Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

Progression of vascular remodeling in pulmonary vein obstruction.

OBJECTIVES: Pulmonary vein obstruction (PVO) frequently occurs after repair of total anomalous pulmonary vein connection with progression of intimal hyperplasia from the anastomotic site toward upstream pulmonary veins (PVs). However, the understanding of mechanism in PVO progression is constrained by lack of data derived from a physiological model of the disease, and no prophylaxis has been established. We developed a new PVO animal model, investigated the mechanisms of PVO progression, and examined a new prophylactic strategy. METHODS: We developed a chronic PVO model using infant domestic pigs by cutting and resuturing the left lower PV followed by weekly hemodynamic parameter measurement and angiographic assessment of the anastomosed PV. Subsequently, we tested a novel therapeutic strategy with external application of rapamycin-eluting film to the anastomotic site. RESULTS: We found the pig PVO model mimicked human PVO hemodynamically and histopathologically. This model exhibited increased expression levels of Ki-67 and phospho-mammalian target of rapamycin in smooth muscle-like cells at the anastomotic neointima. In addition, contractile to synthetic phenotypic transition; that is, dedifferentiation of smooth muscle cells and mammalian target of rapamycin pathway activation in the neointima of upstream PVs were observed. Rapamycin-eluting films externally applied around the anastomotic site inhibited the activation of mammalian target of rapamycin in the smooth muscle-like cells of neointima, and delayed PV anastomotic stenosis. CONCLUSIONS: We demonstrate the evidence on dedifferentiation of smooth muscle-like cells and mammalian target of rapamycin pathway activation in the pathogenesis of PVO progression. Delivery of rapamycin to the anastomotic site from the external side delayed PV anastomotic stenosis, implicating a new therapeutic strategy to prevent PVO progression.

Targeted therapy in pulmonary veno-occlusive disease: time for a rethink?

BACKGROUND: Pulmonary veno-occlusive disease (PVOD) is a rare condition with poor prognosis, and lung transplantation is recommended as the only curative therapy. The role of pulmonary arterial hypertension targeted therapy in PVOD remains controversial, and long-term effects of targeted therapy have been rarely reported. This study aims to retrospectively evaluate the role of targeted therapy in PVOD patients and the long-term outcome. METHODS: PVOD patients with good responses to targeted therapies were analyzed, and data pre- and post- targeted therapies were compared. An overview of the effects of targeted therapies on PVOD patients was also conducted. RESULTS: Five genetically or histologically confirmed PVOD patients received targeted therapies and showed good responses. Their mean pulmonary arterial pressure by right heart catheterization was 62.0 ± 11.7 mmHg. Two receiving monotherapy got stabilized, and three receiving sequential combination therapy got improved, cardiac function and exercise capacity significantly improved after treatments. No pulmonary edema occurred. The mean time from the first targeted therapy to the last follow up was 39.3 months, and the longest was 9 years. A systematic review regarding the effects of targeted therapies on PVOD patients indicated majorities of patients got hemodynamics or 6-min walk distance improved, and 26.7% patients developed pulmonary edema. The interval from targeted drugs use to death ranged from 71 min to over 4 years. CONCLUSIONS: Cautious use of targeted therapy could safely and effectively improve or stabilize hemodynamics and exercise capacity of some patients without any complications. PVOD patients could live longer than expected.

EIF2AK4 mutation in pulmonary veno-occlusive disease: A case report and review of the literature.

BACKGROUND: Pulmonary veno-occlusive disease (PVOD) is a rare and devastating cause of pulmonary arterial hypertension with a non-specific clinical presentation and a relatively specific presentation in high-resolution thoracic CT scan images. Definitive diagnosis is made by histological examination in previous. According to the 2015 ESC/ERS Guidelines, detection of a mutation in the eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) without histological confirmation is recommended to validate the diagnosis of PVOD. METHODS: We report the case of a 27-year-old man who was admitted for persistent cough and dyspnea that had lasted for 5 months and had developed and experienced progressive dyspnea for the last 2 months. The echocardiogram and right heart catheterization without vasodilator challenge confirmed the diagnosis of pulmonary arterial hypertension. Other tests, such as high-resolution thoracic CT scan, V/Q scan, pulmonary function test with diffusion capacity, and blood tests, excluded other associated diseases which could have caused pulmonary hypertension. RESULTS: The initial diagnosis at admission was idiopathic pulmonary arterial hypertension and an oral vasodilator (sildenafil) was given. However, the dyspnea subsequently worsened, and the patient was transferred to a regional lung transplant center, where he died of heart failure 1 week later. Using exome sequencing, we found an EIF2AK4 mutation, which was sufficient to confirm the diagnosis of PVOD. CONCLUSION: This is the first reported case of EIF2AK4 mutation in PVOD in a Chinese patient population. We found the frameshift EIF2AK4 mutation c.1392delT (p.Arg465fs) in this case. Up to now, there has been a paucity of data on this rare disease, and the exact role of EIF2AK4 loss-of-function mutations in the pathogenesis of PVOD is still unknown. More investigations should be conducted in the future.

