Histopathological Evaluation of Pulmonary Thromboendarterectomy Specimens of Chronic Thromboembolic Pulmonary Hypertension

Fig.1. Example of FFPE preparation and RNA sampling from PEA specimen and normalization of histological findings. Samples collected and cryopreserved from fresh PEA tissue were used for RNA analysis (red square), and FFPE was prepared at the same site to confirm the histological findings (blue square). Observable lesions in all FFPE samples were counted and normalized by dividing by the total number of slices. Pulmonary thromboendarterectomy: PEA; formalin-fixed and paraffin-embedded: FFPE

Fig.2. Histopathological findings of PEA tissue. a, c, d, e, f: HE stains, b: EVG stain Colander-like lesion (aggregation of the recanalized vascular lesion) (a–c), the early stage of organization (d), fresh thrombus (e), and atherosclerosis (f). In the low-power view of the colander-like lesion (a, b), an organizing thrombus containing recanalized vessels can be observed, surrounded by the preexisting pulmonary artery tunica media. The recanalized vessels are surrounded by SMCs (c). In the early stage of organization (d), fine recanalized vessels, fibroblasts, and macrophages can be found in the fibrous region bordering the fresh thrombus and intima. The fresh thrombus (e) consists mainly of erythrocytes, adhering to the intima without obvious organization. Atherosclerosis (f) is accompanied by lipid deposition and various degrees of macrophage aggregation. Pulmonary thromboendarterectomy: PEA; hematoxylin and eosin: HE; Elastica van Gieson: EVG; smooth muscle cell: SMC

Fig.3. Comparison of the PEA tissue histological findings between the good and poor clinical course groups. The good and poor clinical course groups included patients with a postoperative PVR＜500 and PVR ≥ 500 and cases of death, respectively. The total number of lesions in the specimen was normalized by dividing it by the total slice number. There were significantly more foci of colander-like lesions in the good clinical course group than in the poor clinical course group (a). No significant differences were found between the two groups in the early organization, fresh thrombi, and atherosclerosis (c, d, e). Mann–Whitney U test, comparison between groups, ＊＊P＜0.01 Pulmonary thromboendarterectomy: PEA; pulmonary vascular resistance: PVR

Fig.4. Immunohistochemical findings of the early phase of organization. HE (a), αSMA (b), h-caldesmon (c), S100A4 (d), smoothelin (e), ET-a (f), TRX (g), TXNIP (h), CD68 (i), and endothelin-1 (j). CD68-positive macrophages and αSMA-positive undifferentiated smooth muscle cells are distributed within the lesion. Undifferentiated smooth muscle cells show few cells positive for h-caldesmon and S100A4, whereas smoothelin and ET-a are both negative. TXNIP and TRX were expressed in the infiltrating macrophages, endothelial cells, and smooth muscle cells. Endothelin-1 was positive in neovascular endothelial cells, αSMA-positive undifferentiated smooth muscle cells, and macrophages present in the early stages of organization. Hematoxylin and eosin: HE; α smooth muscle actin: αSMA; endothelin receptor A: ET-a; thioredoxin: TRX; thioredoxin-interacting protein: TXNIP

Fig.5. Immunohistochemical findings in colander-like lesions. HE (a), αSMA (b), h-caldesmon (c), S100A4 (d), smoothelin (e), ET-a (f), TRX (g), TXNIP (h), CD68 (i), and endothelin-1 (j). The proliferated smooth muscle cells were transformed into well-differentiated smooth muscle cells with h-caldesmon, S100A4, smoothelin, positive for ET-a, and with contractile ability. With the thrombus organization, CD68-positive macrophages are rarely found. TXNIP and TRX were expressed in endothelial cells but tended to decrease in the differentiated smooth muscle cells (white arrowhead). Endothelin-1 was positive in the recanalized vascular endothelial cells and surrounding smooth muscle cells, but the positive finding was more significant in the recanalized vascular endothelium. Hematoxylin and eosin: HE; α smooth muscle actin: αSMA; endothelin receptor A: ET-a; thioredoxin: TRX; thioredoxin-interacting protein: TXNIP

Fig.6. Double immunofluorescence staining in the early phase of organization and colander-like lesions. The early phase of organization (a–d) and colander-like lesions (e–h) were shown. TRX shows a green signal (Alexa Fluor 488). Smoothelin (a, e), αSMA (b, f), CD31 (c, g), and CD68 (d, h) show a red signal (Alexa Fluor 568). All figures show merged images with nuclear staining (DAPI, blue signal). TRX localized in αSMA-positive undifferentiated smooth muscle cells (dashed line area), CD31-positive neovascular endothelial cells (white arrow), and CD68-positive macrophages (arrowhead) in the early phase of organization (b–d). Smoothelin-positive well-differentiated smooth muscle cells were not observed (a). TRX localization was observed in the perivascular αSMA-positive smooth muscle cells (dashed line area) and CD31-positive endothelial cells of the recanalized vessels (white arrow) but attenuated or absent TRX was observed in the smoothelin-positive smooth muscle cells (arrowhead) in colander-like lesions, (e–g). Macrophages were rarely observed (h). α smooth muscle actin: αSMA; thioredoxin: TRX; DAPI: 4′,6-diamidino-2-phenylindole

Fig.7. mRNA expression of endothelin-1, TXNIP, and TRX in the three groups: endothelin-1 (a), TXNIP (b), and TRX (c) intima in the atherosclerosis group, fresh thrombus/early organization group, and colander-like lesion group. Mann–Whitney U test, comparison between groups, ＊＊P＜0.01, ＊P＜0.05 thioredoxin-interacting protein: TXNIP; thioredoxin: TRX

Fig.8. Hypotheses on smooth muscle cell differentiation and oxidative and antioxidative mechanisms in thrombus organization. The antioxidant TRX, which is secreted from endothelial cells, and others in the early stage of organization are involved in the proliferation of smooth muscle cells. The oxidant TXNIP also increased accordingly. The proliferated smooth muscle cells were transformed into well-differentiated smooth muscle cells with smoothelin, positive for ET-a, and with contractile ability by the influence of blood pressure. thioredoxin: TRX; thioredoxin-interacting protein: TXNIP; endothelin receptor A: ET-a