Chronic Hypoxia In Mice Research Articles

Circ‐myh8 Promotes Pulmonary Hypertension by Recruiting KAT7 to Govern Hypoxia‐Inducible Factor‐1α Expression

Background Aberrant expression of circular RNAs (circRNAs) contributes to the initiation and progression of pulmonary hypertension (PH). Hypoxia-inducible factor (HIF) is a well-known modulator of hypoxia-induced PH. The role and underlying mechanism of circRNAs in the regulation of HIF expression remains elusive. Methods and Results We profiled pulmonary artery transcriptomes using RNA sequencing and screened circRNAs associated with hypoxia treatment. The expression of a novel circRNA, circ_chr11_67292179-67294612 (circ-myh8), was increased by hypoxia in a time-dependent manner. We evaluated the effects of circ-myh8 overexpression by adeno-associated virusor inhibition by short hairpin RNA on proliferation and cell cycling in mice and pulmonary artery smooth muscle cells. Overexpression of circ-myh8 promotes PH under normoxia, and disruption of circ-myh8 by short hairpin RNA mitigates PH in chronic hypoxic mice. Biologically, circ-myh8 induces the proliferation and cell-cycle progression of pulmonary artery smooth muscle cells in vivo and in vitro. Mechanistically, RNA pull-down and RNA immunoprecipitation assays were used to examine the interaction of circRNAs with the binding protein KAT7 (lysine acetyltransferase 7). The acetylation level of lysine 5 of histone H4 in the transcriptional initiation region of HIF1α was determined by chromatin immunoprecipitation assay followed by reverse transcription-quantitativepolymerase chain reaction. Circ-myh8 acts as a modular scaffold to recruit histone acetyltransferase KAT7 to the promoters of HIF1α, which elicits acetylation of lysine 5 of histone H4 in their promoters. Conclusions Our findings not only reveal the pivotal roles of circ-myh8 in governing histone modification in anti-PH treatment but also advocate triggering the circ-myh8/KAT7/HIF1α pathway to combat PH.

Calpain-1 mediates vascular remodelling and fibrosis via HIF-1α in hypoxia-induced pulmonary hypertension.

Calpain‐1, a calcium‐activated neutral cysteine proteases, has been reported to be involved in the formation of pulmonary hypertension. HIF‐1α, an oxygen‐sensitive transcription factor, has been reported to activate genes involved in cell proliferation and extracellular matrix recombination. This study was designed to investigate the effect of calpain‐1 in hypoxic pulmonary hypertension (HPH) and to explore whether there is a relationship between calpain‐1 and HIF‐1α in this disease. In the hypoxia‐induced model of HPH, we found that hypoxia resulted in increased right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodelling and collagen deposition in lung tissues of mice. The levels of calpain‐1 and HIF‐1α were up‐regulated in the lung tissues of hypoxia‐treated mice and pulmonary arterial smooth muscle cells (PASMCs). Knock‐out of calpain‐1 restrained haemodynamic and histological changes induced by chronic hypoxia in mice, and inhibition of calpain‐1 also repressed the abnormal proliferation and migration of PASMCs. Besides, knock‐out or inhibition of calpain‐1 suppressed hypoxia‐induced expression of HIF‐1α, VEGF, PCNA, TGF‐β1, MMP2 and collagen I in vivo and in vitro. While inhibition of HIF‐1α abolished the above effects of calpain‐1. Furthermore, we found that calpain‐1 mediates the expression of HIF‐1α through NF‐κB (P65) under hypoxia conditions. In conclusion, our results suggest that calpain‐1 plays a pivotal role in hypoxia‐induced pulmonary vascular remodelling and fibrosis through HIF‐1α, providing a better understanding of the pathogenesis of HPH.

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

Cytochrome P450 Epoxygenase-Dependent Activation of TRPV4 Channel Participates in Enhanced Serotonin-Induced Pulmonary Vasoconstriction in Chronic Hypoxic Pulmonary Hypertension.

