Obstruction of Small Arterioles in Patients with Critical Limb Ischemia due to Partial Endothelial-to-Mesenchymal Transition

Abstract

Critical limb ischemia (CLI) is a hazardous manifestation of atherosclerosis and treatment failure is common. Abnormalities in the arterioles might underlie this failure but the cellular pathobiology of microvessels in CLI is poorly understood. We analyzed 349 intramuscular arterioles in lower limb specimens from individuals with and without CLI. Arteriolar densities were 1.8-fold higher in CLI muscles. However, 33% of small (<20 μm) arterioles were stenotic and 9% were completely occluded. The lumens were closed by bulky, re-oriented endothelial cells expressing abundant N-cadherin that uniquely localized between adjacent and opposing endothelial cells. S100A4 and SNAIL1 were also expressed, supporting an endothelial-to-mesenchymal transition. SMAD2/3 was activated in occlusive endothelial cells and TGFβ1 was increased in the adjacent mural cells. These findings identify a microvascular closure process based on mesenchymal transitions in a hyper-TGFß environment that may, in part, explain the limited success of peripheral artery revascularization procedures.

Keywords: Clinical Finding; Human Specimen; Pathophysiology.

Graphical abstract

Figure 1

Histopathology of Human Skeletal Muscle in Patients with CLI (A–C) H&E-stained sections of non-PAD muscle (A) and CLI muscles (B and C). CLI muscle features include a triangular, shrunken myofiber (asterix, B), interstitial inflammation (arrow, B), a myofiber with an internal nucleus (arrowhead, B), myofiber degeneration and necrosis (asterix, C), and intra-myofiber inflammatory cell infiltration (arrow, C). (D–F) Mason's-trichrome-stained sections of non-PAD muscle (D) and CLI muscles (E and F), the latter showing interstitial accumulation of collagen-containing extracellular matrix (E) and myofibers replaced by collagen (F). (G–I) Images of picrosirius red-stained sections of non-PAD muscle (G) and CLI muscles (H and I) imaged with circularly polarized light and color mapped based on light retardation. Thin, weakly birefringent collagen fibrils can be seen surrounding non-PAD myofibers (G). CLI muscles display accumulation of interstitial collagen fibrils (H) and regions of scarring with replacement fibrosis (I). The light retardation color map is shown, and all images are captured and level adjusted with identical settings. See also Figure S1 and Table S2.

Figure 2

Capillary and Arteriole Density in Skeletal Muscle of Patients with CLI (A) Fluorescence micrographs of human skeletal muscle immunostained for CD31 (red) showing capillaries (arrows) between myofibers (dotted lines) in non-PAD (left) and CLI (right) muscles. Nuclei were counterstained with DAPI (blue). (B) Fluorescence micrographs of human skeletal muscle double immunostained for CD31 (red) and SM α-actin (green) showing arterioles in non-PAD and CLI muscles. (C) Graph depicting capillary densities in skeletal muscles of non-PAD subjects (n = 6) and CLI subjects (n = 10). (D) Graph depicting arteriole density in skeletal muscle from non-PAD and CLI subjects. Means ± standard deviations are shown.

Figure 3

Arteriolar Lumen and Endothelial Cell Morphometry in CLI Muscles (A) Confocal microscope images of pre-terminal (∼25 μm diameter) and terminal (∼10 μm) arterioles in human skeletal muscle immunostained for CD31 (red) and SM α-actin (green). Nuclei were counterstained with DAPI (blue). The lumen of CLI arterioles can be open with a flattened endothelial monolayer, narrowed by thickened and re-oriented endothelial cells, or entirely occluded by swollen endothelial cells with bulky nuclei. (B) Lumen area of small arterioles (circumference at the outer endothelial cell border <60 μm) from CLI subjects (n = 10, total 314 arterioles measured) and non-PAD subjects (n = 6, total 35 arterioles). Lumen areas are averaged per subject. (C) Mean lumen area of small arterioles from CLI and non-PAD subjects binned based on vessel circumference. Overall p < 0.0001; post-hoc p = 0.035, 0.037, 0.226, and 0.038, respectively. (D) Graph depicting fractional endothelial cell area of arterioles in non-PAD and CLI subjects. (E) Nuclear aspect ratio (width-to-height) of endothelial cells of small arterioles in non-PAD and CLI subjects. A total of 119 and 1,450 nuclei were measured, respectively, and the data are presented as average per subject. Shapes depict the nuclei with mean aspect ratios. Means ± standard deviations are shown. See also Figures S2–S4.

