Objective: Pulmonary hypertension remains a major clinical problem despite current therapies. In this study, we examine for the first time a novel pharmacological target, smooth muscle myosin, and determine if the smooth muscle myosin inhibitor, CK-2019165 (CK-165) ameliorates pulmonary hypertension. Materials and Methods: Six domestic female pigs were surgically instrumented to measure pulmonary blood flow and systemic and pulmonary vascular dynamics. Pulmonary hypertension was induced by hypoxia, or infusion of the thromboxane analog (U-46619, 0.1 mu g/kg/min, i.v.). In rats, chronic pulmonary hypertension was induced by monocrotaline. Results: CK-165 (4 mg/kg, i.v.) reduced pulmonary vascular resistance by 22 +/- 3 and 28 +/- 6% from baseline in hypoxia and thromboxane pig models, respectively (p<0.01 and 0.01), while mean arterial pressure also fell and heart rate rose slightly. When CK-165 was delivered via inhalation in the hypoxia model, pulmonary vascular resistance fell by 17 +/- 6% (p<0.05) while mean arterial pressure and heart rate were unchanged. In the monocrotaline model of chronic pulmonary hypertension, inhaled CK-165 resulted in a similar (18.0 +/- 3.8%) reduction in right ventricular systolic pressure as compared with sildenafil (20.3 +/- 4.5%). Conclusion: Inhibition of smooth muscle myosin may be a novel therapeutic target for treatment of pulmonary hypertension.
We performed nocturnal polysomnography in patients with pulmonary hypertension (PH) of varying etiologies to determine the association of metrics describing sleep-disordered breathing (SDB) with measures of PH severity.Consecutive patients referred for evaluation of dyspnea on exertion and elevated pulmonary arterial pressure >30 mmHg on echocardiography, who underwent right and left heart catheterization and polysomnography, were included. Patients were not pre-selected for symptoms of sleep-disordered breathing.Twenty-eight patients including 22 females and six males with a mean age of 55.2 ± 11.9 years were evaluated. Etiologies of PH were idiopathic (32%) and PH associated with other diseases (68%). Most were World Health Organization (WHO) Functional class II (39%) and III (39%). The group mean pulmonary arterial pressure (mPAP) was 40.9 ± 15.1 mmHg. Diurnal resting and exercise arterial oxygen saturations (SaO2) were 94.9 ± 3.7% and 88.3 ± 8.9%. The mean apnea–hypopnea index (AHI) was 11.4 ± 19.8/h; 50% of all patients had an AHI ≥ 5/h; 30.6 ± 36.0% of total sleep time was spent with SaO2 < 90% (T90%); 66% of subjects with an AHI ≥ 5/h of sleep reported snoring, and 60% noted daytime somnolence; however, only 29% had an Epworth Sleepiness Scale ≥10. Right atrial pressure and mPAP were significantly correlated with AHI and T90%. The best predictive model relating PH severity to metrics of SDB was a highly significant association (p = 0.005) between mPAP and a linear combination of AHI and T90%.SDB comprised of obstructive apneas, hypopneas, and nocturnal hypoxemia is prevalent in PH and cannot be accurately predicted by sleep apnea signs and symptoms or diurnal rest and exercise SaO2. The association of AHI and T90% with mPAP suggests a potential relationship between the pathophysiology of sleep-disordered breathing and PH.
