Rodent Models Of Pulmonary Arterial Hypertension Research Articles

Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension.

Background Cardiac failure is the primary cause of death in most patients with pulmonary arterial hypertension (PH). As pleiotropic cytokines, human resistin (Hresistin) and its rodent homolog, resistin-like molecule α, are mechanistically critical to pulmonary vascular remodeling in PH. However, it is still unclear whether activation of these resistin-like molecules can directly cause PH-associated cardiac dysfunction and remodeling. Methods and Results In this study, we detected Hresistin protein in right ventricular (RV) tissue of patients with PH and elevated resistin-like molecule expression in RV tissues of rodents with RV hypertrophy and failure. In a humanized mouse model, cardiac-specific Hresistin overexpression was sufficient to cause cardiac dysfunction and remodeling. Dilated hearts exhibited reduced force development and decreased intracellular Ca2+ transients. In the RV tissues overexpressing Hresistin, the impaired contractility was associated with the suppression of protein kinase A and AMP-activated protein kinase. Mechanistically, Hresistin activation triggered the inflammation mediated by signaling of the key damage-associated molecular pattern molecule high-mobility group box 1, and subsequently induced pro-proliferative Ki67 in RV tissues of the transgenic mice. Intriguingly, an anti-Hresistin human antibody that we generated protected the myocardium from hypertrophy and failure in the rodent PH models. Conclusions Our data indicate that Hresistin is expressed in heart tissues and plays a role in the development of RV dysfunction and maladaptive remodeling through its immunoregulatory activities. Targeting this signaling to modulate cardiac inflammation may offer a promising strategy to treat PH-associated RV hypertrophy and failure in humans.

Abstract 10248: Regulation of the Methylation and Expression Levels of the Bmpr2 Gene by Sin3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension

Background: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of Switch-Independent 3a (SIN3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. Methods: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells (hPASMC). Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific Adeno-Associated Virus serotype 1 (AAV1) or a lentivirus encoding for human SIN3a in vivo . Results: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. Interestingly, we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. Interestingly, SIN3a overexpression inhibited hPASMC proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 and EZH2 while promoting the expression of the DNA demethylase TET1. Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF binding to the BMPR2 promoter. Finally, we identified intratracheal delivery of AAV1.hSIN3a to be a beneficial therapeutic approach in PAH- by attenuating pulmonary vascular and RV remodeling, decreasing RVSP and mPAP pressure, and restoring BMPR2 expression in rodent models of PAH. Conclusions: Altogether, our study unveiled the protective/beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in hPASMC.

Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

Abstract 13932: Lung-targeted Sin3a Gene Therapy as a Promising Strategy to Restore Bmpr2 Expression in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a fatal lung disease of multifactorial etiology, with no curative treatment. Several studies have previously suggested that hypermethylation of the BMPR2 promoter may be associated with gene repression and disease progression. However, the underlying mechanisms have yet to be discovered. Sin3A/B (Switch-Independent 3) plays a critical role in the transcriptional regulation of genes through various epigenetic mechanisms. Here, we investigated for the first time the role of SIN3a in the regulation of BMPR2 methylation and expression in PAH. Methods: Expression of SIN3a was analyzed by qRT-PCR and western blot in lung tissues from PAH patients and rodent models of PAH. Using a gain- and loss-of-function approach, we investigated the role of SIN3a on cell proliferation (BrdU assay) and migration (Boyden chamber assay), and BMPR2 levels in primary human pulmonary arterial smooth muscle cells (hPASMC) and endothelial cells (hPAEC). The methylation level was analyzed by MS-PCR. The therapeutic potential of SIN3a was tested in vivo in the Sugen/Hypoxia (SuHx) mouse and monocrotaline (MCT) rat models of PAH using an adeno-associated virus 1 encoding human SIN3a. Results: We found a significant downregulation of SIN3a expression in the lung samples from PAH patients, SuHx mice, and MCT rats. In hPASMC and hPAEC, our results showed that SIN3a inhibits cell proliferation, migration, and upregulates BMPR2 through two distinct pathways. In hPASMC, our data showed that SIN3a upregulates BMPR2 expression by inhibiting the methylation level of the BMPR2 promoter. In hPAEC, SIN3a restored BMPR2 expression independently of the methylation status by upregulating the FOXK2 transcription factor. In vivo , our results showed that restoring SIN3a expression by gene therapy significantly decreased MCT- and SuHx-induced PAH as illustrated by decreased vascular and RV remodeling, hypertrophy, PAP and RVSP. Conclusions: Altogether, our study revealed that SIN3a plays a critical role in the regulation of BMPR2 expression by modulating the lung epigenetic landscape. Additionally, our study identifies lung-targeted SIN3a gene therapy as a new promising therapeutic strategy for treating PAH patients.

