Rat Model Of Pulmonary Hypertension Research Articles

Echocardiographic markers of pulmonary hemodynamics and right ventricular hypertrophy in rat models of pulmonary hypertension.

Echocardiography is the gold standard non-invasive technique to diagnose pulmonary hypertension. It is also an important modality used to monitor disease progression and response to treatment in patients with pulmonary hypertension. Surprisingly, only few studies have been conducted to validate and standardize echocardiographic parameters in experimental animal models of pulmonary hypertension. We sought to define cut-off values for both invasive and non-invasive measures of pulmonary hemodynamics and right ventricular hypertrophy that would reliably diagnose pulmonary hypertension in three different rat models. The study was designed in two phases: (1) a derivation phase to establish the cut-off values for invasive measures of right ventricular systolic pressure, Fulton's index (right ventricular weight/left ventricle + septum weight), right ventricular to body weight ratio, and non-invasive echocardiographic measures of pulmonary arterial acceleration time, pulmonary arterial acceleration time to ejection time ratio and right ventricular wall thickness in diastole in the hypoxic and monocrotaline rat models of pulmonary hypertension and (2) a validation phase to test the performance of the cut-off values in predicting pulmonary hypertension in an independent cohort of rats with Sugen/hypoxia-induced pulmonary hypertension. Our study demonstrates that right ventricular systolic pressure ≥35.5 mmHg and Fulton's Index ≥0.34 are highly sensitive (>94%) and specific (>91%) cut-offs to distinguish animals with pulmonary hypertension from controls. When pulmonary arterial acceleration time/ejection time and right ventricular wall thickness in diastole were both measured, a result of either pulmonary arterial acceleration time/ejection time ≤0.25 or right ventricular wall thickness in diastole ≥1.03 mm detected right ventricular systolic pressure ≥35.5 mmHg or Fulton's Index ≥0.34 with a sensitivity of 88% and specificity of 100%. With properly validated non-invasive echocardiography measures of right ventricular performance in rats that accurately predict invasive measures of pulmonary hemodynamics, future studies can now utilize these markers to test the efficacy of different treatments with preclinical therapeutic modeling.

Drag-reducing polymers attenuates pulmonary vascular remodeling and right ventricular dysfunction in a rat model of chronic hypoxia-induced pulmonary hypertension.

Drag-reducing polymers (DRPs) was previously demonstrated to increase blood flow, tissue perfusion, and reduce vascular resistance. The purpose of this study was to investigate the effect of DRPs on pulmonary vascular remodeling and right ventricular dysfunction in a rat model of chronic hypoxia-induced pulmonary hypertension (HPH). A total of forty male Wistar rats were randomly and equally assigned into four experimental groups (Group I: normoxia + saline, Group II: normoxia + PEO, Group III: hypoxia + saline, Group IV: hypoxia + PEO) and maintained in normoxia (21% O2) or hypobaric hypoxia (10% O2). After four weeks, comparisons were made of the following aspects: the mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), right ventricular hypertrophy, wall thickness of pulmonary trunk and arteries, internal diameter of pulmonary arteries, cardiomyocyte cross-sectional area (CM CSA), and ultrastructure of right ventricular. Treatment with PEO in Group IV attenuated the increases in RVSP and mPAP (40.5±7.2 and 34.7±7.0 mmHg, respectively, both P < 0.05), compared with Group III. Distal vascular remodeling was visible as a significant increase in medial wall thickness (64.2±12.3% vs. 43.95±7.0%, P < 0.01) and a remarkable decrease in internal diameter of small pulmonary arteries (35.2±9.7μ m vs. 50.4±14.7μ m, P < 0.01) in Group III, to a greater extent than that detected in Group IV. Nevertheless, no significant histopathological differences in medial wall thickness was observed in pulmonary trunk between Group III and Group IV (P > 0.05), denoting that PEO chiefly attenuated the remodeling of small pulmonary arteries rather than main arteries in hypoxic environment. Infusion of DRPs (intravenous injection twice weekly) also attenuated the index of right ventricular hypertrophy, protected against the increase of cardiomyocyte cross-sectional area, and provided protection for cardiac ultrastructure. DRP treatment with intravenous injection elicited a protective effect against pulmonary vascular remodeling and right ventricular dysfunction in the rat model of HPH. DRPs may offer a new potential approach for the treatment of HPH, which may have theoretical significance and application value to society.