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic trpv4−/− mice. These results suggest that TRPV4 contributes to the enhanced 5-HT-induced vasoconstriction in chronic hypoxic PAs, in part via the CYP-EET-TRPV4 pathway. Our results further support the notion that manipulation of TRPV4 function may offer a novel therapeutic strategy for the treatment of hypoxia-related pulmonary hypertension.

CD146-HIF-1\u03b1 hypoxic reprogramming drives vascular remodeling and pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease of cardiopulmonary units. No cure is currently available due to an incomplete understanding of vascular remodeling. Here we identify CD146-hypoxia-inducible transcription factor 1 alpha (HIF-1α) cross-regulation as a key determinant in vascular remodeling and PAH pathogenesis. CD146 is markedly upregulated in pulmonary artery smooth muscle cells (PASMCs/SMCs) and in proportion to disease severity. CD146 expression and HIF-1α transcriptional program reinforce each other to physiologically enable PASMCs to adopt a more synthetic phenotype. Disruption of CD146-HIF-1α cross-talk by genetic ablation of Cd146 in SMCs mitigates pulmonary vascular remodeling in chronic hypoxic mice. Strikingly, targeting of this axis with anti-CD146 antibodies alleviates established pulmonary hypertension (PH) and enhances cardiac function in two rodent models. This study provides mechanistic insights into hypoxic reprogramming that permits vascular remodeling, and thus provides proof of concept for anti-remodeling therapy for PAH through direct modulation of CD146-HIF-1α cross-regulation.

Basic research: Suffocating the heart to stimulate regeneration.

Ischaemic cardiomyopathy leads to destruction of cardiomyocytes beyond the regenerative potential of the adult human heart. The murine heart can regenerate in utero and shortly after birth, but oxidative stress eventually arrests cardiomyocyte division. Chronic hypoxia in mice has now been shown to induce the cell cycle in cardiomyocytes, resulting in cardiac regeneration.

Abstract 13513: Roles of Endothelium-Dependent Hyperpolarization in Pulmonary Microcirculation in Mice

Background: The endothelium modulates vascular tone by synthesizing and releasing endothelium-derived relaxing factors, including prostacycline (PGI 2 ), nitric oxide (NO), and endothelium-dependent hyperpolarization (EDH). Although it is widely known that EDH plays an important role in systemic microcirculation, its role in pulmonary microcirculation remains largely unknown. Furthermore, although pulmonary hypertension (PH) is recognized as a progressive vasculopathy, the initiating mechanisms of the vasculopathy also remain to be elucidated. In this study, we thus aimed to elucidate the role of EDH in pulmonary microcirculation. Methods and Results: We performed experiments with isolated perfused mouse lungs. Isolated whole lungs were prepared from control mice and chronic hypoxic mice (10% O 2 for 1, 2 and 28 days). We evaluated the contribution of PGI 2 , NO, and EDH in isolated lungs by the inhibitory effect of indomethacin (Indo, 10 -5 mol/L), N ω -nitro-L-arginine (L-NNA, 10 -4 mol/L), and apamin (Apa, 10 -6 mol/L) plus charybdotoxin (CTx, 10 -7 mol/L), respectively. In control mice, endothelium-dependent relaxations to bradykinin (BK, 10 -5 mol/L) were resistant to Indo or L-NNA, but were highly sensitive to the combination of Apa and CTx, indicating the primary role of EDH in pulmonary microcirculation. Endothelium-dependent relaxations to BK were significantly reduced in the presence of L-NNA in mice exposed to hypoxia for one day, indicating a transition from EDH to NO in BK-mediated relaxations before developing PH. Importantly, endothelium-independent relaxations were subsequently reduced in the lungs from hypoxic mice for 2 and 28 days compared with normoxic lungs. Conclusions: These results indicate that EDH-mediated relaxations play primary roles in pulmonary microcirculation under physiological conditions and that hypoxia rapidly impairs EDH-mediated responses followed by vascular smooth muscle dysfunctions in pulmonary microcirculation. These findings may provide clues to elucidate the initiating mechanisms of hypoxia-induced PH.