Loss of DP1 Aggravates Vascular Remodeling in Pulmonary Arterial Hypertension via mTORC1 Signaling.

Rationale: Vascular remodeling, including smooth muscle cell hypertrophy and proliferation, is the key pathological feature of pulmonary arterial hypertension (PAH). Prostaglandin I2 analogs (beraprost, iloprost, and treprostinil) are effective in the treatment of PAH. Of note, the clinically favorable effects of treprostinil in severe PAH may be attributable to concomitant activation of DP1 (D prostanoid receptor subtype 1).Objectives: To study the role of DP1 in the progression of PAH and its underlying mechanism.Methods: DP1 levels were examined in pulmonary arteries of patients and animals with PAH. Multiple genetic and pharmacologic approaches were used to investigate DP1-mediated signaling in PAH.Measurements and Main Results: DP1 expression was downregulated in hypoxia-treated pulmonary artery smooth muscle cells and in pulmonary arteries from rodent PAH models and patients with idiopathic PAH. DP1 deletion exacerbated pulmonary artery remodeling in hypoxia-induced PAH, whereas pharmacological activation or forced expression of the DP1 receptor had the opposite effect in different rodent models. DP1 deficiency promoted pulmonary artery smooth muscle cell hypertrophy and proliferation in response to hypoxia via induction of mTORC1 (mammalian target of rapamycin complex 1) activity. Rapamycin, an inhibitor of mTORC1, alleviated the hypoxia-induced exacerbation of PAH in DP1-knockout mice. DP1 activation facilitated raptor dissociation from mTORC1 and suppressed mTORC1 activity through PKA (protein kinase A)-dependent phosphorylation of raptor at Ser791. Moreover, treprostinil treatment blocked the progression of hypoxia-induced PAH in mice in part by targeting the DP1 receptor.Conclusions: DP1 activation attenuates hypoxia-induced pulmonary artery remodeling and PAH through PKA-mediated dissociation of raptor from mTORC1. These results suggest that the DP1 receptor may serve as a therapeutic target for the management of PAH.

Therapeutic Targeting of Vascular Remodeling and Right Heart Failure in Pulmonary Arterial Hypertension with a HIF-2α Inhibitor.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling that leads to right heart failure (RHF) and death. Current therapies do not target vascular remodeling and RHF, and result in only modest improvement of morbidity and mortality. To determine whether targeting HIF-2α (hypoxia-inducible factor-2α) with a HIF-2α-selective inhibitor could reverse PAH and RHF in various rodent PAH models. HIF-2α and its downstream genes were evaluated in lung samples and pulmonary arterial endothelial cells and smooth muscle cells from patients with idiopathic PAH as well as various rodent PAH models. A HIF-2α-selective inhibitor was used in human lung microvascular endothelial cells and in Egln1Tie2Cre mice, and in Sugen 5416/hypoxia- or monocrotaline-exposed rats. Upregulation of HIF-2α and its target genes was observed in lung tissues and isolated pulmonary arterial endothelial cells from patients with idiopathic PAH and three distinct rodent PAH models. Pharmacological inhibition of HIF-2α by the HIF-2α translation inhibitor C76 (compound 76) reduced right ventricular systolic pressure and right ventricular hypertrophy and inhibited RHF and fibrosis as well as obliterative pulmonary vascular remodeling in Egln1Tie2Cre mice and Sugen 5416/hypoxia PAH rats. Treatment of monocrotaline-exposed PAH rats with C76 also reversed right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling; prevented RHF; and promoted survival. These findings demonstrate that pharmacological inhibition of HIF-2α is a promising novel therapeutic strategy for the treatment of severe vascular remodeling and right heart failure in patients with PAH.

