A window for reversibility in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare, chronic lung disease. In patients with PAH, arteries in the lung progressively occlude and cause high pulmonary blood pressure. PAH is a fatal condition that can already present in childhood, for example in children born with a heart defect. Luckily, early diagnosis and correction of the heart defect can completely reverse PAH. But if the surgery is performed too late, the disease has lost its reversibility, the high pulmonary blood pressure will increase and cause heart failure at a young age.It is unknown why PAH loses its reversibility beyond this point of no return, and no treatment is currently available to cure irreversible PAH. In his research, Diederik van der Feen showed that arteries of rats with irreversible PAH contain high numbers of so-called senescent cells: cells that have gone into rapid-ageing and lost their normal function. These cells cannot be cleared, because they are resistant to normal programmed cell death. Accumulation of senescent cells leads to the occlusion and stiffening of the arteries, because these cells continuously excrete inflammatory factors.This research has led to two new experimental treatments, that target the disease process of PAH at different levels. Both drugs were able to reverse the occlusion of the lung vessels and could thus prevent heart failure in rats with irreversible PAH. One of these drugs is currently being tested in a clinical trial in patients with PAH

A neonate with anaemia of prematurity: zinc protoporphyrin identifies iron deficiency anaemia without iron deficiency

Anaemia is a common problem in premature infants and is generally easy to treat with iron supplementation. If the anaemia persists despite appropriate correction of deficiencies, more extensive evaluation is required. We describe a case of a premature male infant with a production-deficient anaemia without metabolic deficiencies, eventually identified as anaemia of prematurity. This type of anaemia is commonly diagnosed but its highly variable and complex aetiology and phenotype are often poorly understood. A probable explanation for the anaemia of prematurity in this case was a transient iron incorporation defect, identifiable by high levels of zinc protoporphyrin

Animal models of right heart failure

Right heart failure may be the ultimate cause of death in patients with acute or chronic pulmonary hypertension (PH). As PH is often secondary to other cardiovascular diseases, the treatment goal is to target the underlying disease. We do however know, that right heart failure is an independent risk factor, and therefore, treatments that improve right heart function may improve morbidity and mortality in patients with PH. There are no therapies that directly target and support the failing right heart and translation from therapies that improve left heart failure have been unsuccessful, with the exception of mineralocorticoid receptor antagonists. To understand the underlying pathophysiology of right heart failure and to aid in the development of new treatments we need solid animal models that mimic the pathophysiology of human disease. There are several available animal models of acute and chronic PH. They range from flow induced to pressure overload induced right heart failure and have been introduced in both small and large animals. When initiating new pre-clinical or basic research studies it is key to choose the right animal model to ensure successful translation to the clinical setting. Selecting the right animal model for the right study is hence important, but may be difficult due to the plethora of different models and local availability. In this review we provide an overview of the available animal models of acute and chronic right heart failure and discuss the strengths and limitations of the different models

Pirfenidone ameliorates pulmonary arterial pressure and neointimal remodeling in experimental pulmonary arterial hypertension by suppressing NLRP3 inflammasome activation

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased pulmonary arterial pressure, inflammation, and neointimal remodeling of pulmonary arterioles. Serum levels of interleukin (IL)-1 beta and IL-18 are elevated in PAH patients and may enhance proinflammatory neointimal remodeling. NLRP3 inflammasome activation induces cleavage of the cytokines IL-1 beta and IL-18, required for their secretion. Pirfenidone (PFD), an antiflbrotic and anti-inflammatory drug, has been suggested to inhibit NLRP3 inflammasome activation. We hypothesized that PFD delays the progression of PAH by suppressing NLRP3 inflammasome activation. We assessed the effects of PFD treatment in a rat model for neointimal PAH induced by monocrotaline and aortocaval shunt using echocardiographic, hemodynamic, and vascular remodeling parameters. We measured inflammasome activation by NLRP3 immunostaining, Western blots for caspase-1, IL-1 beta and IL-18 cleavage, and macrophage IL-1 beta secretion. PFD treatment ameliorated pulmonary arterial pressure, pulmonary vascular resistance, and pulmonary vascular remodeling in PAH rats. In PAH rats, immunostaining of NLRP3 in pulmonary arterioles and caspase-1, IL-1 beta, and IL-18 cleavage in lung homogenates were increased compared to controls, reflecting NLRP3 inflammasome activation in vivo. PFD decreased IL-1 beta and IL-18 cleavage, as well as macrophage IL-1 beta secretion in vitro. Our studies show that PFD ameliorates pulmonary hemodynamics and vascular remodeling in experimental PAH. Although PFD did not affect all NLRP3 inflammasome parameters, it decreased IL-1 beta and IL-18 cleavage, the products of NLRP3 inflammasome activation that are key to its downstream effects. Our findings thus suggest a therapeutic benefit of PFD in PAH via suppression of NLRP3 inflammasome activation

Inhibition of the prolyl isomerase Pin1 improves endothelial function and attenuates vascular remodelling in pulmonary hypertension by inhibiting TGF-β signalling

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl-prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction, and vascular remodelling in PAH is unknown. Here, we report that Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10456-021-09812-7

