Altered Macrophage Polarization Induces Experimental Pulmonary Hypertension and Is Observed in Patients With Pulmonary Arterial Hypertension.

OBJECTIVE: To determine whether global reduction of CD68 (cluster of differentiation) macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and Results: Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow-derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow cytometry. After 6 weeks of macrophage depletion, male but not female MacLow mice developed PAH. Chimeric mice demonstrated a requirement for both MacLow bone marrow and MacLow recipient mice to cause PAH. Immunohistochemical analysis of lung sections demonstrated imbalance in M1/M2 ratio in male MacLow mice only, suggesting that this imbalance may drive the PAH phenotype. M1/M2 imbalance was also seen in male MacLow bone marrow-derived macrophages and PAH patient monocyte-derived macrophages following stimulation with doxycycline and IL (interleukin)-4, respectively. Furthermore, MacLow-derived alveolar macrophages showed characteristic differences in terms of their polarization and expression of diphtheria toxin A chain following stimulation with doxycycline. CONCLUSIONS: These data further highlight a sex imbalance in PAH and further implicate immune cells into this paradigm. Targeting imbalance of macrophage population may offer a future therapeutic option.

A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.

Toll-like Receptor 3 Is a Therapeutic Target for Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. TLR3 (Toll-like receptor 3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. OBJECTIVES: To study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces pulmonary hypertension in preclinical models. METHODS: Lung tissue and endothelial cells from patients with PAH were investigated by polymerase chain reaction, immunofluorescence, and apoptosis assays. TLR3-/- and TLR3+/+ mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic/polycytidylic acid (Poly[I:C]). MEASUREMENTS AND MAIN RESULTS: TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3-/- mice exhibited more severe pulmonary hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA receptors in endothelial cells. This was associated with greater susceptibility to apoptosis, a known driver of pulmonary vascular remodeling. Poly(I:C) increased TLR3 expression via IL-10 in rat endothelial cells. In vivo, high-dose Poly(I:C) reduced pulmonary hypertension in both rat models in proof-of-principle experiments. In addition, Poly(I:C) also reduced right ventricular failure in established pulmonary hypertension. CONCLUSIONS: Our work identifies a novel role for TLR3 in PAH based on the findings that reduced expression of TLR3 contributes to endothelial apoptosis and pulmonary vascular remodeling.

MicroRNA-140-5p and SMURF1 regulate pulmonary arterial hypertension.

Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been demonstrated to promote pulmonary arterial endothelial cell dysfunction and induce pulmonary arterial smooth muscle cell (PASMC) proliferation, leading to the development of pulmonary arterial hypertension (PAH). MicroRNAs (miRs) mediate higher order regulation of cellular function through coordinated modulation of mRNA targets; however, miR expression is altered by disease development and drug therapy. Here, we examined treatment-naive patients and experimental models of PAH and identified a reduction in the levels of miR-140-5p. Inhibition of miR-140-5p promoted PASMC proliferation and migration in vitro. In rat models of PAH, nebulized delivery of miR-140-5p mimic prevented the development of PAH and attenuated the progression of established PAH. Network and pathway analysis identified SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) as a key miR-140-5p target and regulator of BMP signaling. Evaluation of human tissue revealed that SMURF1 is increased in patients with PAH. miR-140-5p mimic or SMURF1 knockdown in PASMCs altered BMP signaling, further supporting these factors as regulators of BMP signaling. Finally, Smurf1 deletion protected mice from PAH, demonstrating a critical role in disease development. Together, these studies identify both miR-140-5p and SMURF1 as key regulators of disease pathology and as potential therapeutic targets for the treatment of PAH.

Pulmonary artery denervation reduces pulmonary artery pressure and induces histological changes in an acute porcine model of pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension is a devastating disease with high morbidity and mortality and limited treatment options. Recent studies have shown that pulmonary artery denervation improves pulmonary hemodynamics in an experimental model and in an early clinical trial. We aimed to evaluate the nerve distribution around the pulmonary artery, to determine the effect of radiofrequency pulmonary artery denervation on acute pulmonary hypertension induced by vasoconstriction, and to demonstrate denervation of the pulmonary artery at a histological level. METHODS AND RESULTS: Histological evaluation identified a circumferential distribution of nerves around the proximal pulmonary arteries. Nerves were smaller in diameter, greater in number, and located in closer proximity to the luminal aspect of the pulmonary arterial wall beyond the pulmonary artery bifurcation. To determine the effect of pulmonary arterial denervation acute pulmonary hypertension was induced in 8 pigs by intravenous infusion of thromboxane A2 analogue. Animals were assigned to either pulmonary artery denervation, using a prototype radiofrequency catheter and generator, or a sham procedure. Pulmonary artery denervation resulted in reduced mean pulmonary artery pressure and pulmonary vascular resistance and increased cardiac output. Ablation lesions on the luminal surface of the pulmonary artery were accompanied by histological and biochemical alteration in adventitial nerves and correlated with improved hemodynamic parameters. CONCLUSIONS: Pulmonary artery denervation offers the possibility of a new treatment option for patients with pulmonary arterial hypertension. Further work is required to determine the long-term efficacy and safety.

