Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

Role of the Aryl Hydrocarbon Receptor/ARNT/Cytochrome P450 System in Pulmonary Vascular Diseases.

RATIONALE: CYPs (cytochrome p450) are critically involved in the metabolism of xenobiotics and toxins. Given that pulmonary hypertension is strongly associated with environmental exposure, we hypothesize that CYPs play a role in the development and maintenance of pathological vascular remodeling. OBJECTIVE: We sought to identify key CYPs that could link drug or hormone metabolism to the development of pulmonary hypertension. METHODS AND RESULTS: We searched in Medline (PubMed) database, as well as http://www.clinicaltrials.gov, for CYPs associated with many key aspects of pulmonary arterial hypertension including, but not limited to, severe pulmonary hypertension, estrogen metabolism, inflammation mechanisms, quasi-malignant cell growth, drug susceptibility, and metabolism of the pulmonary arterial hypertension-specific drugs. CONCLUSIONS: We postulate a hypothesis where the AhR (aryl hydrocarbon receptor) mediates aberrant cell growth via expression of different CYPs associated with estrogen metabolism and inflammation.

Angiotensin converting enzyme 2 and angiotensin (1-7) axis in pulmonary arterial hypertension.

BACKGROUND: In animal models of pulmonary arterial hypertension (PAH), angiotensin-converting enzyme (ACE)2 and angiotensin (Ang)-(1-7) have been shown to have vasodilatory, antiproliferative, antifibrotic and antihypertrophic properties. However, the status and role of the ACE2-Ang(1-7) axis in human PAH is incompletely understood. METHODS: We studied 85 patients with a diagnosis of PAH of distinct aetiologies. 55 healthy blood donors paired for age and sex served as controls. Blood samples were obtained from the pulmonary artery in patients with PAH during right heart catheterisation. Peripheral blood was obtained for both groups. Ang(1-7) and -II were measured using zone capillary electrophoresis. Aldosterone, Ang(1-9), AngA and ACE2 were measured using ELISA, and ACE2 activity was determined enzymatically. RESULTS: Of the 85 patients, 47 had idiopathic PAH, 25 had PAH associated with congenital heart disease and 13 had PAH associated with collagen vascular disease. Compared to controls, patients with PAH had a higher concentration of AngII (median 1.03, interquartile range 0.72-1.88 pmol·mL-1 versus 0.19, 0.10-0.37 pmol·mL-1; p<0.001) and of aldosterone (88.7, 58.7-132 ng·dL-1 versus 12.9, 9.55-19.9 ng·dL-1; p<0.001). Conversely, PAH patients had a lower concentration of Ang(1-7) than controls (0.69, 0.474-0.91 pmol·mL-1 versus 4.07, 2.82-6.73 pmol·mL-1; p<0.001), and a lower concentration of Ang(1-9) and AngA. Similarly, the ACE2 concentration was higher than in controls (8.7, 5.35-13.2 ng·mL-1 versus 4.53, 1.47-14.3 ng·mL-1; p=0.011), whereas the ACE2 activity was significantly reduced (1.88, 1.08-2.81 nmol·mL-1 versus 5.97, 3.1-17.8 nmol·mL-1; p<0.001). No significant differences were found among the three different aetiological forms of PAH. CONCLUSIONS: The AngII-ACE2-Ang(1-7) axis appears to be altered in human PAH and we propose that this imbalance, in favour of AngII, plays a role in the pathogenesis of the severe PAH. Further mechanistic studies are warranted.

HIMF (Hypoxia-Induced Mitogenic Factor) Signaling Mediates the HMGB1 (High Mobility Group Box 1)-Dependent Endothelial and Smooth Muscle Cell Crosstalk in Pulmonary Hypertension.