Neuregulin-1 enhances cell-cycle activity, delays cardiac fibrosis, and improves cardiac performance in rat pups with right ventricular pressure load

Objectives: Right ventricular (RV) failure is a leading cause of death in patients with congenital heart disease. RV failure is kept at bay during childhood. Limited proliferation of cardiomyocytes is present in the postnatal heart. We propose that cardiomyocyte proliferation improves RV adaptation to pressure load (PL). We studied adaptation in response to increased RV PL and the role of increased cardiomyocyte cell cycle activity (CCA) in rat pups growing into adulthood. Methods: We induced RV PL at day of weaning in rats (3 weeks; 30-40 g) by pulmonary artery banding and followed rats into adulthood (300 g). We performed histological analyses and RNA sequencing analysis. To study the effects of increased cardiomyocyte cell cycle activity, we administered neuregulin-1 (NRG1), a growth factor involved in cardiac development. Results: PL induced an increase in CCA, with subsequent decline of CCA (sham/PL at 4 weeks: 0.14%/0.83%; P = .04 and 8 weeks: 0.00%/0.00%; P = .484) and cardiac function (cardiac index: control/PL 4 weeks: 4.41/3.29; P = .468 and 8 weeks: 3.57/1.44; P = .024). RNA sequencing analysis revealed delayed maturation and increased CCA pathways. NRG1 stimulated CCA (PL vehicle/NRG1 at 2 weeks: 0.62%/2.28%; P = .003), improved cardiac function (cardiac index control vs vehicle/NRG1 at 2 weeks: 4.21 vs 3.07/4.17; P = .009/.705) and postponed fibrosis (control vs vehicle/NRG1 at 4 weeks: 1.66 vs 4.82%/2.97%; P = .009/.078) in RV PL rats during childhood. Conclusions: RV PL during growth induces a transient CCA increase. Further CCA stimulation improves cardiac function and delays fibrosis. This proof-of-concept study shows that stimulation of CCA can improve RV adaptation to PL in the postnatal developing heart and might provide a new approach to preserve RV function in patients with congenital heart disease.</p

Neuregulin-1 enhances cell-cycle activity, delays cardiac fibrosis, and improves cardiac performance in rat pups with right ventricular pressure load

Objectives: Right ventricular (RV) failure is a leading cause of death in patients with congenital heart disease. RV failure is kept at bay during childhood. Limited proliferation of cardiomyocytes is present in the postnatal heart. We propose that cardiomyocyte proliferation improves RV adaptation to pressure load (PL). We studied adaptation in response to increased RV PL and the role of increased cardiomyocyte cell cycle activity (CCA) in rat pups growing into adulthood. Methods: We induced RV PL at day of weaning in rats (3 weeks; 30-40 g) by pulmonary artery banding and followed rats into adulthood (300 g). We performed histological analyses and RNA sequencing analysis. To study the effects of increased cardiomyocyte cell cycle activity, we administered neuregulin-1 (NRG1), a growth factor involved in cardiac development. Results: PL induced an increase in CCA, with subsequent decline of CCA (sham/PL at 4 weeks: 0.14%/0.83%; P = .04 and 8 weeks: 0.00%/0.00%; P = .484) and cardiac function (cardiac index: control/PL 4 weeks: 4.41/3.29; P = .468 and 8 weeks: 3.57/1.44; P = .024). RNA sequencing analysis revealed delayed maturation and increased CCA pathways. NRG1 stimulated CCA (PL vehicle/NRG1 at 2 weeks: 0.62%/2.28%; P = .003), improved cardiac function (cardiac index control vs vehicle/NRG1 at 2 weeks: 4.21 vs 3.07/4.17; P = .009/.705) and postponed fibrosis (control vs vehicle/NRG1 at 4 weeks: 1.66 vs 4.82%/2.97%; P = .009/.078) in RV PL rats during childhood. Conclusions: RV PL during growth induces a transient CCA increase. Further CCA stimulation improves cardiac function and delays fibrosis. This proof-of-concept study shows that stimulation of CCA can improve RV adaptation to PL in the postnatal developing heart and might provide a new approach to preserve RV function in patients with congenital heart disease.</p

Volume Load-Induced Right Ventricular Failure in Rats Is Not Associated With Myocardial Fibrosis

Background Right ventricular (RV) function and failure are key determinants of morbidity and mortality in various cardiovascular diseases. Myocardial fibrosis is regarded as a contributing factor to heart failure, but its importance in RV failure has been challenged. This study aims to assess whether myocardial fibrosis drives the transition from compensated to decompensated volume load-induced RV dysfunction. Methods Wistar rats were subjected to aorto-caval shunt (ACS, n = 23) or sham (control, n = 15) surgery, and sacrificed after 1 month, 3 months, or 6 months. Echocardiography, RV pressure-volume analysis, assessment of gene expression and cardiac histology were performed. Results At 6 months, 6/8 ACS-rats (75%) showed clinical signs of RV failure (pleural effusion, ascites and/or liver edema), whereas at 1 month and 3 months, no signs of RV failure had developed yet. Cardiac output has increased two- to threefold and biventricular dilatation occurred, while LV ejection fraction gradually decreased. At 1 month and 3 months, RV end-systolic elastance (Ees) remained unaltered, but at 6 months, RV Ees had decreased substantially. In the RV, no oxidative stress, inflammation, pro-fibrotic signaling (TGF beta 1 and pSMAD2/3), or fibrosis were present at any time point. Conclusions In the ACS rat model, long-term volume load was initially well tolerated at 1 month and 3 months, but induced overt clinical signs of end-stage RV failure at 6 months. However, no myocardial fibrosis or increased pro-fibrotic signaling had developed. These findings indicate that myocardial fibrosis is not involved in the transition from compensated to decompensated RV dysfunction in this model

