Petra Faulhammer

Rapamycin attenuates hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy in mice

BackgroundChronic hypoxia induces pulmonary arterial hypertension (PAH). Smooth muscle cell (SMC) proliferation and hypertrophy are important contributors to the remodeling that occurs in chronic hypoxic pulmonary vasculature. We hypothesized that rapamycin (RAPA), a potent cell cycle inhibitor, prevents pulmonary hypertension in chronic hypoxic mice.MethodsMice were held either at normoxia (N; 21% O2) or at hypobaric hypoxia (H; 0.5 atm; ~10% O2). RAPA-treated animals (3 mg/kg*d, i.p.) were compared to animals injected with vehicle alone. Proliferative activity within the pulmonary arteries was quantified by staining for Ki67 (positive nuclei/vessel) and media area was quantified by computer-aided planimetry after immune-labeling for α-smooth muscle actin (pixel/vessel). The ratio of right ventricle to left ventricle plus septum (RV/[LV+S]) was used to determine right ventricular hypertrophy.ResultsProliferative activity increased by 34% at day 4 in mice held under H (median: 0.38) compared to N (median: 0.28, p = 0.028) which was completely blocked by RAPA (median HO+RAPA: 0.23, p = 0.003). H-induced proliferation had leveled off within 3 weeks. At this time point media area had, however, increased by 53% from 91 (N) to 139 (H, p < 0.001) which was prevented by RAPA (H+RAPA: 102; p < 0.001). RV/[LV+S] ratio which had risen from 0.17 (N) to 0.26 (H, p < 0.001) was attenuated in the H+RAPA group (0.22, p = 0.041). For a therapeutic approach animals were exposed to H for 21 days followed by 21 days in H ± RAPA. Forty two days of H resulted in a media area of 129 (N: 83) which was significantly attenuated in RAPA-treated mice (H+RAPA: 92). RV/[LV+S] ratios supported prevention of PH (N 0.13; H 0.27; H+RAPA 0.17). RAPA treatment of N mice did not influence any parameter examined.ConclusionTherapy with rapamycin may represent a new strategy for the treatment of pulmonary hypertension.

Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices.

BackgroundAcute alveolar hypoxia causes pulmonary vasoconstriction (HPV) which serves to match lung perfusion to ventilation. The underlying mechanisms are not fully resolved yet. The major vascular segment contributing to HPV, the intra-acinar artery, is mostly located in that part of the lung that cannot be selectively reached by the presently available techniques, e.g. hemodynamic studies of isolated perfused lungs, recordings from dissected proximal arterial segments or analysis of subpleural vessels. The aim of the present study was to establish a model which allows the investigation of HPV and its underlying mechanisms in small intra-acinar arteries.MethodsIntra-acinar arteries of the mouse lung were studied in 200 μm thick precision-cut lung slices (PCLS). The organisation of the muscle coat of these vessels was characterized by α-smooth muscle actin immunohistochemistry. Basic features of intra-acinar HPV were characterized, and then the impact of reactive oxygen species (ROS) scavengers, inhibitors of the respiratory chain and Krebs cycle metabolites was analysed.ResultsIntra-acinar arteries are equipped with a discontinuous spiral of α-smooth muscle actin-immunoreactive cells. They exhibit a monophasic HPV (medium gassed with 1% O2) that started to fade after 40 min and was lost after 80 min. This HPV, but not vasoconstriction induced by the thromboxane analogue U46619, was effectively blocked by nitro blue tetrazolium and diphenyleniodonium, indicating the involvement of ROS and flavoproteins. Inhibition of mitochondrial complexes II (3-nitropropionic acid, thenoyltrifluoroacetone) and III (antimycin A) specifically interfered with HPV, whereas blockade of complex IV (sodium azide) unspecifically inhibited both HPV and U46619-induced constriction. Succinate blocked HPV whereas fumarate had minor effects on vasoconstriction.ConclusionThis study establishes the first model for investigation of basic characteristics of HPV directly in intra-acinar murine pulmonary vessels. The data are consistent with a critical involvement of ROS, flavoproteins, and of mitochondrial complexes II and III in intra-acinar HPV. In view of the lack of specificity of any of the classical inhibitors used in such types of experiments, validation awaits the use of appropriate knockout strains and siRNA interference, for which the present model represents a well-suited approach.

Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability

Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1alpha on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1alpha enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage.

Muscarinic receptor subtypes in cilia-driven transport and airway epithelial development

Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1–M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect, most likely through the M1 receptor, and normalised the ATP response. M1, M4 and M5 receptor-deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptors are missing. None of the receptors is necessary for epithelial development.

Mitochondrial complex II is essential for hypoxia-induced ROS generation and vasoconstriction in the pulmonary vasculature.

Hypoxia induces an increase in the ROS generation by cells of small intrapulmonary vessels. Based on our results we suppose that this is caused by a switch in the catalytic activity of mitochondrial complex II from succinate dehydrogenase to fumarate reductase. Functional complex II is also necessary for hypoxic pulmonary vasoconstriction.

Cilia-driven particle and fluid transport over mucus-free mice tracheae

To date, there is only a fragmentary understanding of the fundamental mechanisms of airway mucociliary transport. Application of the latest measurement techniques can aid in deciphering the complex interplay between ciliary beat and airway surface liquid (ASL) transport. In the present study, direct, quasi-simultaneous measurements of the cilia-induced fluid and bead transport were performed to gain a better insight into both transport mechanisms. In this study cilia-induced periciliary liquid (PCL) transport is measured by means of micro Particle Image Velocimetry (μPIV) with neutrally buoyant tracers. Particle Tracking Velocimetry (PTV) with heavier polystyrene-ferrite beads is performed to simulate particle transport. Contrary to recent literature, in which the presence of mucus was deemed necessary to maintain periciliary liquid (PCL) transport, effective particle and fluid transport was measured in our experiments in the absence of mucus. In response to muscarine or ATP stimulation, maximum fluid transport rates of 250 μm/s at 15 μm distance to the tracheal epithelia were measured while bead transport rates over the epithelia surfaces reached 200 μm/s. We estimated that the mean bead transport is dominated by viscous drag compared to inertial fluid forces. Furthermore, mean bead transport velocities appear to be two orders of magnitude larger compared to bead sedimentation velocities. Therefore, beads are expected to closely follow the mean PCL flow in non-ciliated epithelium regions. Based on our results, we have shown that PCL transport can be directly driven by the cilia beat and that the PCL motion may be capable of driving bead transport by fluid drag.

Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries

AIMS Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-)). METHODS AND RESULTS Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice. CONCLUSION SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

Impact of Modulators of Mitochondrial ATP-Sensitive Potassium Channel (mitoKATP) on Hypoxic Pulmonary Vasoconstriction

Previously, we demonstrated that hypoxic pulmonary vasoconstriction (HPV) of intra-acinar arteries (IAA) requires mitochondrial complex II (= succinate dehydrogenase, SDH) activity (citeauthor ch41:paddenberg2006, Respir Res, 7:93, citeyear ch41:paddenberg2006). Interestingly, SDH subunits A and B have recently been described as components of a multiprotein mitochondrial ATP-sensitive potassium channel (mitoK(ATP)), together with mitochondrial ATP-binding cassette protein-1, adenine nucleotide translocator (ANT), ATP synthase, and phosphate carrier (citeauthor ch41:ardehali2004, Proc Natl Acad Sci USA, 101(32):11880-5, citeyear ch41:ardehali2004). Hence, we tested the hypothesis that such an SDH-containing mitoK(ATP) is involved in HPV. For this purpose, the impact of modulators of mitoK(ATP) on HPV of IAA was studied videomorphometrically in precision cut murine lung slices. Inhibitors of mitoK(ATP) (glibenclamide, 5-hydroxydecanoate) completely suppressed HPV, mitoK(ATP) activators (pinacidil, diazoxide) even induced vasodilatation, and ANT inhibitors (bongkrekic acid, atractyloside) attenuated HPV. This pharmacological profile differs clearly from that described for mitoK(ATP). Accordingly, co-immunoprecipitation experiments provided no evidence for association of complex II subunits SDH-A, -B and -C with ANT, ATP synthase or cytochrome c oxidase in murine heart mitochondria. Hence, it is likely that the inhibitory effects on HPV that we observed in our experiments result from modulation of several mitochondrial protein complexes independently involved in the signalling cascade such as ROS-producing complex II and ANT-regulated mitochondrial permeability transition pore.