Pulmonary Veno-Occlusive Disease: A Newly Recognized Cause of Severe Pulmonary Hypertension in Dogs.

Pulmonary hypertension is a well-known though poorly characterized disease in veterinary medicine. In humans, pulmonary veno-occlusive disease (PVOD) is a rare cause of severe pulmonary hypertension with a mean survival time of 2 years without lung transplantation. Eleven adult dogs (5 males, 6 females; median age 10.5 years, representing various breeds) were examined following the development of severe respiratory signs. Lungs of affected animals were evaluated morphologically and with immunohistochemistry for alpha smooth muscle actin, desmin, CD31, CD3, CD20, and CD204. All dogs had pulmonary lesions consistent with PVOD, consisting of occlusive remodeling of small- to medium-sized pulmonary veins, foci of pulmonary capillary hemangiomatosis (PCH), and accumulation of hemosiderophages; 6 of 11 dogs had substantial pulmonary arterial medial and intimal thickening. Ultrastructural examination and immunohistochemistry showed that smooth muscle cells contributed to the venous occlusion. Increased expression of CD31 was evident in regions of PCH indicating increased numbers of endothelial cells in these foci. Spindle cells strongly expressing alpha smooth muscle actin and desmin co-localized with foci of PCH; similar cells were present but less intensely labeled elsewhere in non-PCH alveoli. B cells and macrophages, detected by immunohistochemistry, were not co-localized with the venous lesions of canine PVOD; small numbers of CD3-positive T cells were occasionally in and around the wall of remodeled veins. These findings indicate a condition in dogs with clinically severe respiratory disease and pathologic features resembling human PVOD, including foci of pulmonary venous remodeling and PCH.

Respuesta paradójica a prostanoides en paciente con hipertensión pulmonar severa / No disponible

La enfermedad veno oclusiva pulmonar (EVOP) es un síndrome clínico-patológico infrecuente que se asocia a hipertensión arterial pulmonar (HAP). La respuesta paradójica a prostanoides nos debe hacer sospechar en esta entidad, aunque esta situación es muy infrecuente. Además, el planteamiento terapéutico de este tipo de pacientes es muy controvertido porque los pacientes pueden presentar empeoramiento clínico con al realizar tratamiento con vasodilatadores. Presentamos un caso de un varón de 39 años diagnosticado de EVOP con respuesta paradójica a prostanoides y revisamos las controversias en cuanto a la actitud terapéutica

Pulmonary veno-occlusive disease (PVOD) is a rare clinic-pathologic syndrome that is associated with pulmonary arterial hypertension (PAH). The paradoxical response to prostanoids should make us suspect this entity, although this is unusual. The therapeutic approach to these patients is highly controversial because they can develop clinical worsening using vasodilators. We present a case of a 39 years old male diagnosed with (PVOD) and paradoxical response to prostanoids. Also, we review the controversies regarding the therapeutic approach

Haemosiderin-laden sputum macrophages for diagnosis in pulmonary veno-occlusive disease.

AIMS: Pulmonary veno-occlusive disease (PVOD) is a rare condition of pulmonary arterial hypertension (PAH), in which post-capillary veins are affected. Since the therapeutic approach in PVOD differs from other forms of PAH, it is crucial to establish the diagnosis. Due to the fact that affected patients are often hemodynamically unstable, minimal invasive procedures are necessary for the diagnostic work-up. Chronic alveolar haemorrhage has been observed during bronchoalveolar lavage in PVOD cases. This study therefore investigates whether signs of alveolar haemorrhage can also be found in the sputum of these patients. METHODS AND RESULTS: Six patients suffering from PVOD were included in this analysis. As controls, patients with idiopathic PAH (n = 11), chronic thromboembolic PH (n = 9) and with sclerodermia-associated PH (n = 10) were assessed. Sputum from every patient was obtained by a non-invasive manner. The amount of haemosiderin-laden macrophages was determined using the Golde score. There were statistically significant more haemosiderin-laden macrophages in the sputum of patients suffering from PVOD as compared to the other groups (P<0.05). Assuming a cut-off of 200 on the Golde score, all of the 6 PVOD patients surpassed this value compared with only 1 out of the 30 cases with precapillary PH. Thus, sensitivity and specificity with respect to the diagnosis of PVOD was 100% and 97%, respectively. CONCLUSION: The content of haemosiderin-laden macrophages in the sputum of patients suffering from PVOD is significantly higher as compared to other forms of PH and may be useful in the non-invasive diagnostic work-up of these patients.

