Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

Alpha-1-antitrypsin aerosolized augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro

Background: Neutrophil elastase (NE) activity is increased in lung diseases such as a1-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy.

Methods: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT.

Results: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy.

Conclusion: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.

Evaluation and outcome of patients with chronic non-productive cough using a comprehensive diagnostic protocol

Background-Asthma, post-nasal drip syndrome (PNDS), and gastrooesophageal reflux (GOR) account for many cases of chronic non-productive cough (CNPC). Each may simultaneously contribute to cough even when clinically silent, and failure to recognise their contribution may lead to unsuccessful treatment.

Methods—Patients (all lifetime non-smokers with normal chest radiographs and spirometric measurements) referred with CNPC persisting for more than three weeks as their sole respiratory symptom underwent histamine challenge, home peak flow measurements, ear, nose and throat (ENT) examination, sinus CT scanning, and 24 hour oesophageal pH monitoring. Treatment was prescribed on the basis of diagnoses informed by investigation results.

RESULTS—Forty three patients (29 women) of mean age 47.5 years (range 18-77) and mean cough duration 67 months (range 2-240) were evaluated. On the basis of a successful response to treatment, a cause for the cough was identified in 35 patients (82%) as follows: cough variant asthma (CVA) (10 cases), PNDS (9 cases), GOR (8cases), and dual aetiologies (8 cases). Histamine challenge correctly predicted CVA in 15 of 17 (88%) positive tests. ENT examination and sinus CT scans each had low positive predictive values for PNDS (10 of 16 (63%) and 12 of 18 (67%) positive cases, respectively), suggesting that upper airways disease frequently co-exists but does not always contribute to cough. When negative, histamine challenge and 24 hour oesophageal pH monitoring effectively ruled out CVA and GOR, respectively, as a cause for cough.

CONCLUSION—This comprehensive approach aids the accurate direction of treatment and, while CVA, PNDS and GOR remain the most important causes of CNPC to consider, a group with no identifiable aetiology remains.

Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses but the role of different ROS sources remains unclear. Here, we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level which increased in cardiomyocytes during stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte Hif1 and the release of VEGF, resulting in an increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a novel inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of non-specific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

ZapA, a virulence factor in a rat model of Proteus mirabilis-induced acute and chronic prostatitis.

Our knowledge of pathogenesis has benefited from a better understanding of the roles of specific virulence factors in disease. To determine the role of the virulence factor ZapA, a 54-kDa metalloproteinase of Proteus mirabilis, in prostatitis, rats were infected with either wild-type (WT) P. mirabilis or its isogenic ZapA- mutant KW360. The WT produced both acute and chronic prostatitis showing the typical histological progressions that are the hallmarks of these diseases. Infection with the ZapA- mutant, however, resulted in reduced levels of acute prostatitis, as determined from lower levels of tissue damage, bacterial colonization, and inflammation. Further, the ZapA- mutant failed to establish a chronic infection, in that bacteria were cleared from the prostate, inflammation was resolved, and tissue was seen to be healing. Clearance from the prostate was not the result of a reduced capacity of the ZapA- mutant to form biofilms in vitro. These finding clearly define ZapA as an important virulence factor in both acute and chronic bacterial prostatitis.

Development of a novel DNA microarray to detect bacterial pathogens in patients with chronic obstructive pulmonary disease (COPD)

A novel microarray was constructed with DNA PCR product probes targeting species specific functional genes of nine clinically significant respiratory pathogens, including the Gram-positive organisms (Streptococcus pneumoniae, Streptococcus pyogenes), the Gram-negative organisms (Chlamydia pneumoniae, Coxiella burnetii Haemophilus spp., Legionella pneumophila, Moraxella catarrhalis, and Pseudomonas aeruginosa), as well as the atypical bacterium, Mycoplasma pneumoniae. In a "proof-of-concept" evaluation of the developed microarray, the microarray was compared with real-time PCR from 14 sputum specimens from COPD patients. All of the samples positive for bacterial species in real-time PCR were also positive for the same bacterial species using the microarray. This study shows that a microarray using PCR probes is a potentially useful method to monitor the populations of bacteria in respiratory specimens and can be tailored to specific clinical needs such as respiratory infections of particular patient populations, including patients with cystic fibrosis and bronchiectasis. (C) 2010 Elsevier B.V. All rights reserved.