OBJECTIVE: HIMF (hypoxia-induced mitogenic factor; also known as FIZZ1 [found in inflammatory zone-1] or RELM [resistin-like molecule-α]) is an etiological factor of pulmonary hypertension (PH) in rodents, but its underlying mechanism is unclear. We investigated the immunomodulatory properties of HIMF signaling in PH pathogenesis. Approach and Results: Gene-modified mice that lacked HIMF (KO [knockout]) or overexpressed HIMF human homolog resistin (hResistin) were used for in vivo experiments. The pro-PH role of HIMF was verified in HIMF-KO mice exposed to chronic hypoxia or sugen/hypoxia. Mechanistically, HIMF/hResistin activation triggered the HMGB1 (high mobility group box 1) pathway and RAGE (receptor for advanced glycation end products) in pulmonary endothelial cells (ECs) of hypoxic mouse lungs in vivo and in human pulmonary microvascular ECs in vitro. Treatment with conditioned medium from hResistin-stimulated human pulmonary microvascular ECs induced an autophagic response, BMPR2 (bone morphogenetic protein receptor 2) defects, and subsequent apoptosis-resistant proliferation in human pulmonary artery (vascular) smooth muscle cells in an HMGB1-dependent manner. These effects were confirmed in ECs and smooth muscle cells isolated from pulmonary arteries of patients with idiopathic PH. HIMF/HMGB1/RAGE-mediated autophagy and BMPR2 impairment were also observed in pulmonary artery (vascular) smooth muscle cells of hypoxic mice, effects perhaps related to FoxO1 (forkhead box O1) dampening by HIMF. Experiments in EC-specific hResistin-overexpressing transgenic mice confirmed that EC-derived HMGB1 mediated the hResistin-driven pulmonary vascular remodeling and PH. CONCLUSIONS: In HIMF-induced PH, HMGB1-RAGE signaling is pivotal for mediating EC-smooth muscle cell crosstalk. The humanized mouse data further support clinical implications for the HIMF/HMGB1 signaling axis and indicate that hResistin and its downstream pathway may constitute targets for the development of novel anti-PH therapeutics in humans.

Consequences of chronic frequent premature atrial contractions: Association with cardiac arrhythmias and cardiac structural changes.

INTRODUCTION: Frequent premature ventricular contractions (PVCs) can cause cardiomyopathy (CM). Postextrasystolic potentiation (PESP) and irregularity have been in implicated as triggers of PVC-CM. Because both phenomena can also be found in premature atrial contractions (PACs), it is speculated that frequent PACs have similar consequences. METHODS AND RESULTS: A single-center, retrospective study included all consecutive patients undergoing a 14-day Holter monitors (November 2014 to October 2016). Patients were divided into four groups by ectopy burden group 1 (<1%) and remaining by tertiles (group 2-4). Echocardiographic and arrhythmic data were compared between PAC and PVC burdens. In addition, a translational PAC animal model was used to assess the chronic effects of frequent PACs. A total 846 patients were reviewed. In contrast to PVCs, we found no difference in left ventricular ejection fraction (LVEF), end-systolic and end-diastolic dimensions and presence of CM (LVEF <50%) between different PAC groups. Multivariate regression analysis demonstrated that only PVC burden predicted low EF (odds ratio, 1.1; confidence interval, 1.03-1.13; P = .001). While there was a weak correlation between PAC burden and supraventricular tachycardia (SVT) episodes and atrial fibrillation (AF) burden (r = 0.19; P < .001), there was no correlation between PAC burden and LVEF or CM. Finally, atrial bigeminy in our animal model did not significantly decrease LVEF after 3 months. CONCLUSION: PAC burden is associated with increased AF and SVT episodes. In contrast to a high PVC burden, a high PAC burden is not associated with CM. Our findings suggest that heart rate irregularity and/or PESP may play a minimal role in the pathophysiology of PVC-CM.

Iloprost reverses established fibrosis in experimental right ventricular failure.

Prostacyclin and its analogues improve cardiac output and functional capacity in patients with pulmonary arterial hypertension (PAH); however, the underlying mechanism is not fully understood. We hypothesised that prostanoids have load-independent beneficial effects on the right ventricle (RV). Angio-obliterative PAH and RV failure were induced in rats with a single injection of SU5416 followed by 4 weeks of exposure to hypoxia. Upon confirmation of RV dysfunction and PAH, rats were randomised to 0.1 µg·kg(-1) nebulised iloprost or drug-free vehicle, three times daily for 2 weeks. RV function and treadmill running time were evaluated pre- and post-iloprost/vehicle treatment. Pulmonary artery banded rats were treated 8 weeks after surgery to allow for significant RV hypertrophy. Inhaled iloprost significantly improved tricuspid annulus plane systolic excursion and increased exercise capacity, while mean pulmonary artery pressure and the percentage of occluded pulmonary vessels remained unchanged. Rats treated with iloprost had a striking reduction in RV collagen deposition, procollagen mRNA levels and connective tissue growth factor expression in both SU5416/hypoxia and pulmonary artery banded rats. In vitro, cardiac fibroblasts treated with iloprost showed a reduction in transforming growth factor (TGF)-ß1-induced connective tissue growth factor expression, in a protein kinase A-dependent manner. Iloprost decreased TGF-ß1-induced procollagen mRNA expression as well as cardiac fibroblast activation and migration. Iloprost significantly induced metalloproteinase-9 gene expression and activity and increased the expression of autophagy genes associated with collagen degradation. Inhaled iloprost improves RV function and reverses established RV fibrosis partially by preventing collagen synthesis and by increasing collagen turnover.