NPY/Y₁ receptor-mediated vasoconstrictory and proliferative effects in pulmonary hypertension.

Pulmonary arteries (PAs) are innervated, but little is known about the role of neuronal axis in pulmonary hypertension (PH). Here, we have examined the role of the neuropeptide Y (NPY) and its Y₁ receptor in PH pathogenesis. NPY was localized by immunofluorescence. Expression of NPY and Y₁ receptor were determined by quantitative PCR. Cellular response to NPY stimulation was assessed by Western blotting, thymidine incorporation and calcium imaging. Wire myography and isolated perfused mouse lung were applied to study pulmonary vasoactive effects of NPY. Selective receptor antagonists were used to assess the contribution of receptor subtypes in mediating NPY effects. Samples from PH patients showed increased NPYergic innervation within the PA wall and higher Y₁ receptor expression, compared with donors. However, NPY levels were unchanged in both PA and serum. In the chronic hypoxic mouse model, Y₁ receptor were up-regulated, while expression of both NPY and Y₁ receptor was increased in the lungs of monocrotaline and SU5416-hypoxia rats. On a functional level, NPY acutely increased intracellular calcium levels and enhanced vasoconstriction of lung vessels preconstricted with adrenaline. Furthermore, NPY stimulated proliferation of human pulmonary arterial smooth muscle cells and activated p38 and PKD pathways. Correspondingly, higher phosphorylation of PKD was observed in remodelled vessels from PH patients. The selective Y₁ receptor antagonist, BIBO 3304, concentration-dependently inhibited vasoconstrictive and proliferative effects of NPY. NPY and Y₁ receptor are possible mediators of both vasoconstriction and pulmonary vascular remodelling in PH.

Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels

ObjectiveIn vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.Approach and ResultMale wild type and KCa3.1−/−/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1−/−/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.ConclusionDespite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.

Supplementing exposure to hypoxia with a copper depleted diet does not exacerbate right ventricular remodeling in mice.

BackgroundPulmonary hypertension and subsequent right ventricular (RV) failure are associated with high morbidity and mortality. Prognosis is determined by occurrence of RV failure. Currently, adequate treatment for RV failure is lacking. Further research into the molecular basis for the development of RV failure as well as the development of better murine models of RV failure are therefore imperative. We hypothesize that adding a low-copper diet to chronic hypoxia in mice reinforces their individual effect and that the combination of mild pulmonary vascular remodeling and capillary rarefaction, induces RV failure.MethodsSix week old mice were subjected to normoxia (N; 21% O2) or hypoxia (H; 10% O2) during a period of 8 weeks and received either a normal diet (Cu+) or a copper depleted diet (Cu-). Cardiac function was assessed by echocardiography and MRI analysis.Results and ConclusionHere, we characterized a mouse model of chronic hypoxia combined with a copper depleted diet and demonstrate that eight weeks of chronic hypoxia (10%) is sufficient to induce RV hypertrophy and subsequent RV failure. Addition of a low copper diet to hypoxia did not have any further deleterious effects on right ventricular remodeling.

Endothelial Krüppel-Like Factor 4 Modulates Pulmonary Arterial Hypertension

Krüppel-like factor 4 (KLF4) is a transcription factor expressed in the vascular endothelium, where it promotes anti-inflammatory and anticoagulant states, and increases endothelial nitric oxide synthase expression. We examined the role of endothelial KLF4 in pulmonary arterial (PA) hypertension (PAH). Mice with endothelial KLF4 knockdown were exposed to hypoxia for 3 weeks, followed by measurement of right ventricular and PA pressures, pulmonary vascular muscularization, and right ventricular hypertrophy. The effect of KLF4 on target gene expression was assessed in lungs from these mice, verified in vitro by small interfering RNA (siRNA) knockdown of KLF4, and further studied at the promoter level with cotransfection experiments. KLF4 expression was measured in lung tissue from patients with PAH and normal control subjects. We found that, after hypoxia, right ventricular and PA pressures were significantly higher in KLF4 knockdown animals than controls. Knockdown animals also had more severe pulmonary vascular muscularization and right ventricular hypertrophy. KLF4 knockdown resulted in increased pulmonary expression of endothelin-1 and decreased expression of endothelial nitric oxide synthase, endothelin receptor subtype B, and prostacyclin synthase. Concordant findings were observed in vitro, both with siRNA knockdown of KLF4 and promoter activity assays. Finally, KLF4 expression was reduced in lungs from patients with PAH. In conclusion, endothelial KLF4 regulates the transcription of genes involved in key pathways implicated in PAH, and its loss exacerbates pulmonary hypertension in response to chronic hypoxia in mice. These results introduce a novel transcriptional modulator of PAH, with the potential of becoming a new therapeutic target.

