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We assessed the pulmonary hemodynamic response to vascular endothelial growth factor receptor, type 2, inhibition using SU5416 (SU) with and without chronic hypoxia (CH) in different background strains and colonies of rats. A single subcutaneous injection of SU (20 mg/kg) or vehicle was administered to different substrains of Sprague-Dawley (SD) rats, and they were compared with Lewis and Fischer rats, with and without exposure to CH (10% O2 for 3 wk). Remarkably, a unique colony of SD rats from Charles River Laboratories, termed the SD-hyperresponsive type, exhibited severe pulmonary arterial hypertension (PAH) with SU alone, characterized by increased right ventricular systolic pressure, right ventricular/left ventricular plus septal weight ratio, and arteriolar occlusive lesions at 7-8 weeks (all P < 0.0001 versus vehicle). In contrast, the other SD substrain from Harlan Laboratories, termed SD-typical type, as well as Fischer rats, developed severe PAH only when exposed to SU and CH, whereas Lewis rats showed only a minimal response. All SD-typical type rats survived for up to 13 weeks after SU/CH, whereas SD-hyperresponsive type rats exhibited mortality after SU and SU/CH (35% and 50%, respectively) at 8 weeks. Fischer rats exposed to SU/CH exhibited the greatest mortality at 8 weeks (78%), beginning as early as 4 weeks after SU and preceded by right ventricle enlargement. Of note, a partial recovery of PAH after 8 weeks was observed in the SD-typical type substrain only. In conclusion, variation in strain, even between colonies of the same strain, has a remarkable influence on the nature and severity of the response to SU, consistent with an important role for genetic modifiers of the PAH phenotype.

Rationale: Pulmonary arterial hypertension (PAH) remains a progressive and eventually lethal disease characterized by increased pulmonary vascular resistance because of loss of functional lung microvasculature, primarily at the distal (intracinar) arteriolar level. Cell-based therapies offer the potential to repair and regenerate the lung microcirculation and have shown promise in preclinical evaluation in experimental models of PAH., Objective: The Pulmonary Hypertension and Angiogenic Cell Therapy (PHACeT) trial was a phase 1, dose-escalating clinical study of the tolerability of culture-derived endothelial progenitor cells, transiently transfected with endothelial nitric oxide synthase, in patients with PAH refractory to PAH-specific therapies., Methods and Results: Seven to 50 million endothelial nitric oxide synthase-transfected endothelial progenitor cells, divided into 3 doses on consecutive days, were delivered into the right atrium via a multiport pulmonary artery catheter during continuous hemodynamic monitoring in an intensive care unit setting. Seven patients (5 women) received treatment from December 2006 to March 2010. Cell infusion was well tolerated, with no evidence of short-term hemodynamic deterioration; rather, there was a trend toward improvement in total pulmonary resistance during the 3-day delivery period. However, there was 1 serious adverse event (death) which occurred immediately after discharge in a patient with severe, end stage disease. Although there were no sustained hemodynamic improvements at 3 months, 6-minute walk distance was significantly increased at 1, 3, and 6 months., Conclusion: Delivery of endothelial progenitor cells overexpressing endothelial nitric oxide synthase was tolerated hemodynamically in patients with PAH. Furthermore, there was evidence of short-term hemodynamic improvement, associated with long-term benefits in functional and quality of life assessments. However, future studies are needed to further establish the efficacy of this therapy., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00469027., (© 2015 American Heart Association, Inc.)

Background: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular endothelial cells (ECs). Hereditary PAH (HPAH) is often caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. Therefore, we screened for dysregulated proteins in blood-outgrowth ECs of HPAH patients with BMPR2 mutations compared with healthy control subjects., Methods and Results: A total of 416 proteins were detected with 2-dimensional PAGE in combination with liquid chromatography/tandem mass spectrometry analysis, of which 22 exhibited significantly altered abundance in blood-outgrowth ECs from patients with HPAH. One of these proteins, translationally controlled tumor protein (TCTP), was selected for further study because of its well-established role in promoting tumor cell growth and survival. Immunostaining showed marked upregulation of TCTP in lungs from patients with HPAH and idiopathic PAH, associated with remodeled vessels of complex lesions. Increased TCTP expression was also evident in the SU5416 rat model of severe and irreversible PAH, associated with intimal lesions, colocalizing with proliferating ECs and the adventitia of remodeled vessels but not in the vascular media. Furthermore, silencing of TCTP expression increased apoptosis and abrogated the hyperproliferative phenotype of blood-outgrowth ECs from patients with HPAH, raising the possibility that TCTP may be a link in the emergence of apoptosis-resistant, hyperproliferative vascular cells after EC apoptosis., Conclusion: Proteomic screening identified TCTP as a novel mediator of endothelial prosurvival and growth signaling in PAH, possibly contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis., (© 2014 American Heart Association, Inc.)