The role of vascular endothelial growth factor in pulmonary arterial hypertension. The angiogenesis paradox.

Pulmonary arterial hypertension (PAH) is characterized by dysfunctional angiogenesis leading to lung vessel obliteration. PAH is widely considered a proangiogenic disease; however, the role of angiogenic factors, such as the vascular endothelial growth factor (VEGF) and its receptors, in the pathobiology of PAH remains incompletely understood. This Review attempts to untangle some of the complex multilayered actions of VEGF to provide a VEGF-centered perspective of PAH. Furthermore, we provide a cogent explanation for the paradox of VEGF receptor blockade-induced pulmonary hypertension that characterizes the SU5416-hypoxia rat model of PAH, and attempt to translate the knowledge gained from the experimental model to the human disease by postulating the potential role of endogenous (SU5416-like) VEGF inhibitors. The main objective of this Review is to promote discussion and investigation of the opposing and complementary actions of VEGF in PAH. Understanding the balance between angiogenic and antiangiogenic factors and their role in the pathogenesis of PAH will be necessary before antiangiogenic drugs can be considered for the treatment of PAH.

SuHx rat model: partly reversible pulmonary hypertension and progressive intima obstruction.

The SU5416 combined with hypoxia (SuHx) rat model features angio-obliterative pulmonary hypertension resembling human pulmonary arterial hypertension. Despite increasing use of this model, a comprehensive haemodynamic characterisation in conscious rats has not been reported. We used telemetry to characterise haemodynamic responses in SuHx rats and associated these with serial histology. Right ventricular systolic pressure (RVSP) increased to a mean±sd of 106±7 mmHg in response to SuHx and decreased but remained elevated at 72±8 mmHg upon return to normoxia. Hypoxia-only exposed rats showed a similar initial increase in RVSP, a lower maximum RVSP and near-normalisation of RVSP during subsequent normoxia. Progressive vascular remodelling consisted of a four-fold increase in intima thickness, while only minimal changes in media thickness were found. The circadian range in RVSP provided an accurate longitudinal estimate of vascular remodelling. In conclusion, in SuHx rats, re-exposure to normoxia leads to a partial decrease in pulmonary artery pressure, with persisting hypertension and pulmonary vascular remodelling characterised by progressive intima obstruction.

FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4.

Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/ß-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/ß-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/ß-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.

Chronic carvedilol treatment partially reverses the right ventricular failure transcriptional profile in experimental pulmonary hypertension.

Right ventricular failure (RVF) is the most frequent cause of death in patients with pulmonary arterial hypertension (PAH); however, specific therapies targeted to treat RVF have not been developed. Chronic treatment with carvedilol has been shown to reduce established maladaptive right ventricle (RV) hypertrophy and to improve RV function in experimental PAH. However, the mechanisms by which carvedilol improves RVF are unknown. We have previously demonstrated by microarray analysis that RVF is characterized by a distinct gene expression profile when compared with functional, compensatory hypertrophy. We next sought to identify the effects of carvedilol on gene expression on a genome-wide basis. PAH and RVF were induced in male Sprague-Dawley rats by the combination of VEGF-receptor blockade and chronic hypoxia. After RVF was established, rats were treated with carvedilol or vehicle for 4 wk. RNA was isolated from RV tissue and hybridized for microarray analysis. An initial prediction analysis of carvedilol-treated RVs showed that the gene expression profile resembled the RVF prediction set. However, a more extensive analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes in pathways involved in cardiac hypertrophy, mitochondrial dysfunction, and protein ubiquitination. Genes encoding proteins in the cardiac hypertrophy and protein ubiquitination pathways were downregulated in the RV by carvedilol, while genes encoding proteins in the mitochondrial dysfunction pathway were upregulated by carvedilol. These gene expression changes may explain some of the mechanisms that underlie the functional improvement of the RV after carvedilol treatment.