Hypoxia- or PDGF-BB-dependent paxillin tyrosine phosphorylation in pulmonary hypertension is reversed by HIF-1α depletion or imatinib treatment.

Chronic exposure to hypoxia induces a pronounced remodelling of the pulmonary vasculature leading to pulmonary hypertension (PH). The remodelling process also entails increased proliferation and decreased apoptosis of pulmonary arterial smooth muscle cells (PASMC), processes regulated by the cytoskeletal protein paxillin. In this study, we aimed to examine the molecular mechanisms leading to deregulation of paxillin in PH. We detected a time-dependent increase in paxillin tyrosine 31 (Y31) and 118 (Y118) phosphorylation following hypoxic exposure (1 % O2) or platelet-derived growth factor (PDGF)-BB stimulation of primary human PASMC. In addition, both, hypoxia- and PDGF-BB increased the nuclear localisation of phospho-paxillin Y31 as indicated by immunofluorescence staining in human PASMC. Elevated paxillin tyrosine phosphorylation in human PASMC was attenuated by hypoxia-inducible factor (HIF)-1α depletion or by treatment with the PDGF-BB receptor antagonist, imatinib. Moreover, we observed elevated paxillin Y31 and Y118 phosphorylation in the pulmonary vasculature of chronic hypoxic mice (21 days, 10 % O2) which was reversible by imatinib-treatment. PDGF-BB-dependent PASMC proliferation was regulated via the paxillin-Erk1/2-cyclin D1 pathway. In conclusion, we suggest paxillin up-regulation and phosphorylation as an important mechanism of vascular remodelling underlying pulmonary hypertension.

Precapillary Oxygenation Contributes Relevantly to Gas Exchange in the Intact Lung

Oxygen uptake is the elemental function of the lung. However, current understanding of this process has largely been derived from theoretical considerations and measurements of global pulmonary gas exchange. To report the direct visualization of pulmonary oxygen uptake in vivo and its use for the analysis of temporal and spatial oxygenation profiles along individual arteriovenous pathways in lungs of healthy and chronic hypoxic mice. A murine model for intravital microscopy of the breathing lung under sealed thorax conditions was combined with multispectral oximetry for two-dimensional oxygen saturation mapping. This combination allowed for visualization of the blood oxygenation process from pulmonary arterioles to capillaries and venules in two-dimensional oxygen saturation maps. Temporal and spatial oxygenation profiles revealed that oxygenation occurs within 100 milliseconds over a distance of approximately 130 μm in the pulmonary microvasculature of the anesthetized mouse. About 50% of total oxygen uptake takes place in precapillary arterioles of less than 30 μm in diameter before the blood enters the alveolar capillary bed. In chronic hypoxic mice, precapillary oxygenation was significantly attenuated as a result of the widened transarteriolar diffusion distance. Oxygen saturation mapping in the intact lung yields unique insights into the temporal and spatial characteristics of pulmonary gas exchange in intact and diseased lungs. Precapillary gas exchange contributes importantly to blood oxygenation at rest, but is attenuated in remodeled lung arterioles, which may be of relevance in pulmonary hypertension.