Figure 4

Reconfigured N-cadherin in Endothelial Cells of Arterioles in CLI Muscle (A) Confocal micrographs of human skeletal muscle arterioles immunostained for CD31 (red) and N-cadherin (green), with nuclei counterstained with DAPI (blue). Top row shows diffuse endothelial cell N-cadherin signal in a non-PAD arteriole. Middle row shows an arteriole that is occluded by bulky, pyramidal-shaped endothelial cells, with enriched N-cadherin signal at junctions between adjacent and opposing endothelial cells (arrows). Bottom row shows an arteriole that is substantially narrowed by columnar endothelial cells, with enriched N-cadherin signal between adjacent endothelial cells (arrow) and also at the apical cell surface (arrowhead). (B) Graph depicting N-cadherin signal intensity in arteriolar endothelium in muscles from non-PAD and CLI patients. Pooled data are represented as mean ± standard deviation. (C) N-cadherin signals in endothelium of CLI arterioles with open lumens and CLI arterioles with narrowed or fully occluded lumens. Data from open-lumen and narrowed/occluded-lumen arterioles from a given patient are denoted by the adjoining lines.

Figure 5

Mesenchymal Markers S100A4 and SNAIL1 in Endothelial Cells of Stenotic Arterioles in CLI Muscle (A) Confocal micrographs of arterioles in non-PAD and CLI muscle immunostained for CD31 (red) and S100A4 (green), showing cytoplasmic S100A4 signal in endothelial cells of a CLI arteriole with a narrowed lumen (arrow). (B) Confocal micrographs showing punctate SNAIL1 signal (green) in the nuclei of endothelial cells of a CLI arteriole with a narrowed lumen (arrow). (C) Graph depicting the proportion of endothelial S100A4-positive arterioles in muscles from non-PAD and CLI patients (mean ± standard deviation). (D) Proportion of endothelial S100A4-positive CLI arterioles with open and narrowed/occluded lumens. Data from open-lumen and narrowed/occluded-lumen arterioles from a given patient are denoted by the adjoining lines. (E) Proportion of SNAIL1-positive endothelial cells in arterioles in non-PAD and CLI muscle samples (median ± interquartile range). (F) Proportion of endothelial SNAIL1-positive CLI arterioles with open and narrowed/occluded lumens (F). Data from open-lumen and narrowed/occluded-lumen arterioles from a given patient are denoted by the adjoining lines.

Figure 6

Hyperactive TGFβ Signaling in Occluded Arterioles (A) Confocal micrographs of arterioles in non-PAD and CLI muscle immunostained for CD31 (red) and pSMAD2/3 (green) showing discrete punctate signals of phosphorylated SMAD2/3 (pSMAD2/3) in the nucleus of endothelial cells within the CLI arteriole (arrow). Nuclei were counterstained with DAPI (blue). (B) Confocal micrographs of arterioles in non-PAD and CLI muscle immunostained for CD31 (red), SM α-actin (green), and TGFβ1 (white). Nuclei were counterstained with DAPI (blue). TGFβ1 signal was predominantly observed in SMCs of the CLI arteriole (arrows). (C) Proportion of endothelial pSMAD2/3-positive arterioles in muscle samples from non-PAD and CLI subjects (mean ± standard deviation). (D) Proportion of endothelial pSMAD2/3-positive CLI muscle arterioles with either open or narrowed/occluded lumens. (E) Graph depicting TGFβ1 content (median ± interquartile range) in arterioles in non-PAD and CLI subjects. (F) TGFβ1 content in skeletal muscle arterioles with either open or narrowed/occluded lumens. For D and F, arteriole data from a given patient are depicted by the adjoining lines. A.U., arbitrary units of integrated fluorescence intensity.