Thyroid hormone is highly permissive in angioproliferative pulmonary hypertension in rats.

Epidemiological evidence links pulmonary arterial hypertension (PAH) with thyroid disease, but a mechanistic explanation for this association is lacking. Because a central hallmark of vascular remodelling in pulmonary hypertension is lumen obliteration by endothelial cell growth and because thyroid hormones are known to be angiogenic, we hypothesised that thyroid hormones play a role in the control of endothelial cell proliferation in experimental PAH in rats. Hypothyroidism was induced by subtotal thyroidectomy and treatment with propylthiouracil (PTU) in rats with experimental PAH after combined exposure to vascular endothelial growth factor receptor inhibition and hypoxia (the Sugen-chronic hypoxia (SuHx) model). Subtotal thyroidectomy prevented and PTU treatment reversed the development of severe experimental PAH. Thyroxin repletion restored the PAH phenotype in thyroidectomised SuHx rats. The prevention of PAH by thyroidectomy was associated with a reduced rate of cell turnover, reduced extracellular signal-regulated protein kinases 1 and 2 phosphorylation, and reduced expression of α(v)ß(3) integrin, fibroblast growth factor (FGF)-2 and FGF receptor. Thyroidectomy mitigated hypoxia-induced pulmonary hypertension, but this effect was not associated with a decreased pulmonary vascular resistance. These data suggest that thyroid hormone permits endothelial cell proliferation in PAH. A causal link between thyroid diseases and the onset or progression of vascular remodelling in PAH patients remains to be determined.

Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α.

Introduction: Forkhead Box F1 (FOXF1) transcription factor plays a critical role in lung angiogenesis during embryonic development and lung repair after injury. FOXF1 expression is decreased in endothelial cells after lung injury; however, molecular mechanisms responsible for the FOXF1 transcript changes in injured lung endothelium remain unknown. Methods: We used immunostaining of injured mouse lung tissues, FACS-sorted lung endothelial cells from hypoxia-treated mice, and data from patients diagnosed with hypoxemic respiratory failure to demonstrate that hypoxia is associated with decreased FOXF1 expression. Endothelial cell cultures were used to induce hypoxia in vitro and identify the upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression. Results: Bleomycin-induced lung injury induced hypoxia in the mouse lung tissue which was associated with decreased Foxf1 expression. Human FOXF1 mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure. Mice exposed to hypoxia exhibited reduced Foxf1 expression in the lung tissue and FACS-sorted lung endothelial cells. In vitro, hypoxia (1% of O2) or treatment with cobalt (II) chloride increased HIF-1α protein levels but inhibited FOXF1 expression in three endothelial cell lines. Overexpression of HIF-1α in cultured endothelial cells was sufficient to inhibit Foxf1 expression. siRNA-mediated depletion of HIF-1α prevented the downregulation of Foxf1 gene expression after hypoxia or cobalt (II) chloride treatment. Conclusion: Hypoxia inhibits FOXF1 expression in endothelial cells in a HIF-1α dependent manner. Our data suggest that endothelial cell-specific inhibition of HIF-1α via gene therapy can be considered to restore FOXF1 and improve lung repair in patients with severe lung injury.

Corrigendum: Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma via the combination therapy with FOXM1 inhibitor RCM1.

[This corrects the article DOI: 10.3389/fonc.2023.1112859.].

Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma via the combination therapy with FOXM1 inhibitor RCM1.