Smooth Muscle-Specific BCL6+/- Knockout Abrogates Sex Bias in Chronic Hypoxia-Induced Pulmonary Arterial Hypertension in Mice.

The “estrogen paradox” in pulmonary arterial hypertension (PAH) refers to observations that while there is a higher incidence of idiopathic PAH in women, rodent models of PAH show male dominance and estrogens are protective. To explain these differences, we previously proposed the neuroendocrine-STAT5-BCL6 hypothesis anchored in the sex-biased and species-specific patterns of growth hormone (GH) secretion by the pituitary, the targeting of the hypothalamus by estrogens to feminize GH secretion patterns, and the role of the transcription factors STAT5a/b and BCL6 as downstream mediators of this patterned GH-driven sex bias. As a test of this hypothesis, we previously reported that vascular smooth muscle cell- (SMC-) specific deletion of the STAT5a/b locus abrogated the male-dominant sex bias in the chronic hypoxia model of PAH in mice. In the present study, we confirmed reduced BCL6 expression in pulmonary arterial (PA) segments in both male and female SMC:STAT5a/b−/− mice. In order to test the proposed contribution of BCL6 to sex bias in PAH, we developed mice with SMC-specific deletion of BCL6+/− by crossing SM22α-Cre mice with BCL6-floxed mice and investigated sex bias in these mutant mice in the chronic hypoxia model of PAH. We observed that the male-bias observed in wild-type- (wt-) SM22α-Cre-positive mice was abrogated in the SMC:BCL6+/− knockouts—both males and females showed equivalent enhancement of indices of PAH. The new data confirm BCL6 as a contributor to the sex-bias phenotype observed in hypoxic PAH in mice and support the neuroendocrine-STAT5-BCL6 hypothesis of sex bias in this experimental model of vascular disease.

Inhibition of CRTH2-mediated Th2 activation attenuates pulmonary hypertension in mice.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary artery (PA) remodeling. T helper 2 cell (Th2) immune response is involved in PA remodeling during PAH progression. Here, we found that CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cell) expression was up-regulated in circulating CD3+CD4+ T cells in patients with idiopathic PAH and in rodent PAH models. CRTH2 disruption dramatically ameliorated PA remodeling and pulmonary hypertension in different PAH mouse models. CRTH2 deficiency suppressed Th2 activation, including IL-4 and IL-13 secretion. Both CRTH2+/+ bone marrow reconstitution and CRTH2+/+ CD4+ T cell adoptive transfer deteriorated hypoxia + ovalbumin-induced PAH in CRTH2-/- mice, which was reversed by dual neutralization of IL-4 and IL-13. CRTH2 inhibition alleviated established PAH in mice by repressing Th2 activity. In culture, CRTH2 activation in Th2 cells promoted pulmonary arterial smooth muscle cell proliferation through activation of STAT6. These results demonstrate the critical role of CRTH2-mediated Th2 response in PAH pathogenesis and highlight the CRTH2 receptor as a potential therapeutic target for PAH.

Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension.

Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr1]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and β-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in monocrotaline-exposed rats. These results show that ELA is an endogenous agonist of the human apelin receptor, exhibits a cardiovascular profile comparable to apelin, and is downregulated in human disease and rodent PAH models, and exogenous peptide can reduce the severity of cardiopulmonary remodeling and function in PAH in rats. This study provides additional proof of principle that an apelin receptor agonist may be of therapeutic use in PAH in humans.

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Reactive oxygen species effect PASMCs apoptosis via regulation of dynamin-related protein 1 in hypoxic pulmonary hypertension.