Alterations of N‐3 Polyunsaturated Fatty Acid‐Activated K2P Channels in Hypoxia‐Induced Pulmonary Hypertension

Polyunsaturated fatty acid (PUFA)-activated two-pore domain potassium channels (K2P ) have been proposed to be expressed in the pulmonary vasculature. However, their physiological or pathophysiological roles are poorly defined. Here, we tested the hypothesis that PUFA-activated K2P are involved in pulmonary vasorelaxation and that alterations of channel expression are pathophysiologically linked to pulmonary hypertension. Expression of PUFA-activated K2P in the murine lung was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), by patch clamp (PC) and myography. K2P -gene expression was examined in chronic hypoxic mice. qRT-PCR showed that the K2P 2.1 and K2P 6.1 were the predominantly expressed K2P in the murine lung. IHC revealed protein expression of K2P 2.1 and K2P 6.1 in the endothelium of pulmonary arteries and of K2P 6.1 in bronchial epithelium. PC showed pimozide-sensitive K2P -like K(+) -current activated by docosahexaenoic acid (DHA) in freshly isolated endothelial cells as well as DHA-induced membrane hyperpolarization. Myography on pulmonary arteries showed that DHA induced concentration-dependent instantaneous relaxations that were resistant to endothelial removal and inhibition of NO and prostacyclin synthesis and to a cocktail of blockers of calcium-activated K(+) channels but were abolished by high extracellular (30mM) K(+) -concentration. Gene expression and protein of K2P 2.1 were not altered in chronic hypoxic mice, while K2P 6.1 was up-regulated by fourfold. In conclusion, the PUFA-activated K2P 2.1 and K2P 6.1 are expressed in murine lung and functional K2P -like channels contribute to endothelium hyperpolarization and pulmonary artery relaxation. The increased K2P 6.1-gene expression may represent a novel counter-regulatory mechanism in pulmonary hypertension and suggest that arterial K2P 2.1 and K2P 6.1 could be novel therapeutic targets.

Chronic hypoxia selectively enhances L- and T-type voltage-dependent Ca2+ channel activity in pulmonary artery by upregulating Cav1.2 and Cav3.2

Hypoxia-induced pulmonary hypertension (HPH) is characterized by sustained pulmonary vasoconstriction and vascular remodeling, both of which are mediated by pulmonary artery smooth muscle cell (PASMC) contraction and proliferation, respectively. An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation in PASMCs. Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) is an important pathway for the regulation of [Ca²⁺]cyt. The potential role for L- and T-type VDCC in the development of HPH is still unclear. Using a hypoxic-induced pulmonary hypertension mouse model, we undertook this study to identify if VDCC in pulmonary artery (PA) are functionally upregulated and determine which type of VDCC are altered in HPH. Mice subjected to chronic hypoxia developed pulmonary hypertension within 4 wk, and high-K⁺- and U-46619-induced contraction of PA was greater in chronic hypoxic mice than that in normoxic control mice. Additionally, we demonstrate that high-K⁺- and U-46619-induced Ca²⁺ influx in PASMC is significantly increased in the hypoxic group. The VDCC activator, Bay K8864, induced greater contraction of the PA of hypoxic mice than in that of normoxic mice in isometric force measurements. L-type and T-type VDCC blockers significantly attenuated absolute contraction of the PA in hypoxic mice. Chronic hypoxia did not increase high-K⁺- and U-46619-induced contraction of mesenteric artery (MA). Compared with MA, PA displayed higher expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) upon exposure to chronic hypoxia. In conclusion, both L-type and T-type VDCC were functionally upregulated in PA, but not MA, in HPH mice, which could result from selectively increased expression of Cav1.2 and Cav3.2.

