Aneurysm-type plexiform lesions form in supernumerary arteries in pulmonary arterial hypertension: potential therapeutic implications

Abstract

Histological observations in human pulmonary arterial hypertension (PAH) suggest a link between plexiform lesions and pulmonary supernumerary arteries. Pulmonary microvascular endothelial cells are characterized as hyperproliferative and progenitor-like. This study investigates the hypothesis that aneurysm-type plexiform lesions form in pulmonary supernumerary arteries because of their anatomical properties and endothelial characteristics similar to pulmonary microvascular endothelial cells. To induce PAH, rats were injected with Sugen5416, and exposed to hypoxia (10% O₂) for 3 days (early stage) or 3 wk (mid-stage), or 3 wk of hypoxia with an additional 10 wk of normoxia (late-stage PAH). We examined morphology of pulmonary vasculature and vascular remodeling in lung serial sections from PAH and normal rats. Aneurysm-type plexiform lesions formed in small side branches of pulmonary arteries with morphological characteristics similar to supernumerary arteries. Over the course of PAH development, the number of Ki67-positive cells increased in small pulmonary arteries, including supernumerary arteries, whereas the number stayed consistently low in large pulmonary arteries. The increase in Ki67-positive cells was delayed in supernumerary arteries compared with small pulmonary arteries. In late-stage PAH, ~90% of small unconventional side branches that were likely to be supernumerary arteries were nearly closed. These results support our hypothesis that supernumerary arteries are the predominant site for aneurysm-type plexiform lesions in Sugen5416/hypoxia/normoxia-exposed PAH rats partly because of the combination of their unique anatomical properties and the hyperproliferative potential of endothelial cells. We propose that the delayed and extensive occlusive lesion formation in supernumerary arteries could be a preventive therapeutic target in patients with PAH.

Keywords: cell proliferation; dilatation lesion; occlusive lesion; plexiform lesions; supernumerary arteries.

Fig. 1.

Examples of diameter measurements for parent and arterial branches. Red double-headed arrows/lines indicate the measured axes. A: cross-sectional parent arteries. B: longitudinally cut parent arteries. C: longitudinally cut branches without severe occlusive lesion. D: longitudinally cut branches with severe occlusive (plexiform) lesion with a continuously open part (red line). PA, parent artery. E: longitudinally cut branches with severe occlusive (plexiform) and dilatation (red line) lesions. Scale bars, 50 μm.

Fig. 2.

Examples of branching patterns observed in serial sections. A1–4: dichotomous type. B1–4: vertical type. C1–4: unclassifiable type. Images shown are serial sections. P, parent artery; black arrow, daughter branch; black arrowhead, plexiform lesion.

Fig. 3.

Representative photomicrographs of von Willebrand factor-stained aneurysm-type plexiform lesions in late-stage (13-wk time point) pulmonary arterial hypertension (PAH) rat lungs. A and B: typical mature lesions. C: small cellular lesion growing inside a thin-walled small branch artery with almost all plexiform cellular features except for the typical slit-like channel formation (plexiform-like lesion). C’: magnified area outlined in C. P, parent pulmonary artery; arrows indicate the complex lesion. Scale bars, 50 μm.

Fig. 4.

Representative photomicrographs of von Willebrand factor-stained different types of vessels connected distally to aneurysm-type plexiform lesions in the late-stage (13-wk time point) pulmonary arterial hypertension (PAH) rat lungs. A and B: very short types of distal vessels without (A) and with (B) a dilation lesion. C: a long type of distal vessel with veinlike structure. P, parent pulmonary artery; white arrows with black shade, complex lesion; white arrow heads with black shade, capillaries; red arrows and arrowheads, dilatation lesions connected and unconnected to the complex lesion, respectively; blue arrowheads, veinlike vessel. Scale bars, 50 μm.

Fig. 5.

Quantification of occlusion grades and types and branch patterns of pulmonary arterial hypertension (PAH) rat (n = 5) pulmonary arteries. A: quantification of occlusion grades in relation to branch types. B: occlusion types in relation to branch types. Grade 0 lesions were not included in this bar graph. D, dichotomous; V, vertical; U, unclassified.

Fig. 6.

Representative photomicrographs of two additional types of von Willebrand factor-stained occlusive lesions at the branching point of supernumerary arteries of the late-stage (13-wk time point) pulmonary arterial hypertension (PAH) rat lungs. A and B: serial sections of a minuscule type of occlusive lesion with longer veinlike distal branch [A1 and A2 and their respective high magnification of the branching point (A1’ and A2’)] and disappearing/collapsed branches [B1 and B2 and their respective high magnification of the branching point (B1’ and B2’)]. Note that there was accumulation of “core cell”-like cells at the branching points (red arrows). C: serial section of nonplexiform type of occlusive lesion [C1 and C2 and their respective high magnification of the branching point (C1’ and C2’)]. Note the contrast of thin veinlike (black arrowhead in A2’) and thickened walls of the branches (red arrowhead in C2’). Scale bars, 50 μm.

Fig. 7.

Top: time course of changes in percentages of Ki67-positive cells in innermost layer of large (Large, A) and small conventional pulmonary (Small, B) and supernumerary arteries (SA, C) at before (CON, black circle), 3 days after (red square), and 3 wk (red triangle) after Sugen5416/hypoxia-exposure. One-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05 vs. small CON, **vs. SA CON, ***vs. SA 3-day, #vs. large 3-day, §vs. large 3-wk, ¶vs. SA 3-day; n = 4–5. Each data point represents a vessel. Bottom: representative photomicrographs of Ki67-stained large (A1–A3) and small (B1–B3) pulmonary arteries, and supernumerary and its parent arteries (C1–C4 and C4’). Black arrow, supernumerary branch. Scale bars indicate 50 μm.