The high level of reactive oxygen species and up-regulation of mitochondrial fission protein dynamin-related protein-1, both of which involved in pulmonary artery smooth muscle cells (PASMCs) apoptosis, have been detected in the lungs of rodent pulmonary arterial hypertension models. However, the regulatory mechanisms between ROS and DRP1 are poorly understood. In this study, ROS inhibitor, hypoxic rodent PAH models, small interfering RNA, polymerase chain reaction, Western blot, flow cytometry, immunohistochemistry and immunofluorescence were used. We determined that ROS, mainly derive from mitochondria, mediate mitochondria fission of PASMCs contributing to pulmonary vascular remodeling. Meanwhile, we also observed that hypoxia-induced DRP1 expression depends on ROS generation, especially mitochondrial ROS (mROS). Moreover, the levels of ROS and mROS evoked by hypoxia were regulated by DRP1. Furthermore, we verified the apoptosis suppression of PASMCs under hypoxia due to the interaction between ROS/mROS and DRP1. Our study reveals a novel mechanism of hypoxia-induced pulmonary vascular remodeling, suggesting a new therapeutic strategy which is targeting on the positive feedback of ROS/mROS-DRP1 for the treatment of PAH.

TRAIL Deficient Mice Are Protected from Sugen/Hypoxia Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive lung disease diagnosed by an increase in pulmonary arterial blood pressure that is driven by a progressive vascular remodelling of small pulmonary arterioles. We have previously reported that tumor necrosis factor apoptosis-inducing ligand (TRAIL) protein expression is increased in pulmonary vascular lesions and pulmonary artery smooth muscle cells (PASMC) of patients with idiopathic PAH. The addition of recombinant TRAIL induces the proliferation and migration of PASMCs in vitro. TRAIL is required for hypoxia-induced pulmonary hypertension in mice, and blockade of TRAIL prevents and reduces disease development in other rodent models of PAH. Due to the availability of knockout and transgenic mice, murine models of disease are key to further advances in understanding the complex and heterogeneous pathogenesis of PAH. However, murine models vary in their disease severity, and are often criticized for lacking the proliferative pulmonary vascular lesions characteristic of PAH. The murine Sugen-hypoxic (SuHx) mouse model has recently been reported to have a more severe PAH phenotype consisting advanced pulmonary vascular remodelling. We therefore aimed to determine whether TRAIL was also required for the development of PAH in this model. C57BL/6 and TRAIL−/− mice were exposed to normoxia, Sugen5416 alone, hypoxia or both Sugen5416 and hypoxia (SuHx). We report here that SuHx treated C57BL/6 mice developed more severe PAH than hypoxia alone, and that TRAIL−/− mice were protected from disease development. These data further emphasise the importance of this pathway and support the use of the SuHx mouse model for investigating the importance of potential mediators in PAH pathogenesis.

Transient but not genetic loss of miR-451 is protective in the development of pulmonary arterial hypertension.

MicroRNAs are small noncoding RNAs involved in the regulation of gene expression and have recently been implicated in the development of pulmonary arterial hypertension (PAH). Previous work has established that miR-451 is upregulated in rodent models of PAH. The role of miR-451 in the pulmonary circulation is unknown. We therefore sought to assess the involvement of miR-451 in the development of PAH. Silencing of miR-451 was performed in vivo using miR-451 knockout mice and an anti-miR targeting mature miR-451 in rats. Coupled with exposure to hypoxia, indices of PAH were assessed. The effect of modulating miR-451 on human pulmonary artery smooth muscle cell proliferation and migration was analyzed. We observed a reduction in systolic right ventricular pressure in hypoxic rats pretreated with anti-miR-451 compared with hypoxia alone ([Formula: see text] mmHg and [Formula: see text] mmHg, respectively; [Formula: see text]). In miR-451 knockout mice, compared with wild-type hypoxic mice, no significant differences were observed following exposure to chronic hypoxia. In vitro analysis demonstrated that overexpression of miR-451 in human pulmonary artery smooth muscle cells promoted migration under serum-free conditions. No effect on cellular proliferation was observed. In conclusion, transient inhibition of miR-451 attenuated the development of PAH in hypoxia-exposed rats. Genetic deletion of miR-451 had no beneficial effect on indices of PAH, potentially because of pathway redundancy compensating for the loss of miR-451.

