Changaram S. Venugopal

The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension.

RATIONALE An activated vasoconstrictive, proliferative, and fibrotic axis of the renin angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin [Ang]II/AngII type 1 receptor) has been implicated in the pathophysiology of pulmonary fibrosis (PF) and pulmonary hypertension (PH). The recent discovery of a counterregulatory axis of the renin angiotensin system composed of ACE2/Ang-(1-7)/Mas has led us to examine the role of this vasoprotective axis on such disorders. OBJECTIVES We hypothesized that Ang-(1-7) treatment would exert protective effects against PF and PH. METHODS Lentiviral packaged Ang-(1-7) fusion gene or ACE2 cDNA was intratracheally administered into the lungs of male Sprague Dawley rats. Two weeks after gene transfer, animals received bleomycin (2.5 mg/kg). In a subsequent study, animals were administered monocrotaline (MCT, 50 mg/kg). MEASUREMENTS AND MAIN RESULTS In the PF study, bleomycin administration resulted in a significant increase in right ventricular systolic pressure, which was associated with the development of right ventricular hypertrophy. The lungs of these animals also exhibited excessive collagen deposition, decreased expression of ACE and ACE2, increased mRNA levels for transforming growth factor β and other proinflammatory cytokines, and increased protein levels of the AT₁R. Overexpression of Ang-(1-7) significantly prevented all the above-mentioned pathophysiological conditions. Similar protective effects were also obtained with ACE2 overexpression. In the PH study, rats injected with MCT developed elevated right ventricular systolic pressure, right ventricular hypertrophy, right ventricular fibrosis, and pulmonary vascular remodeling, all of which were attenuated by Ang-(1-7) overexpression. Blockade of the Mas receptor abolished the beneficial effects of Ang-(1-7) against MCT-induced PH. CONCLUSIONS Our observations demonstrate a cardiopulmonary protective role for the ACE2/Ang-(1-7)/Mas axis in the treatment of lung disorders.

Insulin resistance in equine digital vessel rings: An in vitro model to study vascular dysfunction in equine laminitis

REASONS FOR PERFORMING STUDY One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. HYPOTHESIS AND OBJECTIVES Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. METHODS Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrodes solution at 37°C, 2 g tension was applied and the rings allowed to equilibrate for 45 min. Of the 4 rings of each vessel type, one was used as a control. One each of the remaining 3 rings was used for incubation with insulin (to induce resistance), wortmannin (to block PI3-kinase) and PD-098059 (to block MAP-kinase), respectively, for 30 min. After the incubation period, the rings were contracted with phenylephrine. When the response reached a plateau, a single dose of insulin was added to the baths and the response of each ring monitored for 30 min. RESULTS Insulin relaxed the control rings and those treated with PD 098059 but contracted those pretreated with insulin and wortmannin. Normal relaxation responses of the rings were converted to contractions by insulin resistance. Insulin resistance was confirmed by the qualitative response of insulin-incubated and wortmannin-incubated rings. CONCLUSIONS This study demonstrated successful induction of insulin resistance in both arterial and venous rings. It also suggested that the MAP-kinase pathway plays a minor role in controlling vasomotor tone under normal physiological conditions. POTENTIAL RELEVANCE The study suggests that the induction of insulin resistance in equine palmar digital vessel rings is reliable and provides a good in vitro model for studying the vascular insulin resistance which may occur in equine laminitis.

Plasma and pulmonary fluid endothelin in horses with seasonal recurrent airway obstruction.

BACKGROUND Summer pasture-associated recurrent airway obstruction (SPA-RAO), a seasonal airway obstructive disease of horses, is characterized by clinical exacerbation after exposure to pasture during warm months of the year. Endothelin (ET)-1, potent bronchoconstrictor, mitogen, secretagogue, and proinflammatory mediator, has been implicated in the pathogenesis of asthma and equine heaves. HYPOTHESIS Immunoreactive ET-1 concentrations increase during clinical exacerbation and return to basal values during periods of disease remission. ANIMALS Twelve horses, 6 affected with SPA-RAO and 6 nonaffected. METHODS Prospective, observational study. Bronchoalveolar lavage fluid (BALF), arterial and venous plasma samples, and clinical variables were obtained from affected horses during clinical exacerbation and remission. Samples and data of nonaffected horses were collected during the summer and winter on dates similar to affected horses. Immunoreactive ET-1 was determined using a commercial ELISA. RESULTS The median and range ET-1 concentrations (pg/ml) in arterial (1.3, 0.7-1.8) and venous (1.3, 1.2-1.7) plasma and in BALF (0.3, 0.2-0.4), and pulmonary epithelial lining fluid (PELF) (25.5, 21-50) were greater in affected horses during clinical exacerbation compared with remission (P < .01). The concentrations of immunoreactive ET-1 were greater in affected horses during clinical exacerbation compared with nonaffected horses (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE During clinical exacerbation of SPA-RAO, ET-1 is increased in circulation and pulmonary secretions. Intervention with ET receptor antagonists should provide further information on the role of ET-1 in SPA-RAO.