The role of inflammation in a rat model of chronic thromboembolic pulmonary hypertension induced by carrageenan.

BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening condition arising from the thrombus and obstructive remodeling of the pulmonary arteries, which causes a significant morbidity and mortality. Although the modern treatment in CTEPH has been significant advanced both in surgical and medical treatment, none can claim to cure the disease, largely because of our limited understanding of the underlying pathogenesis of the disease and lack of a reliable CTEPH animal model to study for. Recently, inflammation has been accepted as a common pathway through which various risk factors trigger venous thrombo-embolism (VTE) formation, we describe a novel mouse model of CTEPH which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of CTEPH in humans, to open a new horizons of inflammation in CTEPH.MethodsBy administering a pulmonary embolism (PE) protocol (comprising 3 sequential left jugular vein injections of autologous blood clots) to 8-week-old male Sprague Dawley (SD) rats using tranexamic acid (200 mg/kg.d) to inhibit fibrinolysis and injecting additional carrageenan (20 mg/kg, once a week) to create perivascular inflammation, we successfully generated a CTEPH animal model. By monitoring the mean pulmonary artery pressure (mPAP) and the histopathological change to evaluate the CTEPH model. By detecting the RT-PCR, western blot, TUNEL, and immunohistochemistry in the sub-groups to find the potential mechanism of inflammation may work in the pulmonary vascular remolding.ResultsIn this study, rats with CTEPH exhibited pronounced pulmonary vascular remolding with higher vessel wall area/total area (WA/TA) ratio in comparison to the control rats (85.41%±7.37% vs. 76.41%±5.97%, P<0.05), the mPAP (25.51±1.13 vs. 15.92±1.13 mmHg, P<0.05). Significant differences in mean pulmonary artery pressure (mPAP) values were observed between rats injected solely with clots and those injected with both clots and carrageenan (25.51±1.13 vs. 29.82±1.26 mmHg, P<0.05, respectively). Furthermore, following the third embolization, thrombi and intimal hyperplasia occurred in the pulmonary artery. In addition, repeated embolization elevated mRNA and protein levels of tumor necrosis factor-α (TNF-α), NF-κB/p65, and B-cell lymphoma-2 (BCL-2), but decreased BAX expression in a time-dependent manner.ConclusionsTake advantage of the inflammation to trigger VTE formation, we successfully generated a CTEPH animal model. Inflammation may play a crucial role in the pathogenesis and progression of CTEPH by inhibiting endothelial cell apoptosis. Understanding the role of inflammation in CTEPH may not only help to determine the optimal treatment options but also may aid in the development of future preventative strategies, since current anticoagulation treatment regimens are not designed to inhibit inflammation.

In vitro Species Different Metabolism and CYP Phenotyping of Zamicastast

BackgroundZamicastat (1) is an inhibitor of dopamine‐β‐hydroxylase, enzyme that catalyzes the hydroxylation of dopamine to norepinephrine in sympathetic nerves and adrenal medulla, and it was shown to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.ObjectivesThe purpose of this study was to evaluate zamicastat clearance and metabolic profile in hepatocytes from different species (human, rat, dog, mouse and monkey) and to identify the CYP450 enzymes isoforms that are involved in zamicastat metabolism. Methods. Metabolite profiling of zamicastat (10 μM) was conducted in human, rat, dog, mouse and monkey cryopreserved hepatocytes. Samples were harvested up to 120 min and analyzed by High Resolution Mass Spectrometry (HR‐MS). The Intrinsic Clearance (CLint) values for zamicastat were assessed in all species. For CYP reaction phenotyping, the study was conducted on human CYP450 recombinant enzymes incubated with zamicastat (5 μM) and NADPH, with samples being harvested up to 45 min and analyzed by LC‐MS/MS.ResultsThe CLint for zamicastat was estimated at 40.3, 32.6, 13.6, 44.2 and 6.29 μl/min/106 cells in mouse, rat, dog, monkey and human hepatocytes, respectively. A total of 21 different metabolites were found where 11 were detected in human, 14 in rat, 7 in dog and 13 in mouse and monkey. In general, the metabolites observed in hepatocytes across species were formed as a result of multiple biotransformation processes, including oxidations, desaturations, acetylations, loss of sulfur, thioureas to ureas and glucuronidations. A potential biotransformation that includes S‐Glutathione conjugation (M21) was only detected in rat species whereas oxidation plus desaturation (M5), loss of sulfur (M7 and M8) and glucuronidation (M20) were detected in all species. Under these experimental conditions, zamicastat was mainly metabolized by CYP2D6. The extent of metabolism, in decreasing order was CYP2D6 &gt; CYP1A2 &gt; CYP2C19 &gt; CYP2C8 &gt; CYP3A4. No zamicastat metabolism was detected when incubated with CYP2B6 or CYP2C9 recombinant enzymes.ConclusionAll the metabolites detected in human hepatocytes were also detected in the pre‐clinical species suggesting that, under the conditions tested no human specific metabolite was observed. The major cytochromes P450 involved in zamicastast metabolism are CYP2D6, CYP1A2 and 2C19.

