Simon C. Rowan

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.

The pathophysiological basis of chronic hypoxic pulmonary hypertension in the mouse: vasoconstrictor and structural mechanisms contribute equally.

Chronic hypoxic pulmonary hypertension is characterized by a sustained increase in pulmonary arterial pressure due to abnormally elevated pulmonary vascular resistance. This increased vascular resistance was previously thought to be due largely to changes in the structure of the pulmonary vasculature, i.e. lumen narrowing due to wall hypertrophy and loss of vessels. Recently, this model has been challenged by the demonstration that hypoxic pulmonary hypertension in the rat is caused almost completely by sustained vasoconstriction. The contribution of this vasocontriction to hypoxic pulmonary hypertension has not been examined directly in other species. We exposed groups of mice to hypoxia (10% O2) or normoxia for 3 weeks, following which the lungs were removed post mortem, and vascular resistance was measured in an isolated, ventilated, perfused preparation. Mean pulmonary vascular resistance was significantly increased in hypoxic compared with control normoxic lungs. The rho kinase inhibitor Y27635 (10−4m) (Tocris Bioscience, Bristol, United Kingdom.) significantly reduced the mean (±SEM) hypoxia induced increase by 45.4 (10.8)%, implying that structural vascular changes acounted for the remainder of the hypoxic increase. Stereological quantification showed a significant reduction in the mean lumen diameter of the fully relaxed vessels in hypoxic lungs compared with normoxic control lungs; there was no intra‐acinar vessel loss. Thus, in contrast to the rat, hypoxic pulmonary hypertension in the mouse is due to two mechanisms contributing equally: sustained vasoconstriction and structural lumen narrowing of intra‐acinar vessels. These important species diferences must be considered when using genetically mutated mice to investigate the mechanisms underlying pulmonary hypertension.

Hypoxic pulmonary hypertension in chronic lung diseases: novel vasoconstrictor pathways

Pulmonary hypertension is a well recognised complication of chronic hypoxic lung diseases, which are among the most common causes of death and disability worldwide. Development of pulmonary hypertension independently predicts reduced life expectancy. In chronic obstructive pulmonary disease, long-term oxygen therapy ameliorates pulmonary hypertension and greatly improves survival, although the correction of alveolar hypoxia and pulmonary hypertension is only partial. Advances in understanding of the regulation of vascular smooth muscle tone show that chronic vasoconstriction plays a more important part in the pathogenesis of hypoxic pulmonary hypertension than previously thought, and that structural vascular changes contribute less. Trials of existing vasodilators show that pulmonary hypertension can be ameliorated and systemic oxygen delivery improved in carefully selected patients, although systemic hypotensive effects limit the doses used. Vasoconstrictor pathways that are selective for the pulmonary circulation can be blocked to reduce hypoxic pulmonary hypertension without causing systemic hypotension, and thus provide potential targets for novel therapeutic strategies.

Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

Hypoxic pulmonary hypertension: the paradigm is changing

Sustained hypoxia caused by migration of native sea-level dwellers to high altitude or by chronic lung disease leads to the development of increased pulmonary vascular resistance and pulmonary hypertension, a response to hypoxia that is unique to the pulmonary circulation. In susceptible individuals at high altitude (subacute and chronic mountain sickness) and in lung disease, the resultant pulmonary hypertension causes significant disability and reduced life expectancy. For these reasons, investigation of the underlying mechanisms is an area of intense research activity. Classically, the elevation in pulmonary vascular resistance in chronic hypoxic pulmonary hypertension has been attributed to structural remodelling of the vessels, leading to thickening of the walls of the pulmonary arterioles, which encroach into and narrow the vascular lumen; any vasoconstrictor component has been considered minor. However, more recent morphological evidence from experiments in chronically hypoxic rats suggested that lumen narrowing could not account for the increase in pulmonary vascular resistance or, at the very least, that the effect of this lumen narrowing may have been overestimated. The identification of the RhoA/Rho-kinase (ROCK) signalling cascade as an important regulator of smooth muscle contraction and the development of the potent and selective ROCK inhibitors, including Y27632, provided the opportunity to gain new insights into processes in which altered smooth muscle contractility plays a key role, both physiologically and in disease (Uehata et al. 1997). Activation of the RhoA–ROCK pathway inhibits myosin light chain phosphatase activity, thus enhancing the level of myosin light chain phosphorylation, promoting actin–myosin interaction and causing increased contraction. Effectively, this increases the sensitivity of the contractile apparatus to Ca2+ and augments tension development at a given level of cytosolic Ca2+ (Uehata et al. 1997). Prompted by these new discoveries, we began, in the early 2000s, to explore the hypothesis that vasoconstrictor mechanisms, selectively altered in the lung, were significant contributors to the increase in pulmonary vascular resistance in pulmonary hypertension. We found that in the normal rat pulmonary circulation, the RhoA–ROCK pathway is a greater contributor to vasoconstriction than it is in systemic vessels, demonstrating an important phenotypic difference in the regulation of vascular tone in the two circulations (Hyvelin et al. 2004). Almost simultaneously, Fagan et al. (2004) reported the results of experiments in which Y27632 was used to examine the contribution of the RhoA–ROCK pathway to the elevation in pulmonary vascular resistance in the hypoxic mouse lung. They found that ROCK activation makes an important contribution to acute hypoxic pulmonary vasoconstriction (Fagan et al. 2004). Moreover, sustained ROCK inhibition in vivo attenuated the development of chronic hypoxic pulmonary hypertension (Fagan et al. 2004). Taken together, these findings suggested, for the first time, that sustained vasoconstriction might play a more significant role in chronic hypoxic pulmonary hypertension than previously thought and that the RhoA–ROCK pathway might be important in this. Therefore, we undertook a series of experiments to investigate the exact contributions of vasoconstriction and remodelling to the chronic hypoxia-induced elevation in pulmonary vascular resistance in mice (Cahill et al. 2012). Using an isolated, ventilated lung preparation, we found that following long-term hypoxic exposure, which established ‘fixed’ pulmonary hypertension (i.e. not reversed by acute restoration of normal alveolar oxygen), acute ROCK inhibition using Y27632 reduced by half the increased pulmonary vascular resistance. Combining haemodynamic techniques with quantitative stereological analysis of the pulmonary vascular remodelling enabled us also to determine the degree to which structural remodelling of the pulmonary vascular wall and encroachment of the vessel wall into the lumen contributed to the increased resistance (Cahill et al. 2012). We found that the increase was due to equal contributions of sustained ROCK-dependent vasoconstriction and structural narrowing of the vascular lumen. This suggested that the role of sustained vasoconstriction in hypoxic pulmonary hypertension is considerably more important than previously thought. Building on this identification of the importance of vasoconstriction in chronic hypoxic pulmonary hypertension, Wan et al. (2013) explored the effect of hypoxia on the regulation of cytosolic calcium in pulmonary vascular smooth muscle. Increased cyctosolic Ca2+ is an important trigger for both pulmonary vasoconstriction and pulmonary artery smooth muscle cell proliferation; the Ca2+ influx through voltage-dependent calcium channels (VDCCs) plays an important role in the regulation of cytoplasmic Ca2+ activity. Wan et al. (2013) demonstrated that sustained hypoxia induced functional upregulation of CaV1.2 (an L-type VDCC) and CaV3.2 (a T-type VDCC) in the pulmonary circulation without changing the expression of these channels in systemic vessels. Furthermore, they found that chronic hypoxia increased depolarization and agonist-induced pulmonary vasoconstriction when compared with normoxic pulmonary vessels and that this increase was associated with higher cytosolic Ca2+ than in normoxic vessels. Pharmacological blockade of these two channels reduced the contractile responses to a greater extent in chronically hypoxic vessels than in control normoxic vessels (Wan et al. 2013). Their findings provide further experimental support for the hypothesis that chronic vasoconstriction is a major contributory mechanism in hypoxic pulmonary hypertension. Taken together, these recent reports suggest that the mechanism of this chronic vasoconstriction is a lung-selective increase in vascular smooth muscle cytosolic Ca2+ combined with an increased calcium sensitivity of the contractile apparatus mediated by the RhoA–ROCK pathway. Interestingly, both pathways also mediate smooth muscle proliferation and may therefore contribute to the remodelling component of increased pulmonary vascular resistance. Thus, the classic ‘textbook’ paradigm of chronic hypoxic pulmonary hypertension may need revision. Sustained vasoconstriction is an important contributor to the underlying increase in pulmonary vascular resistance, although structural remodelling of the vessel also contributes. Not only do these findings have important implications for our basic understanding of this phenomenon, but they also identify potential novel approaches to the treatment of pulmonary hypertension in disease. New pharmacological agents targeting the RhoA–ROCK pathway and calcium entry through the L- and T-type calcium channels whose expression and function are selectively enhanced in the hypertensive pulmonary circulation may be useful in the treatment of hypoxic pulmonary hypertension. Blockade could lead to an initial rapid vasodilatation and reduction in resistance, followed by a later, more slowly developing, further reduction due to inhibition of remodelling. A potential advantage of inhibition of such selectively upregulated pathways, either alone or in combination, is that this may permit effective therapy while minimizing systemic vasodilatation and hypotension, a major limiting side-effect of current therapies. Administration of antagonists by the inhaled route might further increase pulmonary selectivity. Additional studies are needed to establish the contribution of vasoconstriction to hypoxic pulmonary hypertension in humans and to identify the mechanisms that selectively regulate the underlying mechanisms in the hypoxic lung.

