Mo Rezaiekhaligh

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.

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.

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.

Responses of pulmonary platelet-derived growth factor peptides and receptors to hyperoxia and nitric oxide in piglet lungs.

The peptides platelet-derived growth factor-A (PDGF-A) and especially -B have important roles in lung development. The effect of hyperoxic exposure with and without inhaled nitric oxide (iNO) on lung expression of PDGF and its receptors is unknown. We hypothesized that hyperoxia exposure would suppress mRNA expression and protein production of these ligands and their receptors. The addition of iNO to hyperoxia may further aggravate the effects of hyperoxia. Thirteen-day-old piglets were randomized to breathe 1) room air (RA); 2) 0.96 fraction of inspired oxygen (O2), or 3) 0.96 fraction of inspired oxygen plus 50 ppm of NO (O2 + NO), for 5 d. Lungs were preserved for mRNA, Western immunoblot, and immunohistochemical analyses for PDGF-A and -B and their receptors PDGFR-α and -β. PDGF-B mRNA expression was greater than that of PDGF-A or PDGFR-α and -β in RA piglet lungs (p < 0.05). Hyperoxia with or without iNO reduced lung PDGF-B mRNA and protein expression relative to the RA group lungs (p < 0.01). PDGF-B immunostain intensity was significantly increased in the alveolar macrophages, which were present in greater numbers in the hyperoxia-exposed piglet lungs, with or without NO (p < 0.01). PDGFR-β immunostaining was significantly increased in airway epithelial cells in O2- and O2 + NO–exposed piglets. PDGF-A and PDGFR-α immunostain intensity and distribution pattern were unchanged relative to the RA group. Sublethal hyperoxia decreases PDGF-B mRNA and protein expression but not PDGF-A or their receptors in piglet lungs. iNO neither aggravates nor ameliorates this effect.

ISOPROSTANES IN NEONATAL PULMONARY DISEASES: POSSIBLE MARKER OF LUNG INJURY. |[dagger]| 1980

The contribution to neonatal pulmonary injury by reactive oxygen species has been difficult to assess. The recent discovery of a novel class of prostaglandins, the isoprostanes, may help as these substances appear to be generated in response to lipid peroxidation. Isoprostanes are noncylooxygenase derived and are produced in vivo. They may contribute to pulmonary pathophysiology by their vasoconstrictive properties. We determined if 8-epi-PGF2α, a member of the isoprostane family, would be detectable in the urine or tracheal aspirates of infants ≥ 37 weeks GA with pulmonary disorders treated with assisted ventilation and FiO2 of 1.0 and in control infants without pulmonary disorders, breathing room air. Isoprostane levels were measured by enzyme immune assay (EIA). Urine levels were normalized by the urine creatinine and tracheal aspirate levels by free Secretory Component (fSC) of IgA, also measured by EIA. Results are as follows: Table The 24 and 48 hr tracheal aspirate values in sick infants were not different. When sick and control groups combined data were compared, there was a significant difference (p<0.02). No differences were found in urine values. Serial urine samples were obtained from a separate group of 15 term healthy infants to analyze isoprostane levels at different age periods. Table

DNA methylation levels associated with race and childhood asthma severity

ABSTRACT Objective: Asthma is a common chronic childhood disease worldwide. Socioeconomic status, genetic predisposition and environmental factors contribute to its incidence and severity. A disproportionate number of children with asthma are economically disadvantaged and live in substandard housing with potential indoor environmental exposures such as cockroaches, dust mites, rodents and molds. These exposures may manifest through epigenetic mechanisms that can lead to changes in relevant gene expression. We examined the association of global DNA methylation levels with socioeconomic status, asthma severity and race/ethnicity. Methods: We measured global DNA methylation in peripheral blood of children with asthma enrolled in the Kansas City Safe and Healthy Homes Program. Inclusion criteria included residing in the same home for a minimum of 4 days per week and total family income of less than 80% of the Kansas City median family income. DNA methylation levels were quantified by an immunoassay that assessed the percentage of 5-methylcytosine. Results: Our results indicate that overall, African American children had higher levels of global DNA methylation than children of other races/ethnicities (p = 0.029). This difference was more pronounced when socioeconomic status and asthma severity were coupled with race/ethnicity (p = 0.042) where low-income, African American children with persistent asthma had significantly elevated methylation levels relative to other races/ethnicities in the same context (p = 0.006, Hedges g = 1.14). Conclusion: Our study demonstrates a significant interaction effect among global DNA methylation levels, asthma severity, race/ethnicity, and socioeconomic status.

Hyperoxia and Inhaled NO: Evidence for Extracellular Matrix Damage† 1646

Inhaled nitric oxide (NO) is now established as a selective pulmonary vasodilator. Toxicity potential of NO however remains a great concern. The postulated injurious mechanisms include NO-induced activation of matrix metalloproteinases (MMP) with consequent breakdown of the alveolar extracellular matrix. Endothelin-1 (ET-1) is reportedly a marker of acute pulmonary injury. 8-Isoprostanes are lipid peroxidation products of oxyradical-induced cell membrane injury. We hypothesized that lung lavage fluid from animals exposed to hyperoxia and NO will show increased laminin and hydroxyproline contents as early markers of alveolar capillary basement membrane and matrix breakdown. Additionally, we reasoned that lung lavage fluid ET-1 and 8-isoprostane concentration will correlate with laminin and hydroxyproline concentration. To test this hypothesis, we designed the following study. Twenty-eight newborn piglets were randomized into four exposure groups to breathe for five days in a 200-liter exposure chamber. The groups were (i) room air (RA), (ii) RA+50 ppm NO, (RA+NO), (iii) hyperoxia(FiO2≥0.96) (O2), and (iv) hyperoxia + 50 ppm NO(O2+NO). Lung lavage fluid was obtained and analyzed by ELISA to quantitate laminin, ET-1, and 8-isoprostane concentrations. Lavage fluid hydroxyproline was measured by a colorimetric method. Mean hydroxyproline levels were about twenty fold higher in the O2 and O2 + NO group as compared to other groups. ET-1 concentration was higher in the O2 + NO group than in any other group (P <.02). Lung lavage fluid Laminin and 8-Isoprostane concentration did not differ significantly amongst the groups. We conclude, hyperoxia alone or in combination with 50 ppm NO increases lung lavage fluid hydroxyproline levels. This may reflect increased extracellular matrix breakdown. NO did not exacerbate nor ameliorate this effect of oxygen. Elevated ET-1 concentration may reflect inflammatory induction and airway hyperreactivity.