The capacity of imatinib mesylate to reverse established pulmonary arterial hypertension (PAH) has been attributed to a reduction in pulmonary arterial muscularization via inhibition of platelet-derived growth factor receptor-β on vascular smooth muscle cells. However, there is also a significant immunomodulatory component to the action of imatinib that may account for its efficacy in PAH. We found that monocrotaline-induced pulmonary hypertension was associated with a significant decrease in pulmonary natural killer (NK) cells and T lymphocytes and the accumulation of macrophages in the lungs of F344 rats. The prevention of pulmonary hypertension by imatinib blocked these changes in pulmonary leukocyte content and induced elevations in pulmonary interferon-γ, tumor necrosis factor α, and IL-10, corresponding to the enhanced activity of splenic NK cells ex vivo. Treatment with anti-asialo GM1 antiserum (ASGM1), which ablated circulating NK cells and depleted T cells, eliminated the therapeutic benefit of imatinib. ASGM1-treated animals also exhibited significant pulmonary arteriolar muscularization in response to monocrotaline challenge compared with immunocompetent controls despite daily imatinib administration to both groups. In the athymic rat, imatinib decreased right ventricular hypertrophy and pulmonary arteriolar muscularization in monocrotaline-challenged animals versus saline-treated controls but did not prevent pulmonary macrophage accumulation or the development of pulmonary hypertension. These data demonstrate that the immunomodulatory effects of imatinib are critical to its therapeutic action in experimental PAH., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Direct injection of endothelial progenitor cells (EPCs) into the circulation has shown therapeutic benefit in both experimental models and clinical studies of pulmonary arterial hypertension (PAH). Using the monocrotaline (MCT)-induced rat model of PAH, we investigated the role of innate immunity in the therapeutic activity of two types of putative EPCs derived from human peripheral blood mononuclear cells: an early population of endothelial-like, culture-modified monocytes (E-CMMs) and late-outgrowth EPCs (L-EPCs), which exhibit a strong endothelial phenotype. In the athymic nude rat, E-CMMs prevented MCT-induced increases in right ventricular systolic pressure (P < 0.001) and right ventricular hypertrophy (P < 0.01) when administered 3 days after MCT challenge, whereas L-EPCs were ineffective. However, in both cases, there was a lack of cell persistence within the lungs at 24 hours after injection, likely due to residual natural killer (NK) cell activity in the model. Although ablation of NK and NK-T cells with anti-asialo-GM-1 antiserum enhanced the retention of both E-CMMs and L-EPCs, still no benefit was seen with L-EPCs, and the efficacy of E-CMMs was lost. In vitro characterization revealed that E-CMMs resemble a regulatory subtype of dendritic cells, producing IL-10, but not IL-12, in response to inflammatory stimuli. Coculture studies demonstrated the capacity of E-EPCs to stimulate autologous human and nude rat NK cells in vitro. These data support a novel mode of action for human E-CMMs in the prevention of PAH, whereby they act through an immune-dependent mechanism, potentially involving the stimulation of NK cells.

Hyaluronan (HA) degradation fragments have been linked to inflammation in a wide range of lung diseases. In idiopathic pulmonary arterial hypertension, HA accumulation has been associated with advanced disease. In this study, we investigated the potential role of HA degradation in the early stages of disease by examining HA distribution, molecular mass, synthesis, and enzymatic degradation at different stages of disease progression in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). At 28 days post-MCT, severe PH was associated with increased total lung HA (P = 0.04). In contrast, a significant decrease in total lung HA was observed on day 10, before the onset of PH (P = 0.02). Molecular mass analysis revealed a loss of high molecular mass (HMM) HA at 10 and 24 days post-MCT, followed by an increase in HMM HA at 28 days. Expression of HA synthase 2 (HAS2) was elevated in MCT-challenged animals at 24 and 28 days, consistent with increased synthesis of HMM HA. Analysis by Morgan Elson assay and zymography demonstrated increased hyaluronidase-1 activity in the lungs of MCT-challenged rats, indicating that the observed increases in HAS2 expression and HA synthesis were counterbalanced, in part, by enhanced degradation. The present data demonstrate that, in the MCT model, early-stage PH is associated with enhanced hyaluronidase-1 activity, while both degradation and synthesis are increased at later stages. Thus an early increase in the generation of proinflammatory HA fragments may play a role in the onset and progression of pulmonary arterial hypertension.