In 70 consecutive male patients with obstructive sleep apnea (OSA) diagnosed at the Northport VA Medical Center Sleep Disorders Center, we have characterized the association between obesity, OSA, and pulmonary hypertension (PH).By including anthropometric, pulmonary function, and sleep study parameters in a multivariate logistic regression model, we found that a BMI of >40 kg/m2 and the minimum oxygen saturation in non-rapid eye movement (NREM) sleep predicted the presence of pretibial edema in this sleep apnea population. We then characterized the hemodynamics of those OSA patients that had lower extremity edema. Twenty-nine of the 70 consecutive patients with sleep apnea (41%) had pretibial edema, and right heart catheterization data was obtained for 28 (97%) of these patients.Ninety-three percent (26/28) of the patients had right heart failure (mean RAP > 5 mm Hg; RAP range = 0–32 mmHg) and PH (PA mean ≥ 20 mm Hg) was present in 86% (24/28.) The OSA patients with lower extremity edema had an increased cardiac output (7.0 + 1.4 l/min) with a normal cardiac index (2.9 + 0.5 l/min/m2) in the setting of an elevated pulmonary capillary wedge pressure (PCWP 17 ± 7 mmHg) and a normal pulmonary vascular resistance (122 + 70 dynes s cm−5). While PCWP, FEV1% predicted, and the minimum oxygen saturation in NREM sleep all independently predicted PH, PCWP was the most important predictor of PH.We conclude that pulmonary hypertension is commonly seen in patients with OSA with pretibial edema and that pretibial edema is a highly specific sign of PH in OSA patients. Pulmonary hypertension appears to result from an elevated back pressure and diastolic dysfunction with contributions from lung function and nocturnal oxygen saturation.
The purpose of this study was to determine the frequency of central and obstructive sleep apnea in adult patients who have echocardiographic evidence of left ventricular dysfunction and pulmonary hypertension. Subjects with left ventricular dysfunction, pulmonary hypertension (pulmonary artery systolic pressure >30 mm Hg) and no lung disease were evaluated for risk factors associated with pulmonary hypertension. Of eight eligible adults, six completed the study. Subjects were from suburban and inner city family practices. Spirometric assessment, pulse oximetry on room air, rheumatologic evaluation, polysomnography, and additional history were taken. All six subjects had sleep apnea (apnea-plus-hypopnea index, or AHI, ≥20): obstructive, central, or mixed. All were obese, and almost all the subjects had a restrictive pattern on spirometry, which is consistent with obesity. All had a pulmonary artery systolic blood pressure of 35 mm Hg or greater. None had daytime hypoxemia or collagen vascular disease, and none had ever used appetite suppressants. This study found a strong association between pulmonary hypertension and obstructive or central sleep apnea in obese patients with congestive heart failure (CHF). We propose that a pulmonary artery systolic pressure of 35 mm Hg or greater in ambulatory patients with CHF may signify an increased risk of sleep apnea.
Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition.
Abstract Cerium oxide (CeO2 ) nanoparticles have been posited to exhibit potent anti-oxidant activity which may allow for the use of these materials in biomedical applications. Herein, we investigate whether CeO2 nanoparticle administration can diminish right ventricular (RV) hypertrophy following four weeks of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Male Sprague Dawley rats were randomly divided into three groups: control, MCT only (60 mg/kg), or MCT + CeO2 nanoparticle treatment (60 mg/kg; 0.1 mg/kg). Compared to the control group, the RV weight to body weight ratio was 45% and 22% higher in the MCT and MCT + CeO2 groups, respectively ( p < 0.05). Doppler echocardiography demonstrated that CeO2 nanoparticle treatment attenuated monocrotaline-induced changes in pulmonary flow and RV wall thickness. Paralleling these changes in cardiac function, CeO2 nanoparticle treatment also diminished MCT-induced increases in right ventricular (RV) cardiomyocyte cross sectional area, β-myosin heavy chain, fibronectin expression, protein nitrosylation, protein carbonylation and cardiac superoxide levels. These changes with treatment were accompanied by a decrease in the ratio of Bax/Bcl2, diminished caspase-3 activation and reduction in serum inflammatory markers. Taken together, these data suggest that CeO2 nanoparticle administration may attenuate the hypertrophic response of the heart following PAH.