Losartan ameliorates "upstream" pulmonary vein vasculopathy in a piglet model of pulmonary vein stenosis.

OBJECTIVES: Pulmonary vein stenosis (PVS) is a relentless disease with a poor prognosis. Although surgical repair can effectively treat "downstream" (near left atrial junction) PVS, residual "upstream" (deep in lung parenchyma) PVS commonly dictates long-term survival. Our initial studies revealed an association between PVS and transforming growth factor-ß signaling, which led us to investigate the effect of losartan on upstream pulmonary vein vasculopathy in a piglet model of PVS. METHODS: Neonatal Yorkshire piglets underwent sham surgical banding (sham, n = 6), staged bilateral pulmonary vein banding of all pulmonary veins except the right middle pulmonary vein (banded, n = 6), and staged pulmonary vein banding with losartan treatment (losartan, 1 mg/kg/d, n = 7). After 7 weeks, the hemodynamic data were obtained and the piglets killed. RESULTS: Pulmonary vein banding (compared with sham) was associated with continuous turbulent flow in banded pulmonary veins, pulmonary hypertension (pulmonary artery/systemic blood pressure ratio 0.51 ± 0.06 vs 0.23 ± 0.02, P < .001), and diffuse pulmonary vein intimal hyperplasia in the upstream pulmonary veins (P < .001). Losartan administration decreased the pulmonary artery/systemic blood pressure ratios compared with those in the banded piglets (0.36 ± 0.08 vs 0.51 ± 0.06, P = .007) but it remained greater than those in the sham group (P = .001). Losartan was also associated with diminished pulmonary vein intimal hyperplasia compared with that in the banded piglets (P < .001) but still remained more than that in the sham group (P = .035). Pulmonary vein banding reduced vascular endothelial-cadherin expression, indicative of diminished endothelial integrity, which was restored with losartan. CONCLUSIONS: Losartan treatment improved PVS-associated pulmonary hypertension and intimal hyperplasia and might be a beneficial prophylactic therapy for patients at high risk of developing PVS after pulmonary vein surgery.

Pulmonary vein stenosis and the pathophysiology of "upstream" pulmonary veins.

BACKGROUND: Surgical and catheter-based interventions on pulmonary veins are associated with pulmonary vein stenosis (PVS), which can progress diffusely through the "upstream" pulmonary veins. The mechanism has been rarely studied. We used a porcine model of PVS to assess disease progression with emphasis on the potential role of endothelial-mesenchymal transition (EndMT). METHODS: Neonatal piglets underwent bilateral pulmonary vein banding (banded, n = 6) or sham operations (sham, n = 6). Additional piglets underwent identical banding and stent implantation in a single-banded pulmonary vein 3 weeks postbanding (stented, n = 6). At 7 weeks postbanding, hemodynamics and upstream PV pathology were assessed. RESULTS: Banded piglets developed pulmonary hypertension. The upstream pulmonary veins exhibited intimal thickening associated with features of EndMT, including increased transforming growth factor (TGF)-ß1 and Smad expression, loss of endothelial and gain of mesenchymal marker expression, and coexpression of endothelial and mesenchymal markers in banded pulmonary vein intimal cells. These immunopathologic changes and a prominent myofibroblast phenotype in the remodeled pulmonary veins were consistently identified in specimens from patients with PVS, in vitro TGF-ß1-stimulated cells isolated from piglet and human pulmonary veins, and human umbilical vein endothelial cells. After stent implantation, decompression of a pulmonary vein was associated with reappearance of endothelial marker expression, suggesting the potential for plasticity in the observed pathologic changes, followed by rapid in-stent restenosis. CONCLUSIONS: Neonatal pulmonary vein banding in piglets recapitulates critical aspects of clinical PVS and highlights a pathologic profile consistent with EndMT, supporting the rationale for evaluating therapeutic strategies designed to exploit reversibility of upstream pulmonary vein pathology.

Platelet-derived growth factor receptor-ß and epidermal growth factor receptor in pulmonary vasculature of systemic sclerosis-associated pulmonary arterial hypertension versus idiopathic pulmonary arterial hypertension and pulmonary veno-occlusive disease: a case-control study.