Acute and chronic effects of dietary fatty acids on cholecystokinin expression, storage and secretion in enteroendocrine STC-1 cells

Cholecystokinin (CCK) is a peptide hormone secreted from the I-cells of the intestine and it has important physiological actions related to appetite regulation and satiety. In this study we used STC-1 cells to investigate the effects of common dietary-derived fatty acids (FAs) on I-cell secretory function and metabolism. We extend earlier studies by measuring the acute and chronic effects of 11 FAs on CCK secretion, cellular CCK content, CCK mRNA levels, cellular DNA synthesis, cellular viability and cytotoxicity. FAs were selected in order to assess the importance of chain length, degree of saturation, and double bond position and conformation. The results demonstrate that secretory responses elicited by dietary FAs are highly selective. For example, altering the conformation of a double bond from cis to trans (i.e. oleic acid versus elaidic acid) completely abolishes CCK secretion. Lauric acid appears to adversely affect I-cell metabolism and arachidonic acid suppresses DNA synthesis. Our studies reveal for the first time that conjugated linoleic acid isoforms are particularly potent CCK secretagogues, which also boost intracellular stores of CCK. These actions of conjugated linoleic acid may explain satiating actions observed in dietary intervention studies.

Chronic treatment with a stable obestatin analogue significantly alters plasma triglyceride levels but fails to influence food intake, fluid intake, body weight, or body composition in rats.

Obestatin (OB(1-23) is a 23 amino acid peptide encoded on the preproghrelin gene, originally reported to have metabolic actions related to food intake, gastric emptying and body weight. The biological instability of OB(1-23) has recently been highlighted by studies demonstrating its rapid enzymatic cleavage in a number of biological matrices. We assessed the stability of both OB(1-23) and an N-terminally PEGylated analogue (PEG-OB(1-23)) before conducting chronic in vivo studies. Peptides were incubated in rat liver homogenate and degradation monitored by LC-MS. PEG-OB(1-23) was approximately 3-times more stable than OB(1-23). Following a 14 day infusion of Sprague Dawley rats with 50 mol/kg/day of OB(1-23) or a N-terminally PEGylated analogue (PEG-OB(1-23)), we found no changes in food/fluid intake, body weight and plasma glucose or cholesterol between groups. Furthermore, morphometric liver, muscle and white adipose tissue (WAT) weights and tissue triglyceride concentrations remained unaltered between groups. However, with stabilised PEG-OB(1-23) we observed a 40% reduction in plasma triglycerides. These findings indicate that PEG-OB(1-23) is an OB(1-23) analogue with significantly enhanced stability and suggest that obestatin could play a role in modulating physiological lipid metabolism, although it does not appear to be involved in regulation of food/fluid intake, body weight or fat deposition.

Bortezomib induces apoptosis in primitive chronic myeloid leukemia cells including LTC-IC and NOD/SCID repopulating cells

Chronic myeloid leukemia (CML) is treated effectively with tyrosine kinase inhibitors (TKIs); however, 2 key problems remain-the insensitivity of CML stem and progenitor cells to TKIs and the emergence of TKI-resistant BCR-ABL mutations. BCR-ABL activity is associated with increased proteasome activity and proteasome inhibitors (PIs) are cytotoxic against CML cell lines. We demonstrate that bortezomib is antiproliferative and induces apoptosis in chronic phase (CP) CD34(+) CML cells at clinically achievable concentrations. We also show that bortezomib targets primitive CML cells, with effects on CD34(+)38(-), long-term culture-initiating (LTC-IC) and nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells. Bortezomib is not selective for CML cells and induces apoptosis in normal CD34(+)38(-) cells. The effects against CML cells are seen when bortezomib is used alone and in combination with dasatinib. Bortezomib causes proteasome but not BCR-ABL inhibition and is also effective in inhibiting proteasome activity and inducing apoptosis in cell lines expressing BCR-ABL mutations, including T315I. By targeting both TKI-insensitive stem and progenitor cells and TKI-resistant BCR-ABL mutations, we believe that bortezomib offers a potential therapeutic option in CML. Because of known toxicities, including myelosuppression, the likely initial clinical application of bortezomib in CML would be in resistant and advanced disease. (Blood. 2010;115:2241-2250)