Acute lung injury (ALI) is associated with severe alterations in lung structure and function and is characterized by hypoxemia, pulmonary edema, low lung compliance and widespread capillary leakage. Asymmetric dimethylarginine (ADMA), a known cardiovascular risk factor, has been linked to endothelial dysfunction and the pathogenesis of a number of cardiovascular diseases. However, the role of ADMA in the pathogenesis of ALI is less clear. ADMA is metabolized via hydrolytic degradation to L-citrulline and dimethylamine by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Recent studies suggest that lipopolysaccharide (LPS) markedly increases the level of ADMA and decreases DDAH activity in endothelial cells. Thus, the purpose of this study was to determine if alterations in the ADMA/DDAH pathway contribute to the development of ALI initiated by LPS-exposure in mice. Our data demonstrate that LPS exposure significantly increases ADMA levels and this correlates with a decrease in DDAH activity but not protein levels of either DDAH I or DDAH II isoforms. Further, we found that the increase in ADMA levels cause an early decrease in nitric oxide (NO x ) and a significant increase in both NO synthase (NOS)-derived superoxide and total nitrated lung proteins. Finally, we found that decreasing peroxynitrite levels with either uric acid or Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTymPyp) significantly attenuated the lung leak associated with LPS-exposure in mice suggesting a key role for protein nitration in the progression of ALI. In conclusion, this is the first study that suggests a role of the ADMA/DDAH pathway during the development of ALI in mice and that ADMA may be a novel therapeutic biomarker to ascertain the risk for development of ALI.

erator-activated receptor type gamma (PPAR␥) is a subgroup of the PPAR transcription factor family. Recent studies indicate that loss of PPAR␥ is associated with the development of pulmonary hypertension (PH). We hypothesized that the endothelial dysfunction associated with PPAR␥ inhibition may play an important role in the disease process by altering cellular gene expression and signaling cascades. We utilized microarray analysis to determine if PPAR␥ inhibition induced changes in gene expression in pulmonary arterial endothelial cells (PAEC). We identified 100 genes and expressed sequence tags (ESTs) that were upregulated by Ͼ1.5-fold and 21 genes and ESTs that were downregulated by Ͼ1.3-fold (P Ͻ 0.05) by PPAR␥ inhibition. The upregulated genes can be broadly classified into four functional groups: cell cycle, angiogenesis, ubiquitin system, and zinc finger proteins. The genes with the highest fold change in expression: hyaluronan-mediated motility receptor (HMMR), VEGF receptor 2 (Flk-1), endothelial PAS domain protein 1 (EPAS1), basic fibroblast growth factor (FGF-2), and caveolin-1 in PAEC were validated by real time RT-PCR. We further validated the upregulation of HMMR, Flk-1, FGF2, and caveolin-1 by Western blot analysis. In keeping with the microarray results, PPAR␥ inhibition led to re-entry of cell cycle at G1/S phase and cyclin C upregulation. PPAR␥ inhibition also exacerbated VEGF-induced endothelial barrier disruption. Finally we confirmed the downregulation of PPAR␥ and the upregulation of HMMR, Flk-1, FGF2, and Cav-1 proteins in the peripheral lung tissues of an ovine model of PH. In conclusion, we have identified an array of endothelial genes modulated by attenuated PPAR␥ signaling that may play important roles in the development of PH. microarray; cell signaling; peroxisome proliferator-activated receptor PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-␥ (PPAR␥) was initially identified in adipose tissue and is a key regulator of lipid metabolism and glucose homeostasis (47, 50). Later, it was shown that PPAR␥ is also expressed in the vasculature including endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and monocytes/macrophages (13, 43). Upon activation by its ligands (fatty acids, arachidonic acid metabolites, and thiazolidinediones, etc), PPAR␥ forms heterodimers with the retinoid X receptor (RXR), and binds to specific PPAR response elements (PPRE) in the promoter region of its target genes, thereby regulating downstream gene expression (15,46). PPAR␥-mediated gene regulation can also be modulated by its interactions with specific co-activators and co-repressors (15, 26). PPAR␥ activation has been shown to alleviate atherosclerotic lesion formation and tumor progression (25, 30). The antiproliferative effects on VSMCs and antiangiogenic effects on tumor vasculogenesis by PPAR␥ activation seem to play important roles in these beneficial effects (9,31,53).Recently, it has been shown that loss of PPAR␥ is associated with pulmonary hypertension (PH) (35). PPAR␥ expression has been sho...

We showed that nitric oxide (NO) signaling is decreased in the pulmonary vasculature before the development of endothelial dysfunction in a lamb model of congenital heart disease and increased pulmonary blood flow (Shunt). The elucidation of the molecular mechanism by which this occurs was the purpose of this study. Here, we demonstrate that concentrations of the endogenous NO synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), are elevated, whereas the NOS cofactor tetrahydrobiopterin (BH(4)) is decreased in Shunt lambs. Our previous studies demonstrated that ADMA decreases heat shock protein-90 (Hsp90) chaperone activity, whereas other studies suggest that guanosine-5'-triphosphate cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the generation of BH(4), may be a client protein for Hsp90. Thus, we determined whether increases in ADMA could alter GCH1 protein and activity. Our data demonstrate that ADMA decreased GCH1 protein, but not mRNA concentrations, in pulmonary arterial endothelial cells (PAECs) because of the ubiquitination and proteasome-dependent degradation of GCH1. We also found that Hsp90-GCH1 interactions were reduced, whereas the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) increased in ADMA-exposed PAECs. The overexpression of CHIP potentiated, whereas a CHIP U-box domain mutant attenuated, ADMA-induced GCH1 degradation and reductions in cellular BH(4) concentrations. We also found in vivo that Hsp90/GCH1 interactions are decreased, whereas GCH1-Hsp70 and GCH1-CHIP interactions and GCH1 ubiquitination are increased. Finally, we found that supplementation with l-arginine restored Hsp90-GCH1 interactions and increased both BH(4) and NO(x) concentrations in Shunt lambs. In conclusion, increased concentrations of ADMA can indirectly alter NO signaling through decreased cellular BH(4) concentrations, secondary to the disruption of Hsp90-GCH1 interactions and the CHIP-dependent proteasomal degradation of GCH1.

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Sublethal plasma membrane disruption (PMD) is an established mechanism for signaling in several cell types, including endothelial cells and skeletal muscle. We used a rat model of orthodontic tooth movement to test the hypothesis that periodontal ligament (PDL) cells communicate stretch to changes in bone cell activity in part via PMD. To produce PMD, we used a 50-g load from a spring activated in the buccal direction against the maxillary first molars for 5 min. Uptake of endogenous serum albumin was used as a PMD marker. Immunohistochemistry demonstrates albumin in PDL cells surrounding moved first molar tips. Image analysis shows significantly more albumin in cells of the buccal side (tension) of the moved teeth compared with those of the lingual, distal, and mesial sides, and those of the unmoved control. Albumin localization within cells of the PDL, after only 5 min of mechanical loading, suggests that PMD could promote uptake or release of signaling molecules.