An Experimental Study on the Establishment of Pulmonary Hypertension Model in Rats induced by Monocrotaline

Pulmonary hypertension is called PH for short. It is caused by the pulmonary artery vascular disease leading to pulmonary vascular resistance, and the increase right lung compartment load, which resulting in weakening or even collapse of the right ventricular function. The establishment of rat PH model under the action of monocrotaline is a repeatable, simple and accessible operation technique, which has been widely used in the treatment of pulmonary hypertension. This paper discusses the principle and properties of the PH model on rats under the monocrotaline action.

Protein tyrosine phosphatase L1 represses endothelial-mesenchymal transition by inhibiting IL-1β/NF-κB/Snail signaling

Endothelial–mesenchymal transition (EnMT) plays a pivotal role in various diseases, including pulmonary hypertension (PH), and transcription factors like Snail are key regulators of EnMT. In this study we investigated how these factors were regulated by PH risk factors (e.g. inflammation and hypoxia) in human umbilical vein endothelial cells (HUVECs). We showed that treatment with interleukin 1β (IL‐1β) induced EnMT of HUVECs via activation of NF-κB/Snail pathway, which was further exacerbated by knockdown of protein tyrosine phosphatase L1 (PTPL1). We demonstrated that PTPL1 inhibited NF-κB/Snail through dephosphorylating and stabilizing IκBα. IL‐1β or hypoxia could downregulate PTPL1 expression in HUVECs. The deregulation of PTPL1/NF-κB signaling was validated in a monocrotaline-induced rat PH (MCT-PH) model and clinical PH specimens. Our findings provide novel insights into the regulatory mechanisms of EnMT, and have implications for identifying new therapeutic targets for clinical PH.

TMEM16A regulates the cell cycle of pulmonary artery smooth muscle cells in high-flow-induced pulmonary arterial hypertension rat model.

High-flow-induced pulmonary arterial hypertension (PAH) has attained global notoriety, the mechanism of which remains elusive. The present study investigated the regulation of Anoctamin-1, also known as transmembrane member 16A (TMEM16A), in the cell cycle progression of pulmonary artery smooth muscle cells (PASMCs) from a PAH rat model induced by high pulmonary blood flow. A total of 30 Sprague-Dawley rats were randomly assigned into control, sham and shunt groups. A rat model of high pulmonary blood flow-induced PAH was established by surgery using abdominal aorta-inferior vena cava fistula. Right ventricular pressure, right ventricular hypertrophy index and pulmonary arteriole structural remodeling were assessed 11 weeks following operation. The cell cycle statuses of PASMCs was assessed via flow cytometry, whereas western blot analysis was performed to measure the expression of cyclin D1, CDK2, p27KIP and cyclin E in primary PASMCs isolated from rats. The expression of cyclin E and cyclin D1 was revealed to be increased in the shunt group compared with the control group, which was accompanied with an increased expression of TMEM16A in the shunt group. Changes in the ratio of PASMCs in the G0/G1, S and G2/M phases of cycle induced by PAH were reversed by TMEM16A knockdown. The expression of cyclin E and cyclin D1 in the shunt group was significantly higher compared with the control group in vitro, which was reversed by TMEM16A-siRNA transfection. In conclusion, TMEM16A may be involved in high pulmonary blood flow-induced PAH by regulating PASMC cell cycle progression.