Inhibition of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses experimental pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by the progressive narrowing and occlusion of small pulmonary arteries. Current therapies fail to fully reverse this vascular remodeling. Identifying key pathways in disease pathogenesis is therefore required for the development of new-targeted therapeutics. We have previously reported tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) immunoreactivity within pulmonary vascular lesions from patients with idiopathic PAH and animal models. Because TRAIL can induce both endothelial cell apoptosis and smooth muscle cell proliferation in the systemic circulation, we hypothesized that TRAIL is an important mediator in the pathogenesis of PAH. We demonstrate for the first time that TRAIL is a potent stimulus for pulmonary vascular remodeling in human cells and rodent models. Furthermore, antibody blockade or genetic deletion of TRAIL prevents the development of PAH in three independent rodent models. Finally, anti-TRAIL antibody treatment of rodents with established PAH reverses pulmonary vascular remodeling by reducing proliferation and inducing apoptosis, improves hemodynamic indices, and significantly increases survival. These preclinical investigations are the first to demonstrate the importance of TRAIL in PAH pathogenesis and highlight its potential as a novel therapeutic target to direct future translational therapies.

Enhanced macrophage tribbles-1 expression in murine experimental atherosclerosis.

Development of the atherosclerotic plaque involves a complex interplay between a number of cell types and an extensive inter-cellular communication via cell bound as well as soluble mediators. The family of tribbles proteins has recently been identified as novel controllers of pro-inflammatory signal transduction. The objective of this study was to address the expression pattern of all three tribbles proteins in atherosclerotic plaques from a mouse model of atherosclerosis. Each tribbles were expressed in vascular smooth muscle cells, endothelial cells as well as in resident macrophages of mouse atherosclerotic plaques. The role of IL-1 mediated inflammatory events in controlling tribbles expression was also addressed by inducing experimental atherosclerosis in ApoE-/-IL1R1-/- (double knockout) mice. Immunohistochemical analysis of these mice showed a selective decrease in the percentage of trb-1 expressing macrophages, compared to the ApoE-/- cohort (14.7% ± 1.55 vs. 26.3% ± 1.19). The biological significance of this finding was verified in vitro where overexpression of trb-1 in macrophages led to a significant attenuation (~70%) of IL-6 production as well as a suppressed IL-12 expression induced by a proinflammatory stimulus. In this in vitro setting, expression of truncated trb-1 mutants suggests that the kinase domain of this protein is sufficient to exert this inhibitory action.

Simultaneous kissing stent technique to treat left main stem bifurcation disease.

BACKGROUND: While several endovascular techniques have been developed for treating arterial bifurcation lesions, there is, as yet, no single, widely accepted technique for treating left main stem (LMS) bifurcation lesions with stents. The simultaneous kissing stent (SCS) technique seems particularly suited for such lesions. The authors describe a consecutive cohort of patients with LMS bifurcation stenosis treated with this technique and present mechanistic insights from a porcine model. METHODS: Thirty consecutive patients with LMS bifurcation stenosis +/- multivessel disease were treated with SCS technique using paclitaxel-eluting stents. The technique involves simultaneous implantation of two stents (LMS-LAD and LMS-Cx) so that the proximal end of both stents lie at the same level in the body of LMS. Symptom status was recorded and follow-up angiography was performed. In addition, four pigs underwent SCS implantation of the LMS and were studied by direct examination, histology, and scanning electron microscopy. RESULTS: The patients' mean age was 63, 26 were elective and 19 were male. Paclitaxel-eluting stents (Taxus, Boston Scientific) were used. There was one in-hospital death in a highly unstable patient. At 6-month follow-up angiography, two patients required target lesion revascularization. At 13 +/- 3 month follow-up, the remaining patients remain well, with no other major adverse cardiac events. The animals all survived with patent stents. SEM demonstrated full re-endothelialization and histology showed a thin but complete neointima covering the stent struts. CONCLUSION: The SCS technique for treating LMS bifurcation disease with paclitaxel-eluting stents is safe and feasible. Medium term clinical and angiographic results are good. An endothelium and a thin neointima cover the metal struts.

Interleukin-1 receptor antagonist alters the response to vessel wall injury in a porcine coronary artery model.

OBJECTIVE: To determine the influence of IL-1 on the arterial response to experimental injury in porcine models of percutaneous coronary intervention (PCI). METHODS: An intravenous (i.v.) bolus of 0.5 mg/kg followed by a subcutaneous (s.c.) infusion of 2 mg/kg/24 h of human IL-1 receptor antagonist (IL-1ra) inhibited neutrophil recruitment in response to intradermal IL-1. Using this dose regimen, five groups of pigs were studied: Group 1, oversized balloon angioplasty of 2 coronary vessels (14-day infusion, 28th day sacrifice and analysis); Groups 2, 3, 4, and 5, oversized stenting of 2 coronary vessels (Group 2: 14-day infusion, 28th day analysis; Group 3: 14-day infusion, 14th day analysis; Group 4: 28-day infusion, 28th day analysis; Group 5: 28-day infusion, 90th day analysis). Neointimal area was quantified by standard means. RESULTS: In Group 1, IL-1ra resulted in a 23% decrease in neointimal area (p=0.04); in Group 2, a 34% increase (p=0.001); in Group 3, a 38% decrease (p<0.0001); in Group 4, a 34% decrease (p=0.0004); and in Group 5, a 41% decrease (p=0.00001). CONCLUSIONS: IL-1ra was associated with a sustained, significant reduction in neointima after vessel wall injury as long as it is given for the duration of the stimulation of the IL-1 system, in this case at least 28 days. This suggests that therapies based on the antagonism of IL-1 may modulate the coronary artery response to injury.