Marinus A.J. Borgdorff

Differential responses of the right ventricle to abnormal loading conditions in mice: pressure vs. volume load.

Right ventricular (RV) dysfunction is a major determinant of long‐term morbidity and mortality in congenital heart disease. The right ventricle (RV) is genetically different from the left ventricle (LV), but it is unknown as to whether this has consequences for the cellular responses to abnormal loading conditions. In the LV, calcineurin‐activation is a major determinant of pathological hypertrophy and an important target for therapeutic strategies. We studied the functional and molecular adaptation of the RV in mouse models of pressure and volume load, focusing on calcineurin‐activation.

Sildenafil enhances systolic adaptation, but does not prevent diastolic dysfunction, in the pressure-loaded right ventricle

Right ventricular (RV) failure due to pressure or volume overload is a major risk factor for early mortality in congenital heart disease and pulmonary hypertension, but currently treatments are lacking. We aimed to demonstrate that the phosphodiesterase 5A inhibitor sildenafil can prevent adverse remodelling and improve function in chronic abnormal RV overload, independent from effects on the pulmonary vasculature.

The role of disturbed blood flow in the development of pulmonary arterial hypertension: lessons from preclinical animal models

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vasoproliferative disorder characterized by the development of unique neointimal lesions, including concentric laminar intima fibrosis and plexiform lesions. Although the histomorphology of neointimal lesions is well described, the pathogenesis of PAH and neointimal development is largely unknown. After three decades of PAH pathobiology research the focus has shifted from vasoconstriction towards a mechanism of cancer-like angioproliferation. In this concept the role of disturbed blood flow is seen as an important trigger in the development of vascular remodeling. For instance, in PAH associated with congenital heart disease, increased pulmonary blood flow (i.e., systemic-to-pulmonary shunt) is an essential trigger for the occurrence of neointimal lesions and PAH development. Still, questions remain about the exact role of these blood flow characteristics in disease progression. PAH animal models are important for obtaining insight in new pathobiological processes and therapeutical targets. However, as for any preclinical model the pathophysiological mechanism and clinical course has to be comparable to the human disease that it mimics. This means that animal models mimicking human PAH ideally are characterized by: a hit recognized in human disease (e.g., altered pulmonary blood flow), specific vascular remodeling resembling human neointimal lesions, and disease progression that leads to right ventriclular dysfunction and death. A review that underlines the current knowledge of PAH due to disturbed flow is still lacking. In this review we will summarize the current knowledge obtained from PAH animal models associated with disturbed pulmonary blood flow and address questions for future treatment strategies for PAH.

Distinct loading conditions reveal various patterns of right ventricular adaptation

Right ventricular (RV) failure due to chronically abnormal loading is a main determinant of outcome in pulmonary hypertension (PH) and congenital heart disease. However, distinct types of RV loading have been associated with different outcomes. To determine whether the adaptive RV response depends on loading type, we compared hemodynamics, exercise, and hypertrophy in models of pressure overload due to pulmonary artery banding (PAB), pressure overload due to PH, combined pressure and volume overload, and isolated volume load. Ninety-four rats were subjected to either PAB, monocrotaline-induced PH (PH), aortocaval shunt (shunt), or combined monocrotaline and aortocaval shunt (PH + shunt). We performed pressure-volume analysis and voluntary exercise measurements at 4 wk. We compared PAB to PH (part I) and PH + shunt to either isolated PH or shunt (part II). In part I, enhanced contractility (end-systolic elastance and preload recruitable stroke work) was present in PH and PAB, but strongest in PAB. Frank-Starling mechanism was active in both PAB and PH. In PAB this was accompanied by diastolic dysfunction (increased end-diastolic elastance, relaxation constant), clinical signs of RV failure, and reduced exercise. These distinct responses were not attributable to differences in hypertrophy. In part II, in PH + shunt the contractility response was blunted compared with PH, which caused pseudonormalization of parameters. Additional volume overload strongly enhanced hypertrophy in PH. We conclude that different types of loading result in distinct patterns of RV adaptation. This is of importance for the approach to patients with chronically increased RV load and for experimental studies in various types of RV failure.

A cornerstone of heart failure treatment is not effective in experimental right ventricular failure