DANTROLENE PREVENTS RIGHT VENTRICULAR DYSFUNCTION IN PULMONARY HYPERTENSION BY INHIBITING SARCOPLASMIC RETICULUM CA2+ LEAK

Right ventricular failure (RVF) is a major cause of death in pulmonary hypertension (PH), but its mechanism remains poorly understood. We investigate whether sarcoplasmic reticulum (SR) Ca2+ leak through type 2 ryanodine receptor (RyR2) is associated with RVF. In a rat PH model (monocrotaline), we

Decreased expression of prohibitin 1 mediates high-mobility group protein 1-induced pulmonary artery endothelial cell injury.

There is a significant increase of high-mobility group protein B1 (HMGB1) in plasma levels of patients with pulmonary hypertension, but the biological significance is still unclear. Anti-proliferative protein 1 (prohibitin 1, PHB1) is an important protein that maintains the homeostasis of vascular cells. This study aimed to investigate the effect of HMGB1 on pulmonary artery endothelial cells and the role of PHB1. In vivo experiment: A rat model of pulmonary hypertension induced by monocrotaline (MCT) was constructed. The right ventricular systolic pressure (RVSP), and the weight ratio of right ventricle to left ventricle plus ventricular septum were used to evaluate the success of model. ELISA was used to detect the level of HMGB1 in rat's plasma. Western blotting was used to detect the level of PHB1 in rat's lung tissues. CD31 immunofluorescence was used to detect the integrity of pulmonary vascular endothelium. In vitro experiments: Pulmonary artery endothelial cell (PAEC) was incubated with HMGB1 to observe the effect of HMGB1 on PAEC injury. Overexpression and knockdown of PHB1 were conducted, and the role of PHB1 was investigated by detecting the levels of reative oxygen species and cytochrome c (cyto-c), and the activation of caspase-3. Compared with the control group, the level of HMGB1 in the plasma of rats with pulmonary hypertension was significantly increased (P<0.05), and the expression of PHB1 in the lung tissue was decreased accompanied with endothelial dysfunction (P<0.05); HMGB1 incubation damaged the pulmonary artery endothelium and down-regulated PHB1 expression (P<0.05), while overexpression of PHB1 reduced the PAEC damage and oxidative stress induced by HMGB1 (P<0.05). Meanwhile, PHB1 reduced HMGB1-induced cyto-c expression and caspase-3 cleavage by inhibiting oxidative stress (P<0.05). The down-regulation of PHB1 expression mediates HMGB1-induced PAEC injury, which is related to the induction of oxidative stress, the increase of cyto-c release, and the promotion of caspase-3 cleavage.

Cellular inflammation in pulmonary hypertension: Detailed analysis of lung and right ventricular tissue, circulating immune cells and effects of a dual endothelin receptor antagonist.

Growing evidence suggests that inflammation is crucially involved in the pathogenesis of pulmonary hypertension (PH) and consecutive right heart failure. The present study analyzed the inflammatory response in lung and right ventricle in a rat model of PH and evaluated the effects of the dual endothelin receptor antagonist (ERA) Macitentan. PH was induced by monocrotalin (60 mg/kg body weight s.c.) in Sprague-Dawley rats (PH, n = 10) and compared to healthy controls (CON, n = 10) as well as monocrotalin-induced, macitentan-treated rats (THER, n = 10). Detection of Dendritic cells (DCs), regulatory T cells (Tregs) and others as well as RT-PCR based inflammatory gene expression analysis were performed. Circulating DCs and Tregs were quantified by flow cytometry in the rat model and in PH patients (n = 70) compared to controls (n = 52). Inflammatory cells were increased in lung and right ventricular tissue, whereas DCs and Tregs were decreased in blood. Expression of 17 genes in the lung and 20 genes in the right ventricle were relevantly (>2.0 fold) regulated in the PH group. These effects were, at least in part, attenuated in response to Macitentan treatment. In humans as well as rats, immune cells showed significant correlations to clinical, echocardiographic, and haemodynamic parameters. PH is accompanied by a distinct inflammatory response in lung and right but not left ventricular tissue attenuated by Macitentan. Correlations of circulating DCs as well as tissue resident immune cells with parameters reflecting right ventricular function raise the idea of both, promising biomarkers and novel treatment strategies.