Abstract Adventitial fibroblasts (AFs) are key determinants of arterial function and critical mediators of arterial disease progression. The effects of altered stiffness, particularly those observed across individuals during normal vascular function, and the mechanisms by which AFs respond to altered stiffness, are not well understood. To study the effects of matrix stiffness on AF phenotype, cytokine production, and the regulatory pathways utilized to interpret basic cell-matrix interactions, human aortic AFs were grown in 5%, 7.5%, and 10% (w/v%) PEG-based hydrogels with Young's moduli of 1.2, 3.3, and 9.6 kPa, respectively. In 5% gels, AFs had higher proliferation rates, elevated monocyte chemoattractant protein-1 secretion, and enhanced monocyte recruitment. Significantly more AFs were α-smooth muscle actin positive in 7.5% gels, indicating myofibroblast development. AFs in 10% gels had low proliferation rates but produced high levels of interleukin-6 and vascular endothelial growth factor-A. Importantly, these modulus-dependent changes in AF phenotype were accompanied by alterations in the mitogen-activated protein kinase (MAPK) pathways contributing to the production of cytokines. These data indicate that complex cell regulatory changes occur with altered tissue stiffness and suggest that therapeutics affecting MAPK pathways may have altered effects on AFs depending on substrate stiffness.
Background Pulmonary vascular remodeling of pulmonary arterial hypertension (PAH) is characterized by an inappropriate increase of vascular cells. The receptor for advanced glycation end products (RAGE) is a type I single-pass transmembrane protein belonging to the immunoglobulin superfamily and is involved in a broad range of hyperproliferative diseases. RAGE is also implicated in the etiology of PAH and is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with PAH. We examined the role of RAGE in the inappropriate increase of PASMCs in patients with PAH. Methods and results PASMCs were obtained from 12 patients with PAH including 9 patients with idiopathic PAH (IPAH) and 3 patients with heritable PAH (HPAH) (2 patients with BMPR2 mutation and one patient with SMAD9 mutation) who underwent lung transplantation. Western blot analysis and immunofluorescence staining revealed that RAGE and S100A8 and A9, ligands of RAGE, were overexpressed in IPAH and HPAH-PASMCs in the absence of any external growth stimulus. PDGF-BB (10 ng/mL) up-regulated the expression of RAGE in IPAH and HPAH-PASMCs. PAH-PASMCs are hyperplastic in the absence of any external growth stimulus as assessed by 3H-thymidine incorporation. This result indicates overgrowth characterized by continued growth under a condition of no growth stimulation in PAH-PASMCs. PDGF-BB stimulation caused a higher growth rate of PAH-PASMCs than that of non-PAH-PASMCs. AS-1, an inhibitor of TIR domain-mediated RAGE signaling, significantly inhibited overgrowth characterized by continued growth under a condition of no growth stimulation in IPAH and HPAH-PASMCs (P<0.0001). Furthermore, AS-1 significantly inhibited PDGF-stimulated proliferation of IPAH and HPAH-PASMCs (P<0.0001). Conclusions RAGE plays a crucial role in the inappropriate increase of PAH-PASMCs. Inhibition of RAGE signaling may be a new therapeutic strategy for PAH.
Abstract The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics for diseases related to hypoxic abnormal growth of PASMCs.
Abstract The efficient delivery of short interfering RNA (siRNA) is an enormous challenge in the field of gene therapy. Herein, we report a delivery nanosystem based on programmed DNA self-assembly mammalian target of rapamycin (mTOR) siRNA-loaded DNA nanotubes (DNA-NTs). We demonstrate that these siRNA-DNA-NTs can be effectively transfected into pulmonary arterial smooth muscle cells (PASMCs) via endocytosis; and that the loaded mTOR siRNA can induce obvious autophagy and inhibit cell growth under both normal and hypoxic conditions. Moreover, we found that mTOR siRNA can control the autophagy and proliferation of PASMCs under hypoxic condition, suggesting a potential therapeutic application for mTOR siRNA in diseases involving abnormal autophagy in PASMCs.