Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

BACKGROUND: Plexiform lesions, which have a dynamic appearance in structure and cellular composition, are the histological hallmark of severe pulmonary arterial hypertension in humans. The pathogenesis of the lesion development remains largely unknown, although it may be related to local inflammation and dysfunction in early progenitor endothelial cells (eEPCs). We tested the hypothesis that eEPCs contribute to the development of plexiform lesions by differentiating into macrophages in the setting of chronic inflammation.
METHODS: The eEPC markers CD133 and VEGFR-2, macrophage lineage marker mannose receptor C-type 1 (MRC1), TNFα and nuclear factor erythroid 2-related factor 2 (Nrf2) in plexiform lesions in a broiler model were determined by immunohistochemistry. eEPCs derived from peripheral blood mononuclear cells were exposed to TNFα, and macrophage differentiation and angiogenic capacity of the cells were evaluated by phagocytotic and Matrigel plug assays, respectively. The role of Nrf2 in eEPC-to-macrophage transition as well as in MRC1 expression was also evaluated. Intratracheal installation of TNFα was conducted to determine the effect of local inflammation on the formation of plexiform lesions.
RESULTS: Cells composed of the early lesions have a typical eEPC phenotype whereas those in more mature lesions display molecular and morphological characteristics of macrophages. Increased TNFα production in plexiform lesions was observed with lesion progression. In vitro studies showed that chronic TNFα challenge directed eEPCs to macrophage differentiation accompanied by hyperactivation of Nrf2, a stress-responsive transcription factor. Nrf2 activation (Keap1 knockdown) caused a marked downregulation in CD133 but upregulation in MRC1 mRNA. Dual luciferase reporter assay demonstrated that Nrf2 binds to the promoter of MRC1 to trigger its expression. In good agreement with the in vitro observation, TNFα exposure induced macrophage differentiation of eEPCs in Matrigel plugs, resulting in reduced neovascularization of the plugs. Intratracheal installation of TNFα resulted in a significant increase in plexiform lesion density.
CONCLUSIONS: This work provides evidence suggesting that macrophage differentiation of eEPCs resulting from chronic inflammatory stimulation contributes to the development of plexiform lesions. Given the key role of Nrf2 in the phenotypic switching of eEPCs to macrophages, targeting this molecular might be beneficial for intervention of plexiform lesions.

UNASSIGNED: Pulmonary arterial hypertension (PAH) is a progressive and fatal pulmonary vascular disease initiated by endothelial dysfunction. Mesenchymal stromal cells (MSCs) have been shown to ameliorate PAH in various rodent models; however, these models do not recapitulate all the histopathological alterations observed in human PAH. Broiler chickens (Gallus gallus) can develop PAH spontaneously with neointimal and plexogenic arteriopathy strikingly similar to that in human patients. Herein, we examined the protective effects of MSC transplantation on the development of PAH in this avian model.
UNASSIGNED: Mixed-sex broilers at 15 d of age were received 2×106 MSCs or PBS intravenously. One day later, birds were exposed to cool temperature with excessive salt in their drinking water to induce PAH. Cumulative morbidity from PAH and right-to-left ventricle ratio were recorded. Lung histologic features were evaluated for the presence of endothelial damage, endothelial proliferation and plexiform lesions. Expression of proinflammatory mediators and angiogenic factors in the lung was detected. Matrigel tube formation assay was performed to determine the angiogenic potential of endogenous MSCs.
UNASSIGNED: MSC administration reduced cumulative PAH morbidity and attenuated endothelial damage, plexiform lesions and production of inflammatory mediators in the lungs. No significant difference in the expression of paracrine angiogenic factors including VEGF-A and TGF-β was determined between groups, suggesting that they are not essential for the beneficial effect of MSC transplantation. Interestingly, the endogenous MSCs from birds receiving MSC transplantation demonstrated endothelial differentiatial capacity in vitro whereas those from the mock birds did not.
UNASSIGNED: Our results support the therapeutic use of MSC transplantation for PAH treatment and suggest that exogenous MSCs produce beneficial effects through modulating inflammation and endogenous MSC-mediated vascular repair.