Rhabdomyosarcoma (RMS) is a highly metastatic soft-tissue sarcoma that often develops resistance to current therapies, including vincristine. Since the existing treatments have not significantly improved survival, there is a critical need for new therapeutic approaches for RMS patients. FOXM1, a known oncogene, is highly expressed in RMS, and is associated with the worst prognosis in RMS patients. In the present study, we found that the combination treatment with specific FOXM1 inhibitor RCM1 and low doses of vincristine is more effective in increasing apoptosis and decreasing RMS cell proliferation in vitro compared to single drugs alone. Since RCM1 is highly hydrophobic, we developed innovative nanoparticle delivery system containing poly-beta-amino-esters and folic acid (NPFA), which efficiently delivers RCM1 to mouse RMS tumors in vivo. The combination of low doses of vincristine together with intravenous administration of NPFA nanoparticles containing RCM1 effectively reduced RMS tumor volumes, increased tumor cell death and decreased tumor cell proliferation in RMS tumors compared to RCM1 or vincristine alone. The combination therapy was non-toxic as demonstrated by liver metabolic panels using peripheral blood serum. Using RNA-seq of dissected RMS tumors, we identified Chac1 as a uniquely downregulated gene after the combination treatment. Knockdown of Chac1 in RMS cells in vitro recapitulated the effects of the combination therapy. Altogether, combination treatment with low doses of vincristine and nanoparticle delivery of FOXM1 inhibitor RCM1 in a pre-clinical model of RMS has superior anti-tumor effects and decreases CHAC1 while reducing vincristine toxicity.

Demonstration of Safety in Wild Type Mice of npFOXF1, a Novel Nanoparticle-Based Gene Therapy for Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins.

Introduction: Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins (ACDMPV) is a fatal congenital disease resulting from a pulmonary vascular endothelial deficiency of FOXF1, producing abnormal morphogenesis of alveolar capillaries, malpositioned pulmonary veins and disordered development of lung lobes. Affected neonates suffer from cyanosis, severe breathing insufficiency, pulmonary hypertension, and death typically within days to weeks after birth. Currently, no treatment exists for ACDMPV, although recent murine research in the Kalinichenko lab demonstrates nanoparticle delivery improves survival and reconstitutes normal alveolar-capillary architecture. The aim of the present study is to investigate the safety of intravenous administration of FOXF1-expressing PEI-PEG nanoparticles (npFOXF1), our pioneering treatment for ACDMPV. Methods: npFOXF1 was constructed, validated, and subsequently administered in a single dose to postnatal day 14 (P14) mice via retro-orbital injection. Biochemical, serologic, and histologic safety were monitored at postnatal day 16 (P16) and postnatal day 21 (P21). Results: With treatment we observed no lethality, and the general condition of mice revealed no obvious abnormalities. Serum chemistry, whole blood, and histologic toxicity was assayed on P16 and P21 and revealed no abnormality. Discussion: In conclusion, npFOXF1 has a very good safety profile and combined with preceding studies showing therapeutic efficacy, npFOXF1 can be considered as a good candidate therapy for ACDMPV in human neonates.

Severe pulmonary arterial hypertension induced by SU5416 and ovalbumin immunization.

The combination of chronic hypoxia and treatment of rats with the vascular endothelial growth factor (VEGF) receptor blocker, SU5416, induces pulmonary angio-obliteration, resulting in severe pulmonary arterial hypertension (PAH). Inflammation is thought to contribute to the pathology of PAH. Allergic inflammation caused by ovalbumin (OVA) immunization causes muscularization of pulmonary arteries, but not severe PAH. Whether disturbance of the immune system and allergic inflammation in the setting of lung endothelial cell apoptosis causes PAH is unknown. We investigated the effects of OVA-allergic inflammation on the development of PAH initiated by VEGF blockade-induced lung endothelial cell apoptosis. OVA-immunized rats were treated with SU5416 to induce pulmonary vascular endothelial cell apoptosis. The combination of OVA and SU5416 treatment resulted in severe angio-obilterative PAH, accompanied by increased IL-6 expression in the lungs. c-Kit(+) and Sca-1(+) cells were found in and around the lung vascular lesions. Pan-caspase inhibiton, dexamethasone treatment, and depletion of B-lymphocytes using an anti-CD20 antibody suppressed this remodeling. OVA immunization also increased lung tissue hypoxia-induced factor-1α and VEGF expression. Our results also suggest that the increased expression of hypoxia-induced factor-1α and IL-6 induced by the allergic lung inflammation may be a component of the pathogenesis of PAH.

The monocrotaline model of pulmonary hypertension in perspective.

Severe forms of pulmonary arterial hypertension (PAH) are characterized by various degrees of remodeling of the pulmonary arterial vessels, which increases the pulmonary vascular resistance and right ventricular afterload, thus contributing to the development of right ventricle dysfunction and failure. Recent years have seen advances in the understanding of the pathobiology of PAH; however, many important questions remain unanswered. Elucidating the pathobiology of PAH continues to be critical to design new effective therapeutic strategies, and appropriate animal models of PAH are necessary to achieve the task. Although the monocrotaline rat model of PAH has contributed to a better understanding of vascular remodeling in pulmonary hypertension, we question the validity of this model as a preclinically relevant model of severe plexogenic PAH. Here we review pertinent publications that either have been forgotten or ignored, and we reexamine the monocrotaline model in the context of human forms of PAH.