P156 Neutrophil and redox dependent proteolysis of bone morphogenetic protein 9: potential role in the pathogenesis of pulmonary arterial hypertension

IntroductionA critical reduction of bone morphogenetic protein type II receptor (BMPRII) in the pulmonary circulation, either due to the genetic loss-of-function mutations, heightened inflammation or prolonged hypoxia, is one of...

Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH.

Longitudinal In Vivo SPECT/CT Imaging Reveals Morphological Changes and Cardiopulmonary Apoptosis in a Rodent Model of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) has a complex pathogenesis involving both heart and lungs. Animal models can reflect aspects of the human pathology and provide insights into the development and underlying mechanisms of disease. Because of the variability of most animal models of PAH, serial in vivo measurements of cardiopulmonary function, morphology, and markers of pathology can enhance the value of such studies. Therefore, quantitative in vivo SPECT/CT imaging was performed to assess cardiac function, morphology and cardiac perfusion utilizing 201Thallium (201Tl) in control and monocrotaline-treated rats. In addition, lung and heart apoptosis was examined with 99mTc-Annexin V (99mTc-Annexin) in these cohorts. Following baseline imaging, rats were injected with saline or monocrotaline (50 mg/kg, i.p.) and imaged weekly for 6 weeks. To assess a therapeutic response in an established pulmonary hypertensive state, a cohort of rats received resveratrol in drinking water (3 mg/kg/day) on days 28–42 post-monocrotaline injection to monitor regression of cardiopulmonary apoptosis. PAH in monocrotaline-treated rats was verified by conventional hemodynamic techniques on day 42 (right ventricular systolic pressure (RSVP) = 66.2 mmHg in monocrotaline vs 28.8 mmHg in controls) and in terms of right ventricular hypertrophy (RV/LVS = 0.70 in monocrotaline vs 0.32 in controls). Resveratrol partially reversed both RVSP (41.4 mmHg) and RV/LVS (0.46), as well as lung edema and RV contractility +dP/dtmax. Serial 99mTc-Annexin V imaging showed clear increases in pulmonary and cardiac apoptosis when compared to baseline, which regressed following resveratrol treatment. Monocrotaline induced modest changes in whole-heart perfusion as assessed by 201TI imaging and cardiac morphological changes consistent with septal deviation and enlarged RV. This study demonstrates the utility of functional in vivo SPECT/CT imaging in rodent models of PAH and further confirms the efficacy of resveratrol in reversing established monocrotaline-induced PAH presumably by attenuation of cardiopulmonary apoptosis.