BACKGROUND Right ventricular (RV) failure due to increased pressure load causes significant morbidity and mortality in patients with congenital heart diseases and pulmonary arterial hypertension. It is unknown whether renin-angiotensin-aldosterone-system (RAAS) inhibition (the cornerstone of left ventricular failure treatment) is effective in RV failure. We investigated the effects of combination treatment of aldosterone-blocker eplerenone+angiotensin II receptor blocker losartan (Ep/Lo) on RV remodeling and function in a model of RV failure due to increased pressure load. METHODS AND RESULTS Rats (n=48) were randomized for pulmonary artery banding (PAB) or sham surgery and for losartan (20 mg/kg/d)+eplerenone (100 mg/kg/d) treatment (Ep/Lo) or vehicle (VEH). RV function was assessed by echocardiography and pressure-volume analysis at 5 and 11 weeks, or at the occurrence of clinical RV failure symptoms necessitating termination. PAB resulted in RV failure in all rats, as defined by reduced cardiac output, RV stroke volume, increased RV end diastolic pressure and liver congestion as well as RV fibrosis, hypertrophy and reduced capillary density. Clinical RV failure necessitated termination in 5/12 PAB-VEH rats. Angiotensin II type 1-receptor expression in the RV was reduced in PAB rats indicating local RAAS activation. Treatment of PAB rats with Ep/Lo significantly lowered arterial pressures, but had no significant effect on RV function, remodeling or survival compared to PAB-VEH rats. CONCLUSIONS RAAS inhibition does not beneficially affect experimental RV failure due to chronic pressure load. This is of high clinical relevance, because it indicates that the RV response to RAAS inhibition might fundamentally differ from that of the LV.

Egr-1 expression during neointimal development in flow-associated pulmonary hypertension.

In flow-associated pulmonary arterial hypertension (PAH), increased pulmonary blood flow is an essential trigger for neointimal formation. Using microarray analysis, we recently found that the early growth response protein 1 (Egr-1) transcription factor is increased in experimental flow-associated end-stage PAH. Its role in PAH development is unknown. Here, we assessed the spatiotemporal expression of Egr-1 during neointimal development in flow-associated PAH. Flow-associated PAH was produced in rats by combining monocrotaline administration with an aortocaval shunt. Animals were sacrificed 1 day before or 1 day, 1 week, or 4 to 5 weeks after flow addition. Egr-1 expression was spatiotemporally assessed using laser microdissection, quantitative real-time PCR and immunohistochemistry. In addition, Egr-1 expression was assessed in a non-neointimal pulmonary hypertension model and in human PAH associated with congenital shunt. In 4 to 5 weeks, rats subjected to increased flow developed PAH with neointimal lesions. Egr-1 mRNA was increased 1 day after flow addition and in end-stage PAH, whereas monocrotaline only did not result in increased Egr-1 mRNA. Directly after flow addition, Egr-1 was expressed in endothelial cells. During disease development, Egr-1 protein expression increased and migrated throughout the vessel wall. In PAH patients, Egr-1 was expressed in vessels with media hypertrophy and neointimal lesions, including plexiform lesions. Thus, Egr-1 may be an important regulator in the development of pulmonary neointimal lesions induced by increased pulmonary blood flow.

Sildenafil treatment in established right ventricular dysfunction improves diastolic function and attenuates interstitial fibrosis independent from afterload

Right ventricular (RV) function is an important determinant of prognosis in congenital heart diseases, pulmonary hypertension, and heart failure. Preventive sildenafil treatment has been shown to enhance systolic RV function and improve exercise capacity in a model of fixed RV pressure load. However, it is unknown whether sildenafil has beneficial effects when treatment is started in established RV dysfunction, which is clinically more relevant. Our aim was to assess the effects of sildenafil treatment on RV function and fibrosis in a model of established RV dysfunction due to fixed afterload. Rats were subjected to pulmonary artery banding (PAB), which induced RV dysfunction after 4 wk, characterized by reduced exercise capacity, decreased tricuspid annular plane systolic excursion, and RV dilatation. From week 4 onward, 50% of rats were treated with sildenafil (100 mg·kg(-1)·day(-1), n = 9; PAB-SIL group) or vehicle (n = 9; PAB-VEH group). At 8 wk, exercise capacity was assessed using cage wheels, and RV function was assessed using invasive RV pressure-volume measurements under anesthesia. Sildenafil treatment, compared with vehicle, improved RV ejection fraction (44 ± 2% vs. 34 ± 2%, P < 0.05, PAB-SIL vs. PAB-VEH groups), reduced RV end-diastolic pressure (2.3 ± 0.5 vs. 5.1 ± 0.9 mmHg, P < 0.05), and RV dilatation (end-systolic volume: 468 ± 45 vs. 643 ± 71 μl, P = 0.05). Sildenafil treatment also attenuated RV fibrosis (30 ± 6 vs. 17 ± 3‰, P < 0.05) but did not affect end-systolic elastance, exercise capacity, or PKG or PKA activity. In conclusion, sildenafil improves RV diastolic function and attenuates interstitial fibrosis in rats with established RV dysfunction, independent from afterload. These results indicate that sildenafil treatment has therapeutic potential for established RV dysfunction.

