Paul McLoughlin

Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation.

Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases, which increase morbidity and mortality. Hypoxic PH has previously been attributed to structural changes in the pulmonary vasculature including narrowing of the vascular lumen and loss of vessels, which produce a fixed increase in resistance. Using quantitative stereology, we now show that chronic hypoxia caused PH and remodeling of the blood vessel walls in rats but that this remodeling did not lead to structural narrowing of the vascular lumen. Sustained inhibition of the RhoA/Rho-kinase pathway throughout the period of hypoxic exposure attenuated PH and prevented remodeling in intra-acinar vessels without enlarging the structurally determined lumen diameter. In chronically hypoxic lungs, acute Rho kinase inhibition markedly decreased PVR but did not alter the alveolar to arterial oxygen gap. In addition to increased vascular resistance, chronic hypoxia induced Rho kinase–dependent capillary angiogenesis. Thus, hypoxic PH was not caused by fixed structural changes in the vasculature but by sustained vasoconstriction, which was largely Rho kinase dependent. Importantly, this vasoconstriction had no role in ventilation-perfusion matching and optimization of gas exchange. Rho kinase also mediated hypoxia-induced capillary angiogenesis, a previously unrecognized but potentially important adaptive response.

Permissive hypercapnia — role in protective lung ventilatory strategies

Abstract‘Permissive hypercapnia’ is an inherent element of accepted protective lung ventilation. However, there are no clinical data evaluating the efficacy of hypercapnia per se, independent of ventilator strategy. In the absence of such data, it is necessary to determine whether the potential exists for an active role for hypercapnia, distinct from the demonstrated benefits of reduced lung stretch. In this review, we consider four key issues. First, we consider the evidence that protective lung ventilatory strategies improve survival and we explore current paradigms regarding the mechanisms underlying these effects. Second, we examine whether hypercapnic acidosis may have effects that are additive to the effects of protective ventilation. Third, we consider whether direct elevation of CO2, in the absence of protective ventilation, is beneficial or deleterious. Fourth, we address the current evidence regarding the buffering of hypercapnic acidosis in ARDS. These perspectives reveal that the potential exists for hypercapnia to exert beneficial effects in the clinical context. Direct administration of CO2 is protective in multiple models of acute lung and systemic injury. Nevertheless, several specific concerns remain regarding the safety of hypercapnia. At present, protective ventilatory strategies that involve hypercapnia are clinically acceptable, provided the clinician is primarily targeting reduced tidal stretch. There are insufficient clinical data to suggest that hypercapnia per se should be independently induced, nor do outcome data exist to support the practice of buffering hypercapnic acidosis. Rapidly advancing basic scientific investigations should better delineate the advantages, disadvantages, and optimal use of hypercapnia in ARDS.

Chronic hypoxia causes angiogenesis in addition to remodelling in the adult rat pulmonary circulation.

Chronic hypoxia caused by migration of native sea‐level dwellers to high altitude or chronic lung disease leads to the development of increased pulmonary vascular resistance and pulmonary hypertension. This altitude‐induced hypertension offers no obvious benefit and may indeed be maladaptive. A major mechanism thought to contribute to the development of pulmonary hypertension is hypoxia‐induced loss of small blood vessels, sometimes termed rarefaction or pruning. More recent evidence caused us to question this widely accepted concept including the potent angiogenic effect of chronic hypoxia in all other vascular beds and the demonstration that new vessels can form in the pulmonary circulation when stimulated by chronic infection and lung resection. We tested the hypothesis that chronic environmental hypoxia causes angiogenesis in the adult pulmonary circulation by using stereological techniques combined with confocal microscopy to examine the resultant changes in pulmonary vascular structure in rats. We found that chronic hypoxia resulted in increased total pulmonary vessel length, volume, endothelial surface area and number of endothelial cells in vivo. This is the first reported demonstration of hypoxia‐induced angiogenesis in the mature pulmonary circulation, a structural adaptation that may have important beneficial consequences for gas exchange. These findings imply that we must revise the widely accepted paradigm that hypoxia‐induced loss of small vessels is a key structural change contributing to the development of pulmonary hypertension in high altitude adaptation and chronic lung disease.

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.

