Natalie Hopkins

The structural basis of pulmonary hypertension in chronic lung disease: remodelling, rarefaction or angiogenesis?

Chronic lung disease in humans is frequently complicated by the development of secondary pulmonary hypertension, which is associated with increased morbidity and mortality. Hypoxia, inflammation and increased shear stress are the primary stimuli although the exact pathways through which these initiating events lead to pulmonary hypertension remain to be completely elucidated. The increase in pulmonary vascular resistance is attributed, in part, to remodelling of the walls of resistance vessels. This consists of intimal, medial and adventitial hypertrophy, which can lead to encroachment into and reduction of the vascular lumen. In addition, it has been reported that there is a reduction in the number of blood vessels in the hypertensive lung, which could also contribute to increased vascular resistance. The pulmonary endothelium plays a key role in mediating and modulating these changes. These structural alterations in the pulmonary vasculature contrast sharply with the responses of the systemic vasculature to the same stimuli. In systemic organs, both hypoxia and inflammation cause angiogenesis. Furthermore, remodelling of the walls of resistance vessels is not observed in these conditions. Thus it has been generally stated that, in the adult pulmonary circulation, angiogenesis does not occur. Prompted by previous observations that chronic airway inflammation can lead to pulmonary vascular remodelling without hypertension, we have recently shown, using quantitative stereological techniques, that angiogenesis can occur in the adult pulmonary circulation. Pulmonary angiogenesis has also been reported in some other conditions including post‐pneumonectomy lung growth, metastatic disease of the lung and in biliary cirrhosis. Such angiogenesis may serve to prevent or attenuate increased vascular resistance in lung disease. In view of these more recent data, the role of structural alterations in the pulmonary vasculature in the development of pulmonary hypertension should be carefully reconsidered.

Sustained hypercapnic acidosis during pulmonary infection increases bacterial load and worsens lung injury

Objective:Hypercapnic acidosis is commonly permitted in patients with acute respiratory distress syndrome during the use of protective ventilation strategies. Hypercapnic acidosis is also a common complication of multiple lung diseases and is associated with a poor prognosis, although the mechanisms by which it leads to increased mortality is not known. Previous studies using noninfective models of lung injury show that acute (<6 hrs) hypercapnic acidosis reduced lung damage by an anti-inflammatory effect. We hypothesized that this anti-inflammatory effect would be detrimental in vivo in the presence of untreated bacterial infection and sustained hypercapnia (>48 hrs) and, furthermore, that if bacterial reproduction were controlled by antibiotic therapy, then the anti-inflammatory effects of hypercapnic acidosis would no longer prove detrimental. Design:This study was a prospective, randomized animal study. Setting:This study was conducted at a university research laboratory. Subjects:Study subjects were adult male Wistar-Kyoto rats. Interventions:After intratracheal instillation of Escherichia coli under general anesthesia, rats were housed in normocapnic (21% O2, 0% CO2) or hypercapnic (21% O2, 5% CO2) environments for 2 days. Rats were then reanesthetized for assessment of physiological and quantitative stereologic indices of lung damage, quantitative bacterial counts, and neutrophil phagocytosis. Measurements and Main Results:Hypercapnic acidosis was associated with higher lung bacterial colony counts, more structural damage, and lower static lung compliance than normocapnia. Neutrophils isolated from hypercapnic rats demonstrated impaired phagocytosis. In a further separate series of experiments, in which rats were given antibiotic therapy, lung damage was not different between normocapnic and hypercapnic acidosis groups. Conclusions:Prolonged hypercapnic acidosis worsened bacterial infection-induced lung injury. Our findings suggest an immunosuppressive effect of hypercapnic acidosis and have important implications for protective ventilation strategies that permit hypercapnic acidosis in patients with adult respiratory distress syndrome and in the management of hypercapnic acidosis during infective exacerbations of chronic obstructive pulmonary disease and other lung diseases.

Combined confocal microscopy and stereology: a highly efficient and unbiased approach to quantitative structural measurement in tissues

Understanding the relationship of the structure of organs to their function is a key component of integrative physiological research. The structure of the organs of the body is not constant but changes, both during growth and development and under conditions of sustained stress (e.g. high altitude exposure and disease). Recently, powerful new techniques have become available in molecular biology, which promise to provide novel insights into the mechanisms and consequences of these altered structure‐function relationships. Conventionally structure‐function relationships are studied by microscopic examination of tissue sections. However, drawing conclusions about the three‐dimensional structure of an organ based on this two‐dimensional information frequently leads to serious errors. The techniques of stereology allow precise and accurate quantification of structural features within three‐dimensional organs that relate in a meaningful way to integrated function. For example, knowledge of changes in the total surface area of the capillary endothelium in an organ can be related directly to changes in fluid filtration and permeability, or knowledge of total vessel length and mean radius allows deductions about vascular resistance. Confocal microscopy adds enormously to the power of stereological approaches. It reduces the difficulties and labour involved in obtaining suitable images. Moreover, when used in conjunction with new analytical software, it allows convenient application of stereology to small samples and those in which it is essential to maintain a specific orientation for interpretation. The information obtained will allow us to examine in a quantitative manner the altered structure‐function relationships produced by manipulation of single genes and regulatory pathways in whole organisms.

Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs

We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N ω-nitro-l-arginine methyl ester increased the mean perfusion pressure (14.4 ± 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 ± 1.0 mmHg) or noninoculated (5.3 ± 0.8 mmHg) lungs ( P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N(omega)-nitro-L-arginine methyl ester increased the mean perfusion pressure (14.4 +/- 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 +/- 1.0 mmHg) or noninoculated (5.3 +/- 0.8 mmHg) lungs (P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.

Anti-inflammatory effect of augmented nitric oxide production in chronic lung infection.

Chronic infection of the lungs with Pseudomonas aeruginosa complicates many long‐term lung diseases including cystic fibrosis, bronchiectasis, chronic obstructive lung disease, and mechanical ventilation. In acute inflammatory lung diseases, increased nitric oxide synthase (NOS‐2) expression leads to excess nitric oxide (NO) production, resulting in the production of reactive nitrogen intermediates, which contribute to tissue damage. In contrast, the contribution of NO to pulmonary damage in chronic Pseudomonas infection of the lung has not been directly examined and is unclear. Although NOS‐2 expression is increased in this condition, NO production is not abnormally elevated. It was hypothesized that chronic infection of the airways does not cause increased NO production but, in contrast, leads to inappropriately low NO concentrations that are pro‐inflammatory. A rodent model of chronic airway infection was used to examine the effects on lung damage of augmenting or inhibiting NO production after airway infection with P. aeruginosa was well established. Three days post‐infection, L‐arginine, which augments NO production, or L‐NAME, an inhibitor of NO production, was administered in drinking water. Lung damage was assessed 12 days later. L‐arginine treatment reduced tissue damage, inhibited neutrophil recruitment, and reduced the pro‐inflammatory cytokine interleukin (IL)‐1β. Treatment with L‐NAME caused loss of alveolar walls, greater vascular damage, and increased levels of the pro‐inflammatory cytokine IL‐6. Thus, in chronic airway infection, inhibition of NO production worsened lung damage, whereas augmenting NO ameliorated this damage. This is the first demonstration that augmenting endogenous NO production in chronic infective lung disease caused by P. aeruginosa is anti‐inflammatory. Given that infection with this organism complicates many chronic lung diseases, most notoriously cystic fibrosis, these findings have important clinical implications. Copyright

Hypercapnic acidosis reduces oxidative reactions in endotoxin-induced lung injury.

Background:Hypercapnic acidosis frequently occurs when patients with acute lung injury are initially ventilated with low tidal volume “protective” strategies. Hypercapnic acidosis per se, in the absence of any change in tidal volume or airway pressure, is protective when instituted before the onset of injury. However, the mechanisms by which hypercapnic acidosis confers this protection are incompletely understood, in particular, the effects on pulmonary oxidative reactions, which are potent mediators of tissue damage, have not been previously examined in vivo. Methods:After anesthesia, tracheostomy, and the intratracheal instillation of endotoxin to establish lung injury, rats were mechanically ventilated for 6 h in normocapnia (21% O2, 0% CO2). Rats were then randomized to either normocapnic (21% O2, 0% CO2) or hypercapnic (21% O2, 5% CO2) ventilation and a nonspecific nitric oxide synthase inhibitor (NG-monomethyl-l-arginine) or vehicle. Dihydrorhodamine was administered intravenously, and the lungs were removed for determination of the oxidative formation of rhodamine by spectrofluorimetry after 20 min. Thus, rats were randomly assigned to either: normocapnia-endotoxin (n = 12), normocapnia-endotoxin-NG-monomethyl-l-arginine (n = 9), hypercapnia-endotoxin (n = 11), or hypercapnia-endotoxin-NG-monomethyl-l-arginine (n = 10). Results:Hypercapnic acidosis significantly reduced the pulmonary oxidative reactions in the inflamed lung compared with normocapnia. Nitric oxide synthase blockade did not alter endotoxin-induced oxidative reactions. Conclusions:Hypercapnic acidosis reduced oxidative reactions in the acutely injured lung in vivo, within minutes of onset and was not reliant on nitric oxide-dependent peroxynitrite production. This rapid onset antioxidant action is a previously undescribed mechanism by which hypercapnic acidosis could act, even when acute lung injury is well established.

Type 2 nitric oxide synthase and protein nitration in chronic lung infection.

Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production. It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage. However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected. A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions. To test this hypothesis, anaesthetized, specific pathogen‐free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group). Ten to 15 days later, the lungs were isolated and fixed. Pseudomonas organisms were isolated from the lungs of the chronically infected group. These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs. Expression of iNOS was increased in the chronically infected group when compared with the control group. However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs. Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway. These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS. Copyright