Improved method for the catheterization of the right ventricle in a rat model of pulmonary artery hypertension.

Ventricle catheterization in the rat is widely practiced in cardiopulmonary research. The catheters deployed are either fluid filled or solid tip pressure or pressure-volume catheters. The access to the right ventricle is through the right jugular vein, most commonly without direct visualization such as fluoroscopy. Advancement of the catheter tip is aided by visualizing the pressure signals of the monitoring/recording systems used. This approach may present challenges due to various reasons, including the stiffness of new catheters, their dimensions or anatomical changes associated with the animal disease model. In this article, we present a novel approach, which has been optimized, successfully validated surgically and adopted in current projects. It has been shown to improve both the overall quality of the signals recorded and the time to access the right ventricle, thus reducing the overall time of surgery. The method presented in this article is safe, easy to reproduce and does not require additional skills compared to a more 'standard' approach.

Structural and functional definition of the pulmonary vein system in a chronic hypoxia-induced pulmonary hypertension rat model.

Unlike the pulmonary artery (PA), the pathophysiological changes of the pulmonary vein (PV) in the development of pulmonary hypertension (PH) remain largely unknown. In this study, we comprehensively investigated the structural and functional changes in the PV isolated from the chronic hypoxia (CH; 10% O2, 21 days)-induced PH rat model (CHPH). Results showed that CH caused an increase in right ventricular pressure but did not affect the mean pulmonary venous pressure and the left atrial pressure. Similar to the PA, vascular lumen stenosis and medial thickening were also observed in the intrapulmonary veins isolated from the CHPH rats. Notably, CH induced more severe loss in the endothelium of intrapulmonary veins than the arteries. Then, the contractile response to 5-HT and U46619 was significantly greater in the intrapulmonary small veins (ISPV) and arteries (ISPA) isolated from CHPH rats than those from normoxic rats but not in the extrapulmonary and intrapulmonary large veins. Treatment with nifedipine (Nif), SKF96365 (SKF), or ryanodine and caffeine either partially attenuated (Nif) or dramatically abolished (SKF or ryanodine and caffeine) 5-HT-induced maximal contraction in ISPV from both normoxic and CHPH rats. Because of the severe loss of endothelium in the PV of CHPH rats, the decrease in acetylcholine (ACh)-induced endothelium-dependent relaxation was significantly larger in ISPV than ISPA, whereas the sodium nitroprusside-induced endothelium-independent relaxation was not altered in both ISPA and ISPV. In conclusion, our results provide fundamental data to comprehensively define the PV system in CHPH rat model.

Transplantation of Mesenchymal Stem Cells Attenuates Pulmonary Hypertension by Normalizing the Endothelial-to-Mesenchymal Transition.

For decades, stem cell therapies for pulmonary hypertension (PH) have progressed from laboratory hypothesis to clinical practice. Promising preclinical investigations have laid both a theoretical and practical foundation for clinical application of mesenchymal stem cells (MSCs) for PH therapy. However, the underlying mechanisms are still poorly understood. We sought to study the effects and mechanisms of MSCs on the treatment of PH. For in vivo experiments, the transplanted GFP+ MSCs were traced at different time points in the lung tissue of a chronic hypoxia-induced PH (CHPH) rat model. The effects of MSCs on PH pathogenesis were evaluated in both CHPH and sugen hypoxia-induced PH models. For in vitro experiments, primary pulmonary microvascular endothelial cells were cultured and treated with the MSC conditioned medium. The specific markers of endothelial-to-mesenchymal transition (EndMT) and cell migration properties were measured. MSCs decreased pulmonary arterial pressure and ameliorated the collagen deposition, and reduced the thickening and muscularization in both CHPH and sugen hypoxia-induced PH rat models. Then, MSCs significantly attenuated the hypoxia-induced EndMT in both the lungs of PH models and primary cultured rat pulmonary microvascular endothelial cells, as reflected by increased mesenchymal cell markers (fibronectin 1 and vimentin) and decreased endothelial cell markers (vascular endothelial cadherin and platelet endothelial cell adhesion molecule-1). Moreover, MSCs also markedly inhibited the protein expression and degradation of hypoxia-inducible factor-2α, which is known to trigger EndMT progression. Our data suggest that MSCs successfully prevent PH by ameliorating pulmonary vascular remodeling, inflammation, and EndMT. Transplantation of MSCs could potentially be a powerful therapeutic approach against PH.