A brief overview of mouse models of pulmonary arterial hypertension: problems and prospects.

Many chronic pulmonary diseases are associated with pulmonary hypertension (PH) and pulmonary vascular remodeling, which is a term that continues to be used to describe a wide spectrum of vascular abnormalities. Pulmonary vascular structural changes frequently increase pulmonary vascular resistance, causing PH and right heart failure. Although rat models had been standard models of PH research, in more recent years the availability of genetically engineered mice has made this species attractive for many investigators. Here we review a large amount of data derived from experimental PH reports published since 1996. These studies using wild-type and genetically designed mice illustrate the challenges and opportunities provided by these models. Hemodynamic measurements are difficult to obtain in mice, and right heart failure has not been investigated in mice. Anatomical, cellular, and genetic differences distinguish mice and rats, and pharmacogenomics may explain the degree of PH and the particular mode of pulmonary vascular adaptation and also the response of the right ventricle.

Pathobiology of pulmonary arterial hypertension and right ventricular failure.

Pulmonary arterial hypertension (PAH) is no longer an orphan disease. There are three different classes of drugs for the treatment of PAH that are currently being used and an increasing number of patients are being treated with a single drug or combination therapy. During the last 25 yrs, new insights into the pathobiology of PAH have been gained. The classical mechanical concepts of pressure, flow, shear stress, right ventricle wall stress and impedance have been complemented with the new concepts of cell injury and repair and interactions of complex multicellular systems. Integrating these concepts will become critical as we design new medical therapies in order to change the prognosis of patients with these fatal diseases. This review intends to summarise recent pathobiological concepts of PAH and right ventricle failure mainly derived from human studies, which reflect the progress made in the understanding of this complex group of pulmonary vascular diseases.

Distinct Platelet Ribonucleic Acid Signatures in Patients with Pulmonary Hypertension.

Rationale: Pulmonary hypertension encompasses progressive disorders leading to right ventricular dysfunction and early death. Late detection is an important cause of poor clinical outcomes. However, biomarkers that accurately predict the presence of pulmonary hypertension are currently lacking. Objectives: In this study, we provide evidence that blood platelets contain a distinctive ribonucleic acid (RNA) profile that may be exploited for the detection of pulmonary hypertension. Methods: Blood platelet RNA was isolated prospectively from 177 prevalent patients with different subtypes of pulmonary hypertension as well as 195 control subjects clinically not suspected of pulmonary hypertension. Sequencing libraries were created using SMARTer (Switching Mechanism at 5' end of RNA Template) copy desoxyribonucleic acid amplification and sequenced on the Illumina High Throughput Sequencing platform. RNA-sequencing reads were mapped to the human reference genome, and intron-spanning spliced RNA reads were selected. Differential spliced RNA panels were calculated by analysis of variance statistics. A particle swarm optimization-enhanced classification algorithm was built employing a development (n = 213 samples) and independent validation series (n = 159 samples). Results: We detected a total of 4,014 different RNAs in blood platelets from patients with pulmonary hypertension (n = 177) and asymptomatic control subjects (n = 195). Gene ontology analysis revealed enhanced RNA concentrations for genes related to RNA processing, translation, and mitochondrial function. A particle swarm optimization-selected RNA panel of 408 distinctive differentially spliced RNAs mediated detection of pulmonary hypertension with 93% sensitivity, 62% specificity, 77% accuracy, 0.89 (95% confidence interval, 0.83-0.93) area under the curve, and a negative predictive value of 91% in the independent validation series. The prediction score was independent of age, sex, smoking, pulmonary hypertension subtype, and the use of pulmonary hypertension-specific medication or anticoagulants. Conclusions: A platelet RNA panel may accurately discriminate patients with pulmonary hypertension from asymptomatic control subjects. In the light of current diagnostic delays, this study is the starting point for further development and evaluation of a platelet RNA-based blood test to ultimately improve early diagnosis and clinical outcomes in patients with pulmonary hypertension.