Gremlin Plays a Key Role in the Pathogenesis of Pulmonary Hypertension

Background— Pulmonary hypertension occurs in chronic hypoxic lung diseases, significantly worsening morbidity and mortality. The important role of altered bone morphogenetic protein (BMP) signaling in pulmonary hypertension was first suspected after the identification of heterozygous BMP receptor mutations as the underlying defect in the rare heritable form of pulmonary arterial hypertension. Subsequently, it was demonstrated that BMP signaling was also reduced in common forms of pulmonary hypertension, including hypoxic pulmonary hypertension; however, the mechanism of this reduction has not previously been elucidated. Methods and Results— Expression of 2 BMP antagonists, gremlin 1 and gremlin 2, was higher in the lung than in other organs, and gremlin 1 was further increased in the walls of small intrapulmonary vessels of mice during the development of hypoxic pulmonary hypertension. Hypoxia stimulated gremlin secretion from human pulmonary microvascular endothelial cells in vitro, which inhibited endothelial BMP signaling and BMP-stimulated endothelial repair. Haplodeficiency of gremlin 1 augmented BMP signaling in the hypoxic mouse lung and reduced pulmonary vascular resistance by attenuating vascular remodeling. Furthermore, gremlin was increased in the walls of small intrapulmonary vessels in idiopathic pulmonary arterial hypertension and the rare heritable form of pulmonary arterial hypertension in a distribution suggesting endothelial localization. Conclusions— These findings demonstrate a central role for increased gremlin in hypoxia-induced pulmonary vascular remodeling and the increased pulmonary vascular resistance in hypoxic pulmonary hypertension. High levels of basal gremlin expression in the lung may account for the unique vulnerability of the pulmonary circulation to heterozygous mutations of BMP type 2 receptor in pulmonary arterial hypertension.

Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension.

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.

NF-κB Links CO2 Sensing to Innate Immunity and Inflammation in Mammalian Cells

Molecular O2 and CO2 are the primary substrate and product of aerobic metabolism, respectively. Levels of these physiologic gases in the cell microenvironment vary dramatically both in health and in diseases, such as chronic inflammation, ischemia, and cancer, in which metabolism is significantly altered. The identification of the hypoxia-inducible factor led to the discovery of an ancient and direct link between tissue O2 and gene transcription. In this study, we demonstrate that mammalian cells (mouse embryonic fibroblasts and others) also sense changes in local CO2 levels, leading to altered gene expression via the NF-κB pathway. IKKα, a central regulatory component of NF-κB, rapidly and reversibly translocates to the nucleus in response to elevated CO2. This response is independent of hypoxia-inducible factor hydroxylases, extracellular and intracellular pH, and pathways that mediate acute CO2-sensing in nematodes and flies and leads to attenuation of bacterial LPS-induced gene expression. These results suggest the existence of a molecular CO2 sensor in mammalian cells that is linked to the regulation of genes involved in innate immunity and inflammation.

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.

Hypoxia Selectively Activates the CREB Family of Transcription Factors in the In Vivo Lung

RATIONALE Pulmonary hypertension is a common complication of chronic hypoxic lung diseases and is associated with increased morbidity and reduced survival. The pulmonary vascular changes in response to hypoxia, both structural and functional, are unique to this circulation. OBJECTIVES To identify transcription factor pathways uniquely activated in the lung in response to hypoxia. METHODS After exposure to environmental hypoxia (10% O(2)) for varying periods (3 h to 2 wk), lungs and systemic organs were isolated from groups of adult male mice. Bioinformatic examination of genes the expression of which changed in the hypoxic lung (assessed using microarray analysis) identified potential lung-selective transcription factors controlling these changes in gene expression. In separate further experiments, lung-selective activation of these candidate transcription factors was tested in hypoxic mice and by comparing hypoxic responses of primary human pulmonary and cardiac microvascular endothelial cells in vitro. MEASUREMENTS AND MAIN RESULTS Bioinformatic analysis identified cAMP response element binding (CREB) family members as candidate lung-selective hypoxia-responsive transcription factors. Further in vivo experiments demonstrated activation of CREB and activating transcription factor (ATF)1 and up-regulation of CREB family-responsive genes in the hypoxic lung, but not in other organs. Hypoxia-dependent CREB activation and CREB-responsive gene expression was observed in human primary lung, but not cardiac microvascular endothelial cells. CONCLUSIONS These findings suggest that activation of CREB and AFT1 plays a key role in the lung-specific responses to hypoxia, and that lung microvascular endothelial cells are important, proximal effector cells in the specific responses of the pulmonary circulation to hypoxia.