The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREBαΔ mice). Here we report that expression of CREB regulated genes was altered in the lungs of CREBαΔ mice. CREBαΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREBαΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREBαΔ. CREBαΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

EXPRESS: Gremlin1 blocks vascular endothelial growth factor signalling in the pulmonary microvascular endothelium

The bone morphogenetic protein (BMP) antagonist gremlin 1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension (HPH). Recently, non-canonical functions of gremlin 1 have been identified, including specific binding to the vascular endothelial growth factor receptor-2 (VEGFR2). We tested the hypothesis that gremlin 1 modulates VEGFR2 signaling in the pulmonary microvascular endothelium. We examined the effect of gremlin 1 haploinsufficiency on the expression of VEGF responsive genes and proteins in the hypoxic (10% O2) murine lung in vivo. Using human microvascular endothelial cells in vitro we examined the effect of gremlin 1 on VEGF signaling. Gremlin 1 haploinsufficiency (Grem1+/–) attenuated the hypoxia-induced increase in gremlin 1 observed in the wild-type mouse lung. Reduced gremlin 1 expression in hypoxic Grem1+/– mice restored VEGFR2 expression and endothelial nitric oxide synthase (eNOS) expression and activity to normoxic values. Recombinant monomeric gremlin 1 inhibited VEGFA-induced VEGFR2 activation, downstream signaling, and VEGF-induced increases in Bcl-2, cell number, and the anti-apoptotic effect of VEGFA in vitro. These results show that the monomeric form of gremlin 1 acts as an antagonist of VEGFR2 activation in the pulmonary microvascular endothelium. Given the previous demonstration that inhibition of VEGFR2 causes marked worsening of HPH, our results suggest that increased gremlin 1 in the hypoxic lung, in addition to blocking BMP receptor type-2 (BMPR2) signaling, contributes importantly to the development of PH by a non-canonical VEGFR2 blocking activity.

Pulmonary endothelial permeability and tissue fluid balance depend on the viscosity of the perfusion solution

Fluid filtration in the pulmonary microcirculation depends on the hydrostatic and oncotic pressure gradients across the endothelium and the selective permeability of the endothelial barrier. Maintaining normal fluid balance depends both on specific properties of the endothelium and of the perfusing blood. Although some of the essential properties of blood needed to prevent excessive fluid leak have been identified and characterized, our understanding of these remains incomplete. The role of perfusate viscosity in maintaining normal fluid exchange has not previously been examined. We prepared a high-viscosity perfusion solution (HVS) with a relative viscosity of 2.5, i.e., within the range displayed by blood flowing in vessels of different diameters in vivo (1.5-4.0). Perfusion of isolated murine lungs with HVS significantly reduced the rate of edema formation compared with perfusion with a standard solution (SS), which had a lower viscosity similar to plasma (relative viscosity 1.5). HVS did not alter capillary filtration pressure. Increased endothelial shear stress produced by increasing flow rates of SS, to mimic the increased shear stress produced by HVS, did not reduce edema formation. HVS significantly reduced extravasation of Evans blue-labeled albumin compared with SS, indicating that it attenuated endothelial leak. These findings demonstrate for the first time that the viscosity of the solution perfusing the pulmonary microcirculation is an important physiological property contributing to the maintenance of normal fluid exchange. This has significant implications for our understanding of fluid homeostasis in the healthy lung, edema formation in disease, and reconditioning of donor organs for transplantation.