Katherine Howell

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.

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.

An intact canonical NF-κB pathway is required for inflammatory gene expression in response to hypoxia.

Hypoxia is a feature of the microenvironment in a number of chronic inflammatory conditions due to increased metabolic activity and disrupted perfusion at the inflamed site. Hypoxia contributes to inflammation through the regulation of gene expression via key oxygen-sensitive transcriptional regulators including the hypoxia-inducible factor (HIF) and NF-κB. Recent studies have revealed a high degree of interdependence between HIF and NF-κB signaling; however, the relative contribution of each to hypoxia-induced inflammatory gene expression remains unclear. In this study, we use transgenic mice expressing luciferase under the control of NF-κB to demonstrate that hypoxia activates NF-κB in the heart and lungs of mice in vivo. Using small interfering RNA targeted to the p65 subunit of NF-κB, we confirm a unidirectional dependence of hypoxic HIF-1α accumulation upon an intact canonical NF-κB pathway in cultured cells. Cyclooxygenase-2 and other key proinflammatory genes are transcriptionally induced by hypoxia in a manner that is both HIF-1 and NF-κB dependent, and in mouse embryonic fibroblasts lacking an intact canonical NF-κB pathway, there is a loss of hypoxia-induced inflammatory gene expression. Finally, under conditions of hypoxia, HIF-1α and the p65 subunit of NF-κB directly bind to the cyclooxygenase-2 promoter. These results implicate an essential role for NF-κB signaling in inflammatory gene expression in response to hypoxia both through the regulation of HIF-1 and through direct effects upon target gene expression.

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.

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.

Infection-induced lung injury is worsened after renal buffering of hypercapnic acidosis

Objective:Prolonged hypercapnia is commonly encountered during the treatment of acute respiratory distress syndrome and acute respiratory failure attributable to other causes with protective ventilation strategies. In these circumstances, compensatory renal buffering returns pH to normal establishing a condition of buffered hypercapnia. It is also common intensive care practice to correct the pH more rapidly using bicarbonate infusions. Although it is well-established that hypercapnic acidosis has potent anti-inflammatory and protective effects, the effect of buffered hypercapnia on acute lung injury and acute respiratory distress syndrome is unknown. We therefore wished to determine the effects of buffered hypercapnia on acute lung injury induced by endotoxin or Escherichia coli infection in vivo. Design:Prospective, randomized animal study. Setting:University research laboratory. Subjects:Adult male Sprague-Dawley rats. Interventions:We established buffered hypercapnia by exposing rats to a hypercapnic environment for 3 days before the induction of lung injury. Buffered hypercapnia rats (initial pH >7.35, FiCO2 = 0.05) and normocapnic controls (initial pH >7.35, FiCO2 = 0.00) were then anesthetized, mechanically ventilated, and lung injury induced by intra-tracheal inoculation of endotoxin (series I) or Escherichia coli (series II). Measurements and Main Results:Buffered hypercapnia significantly increased both endotoxin and Escherichia coli-induced lung injury when compared to normocapnic controls, as assessed by arterial oxygenation, lung compliance, pro-inflammatory pulmonary cytokine concentrations, and measurements of structural lung damage. In additional in vitro experiments buffered hypercapnia did not alter neutrophil phagocytosis ability but did impaired epithelial wound healing. Conclusions:Our results demonstrate that infection-induced injury in vivo is worsened after renal buffering of hypercapnic acidosis independently of any changes in tidal volume. These findings have important implications for our understanding of the pathogenesis of infection-induced lung injury during the use protective ventilation strategies that permits buffered hypercapnia and during infective exacerbations of chronic lung diseases associated with sustained hypercapnia.

L-Arginine promotes angiogenesis in the chronically hypoxic lung: a novel mechanism ameliorating pulmonary hypertension

Chronic alveolar hypoxia, whether due to residence at high altitude or lung disease, leads to a sustained increase in pulmonary vascular resistance and pulmonary hypertension (PH). Strategies that augment endogenous nitric oxide production or activity, including l-arginine supplementation, attenuate the development of PH. This action has been attributed to inhibition of vessel wall remodeling, thus preventing structural narrowing of the vascular lumen. However, more recent evidence suggests that structural changes are not responsible for the elevated vascular resistance observed in chronic hypoxic PH, calling into question the previous explanation for the action of l-arginine. We examined the effect of dietary l-arginine supplementation on pulmonary vasoconstriction, structurally determined maximum vascular lumen diameter, and vessel length in rats during 2 wk of exposure to hypoxia. l-Arginine attenuated the development of hypoxic PH by preventing increased arteriolar resistance. It did not alter mean maximal vascular lumen diameter, nor did it augment nitric oxide-mediated vasodilatation, in chronically hypoxic lungs. However, the total length of vessels within the gas exchange region of the hypoxic lungs was significantly increased after l-arginine supplementation. These findings suggest that dietary l-arginine ameliorated hypoxic PH, but not by an effect on the structurally determined lumen diameter of pulmonary blood vessels. l-Arginine enhanced angiogenesis in the hypoxic pulmonary circulation, which may attenuate hypoxic PH by producing new parallel vascular pathways through the lung.

Structural basis of hypoxic pulmonary hypertension: the modifying effect of chronic hypercapnia

Exposure to chronic hypoxia causes pulmonary hypertension and pulmonary vascular remodelling. In chronic lung disease, chronic hypercapnia frequently coexists with hypoxia and is associated with worsening of pulmonary hypertension. It is generally stated that pulmonary hypertension in these conditions is secondary to hypoxic vascular remodelling and that hypercapnia augments this remodelling thus worsening the hypertension. We review recent evidence which shows that although chronic hypoxia causes thickening of the walls of pulmonary arterioles, these changes do not lead to structural narrowing of the lumen by encroachment. Moreover, hypoxia leads to new vessel formation within the pulmonary vasculature and not loss of vessels as formerly thought. Such neovascularization may provide a beneficial adaptation by increasing the area of the gas exchange membrane. These novel structural findings are supported by recent reports that inhibitors of the RhoA pathway can acutely reduce pulmonary vascular resistance in chronically hypoxic lungs to near normal values, demonstrating that structural changes are not the dominant mechanisms underling hypoxic pulmonary hypertension. Chronic hypercapnia inhibits the development of hypoxic pulmonary hypertension, pulmonary vascular remodelling and hypoxia‐induced angiogenesis. This last effect might be maladaptive, as it would prevent the potentially beneficial increase in gas exchange membrane area. These findings suggest that structural narrowing of the vascular lumen of resistance vessels is not the mechanism by which hypoxia and hypercapnia cause pulmonary hypertension in chronic lung disease.