Lessons of the month 2: A forgotten cause of transient T-wave inversion.

A 19-year-old patient presented with severe chest pain, which is not typical for cardiac angina. However, his smoking history and the strong family history of ischaemic heart disease coupled with evidence of progressive T-wave changes on his electrocardiogram (ECG) caused dilemma in deciding further management. His blood tests were normal apart from hypophosphataemia, and he had two negative troponin results. His arterial blood gases showed respiratory alkalosis. He was given analgesia for a diagnosis of musculoskeletal chest pain and the next morning his ECG, arterial blood gases and phosphate levels all normalised. He had a normal echocardiogram and was reviewed by the cardiologist who diagnosed musculoskeletal chest pain which led to distress and hyperventilation causing hypophosphataemia and transient T-wave inversion. This case is a reminder of an under-recognised physiological phenomenon involving the cardiac conduction during hyperventilation.

Retinoic Acid Maintains Function of Neural Crest-Derived Ocular and Craniofacial Structures in Adult Zebrafish.

Purpose: Retinoic acid (RA) is required for embryonic formation of the anterior segment of the eye and craniofacial structures. The present study further investigated the role of RA in maintaining the function of these neural crest-derived structures in adult zebrafish. Methods: Morphology and histology were analyzed by using live imaging, methylacrylate sections, and TUNEL assay. Functional analysis of vision and aqueous humor outflow were assayed with real-time imaging. Results: Both decreased and increased RA signaling altered craniofacial and ocular structures in adult zebrafish. Exogenous treatment with all-trans RA for 5 days resulted in a prognathic jaw, while inhibition of endogenous RA synthesis through treatment with 4-diethylaminobenzaldehyde (DEAB) decreased head height. In adult eyes, RA activity was localized to the retinal pigment epithelium, photoreceptors, outer plexiform layer, inner plexiform layer, iris stroma, and ventral canalicular network. Exogenous RA increased apoptosis in the iris stroma and canalicular network in the ventral iridocorneal angle, resulting in the loss of these structures and decreased aqueous outflow. DEAB, which decreased RA activity throughout the eye, induced widespread apoptosis, resulting in corneal edema, cataracts, retinal atrophy, and loss of iridocorneal angle structures. DEAB-treated fish were blind with no optokinetic response and no aqueous outflow from the anterior chamber. Conclusions: Tight control of RA levels is required for normal structure and function of the adult anterior segment. These studies demonstrated that RA plays an important role in maintaining ocular and craniofacial structures in adult zebrafish.

Tubular expression of heat-shock protein 27 inhibits fibrogenesis in obstructive nephropathy.

Morphological changes that occur during kidney injury involve actin skeleton remodeling. Here we tested whether heat-shock protein 27 (HSP27), a small stress response protein involved in cytoskeletal remodeling, protects the kidney from tubulointerstitial fibrosis in obstructive nephropathy. Tubular cell HSP27 immunostaining was significantly increased in human kidneys with ureteropelvic junction obstruction, supporting the clinical relevance of our studies. To develop an animal model for mechanistic studies, we generated transgenic mice that specifically overexpress human HSP27 in renal tubules, under the kidney androgen-regulated protein promoter, and determined the effects of HSP27 overexpression on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis following unilateral ureteral obstruction. This was associated with decreased fibrogenesis as evidenced by significant declines in phosphorylated p38MAPK, collagen III, α-smooth muscle actin, 4-hydroxynonenal, and reduced trichrome staining following obstruction. Notably, E-cadherin and ß-catenin remained at the cell membrane of tubular cells in transgenic mice with an obstructed ureter. Monocyte/macrophage infiltration, however, was not significantly affected in these transgenic mice. Thus, tubular HSP27 inhibits fibrogenesis in obstructive nephropathy. Further studies are needed to determine pathways regulating the interactions between HSP27 and the E-cadherin-ß-catenin complex.

An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury.

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)-linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal-regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death-related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag-immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Phospho-akt expression is associated with a favorable outcome in non-small cell lung cancer.

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037).These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC.

Activation of p38 MAP kinase by asbestos in rat mesothelial cells is mediated by oxidative stress.

Asbestos fibers are biopersistent particles that are capable of stimulating chronic inflammatory responses in the pleura of exposed individuals. Exposure of pleural mesothelial cells, the progenitor cell of malignant mesothelioma, to asbestos induces an array of cellular responses. The present studies investigated whether the p38 mitogen-activated protein kinase cascade was induced under asbestos-exposed conditions. p38 plays a vital role in the response to stressful stimuli and enables the cell to enter an inflammatory state characterized by cytokine production. Western blot and in vitro kinase assays showed increases in dual phosphorylation and actual activity of p38 after exposure to fibrous and nonfibrous (milled) crocidolite; in contrast, polystyrene beads and iron (III) oxide had no such effects. In common with other asbestos-induced events, this was shown to be an oxidative stress-sensitive effect, inasmuch as preincubation with N-acetyl-L-cysteine or -tocopherol (vitamin E) ameliorated the effect. The present studies show that p38 activity is important for crocidolite-induced activator protein-1 DNA binding, inasmuch as an inhibitor of p38, SB-203580, reduced this activity. Crocidolite-induced cytotoxicity was also reduced with SB-203580, indicating a role for p38 in asbestos-mediated cell death. Our studies suggest that p38 activity could be a crucial factor in the chronic immune response elicited by asbestos and may represent a target for future pharmacological intervention.