Trimethoxystilbene Reduces Nuclear Factor Kappa B, Interleukin-6, and Tumor Necrosis Factor-α Levels in Rats with Pulmonary Artery Hypertension.

Pulmonary artery hypertension is a refractory disease that severely affects cardiopulmonary function, mainly resulting in irreversible pulmonary vascular remodeling. Current surgical treatment of this disease is not very effective and drug treatment is targeted at relieving symptoms, improving the quality of life of patients, and preventing disease progression. The purpose of this present study was to reveal the regulatory effects of trimethoxystilbene on the serum levels of nuclear factor kappa B, interleukin-6, and tumor necrosis factor-α in a rat model of pulmonary artery hypertension and to explore the possible underlying mechanisms. Healthy Sprague Dawley rats were randomly assigned to experimental groups and treated with monocrotaline to establish the model, and we found a significant difference in the expression levels of nuclear factor kappa B, interleukin-6, and tumor necrosis factor-α between the experimental and control groups. These results suggest that trimethoxystilbene significantly reduced the inflammatory factor levels in pulmonary hypertensive rats, providing us with new potential strategies for elucidating the mechanisms of action of trimethoxystilbene in the treatment of pulmonary artery hypertension.

Programmed death-ligand 1 triggers PASMCs pyroptosis and pulmonary vascular fibrosis in pulmonary hypertension

Pyroptosis is a pro-inflammatory form of programmed cell death, whose genesis directly depended on caspase-1 activation. Pulmonary hypertension (PH) is a disease characterized, in part, by vascular fibrosis. Up to now, there is no report on the relationship between pyroptosis and vascular fibrosis in PH. Here, we confirmed that pyroptosis had occurred in the media of pulmonary arteries in two PH rat models and hypoxic human pulmonary arterial smooth muscle cells (hPASMCs). Caspase-1 inhibition attenuated the pathogenesis of PH, as assessed by vascular remodeling, right ventricular systolic pressure, right ventricle hypertrophy and hemodynamic parameters of pulmonary vasculature. Moreover, caspase-1 inhibition suppressed pulmonary vascular fibrosis as demonstrated by Masson staining, as well as immunohistochemistry and Western blot analysis of fibrillar collagen. In addition, Programmed death-ligand 1 (PD-L1) was markedly increased in PH, which was regulated by the transcription factor STAT1. Furthermore, PD-L1 knockdown in hPASMCs repressed the onset of hypoxia-induced pyroptosis and fibrosis. Overall, these data identify a critical STAT1-dependent posttranscriptional modification that promotes PD-L1 expression in the pyroptosis of PASMCs to modulate pulmonary vascular fibrosis and accelerate the progression of PH.

Monocrotaline-induced pulmonary arterial hypertension: Time-course of injury and comparative evaluation of macitentan and Y-27632, a Rho kinase inhibitor

Novel pharmacological approaches are needed to improve outcomes of patients with idiopathic pulmonary hypertension. Rho-associated protein kinase (ROCK) inhibitors have shown beneficial effects in preclinical models of pulmonary arterial hypertension (PAH), because of their role in the regulation of pulmonary artery vasoconstrictor tone and remodeling. We compared a ROCK inhibitor, Y-27632, for the first time with the dual endothelin receptor antagonist, macitentan, in a monocrotaline-induced rat pulmonary hypertension model. Different methods (echocardiography, hemodynamics, histology of right ventricle and pulmonary vessels, and circulating biomarkers) showed consistently that 100 mg/kg daily of Y-27632 and 10 mg/kg daily of macitentan slowed the progression of PAH both at the functional and structural levels. Treatments started on day 14 after monocrotaline injection and lasted 14 days.The findings of all experimental methods show that the selective ROCK inhibitor Y-27632 has more pronounced effects than macitentan, but a major limitation to its use is its marked peripheral vasodilating action.

Treatment of Pulmonary Hypertension With Angiotensin II Receptor Blocker and Neprilysin Inhibitor Sacubitril/Valsartan.

