, has no significant independent effect, but its concurrent administration during hyperoxia attenuates the decremental effect of hyperoxia on CCR-2 abundance.

Higher TRIP-1 level explains diminished collagen contraction ability of fetal versus adult fibroblasts

Acute lung injury involving extremely immature lungs often heals without excessive fibrosis unlike later in gestation and in adults. Several factors may be involved, but fibroblast contraction of collagen has been linked to the level of wound fibrosis. To assess whether human lung fibroblasts of fetal versus adult origin differ in ability to contract collagen and define the molecular underpinnings, we performed three-dimensional collagen contraction assay, analyzed their differential mRNA profile, specifically for transforming growth factor-beta (TGF-beta) signaling pathway and extracellular matrix components, studied the cell response to TGF-beta in culture, and used two-dimensional gel electrophoresis followed by mass spectrometry to identify differences in their overall proteomes. Human lung fetal fibroblasts contracted the collagen matrix less than the adults. Smooth muscle actin expression did not differ. TGF-beta stimulation resulted in greater Smad3 phosphorylation in fetal compared with adults. mRNA and proteomic profiling reveal a number of TGF-beta pathways, ECM components, and cytoskeletal regulatory molecules are differentially expressed between the cell types. Of note is TGF-beta receptor interacting protein 1 (TRIP-1), which we show inhibits fibroblast collagen contraction and is higher in fetal than adult fibroblasts. We conclude that human lung fetal fibroblasts are less able to contract collagen than adult lung fibroblasts. The diminished ability is not due to impediment of Smad3 activation but rather, at least in part, due to their higher level of TRIP-1 expression. TRIP-1 is a novel modulator of fibroblast collagen contraction.

TRIP-1 regulates TGF-β1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-β receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-β type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-β1 action. TGF-β1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-β1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-β1 induction of EMT. Mechanistically, a pathway involving increased TGF-β type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

Hyperoxia and tidal volume: Independent and combined effects on neonatal pulmonary inflammation.

Background: Hyperoxia and tidal volume mechanical ventilation are independent factors in the genesis of lung injury, but it remains unclear the extent to which each is responsible or contributes to this process in newborns. Objectives: To study the independent and combined effects of hyperoxia and tidal volume mechanical ventilation on the induction of lung inflammation in a newborn piglet model of ventilator-induced lung injury. Methods: Following exposure to either ambient air or FIO2 = 1.0 for a period of 3 days, newborn piglets were randomized to receive mechanical ventilation with either high tidal volume (20 ml/kg) or low tidal volume (6 ml/kg) for 4 h while controlling for pH. Results: Monocyte chemoattractant protein-1 level in the lungs of animals randomized to hyperoxia with high tidal volume ventilation was significantly elevated, compared to all other groups (p < 0.05). Myeloperoxidase assayed in lung homogenate was found to be significantly higher in nonventilated animals exposed to hyperoxia (p < 0.01). Only in animals previously exposed to hyperoxia did the addition of high tidal volume ventilation further increase the level of myeloperoxidase present (p < 0.05). Pulmonary vascular resistance was significantly elevated after 4 h of mechanical ventilation compared to 1 h (p < 0.001). Conclusions: We conclude that in neonatal piglets undergoing hyperoxic stress, superimposition of high tidal volume ventilation exacerbates the lung inflammation as assessed by lung monocyte chemoattractant protein-1 and level of myeloperoxidase.