In This Issue

HomeCirculation ResearchVol. 110, No. 11In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published25 May 2012https://doi.org/10.1161/RES.0b013e31825dd680Circulation Research. 2012;110:1391MicroRNA-Mediated Reprogramming (p 1465)Jayawardena et al report the direct conversion of fibroblasts to cardiomyocytes by transfection with microRNAs (miRNAs).Download figureDownload PowerPointIf cardiologists could transform the scar tissue formed after myocardial infarction into functional muscle, patients might regain better heart function and avoid heart failure. To that end, researchers have shown that mouse fibroblasts can be directly converted into cardiomyocytes by transfection with three particular transcription factors. However, it had not been shown whether such conversion could take place in vivo. Jayawardena et al have now achieved just that, but they did not use the three transcription factors. Their approach was to use miRNAs, on the basis that these small noncoding RNAs can downregulate the expression of multiple genes at once, and that a number of specific miRNAs have been found to control cardiomyocyte development. In summary, the miRNAs might be more effective. The team transfected individual candidate miRNAs, and combinations thereof, into mouse cardiac fibroblasts. They came up with a combination of four miRNAs that could convert fibroblasts to cardiomyoctyes in vitro and in the hearts of mice after myocardial infarction. The researchers have yet to show whether such in vivo conversion confers functional improvement; nevertheless, they provide proof of principle that in vivo transformation is a possibility.RyR2 Phosphorylation in HF Progression (p 1474)Blocking phosphorylation of RyR2 protects against one sort of heart failure, but not another, say Respress et al.Download figureDownload PowerPointHeart failure is a major killer in the United States and across the globe, and it is known to be associated with an increase in the activity of a kinase called CaMKII. Inhibiting CaMKII can prevent or delay heart failure in animal models of the disease. CaMKII phosphorylates a number of targets, including the calcium channel RyR2, which regulates release of calcium from the sarcoplasmic reticulum in heart cells. Because inappropriate leaks of sarcoplasmic reticulum calcium are a feature of heart failure, the team investigated whether phosphorylation of RyR2 by CaMKII might be abnormal in the disease. Interestingly, they found that only nonischemic heart failure patients had elevated CaMKII phosphorylation of RyR2. In ischemic heart failure, this phosphorylation was normal. Similarly, preventing this particular phosphorylation in mice protected the animals against induced nonischemic, but not ischemic, heart failure. Although it is not yet clear why this CaMKII phosphorylation of RyR2 is triggered only in nonischemic heart failure, the results suggest that patients with this form of the disease could benefit from RyR2 phosphorylation–inhibiting treatments.Mitochondrial Fission in PAH (p 1484)Stopping mitochondria from splitting curbs pathological cell proliferation in pulmonary arterial hypertension (PAH), report Marsboom et al.Download figureDownload PowerPointPAH is a condition in which the pulmonary arteries become constricted, obstructed, and inflamed, causing increased vascular pressure and, ultimately, right ventricle hypertrophy and heart failure. Although the fundamental cause of the condition is still unknown, part of the problem is that the smooth muscle cells of the arteries begin to proliferate out of control. This hyperproliferation is associated with activation of the transcription factor HIF-1 and with fission of mitochondria. Now, Marsboom et al have figured out how these parts of the puzzle fit together. They show that stimulation of HIF-1 leads to the activation of a protein called dynamin-related protein 1 (DRP-1), a known promoter of mitochondrial fission. And this fission, in turn, drives cell-cycle progression. Importantly, inhibiting DRP-1 with a small molecule called Mdivi-1 prevented both mitochondrial fission and proliferation in human pulmonary artery smooth muscle cells. Mdivi-1 also prevented hyperproliferation in rodent models of PAH and significantly improved symptoms. Inhibiting mitochondrial fission with Mdivi-1 or other factors could be an effective treatment for PAH and potentially other proliferative disorders, say the authors. Previous Back to top Next FiguresReferencesRelatedDetails May 25, 2012Vol 110, Issue 11 Advertisement Article InformationMetrics © 2012 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31825dd680 Originally publishedMay 25, 2012 PDF download Advertisement

Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation. We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential. Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1α activation by CoCl(2) or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH. DRP-1-mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Serum Osteoprotegerin is Increased and Predicts Survival in Idiopathic Pulmonary Arterial Hypertension

We previously reported that osteoprotegerin (OPG) is regulated by pathways associated with pulmonary arterial hypertension (PAH), and is present at elevated levels within pulmonary vascular lesions and sera from patients with idiopathic PAH (IPAH). Since OPG is a naturally secreted protein, we investigated the relationship between serum OPG and disease severity and outcome in patients with IPAH and animal models. OPG mRNA expression was measured in pulmonary artery smooth muscle cells (PASMC) from pulmonary arteries of patients with and without IPAH. Serum concentrations of OPG were measured in a retrospective and prospective group of patients. OPG levels were compared with phenotypic data and other putative PAH biomarkers. Prognostic significance was assessed and levels compared with healthy controls. Correlation of OPG and pulmonary vascular remodeling was also performed in rodent models of PAH. OPG mRNA was significantly increased 2-fold in PASMC isolated from explanted PAH lungs compared with control. Serum OPG concentrations were markedly elevated in IPAH compared with controls. In Cohort 1 OPG levels significantly correlated with mean right atrial pressure and cardiac index, while in Cohort 2 significant correlations existed between age-adjusted OPG levels and gas transfer. In both cohorts an OPG concentration above a ROC-derived threshold of 4728 pg/ml predicted poorer survival. In two rodent models, OPG correlated with the degree of pulmonary vascular remodeling. OPG levels are significantly elevated in patients with idiopathic PAH and are of prognostic significance. The role of OPG as a potential biomarker and therapeutic target merits further investigation.