Abstract 18277: Role of the miR-204/miR-145 Communication in the Etiology of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by distal pulmonary arteries remodeling secondary to the proliferation and resistance to apoptosis of the pulmonary artery smooth muscle (PASMC). We recently described that this phenotype is associated with 7 aberrantly expressed miRNAs in PAH-PASMC. We showed that miR-204 was the most downregulated and accounts for the activation of the neoplasic axis STAT3/Pim1/NFAT, inducing PASMC proliferation and resistance to apoptosis. Because miR-204 downregulation precedes the development of PAH in chronic hypoxic mice and monocrotaline rats, we tested whether its downregulation accounts for the deregulation of the 6 others affected miRNAs in PAH. As miR-145 is implicated in systemic vascular remodeling and is the one of the most upregulated miRNA in PAH we focused our research on the regulation of miR-145 by miR-204. Methods/Results: Using lungs biopsies from 8 PAH and 8 control patients we confirmed the downregulation of miR-204 and the upregulation of miR-145 in PAH. Moreover, both miR-204 downregulation and miR-145 upregulation correlate with PAH severity. These findings were confirmed in both lungs and distal PAs of 5-MCT and 5 Sugen/Hypoxia/Normoxia rats compare to 5 control rats in which severity was assessed by CT-scan angiogram. Time to event analysis in both monocrotaline (1; 2; 3 and 4 weeks post MCT injection) and Sugen (8 and 15 weeks post Sugen) rats showed that miR-204 downregulation precedes miR-145 upregulation. In vitro using cultured human PASMC from 5 control and 3 PAH patients we showed that miR-204 upregulation in PAH-PASMC decreases miR-145; while miR-204 downregulation in control PASMC increases miR-145. Finally, in vivo nebulized MCT rats with miR-204 mimic had significantly less miR-145 compared to rats nebulized with mimic negative control. Conclusion: We demonstrated for the first time in PAH that one miRNA (miR-204) can regulate the expression of other miRNA such as miR-145. Because miR-204 downregulation precede miR-145 upregulation and PAH development, miR-204 could be considered as an early molecular marker of PAH and might represent the molecular origin of this lethal disease.

Improved energy supply regulation in chronic hypoxic mouse counteracts hypoxia-induced altered cardiac energetics.

BackgroundHypoxic states of the cardiovacular system are undoubtedly associated with the most frequent diseases of modern time. Therefore, understanding hypoxic resistance encountered after physiological adaptation such as chronic hypoxia, is crucial to better deal with hypoxic insult. In this study, we examine the role of energetic modifications induced by chronic hypoxia (CH) in the higher tolerance to oxygen deprivation.Methodology/Principal FindingsSwiss mice were exposed to a simulated altitude of 5500 m in a barochamber for 21 days. Isolated perfused hearts were used to study the effects of a decreased oxygen concentration in the perfusate on contractile performance (RPP) and phosphocreatine (PCr) concentration (assessed by 31P-NMR), and to describe the integrated changes in cardiac energetics regulation by using Modular Control Analysis (MoCA). Oxygen reduction induced a concomitant decrease in RPP (−46%) and in [PCr] (−23%) in Control hearts while CH hearts energetics was unchanged. MoCA demonstrated that this adaptation to hypoxia is the direct consequence of the higher responsiveness (elasticity) of ATP production of CH hearts compared with Controls (−1.88±0.38 vs −0.89±0.41, p<0.01) measured under low oxygen perfusion. This higher elasticity induces an improved response of energy supply to cellular energy demand. The result is the conservation of a healthy control pattern of contraction in CH hearts, whereas Control hearts are severely controlled by energy supply.Conclusions/SignificanceAs suggested by the present study, the mechanisms responsible for this increase in elasticity and the consequent improved ability of CH heart metabolism to respond to oxygen deprivation could participate to limit the damages induced by hypoxia.

Role of Epidermal Growth Factor Inhibition in Experimental Pulmonary Hypertension