Cellular senescence impairs the reversibility of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) in congenital cardiac shunts can be reversed by hemodynamic unloading (HU) through shunt closure. However, this reversibility potential is lost beyond a certain point in time. The reason why PAH becomes irreversible is unknown. In this study, we used MCT+shunt-induced PAH in rats to identify a dichotomous reversibility response to HU, similar to the human situation. We compared vascular profiles of reversible and irreversible PAH using RNA sequencing. Cumulatively, we report that loss of reversibility is associated with a switch from a proliferative to a senescent vascular phenotype and confirmed markers of senescence in human PAH-CHD tissue. In vitro, we showed that human pulmonary endothelial cells of patients with PAH are more vulnerable to senescence than controls in response to shear stress and confirmed that the senolytic ABT263 induces apoptosis in senescent, but not in normal, endothelial cells. To support the concept that vascular cell senescence is causal to the irreversible nature of end-stage PAH, we targeted senescence using ABT263 and induced reversal of the hemodynamic and structural changes associated with severe PAH refractory to HU. The factors that drive the transition from a reversible to irreversible pulmonary vascular phenotype could also explain the irreversible nature of other PAH etiologies and provide new leads for pharmacological reversal of end-stage PAH

Egr-1 and its effectors in flow-associated pulmonary arterial hypertension.

Background: In children with congenital heart disease (CHD), pulmonary arterial hypertension (PAH) is a serious cause for morbidity and mortality. PAH is a vasoproliferative disease in which the formation of irreversible neointimal lesions leads to progressive obstruction of the pulmonary arterioles. Increased pulmonary blood flow is a known trigger for this disease. In a model of experimental flow-induced PAH, Egr-1 was identified as a flow-specific trigger. It is expressed progressively throughout disease development. In our rat model for flow-PAH, Egr-1 inhibition using ED5 has attenuated neointimal formation. The spatiotemporal expression of Egr-1 in human, pediatric disease is unknown. The objective of this study is to elucidate the pathways through which Egr-1 mediates disease progression in our rat model and to determine Egr-1 expression in both early- and end-stage pediatric PAH. Methods: We used rats in which flow-PAH was created through monocrotaline injection and an aorto-caval shunt. In addition rats were treated with either saline, scrambled ED5 or ED5. Rats were sacrificed 1 week (early-stage) and 3 weeks (end-stage) after surgery. Using immunohistochemistry (IHC), we performed a proliferation and apoptosis assay and assessed the spatiotemporal expression of 5 possible PAH and Egr-1 associated proteins: tissue factor (TF), platelet-derived growth factor-B (PDGF-B), transforming growth factor-β1 (Tgf-β1), interleukin-6 (IL-6) and p53. Human Egr-1 expression was assessed in 29 children with either early- or end-stage CHD-PAH by IHC analysis of lung biopsies taken prior to correction of their cardiac anomaly. Egr-1 expression was then related to patient characteristics and hemodynamic measurements. Results: Main findings are that patterns of proliferation and apoptosis both peak in early-stage PAH. Egr-1 inhibition causes a decrease in proliferation in early-stage PAH and an increase in apoptosis in end-stage PAH. PDGF-B, Tgf-β1, IL-6 and p53 expression increase both in early and end-stage PAH and their expression decreases on Egr-1 inhibition. TF was only up regulated in end-stage PAH and down regulated by Egr-1 inhibition in that stage. In human end-stage PAH, Egr-1 expression is increased more than in early-stage disease. Egr-1 expression did not correlate to age, sex, shunt characteristics or any of the hemodynamic variables. Conclusions: In this study of experimental PAH, Egr-1 inhibition changed vascular remodeling by reducing hyper proliferation and allowing normal apoptosis. PDGF-B and IL-6 may be important effectors of Egr-1, that induce proliferation in both early and end-stage PAH, while TF might be an Egr-1 effector only in end-stage disease. The Tgf-β1 response is possibly not initiated but maintained by Egr-1. p53 might be an Egr-1 effector with a protective factor in PAH development. This renders more insight in the function of Egr-1 in the pathobiology of flow-associated PAH and might provide a starting point for a more specifically directed approach for therapeutic intervention. Furthermore, we confirmed the presence of Egr-1 in pediatric early-stage CHD-PAH and demonstrated a difference with expression in end-stage disease. The factors that might cause this difference remain unclear.