Introduction: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by pulmonary arteriolar narrowing and degeneration associated with in situ thrombosis. We hypothesized that microvascular endothelial injury and apoptosis may be an initiating mechanism in IPAH. Endothelial apoptosis generates endothelial microfragments (EMF), which can activate platelets. Platelets release both VEGF and angiostatin, which depending the balance can inhibit or induce endothelial apoptosis, respectively., Materials and Methods: We measured EMFs from blood of IPAH patients as index of endothelial cell apoptosis/injury and levels of pro- and anti- EC apoptotic factors found in platelets. EMFs and platelets in blood samples from control subjects and patients with IPAH were measured using a 4-color flow cytometry protocol, and platelet levels of VEGF and angiostatin were determined by ELISAs and immunoblotting., Results: Compared to controls, IPAH patients exhibited higher numbers of circulating EMFs and more activated/apoptotic platelets. IPAH patients also exhibited higher levels of platelet angiostatin; however, no significant difference was detected in platelet VEGF levels between the two groups., Conclusions: These results are consistent with an increase in EC dysfunction in patients with IPAH, possibly contributing to the progression of IPAH and its associated thrombosis., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Pulmonary arterial hypertension (PAH) is characterized by widespread loss of pulmonary microvasculature. Therefore we hypothesized that angiogenic gene therapy would reverse established PAH, in part restoring the lung microcirculation. Three weeks after monocrotaline (MCT) treatment, Fisher 344 rats were randomized to receive a total of either 1.5 x 10(6) syngeneic fibroblasts (FB) transfected with vascular endothelial growth factor A (VEGF), endothelial NO synthase (eNOS), or null-plasmid transfected FBs. Right ventricular systolic pressure (RVSP) was similarly increased in all MCT-treated groups at the time of gene transfer. Animals receiving the null-vector progressed to severe PAH by Day 35 (P < 0.001). In contrast, eNOS gene transfer significantly reduced RVSP at Day 35 compared with Day 21, whereas VEGF prevented further increases in RVSP over the subsequent 2 wk but did not reverse established PAH. RV hypertrophy was significantly reduced in both the eNOS-treated and VEGF-treated groups compared with the null-transfected controls. Fluorescent microangiography revealed widespread occlusion of the pre-capillary arterioles 21 d after MCT treatment, and animals receiving eNOS gene transfer exhibited the greatest improvement in the arteriolar architecture and capillary perfusion at Day 35. Cell-based eNOS gene transfer was more effective than VEGF in reversing established PAH, associated with evidence of regeneration of pulmonary microcirculation.

Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor alpha, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 microg of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (P=0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance caused by narrowing and loss of pulmonary microvasculature, which in its late stages becomes refractory to traditional therapies. We hypothesized that bone marrow-derived endothelial progenitor cells (EPCs), which normally function to repair and regenerate blood vessels, would restore pulmonary hemodynamics and increase microvascular perfusion in the rat monocrotaline (MCT) model of PAH. Mononuclear cells were isolated from the bone marrow of syngeneic Fisher-344 rats by Ficoll gradient centrifugation and cultured for 7 to 10 days in endothelial growth medium. Fluorescently labeled endothelial-like progenitor cells (ELPCs) engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCT-injured lung. The administration of ELPCs 3 days after MCT nearly completely prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone (31.5+/-0.95 versus 48+/-3 mm Hg, respectively; P<0.001), whereas injection of skin fibroblasts had no protective effect (50.9+/-5.4 mm Hg). Delayed administration of progenitor cells 3 weeks after MCT prevented the further progression of PAH 2 weeks later (ie, 5 weeks after MCT), whereas only animals receiving ELPCs transduced with human endothelial NO-synthase (eNOS) exhibited significant reversal of established disease at day 35 (31+/-2 mm Hg, P<0.005) compared with day 21 (50+/-3 mm Hg). Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT, whereas arteriolar-capillary continuity and microvascular architecture was preserved with the administration of syngeneic ELPCs. Moreover, the delivery of ELPCs to rats with established PAH resulted in marked improvement in survival, which was greatest in the group receiving eNOS-transduced cells. We conclude that bone marrow-derived ELPCs can engraft and repair the MCT-damaged lung, restoring microvasculature structure and function. Therefore, the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with PAH.