Epidermal growth factor (EGF) and its receptors play a role in cell proliferation and survival and are implicated in the pathobiology of pulmonary arterial hypertension (PAH). To study the role of EGF inhibition on experimental pulmonary hypertension. We investigated (1) the effects of three clinically approved EGF receptor (EGFR) antagonists in vitro on rat pulmonary arterial smooth muscle cell proliferation and in vivo on experimental pulmonary hypertension (PH) induced by monocrotaline injection in rats and by chronic hypoxia in mice, and (2) the expression of EGFR in the lung tissues from experimental and clinical PH. The EGFR inhibitors gefitinib, erlotinib, and lapatinib inhibited the EGF-induced proliferation of pulmonary arterial smooth muscle cells. In rats with established PH, gefitinib and erlotinib significantly reduced right ventricular systolic pressure and right ventricular hypertrophy. In addition, the medial wall thickness and muscularization of pulmonary arteries were improved. In contrast, lapatinib did not provide therapeutic benefit. These EGFR antagonists at their highest tolerable dose did not yield significant improvement in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice with chronic hypoxic PH. Moreover, no significant alteration in the EGFR expression was detected in the lung tissues from patients with idiopathic PAH. The partial therapeutic efficacy of the EGFR antagonists in animal models of pulmonary hypertension and the absence of significant alteration in EGFR expression in the lungs from patients with idiopathic PAH suggest that EGFRs do not represent a promising target for the treatment of pulmonary hypertension.

Heme oxygenase-1-dependent central cardiorespiratory adaptations to chronic hypoxia in mice

Adaptations to chronic hypoxia (CH) could reflect cellular changes within the cardiorespiratory regions of the rostral ventrolateral medulla (RVLM), the C1 region, and the pre-Bötzinger complex (pre-BötC). Previous studies have shown that the hypoxic chemosensitivity of these regions are heme oxygenase (HO) dependent and that CH induces HO-1. To determine the time course of HO-1 induction within these regions and explore its relevance to the respiratory and sympathetic responses during CH, the expression of HO-1 mRNA and protein in the RVLM and measures of respiration, sigh frequency, and sympathetic activity (spectral analysis of heart rate) were examined during 10 days of CH. Respiratory and sympathetic responses to acute hypoxia were obtained in chronically instrumented awake wild-type (WT) and HO-1 null mice. After 4 days of CH, there was a significant induction of HO-1 within the C1 region and pre-BötC. WT mice acclimated to CH by increasing peak diaphragm EMG after 10 days of CH but had no change in the respiratory response to acute hypoxia. There were no significant differences between WT and HO-1 null mice. In WT mice, hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity initially declined before returning toward baseline after 5 days of CH, correlating with the induction of HO-1. In contrast, HO-1 null mice had a persistent decline in hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity. We conclude that induction of HO-1 in these RVLM cardiorespiratory regions may be important for the hypoxic sensitivity of sighs and sympathetic activity during CH.

Circulating fibrocytes in pulmonary vascular remodelling

Einleitung: Circulating fibrocytes (CFs) comprise a recently described new cell type of blood born derived fibroblast like cells that are recruited from the circulation to sites of wound repair, vascular remodelling or fibrotic tissue remodelling. A role of these cells in the pulmonary vascular remodelling in pulmonary hypertension has been proposed. Hence, we focussed on these cells type to decipher their role in pulmonary hypertension. Methoden: FACS, confocal laser scanning microscopy; Western blot, qPCR, Rap pull down assay and in vivo hypoxia induced pulmonary hypertension murine model. Ergebnisse: We observed that freshly isolated human peripheral mononuclear cells (PBMCs) upon treatment with treprostinil exhibited poor cell adhesion and subsequently reduction of cell differentiation in ex-vivo expansion. Our further investigations indicated a cAMP induced integrin impairment that was a PKA independent process transduced via an alternative cAMP-RAP-ERK axis. We created blood chimeric animals by bone marrow transplantation of wild type C57 mice with bone marrow derived from eGFP mice. We found a significant contribution of these cells to pulmonary vascular remodelling of the chronic hypoxic mouse and this cellular recruitment was inhibited upon treatment with continuous treprostinil infusion in the mouse directed by an implanted minipump. Diskussion: We could show that prostanoids, which are approved for clinical therapy of pulmonary hypertension, potently inhibit the in vitro differentiation of CF from blood as well as the in vivo recruitment and integration into pulmonary resistance arteries. We thereby showed that CFs are a new therapeutic target in this devastating disease. The further aims are now to more precisely dissect the pathway of prostanoids action on circulating fibrocytes by introducing mutated Rap, Raf and ERK proteins.