INTRODUCTION: Systemic sclerosis (SSc) complicated by pulmonary arterial hypertension (PAH) carries a poor prognosis, despite pulmonary vascular dilating therapy. Platelet-derived growth factor receptor-ß (PDGFR-ß) and epidermal growth factor receptor (EGFR) are potential therapeutic targets for PAH because of their proliferative effects on vessel remodelling. To explore their role in SScPAH, we compared PDGFR- and EGFR-mmunoreactivity in lung tissue specimens from SScPAH. We compared staining patterns with idiopathic PAH (IPAH) and pulmonary veno-occlusive disease (PVOD), as SScPAH vasculopathy differs from IPAH and sometimes displays features of PVOD. Immunoreactivity patterns of phosphorylated PDGFR-ß (pPDGFR-ß) and the ligand PDGF-B were evaluated to provide more insight into the patterns of PDGFR-b activation. METHODS: Lung tissue specimens from five SScPAH, nine IPAH, six PVOD patients and five controls were examined. Immunoreactivity was scored for presence, distribution and intensity. RESULTS: All SScPAH and three of nine IPAH cases (P = 0.03) showed PDGFR-ß-immunoreactivity in small vessels (arterioles/venules); of five SScPAH vs. two of nine IPAH cases (P = 0.02) showed venous immunoreactivity. In small vessels, intensity was stronger in SScPAH vs. IPAH. No differences were found between SScPAH and PVOD. One of five normal controls demonstrated focally mild immunoreactivity. There were no differences in PDGF-ligand and pPDGFR-b-immunoreactivity between patient groups; however, pPDGFR-b-immunoreactivity tended to be more prevalent in SScPAH small vasculature compared to IPAH. Vascular EGFR-immunoreactivity was limited to arterial and arteriolar walls, without differences between groups. No immunoreactivity was observed in vasculature of normals. CONCLUSIONS: PDGFR-ß-immunoreactivity in SScPAH is more common and intense in small- and post-capillary vessels than in IPAH and does not differ from PVOD, fitting in with histomorphological distribution of vasculopathy. PDGFR-ß immunoreactivity pattern is not paralleled by pPDGFR-ß or PDGF-B patterns. PDGFR-ß- and EGFR-immunoreactivity of pulmonary vessels distinguishes PAH patients from controls.

Alterations in elastin and collagen related to the mechanism of progressive pulmonary venous obstruction in a piglet model. A hemodynamic, ultrastructural, and biochemical study.

We created an animal model to understand better the pathogenesis and underlying mechanism of progressive central pulmonary venous (PV) obstruction, a condition not amenable to current therapy. Twenty piglets underwent banding of their PVs, 18 had a sham operation, and 12 were nonoperated controls. After 1, 3, and 6 weeks hemodynamic data were obtained and correlated with ventricular weights, PV and pulmonary artery (PA) distensibilities (at 1 week), morphometric structural and ultrastructural analyses, and biochemical assessment of elastin determined gravimetrically (and by desmosine level at 1 week), collagen, and elastase activity. At 1 week, PV banding was associated with increased PV compliance (p less than 0.05). At 3 weeks, an increased PA pressure (Ppa) (p less than 0.05) was observed, unaccompanied by a rise in PV pressure (Pcw). In the PV, however, there was breakdown of the internal elastic lamina with apparent migration of smooth muscle cells from media to subendothelium. At 6 weeks, a rise in Pcw (p less than 0.01), a further rise in Ppa (p less than 0.01), and right ventricular hypertrophy (p less than 0.005) were observed. We also observed mild PV intimal thickening (p less than 0.01), complete degradation of elastic laminae (p less than 0.05), and an increase in collagen assessed morphometrically (p less than 0.01). The banding procedure resulted in an overall increase in PV elastin synthesis and in the proportion of elastin determined gravimetrically (p less than 0.05 for both) but not by desmosine level, suggesting the possibility of poor cross-linking of elastin, which might account for the early increased distensibility of the PV. However, our assay could not detect an increase in elastase activity associated with either the increased distensibility or the ultrastructural changes of elastin degradation. The increased Ppa was not associated with significant PA biochemical or structural changes. We speculate that in response to distal venous obstruction, early remodeling of the PVs increases distensibility, protecting the lung from venous congestion and blunting a rise in Pcw. PA hypertension precedes the rise in Pcw, likely because of reflex vasoconstriction. The subsequent modest rise in Pcw is already associated with extensive fibrosis of the PV, suggesting a reason for unsuccessful current therapy and a need for consideration of earlier assessment and intervention.