Effect of potential therapeutic agents in reducing oxidative stress in pulmonary tissues of recurrent airway obstruction-affected and clinically healthy horses.

REASONS FOR PERFORMING STUDY To determine and compare the reactive oxygen and nitrogen species (ROS and RNS) in pulmonary tissues of horses affected with recurrent airway obstruction (RAO) and clinically healthy horses, and to evaluate the effectiveness of potential therapeutic agents in reducing ROS and RNS in the tissues of these horses. OBJECTIVES We hypothesised that RAO-affected horses would have high levels of reactive species and that the test agents would reduce them. The objectives were as follows: 1) to determine the level of ROS and RNS in pulmonary tissues (bronchial and arterial rings) of RAO-affected and clinically healthy horses; and 2) to determine the ability of pentoxifylline, pyrrolidine-dithiocarbamate and a combined use of endothelin A and B receptor antagonists (BQ123 and BQ788, respectively) in reducing reactive species. METHODS Arterial and bronchial rings were collected from the diaphragmatic lung lobe of each horse immediately after euthanasia. The levels of ROS and RNS were measured in control tissues and those incubated with test agents, using an electron paramagnetic resonance instrument. RESULTS The levels of ROS and RNS were significantly greater in arterial and bronchial tissues of RAO-affected than of clinically healthy horses. Pentoxifylline and endothelin antagonists reduced both ROS and RNS in tissues from RAO-affected horses. Basal levels of reactive species in clinically healthy horses were not affected by these agents. No difference in the level of reactive species was observed between arterial and bronchial tissues. CONCLUSIONS Horses affected by RAO had higher ROS and RNS than clinically healthy horses. Pentoxifylline and endothelin antagonists effectively reduced ROS and RNS in pulmonary tissues of RAO-affected horses. POTENTIAL RELEVANCE The study suggested a potential use for pentoxifylline and endothelin antagonists in treating RAO-affected horses. As endothelin is involved in physiological functions, therapeutic use of its antagonists is cautioned.

Transcriptional changes associated with recurrent airway obstruction in affected and unaffected horses.

OBJECTIVE To identify differentially expressed genes in pulmonary tissues of horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD), which is a form of recurrent airway obstruction (RAO), compared with those of unaffected horses. ANIMALS 6 horses with SPAOPD-RAO and 6 unaffected (healthy) horses. PROCEDURES Horses were assigned to 2 groups on the basis of medical history, clinical score, and transpleural pressure. Total RNA from each of the 5 lung lobes of each of the 6 SPAOPD-RAO-affected horses was extracted and pooled. Similarly, total RNA from unaffected horses was pooled. Differential display (DD) PCR assay was performed, and differentially expressed bands were purified and cloned into a plasmid vector. Plasmids were extracted from recombinant colonies, and purified DNA was sequenced. Genes of interest for RAO pathogenesis were identified. Real-time PCR assay was performed to confirm findings for the DD PCR assay. RESULTS 18 differentially expressed genes (17 upregulated and 1 downregulated) were identified. Three genes of particular interest were found to be altered (2 upregulated and 1 downregulated) in horses with SPAOPD-RAO by use of real-time PCR assay, and these findings matched the differential expression found by use of the DD PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE SPAOPD-RAO in horses is a multifactorial, complex disease involving several genes. Upregulated genes, particularly beta2-microglobulin, and the downregulated secretoglobin gene can serve as marker genes that may help to identify SPAOPD-RAO at an early age.

Multixenobiotic resistance mechanism in the catfish intestine

Abstract A mechanism similar to the multidrug resistance phenomenon displayed by mammalian tumor cells has been identified in a variety of lower species, and in a variety of tissues, including the gastrointestinal tract. Xenobiotic efflux transport activity, and the location in membranes of the gastrointestinal tract luminal surfaces, indicates that P-glycoprotein (pgp) may act in modulating the bioavailability of dietary xenobiotics in aquatic animals. The objectives of this study were to investigate the dietary inducibility, depuration, and transport abilities of pgp in the catfish intestine. For this purpose, catfish underwent dietary induction with benzo(a)pyrene, β-naphthoflavone, and 3,4,3′,4′-tetrachlorobiphenyl. Plasma membrane vesicles preparations were used for protein quantitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, and immunohistochemical detection with C219 monoclonal antibody. Transport activity was tested using [3H]-vinblastine (VBL) as a substrate. Results have shown that a catfish intestinal protein which cross-reacts with C219 antibody is inducible following dietary xenobiotic exposure, and that lower levels of this protein are still present 16 days after termination of exposure. In addition, VBL transport was shown to be ATP dependent and inhibited by verapamil. The location, function and responsiveness of pgp to dietary inducers suggest that pgp may play a role in the bioavailability and disposition of dietary xenobiotics.