Angiotensin II has been implicated in maladaptive right ventricular (RV) hypertrophy and fibrosis associated with pulmonary hypertension (PH). Natriuretic peptides decrease RV afterload by promoting pulmonary vasodilation and inhibiting vascular remodeling but are degraded by neprilysin. We hypothesized that angiotensin receptor blocker and neprilysin inhibitor, sacubitril/valsartan (Sac/Val, LCZ696), will attenuate PH and improve RV function by targeting both pulmonary vascular and RV remodeling. PH was induced in rats using the SU5416/hypoxia model (Su/Hx), followed by 6-week treatment with placebo, Sac/Val, or Val alone. There were 4 groups: CON-normoxic animals with placebo (n=18); PH-Su/Hx rats+placebo (n=34); PH+Sac/Val (N=24); and PH+Val (n=16). In animals with PH, treatment with Sac/Val but not Val resulted in significant reduction in RV pressure (mm Hg: PH: 62±4, PH+Sac/Val: 46±5), hypertrophy (RV/LV+S: PH: 0.74±0.06, PH+Sac/Val: 0.46±0.06), collagen content (µg/50 µg protein: PH: 8.2±0.3, PH+Sac/Val: 6.4±0.4), pressures and improvement in RVs (mm/s: PH: 31.2±1.8, PH+Sac/Val: 43.1±3.6) compared with placebo. This was associated with reduced pulmonary vascular wall thickness, increased lung levels of ANP (atrial natriuretic peptide), BNP (brain-type natriuretic peptide), and cGMP, and decreased plasma endothelin-1 compared with PH alone. Also, PH+Sac/Val animals had altered expression of PKC isozymes in RV tissue compared with PH alone. Sac/Val reduces pulmonary pressures, vascular remodeling, as well as RV hypertrophy in a rat model of PH and may be appropriate for treatment of pulmonary hypertension and RV dysfunction.

Chrysin Alleviates Chronic Hypoxia-Induced Pulmonary Hypertension by Reducing Intracellular Calcium Concentration in Pulmonary Arterial Smooth Muscle Cells.

Chrysin (CH), the main ingredient of many medicinal plants, has been reported to be a very potent flavonoid possessing a large number of pharmacological activities. Recent studies have shown that CH significantly improves hemodynamic parameters such as right ventricular pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in a rat model of chronic hypoxia-induced pulmonary hypertension (CHPH). These improvements are through the inhibition of NOX4 expression, reactive oxygen species and malondialdehyde production, pulmonary arterial smooth muscle cell (PASMC) proliferation, and collagen accumulation. In this study, we investigated another mechanism by which CH alleviates CHPH by regulating intracellular calcium concentrations ([Ca]i) in PASMCs, as well as the underlying signaling pathway. The results show that (1) in CHPH model rats, CH substantially attenuated elevated right ventricular pressure, right ventricular hypertrophy, and pulmonary vascular remodeling; (2) in cultured rat distal PASMCs, CH inhibited the hypoxia-triggered promotion of cell proliferation, store-operated Ca entry and [Ca]i; and (3) CH significantly suppressed the hypoxia-upregulated HIF-1α, BMP4, TRPC1, and TRPC6 expression in distal pulmonary arteries (PAs) and cultured rat distal PASMCs. These results indicate that CH likely exerts its CHPH protective activity by regulating [Ca]i, which may result from the downregulation of HIF-1α, BMP4, TRPC1, and TRPC in PASMCs.

The FGL2 prothrombinase contributes to the pathological process of experimental pulmonary hypertension.