Clinical symptoms of right ventricular failure in experimental chronic pressure load are associated with progressive diastolic dysfunction

BACKGROUND Right ventricular failure (RVF) due to pressure load is a major cause of death in congenital heart diseases and pulmonary hypertension. The mechanisms of RVF are unknown. We used an experimental approach based upon clinical signs of RVF to delineate functional and biological processes associated with RVF. METHODS AND RESULTS Wistar rats were subjected to a pulmonary artery banding (PAB n=12) or sham surgery (CON, n=7). After 52±5days, 5/12 PAB rats developed clinical symptoms of RVF (inactivity, ruffled fur, dyspnea, ascites) necessitating termination (PAB+CF). We compared these to PAB rats with RVF without clinical symptoms (PAB-). PAB resulted in reduced cardiac output, RV stroke volume, TAPSE, and increased end diastolic pressure (all p<0.05 vs. CON) in all rats, but PAB+CF rats were significantly more affected than PAB-, despite similar pressure load (p=ns). Pressure-volume analysis showed enhanced contractility (end systolic elastance) in PAB- and PAB+CF, but diastolic function (end diastolic elastance, end diastolic pressure) deteriorated especially in PAB+CF. In PAB+CF capillary density was lower than in PAB-. Gene-array analysis revealed downregulation of both fatty acid oxidation and carbohydrate metabolism in PAB+CF. CONCLUSION Chronic PAB led to different degrees of RVF, with half of the rats developing severe clinical symptoms of RVF, associated with progressive deterioration of diastolic function, hypoxia-prone myocardium, increased response to oxidative stress and suppressed myocardial metabolism. This model represents clinical RVF and allows for unraveling of mechanisms involved in the progression from RV adaptation to RV failure and the effect of intervention on these mechanisms.

A critical role for Egr-1 during vascular remodelling in pulmonary arterial hypertension

AIMS Pulmonary arterial hypertension (PAH) is characterized by the development of unique neointimal lesions in the small pulmonary arteries, leading to increased right ventricular (RV) afterload and failure. Novel therapeutic strategies are needed that target these neointimal lesions. Recently, the transcription factor Egr-1 (early growth response protein 1) was demonstrated to be up-regulated early in experimental neointimal PAH. Its effect on disease development, however, is unknown. We aimed to uncover a novel role for Egr-1 as a molecular inductor for disease development in PAH. METHODS AND RESULTS In experimental flow-associated PAH in rats, we investigated the effects of Egr-1 down-regulation on pulmonary vascular remodelling, including neointimal development, and disease progression. Intravenous administration of catalytic oligodeoxynucleotides (DNA enzymes, DNAzymes) resulted in down-regulation of pulmonary vascular Egr-1 expression. Compared with vehicle or scrambled DNAzymes, DNAzymes attenuated pulmonary vascular remodelling, including the development of occlusive neointimal lesions. Selective down-regulation of Egr-1 in vivo led to reduced expression of vascular PDGF-B, TGF-β, IL-6, and p53, resulting in a reduction of vascular proliferation and increased apoptosis. DNAzyme treatment further attenuated pulmonary vascular resistance, RV systolic pressure, and RV hypertrophy. In contrast, in non-neointimal PH rodents, DNAzyme treatment had no effect on pulmonary vascular and RV remodelling. Finally, pharmacological inhibition of Egr-1 with pioglitazone, a peroxisome proliferator activated receptor-γ ligand, attenuated vascular remodelling including the development of neointimal lesions. CONCLUSIONS These results indicate that Egr-1 governs pulmonary vascular remodelling and the development of characteristic vascular neointimal lesions in flow-associated PAH. Egr-1 is therefore a potential target for future PAH treatment.

Egr-1 identifies neointimal remodeling and relates to progression in human pulmonary arterial hypertension

BACKGROUND Pulmonary arterial hypertension (PAH) is hallmarked by the development of neointimal lesions. The transcription factor Egr-1 seems to play a critical role in neointimal formation in experimental PAH and was identified as a putative target for intervention. In this study we investigated whether Egr-1 is also associated with neointimal-type vascular remodeling in different forms of human PAH or pulmonary hypertension. METHODS Using immunohistochemistry, we studied Egr-1 expression specifically in a wide morphologic spectrum of pulmonary arteries in the lung tissue of 72 patients with different forms and stages of PAH, specifically idiopathic PAH (n = 18), advanced-stage congenital heart disease‒associated PAH (PAH-CHD) (n = 21), early-stage PAH-CHD (n = 19) and non-neointimal hypoxic pulmonary hypertension (PH) (n = 4), and controls (n = 10). RESULTS In PAH patients, pulmonary vascular expression of Egr-1 protein was abundant, whereas it was sporadic in non-neointimal (hypoxic) PH patients and controls. In PAH-CHD, protein expression was more pronounced in patients with advanced vascular lesions compared to those with less advanced lesions, such as medial hypertrophy. CONCLUSIONS Pulmonary vascular Egr-1 expression is significantly increased in patients with PAH, appears specifically associated with neointimal-type vascular remodeling, and correlates with disease progression. These data translate the critical role of Egr-1 in the development of experimental PAH to human pulmonary vascular disease forms.