In pulmonary arterial hypertension, plexiform lesions are associated with severe arterial obstruction and right ventricular failure. Exploring their structure and position is crucial for understanding the interplay between hemodynamics and vascular remodeling. The aim of this research was to use synchrotron-based phase-contrast micro-CT to study the three-dimensional structure of plexiform lesions. Archived paraffin-embedded tissue samples from 14 patients with pulmonary arterial hypertension (13 idiopathic, 1 with known BMPR2-mutation) were imaged. Clinical data showed high-median PVR (12.5 WU) and mPAP (68 mmHg). Vascular lesions with more than 1 lumen were defined as plexiform. Prior radiopaque dye injection in some samples facilitated 3-D rendering. Four distinct types of plexiform lesions were identified: 1) localized within or derived from monopodial branches (supernumerary arteries), often with a connection to the vasa vasorum; 2) localized between pulmonary arteries and larger airways as a tortuous transformation of intrapulmonary bronchopulmonary anastomoses; 3) as spherical structures at unexpected abrupt ends of distal pulmonary arteries; and 4) as occluded pulmonary arteries with recanalization. By appearance and localization, types 1-2 potentially relieve pressure via the bronchial circulation, as pulmonary arteries in these patients were almost invariably occluded distally. In addition, types 1-3 were often surrounded by dilated thin-walled vessels, often connected to pulmonary veins, peribronchial vessels, or the vasa vasorum. Collaterals, bypassing completely occluded pulmonary arteries, were also observed to originate within plexiform lesions. In conclusion, synchrotron-based imaging revealed significant plexiform lesion heterogeneity, resulting in a novel classification. The four types likely have different effects on hemodynamics and disease progression.

In intralobar pulmonary sequestrations, vascular changes similar to those in pulmonary hypertension (PH) are generally observed, such as intimal proliferation and plexiform lesions. However, to our knowledge, a sequestrated lung manifesting vascular changes with both arteritis and a plexiform lesion has never been reported. A 25-year-old man was diagnosed with intralobar pulmonary sequestration. Pathologically, both arteritis and a plexiform lesion were observed in the sequestrated lung. Systemic vasculitis syndrome was clinically excluded, and the pathological findings appeared to be associated with local PH. Arteritis is an extremely rare finding; only one case of arteritis associated with local PH has been reported in intralobar sequestration. In this case, the artery near the plexiform lesion had milder inflammation and fibrosis, suggesting that the arteritis formed prior to the plexiform lesion. This is the first case of arteritis and a plexiform lesion co-occurring in intralobar pulmonary sequestration associated with local PH. This case may shed light on the formation of plexiform lesions and their association with arteritis.

We report the case of an infant girl with incontinentia pigmenti (IP) complicated by fatal pulmonary arterial hypertension (PAH). She was diagnosed with IP, based on the presence of specific skin lesions, neonatal seizures, hypereosinophilia and a maternal family history of IP. At the age of 2 months, she was diagnosed with PAH on systolic heart murmur due to tricuspid valve regurgitation. Despite several treatments for PAH but not including epoprostenol, severe PAH persisted and she died of pulmonary hypertensive crisis at the age of 5 months. On postmortem histopathology the pulmonary artery had severe intimal thickening, with occlusion or stenosis of the vascular lumen of the small pulmonary arteries as well as partial plexiform lesions, all of which were compatible with PAH. Modulation of nuclear factor-κB signaling may be involved in the development of PAH in IP.

Vascular occlusion and complex plexiform lesions are hallmarks of the pathology of severe pulmonary arterial hypertension (PAH) in patients. However, the mechanisms of obliterative vascular remodeling remain elusive; hence, current therapies have not targeted the fundamental disease-modifying mechanisms and result in only modest improvement in morbidity and mortality.
Mice with Tie2Cre-mediated disruption of Egln1 (encoding prolyl-4 hydroxylase 2 [PHD2]; Egln1(Tie2)) in endothelial cells and hematopoietic cells exhibited spontaneous severe PAH with extensive pulmonary vascular remodeling, including vascular occlusion and plexiform-like lesions, resembling the hallmarks of the pathology of clinical PAH. As seen in patients with idiopathic PAH, Egln1(Tie2) mice exhibited unprecedented right ventricular hypertrophy and failure and progressive mortality. Consistently, PHD2 expression was diminished in lung endothelial cells of obliterated pulmonary vessels in patients with idiopathic PAH. Genetic deletions of both Egln1 and Hif1a or Egln1 and Hif2a identified hypoxia-inducible factor-2α as the critical mediator of the severe PAH seen in Egln1(Tie2) mice. We also observed altered expression of many pulmonary hypertension-causing genes in Egln1(Tie2) lungs, which was normalized in Egln1(Tie2)/Hif2a(Tie2) lungs. PHD2-deficient endothelial cells promoted smooth muscle cell proliferation in part through hypoxia-inducible factor-2α-activated CXCL12 expression. Genetic deletion of Cxcl12 attenuated PAH in Egln1(Tie2) mice.
These studies defined an unexpected role of PHD2 deficiency in the mechanisms of severe PAH and identified the first genetically modified mouse model with obliterative vascular remodeling and pathophysiology recapitulating clinical PAH. Thus, targeting PHD2/hypoxia-inducible factor-2α signaling is a promising strategy to reverse vascular remodeling for treatment of severe PAH.