In situ thrombus formation is one of the major pathological features of pulmonary hypertension (PH). The mechanism of in situ thrombosis has not been clearly identified. Fibrinogen-like protein 2 (FGL2) prothrombinase is an immune coagulant that can cleave prothrombin to thrombin, which then converts fibrinogen into fibrin. This mechanism triggers in situ thrombus formation directly, bypassing both the intrinsic and extrinsic coagulation pathways. FGL2 prothrombinase is mainly expressed in endothelial cells and mediates multiple pathological processes. This implies that it may also play a role in PH. In this study, we examined the expression of FGL2 in idiopathic pulmonary arterial hypertension (IPAH) patients, and in monocrotaline-induced rat and hypoxia-induced mouse PH models. Fgl2-/- mice were used to evaluate the development of PH and explore associated pathological changes. These included in situ thrombosis, vascular remodeling, and endothelial apoptosis. Following these analyses, we examined possible signaling pathways downstream of FGL2 in PH. We show FGL2 is upregulated in pulmonary vascular endothelium in human IPAH and in two animal PH models. Genetic knockout of Fgl2 limited the development of PH, indicated by decreased in situ thrombus formation, less vascular remodeling, and reduced endothelial dysfunction. In addition, loss of FGL2 downregulated PAR1 (proteinase-activated receptor 1) expression and decreased the overactivation and consumption of platelets in hypoxia-induced PH. These results indicate FGL2 participate in the development of PH and loss of FGL2 could attenuate PH by reducing in situ thrombosis and suppressing PAR1 signaling. Thus we provide evidence that suggests FGL2 prothrombinase presents a potential therapeutic target for clinical treatment of PH.NEW & NOTEWORTHY This is the first study to demonstrate that fibrinogen-like protein 2 (FGL2) participates in the pathological progression of pulmonary hypertension (PH) in human idiopathic pulmonary arterial hypertension, a monocrotaline rat PH model, and a hypoxia mouse PH model. Genetic knockout of Fgl2 significantly limited the development of PH indicated by reduced in situ thrombosis, vascular remodeling, and endothelial dysfunction, and suppressed PAR1 (proteinase-activated receptor 1) signaling and overactivation of platelets on PH. These results suggest FGL2 presents a potential therapeutic target for clinical treatment of PH.

3311A new rat model of chronic thromboembolic pulmonary hypertension induced by repeated intravenous administration of biodegradable alginate microspheres

Abstract Introduction Chronic thromboembolic pulmonary hypertension (CTEPH) is life-threatening complication of pulmonary embolism (PE) with insufficiently understood mechanisms. Several rodent CTEPH models based on i.v. administration of non-biodegradable microparticles have been validated for preclinical studies. Major limitation of these models is the lack of partial dissolution of emboli following their entrapment in the lung vasculature. Purpose The study was aimed at development and validation of rat CTEPH model based on recurrent embolization of (sub)segmental pulmonary artery branches with biodegradable microspheres. Methods Male Wistar rats were used for the experiments. Pulmonary vasculature was embolized either with sodium alginate microspheres (MS) or with autologous blood clots (AT). The animals were randomized into the following groups: i) controls: saline at a volume of 50 μL was injected 4 times with 8-day interval into the tail vein; the same regimen was used in two next groups; ii) AT; iii) MS4; iv) MS8: MS were administered 8 times with 4-day interval. Histological examination of the lungs was performed after 2 and 6 weeks after the last injection. 6 weeks after the last injection the following analyses were performed: treadmill test, transthoracic echocardiography (TTE), right ventricular catheterization with measurement of right ventricular systolic pressure (RVSP), determination of serum endothelin-1 level. Results The survival rate in the MS8 group was 50%. In the other groups, there were no animal deaths. Multiple emboli were found in the lumen of (sub)segmental pulmonary artery branches 2 weeks after the last injection in MS4 and MS8 groups. Increased diameter and thickening of the bronchial arterial wall were also registered. After 6 weeks, the index of hypertrophy of vessel wall in MS4 and MS8 groups was significantly higher than in controls (p=0.041 and p=0.006, respectively) (Fig. 1). No sign of vascular remodeling was identified in the branches of the pulmonary artery in the AT group. Exercise tolerance was significantly reduced in both MS4 and MS8 groups compared with the controls (p=0.025 and p=0.008, respectively). There were no significant differences in exercise tolerance between the AT and control groups. TTE data indicate a significant increase in the diameter of the pulmonary trunk and the right ventricular outflow tract in the MS8 group compared with controls and AT (p&lt;0.05). Significant increase in RVSP as well as in endothelin-1 level versus controls was found only in the MS8 group. Figure 1. Histological changes in the branches of the pulmonary artery 6 weeks after the last injection of emboli. Conclusion Recurrent (×8) intravenous administration of MS in rats resulted in CTEPH development characterized by specific lung vasculature remodelling, reduced exercise tolerance, and persistent rise in RVSP. The model developed can be used for preclinical testing of promising drug candidates.