c-kit⁺ cells in the angioobliterative lesions were not mast cells.

<p>Representative optical section obtained by confocal microscopy demonstrates the presence of mast cells (c-kit⁺ mast cell tryptase⁺, arrows) around the airway mucosa of SU5416/chronic hypoxia (SuHx) animal at day 21 (positive control). In a naïve control lung, only one isolated c-kit⁺ mast cell tryptase⁻ cell was present in the pulmonary artery (open arrow). One cell with faint granular green autofluorescence (likely a macrophage) was present in the alveolar space (double-headed arrow). No mast cells were seen in the lumen-obliterating lesions of SuHx animals at day 21, only c-kit⁺ mast cell tryptase⁻ cells (open arrows). A single cell with faint granular green autofluorescence was seen in the alveolar space, likely a macrophage (double-headed arrow). Nuclear counterstaining: 4',6-diamidino-2-phenylindole (DAPI). Magnification: 630×. Scale bar: 20 µm.</p

Expression of CXC chemokine receptor 4 (CXCR4) and von Willebrand Factor (vWF) in c-kit⁺ cells.

<p>Representative optical sections obtained by confocal microscopy demonstrating the co-expression of CXCR4, vWF and c-kit in the pulmonary artery wall of a naïve animal and in pulmonary vascular lesion cells of animals with SU541/chronic hypoxia (SuHx) induced severe PAH. The inserts show the area outlined by a box in more detail. Arrows indicate triple positive cells in the inserts. Nuclear counterstaining: 4’-6-diamidino-2-phenylindole (DAPI). Magnification: 400×. Scale bar: 20 µm.</p

AMD3100 prevented severe pulmonary arterial hypertension (PAH) in the SU5416/chronic hypoxia (SuHx) model.

<p>(<b>A</b>) Representative von Willebrand Factor (vWF) immunohistochemistry indicates the occlusion of pulmonary arteries (arrows). These images demonstrate that treatment with the CXC chemokine receptor 4 inhibitor AMD3100 only partially prevented the obliteration of pulmonary arteries. Counterstaining: Mayer’s Hematoxylin. Magnification: 100×. Scale bar: 100 µm. (<b>B</b>) Reduced right ventricular systolic pressure (RVSP) and (<b>C</b>) decreased right ventricle (RV)/(left ventricle [LV]+Septum) ratio. (<b>D</b>) Reduced pulmonary arterial muscularization (external diameter [ED] <100 µm) was detected after AMD3100 treatment. (<b>E-F</b>) The degree of obliteration of pulmonary arteries in AMD3100-treated SuHx animals was partially reduced for small (E) (25 µmP<0.05, ** <i>P</i><0.01, *** <i>P</i><0.0001.</p

Proliferation of c-kit⁺ cells in the lungs of AMD3100-treated SU5416/chronic hypoxia (SuHx) animals.

<p>(<b>A</b>) Representative optical sections (confocal microscopy) demonstrate c-kit⁺ von Willebrand Factor⁺ (vWF⁺) proliferating cell nuclear antigen⁺ (PCNA⁺) cells (upper row) or c-kit⁺ α-smooth muscle actin⁺ (α-SMA⁺) PCNA⁺ cells (lower row) in pulmonary arteries of SuHx + vehicle and SuHx + AMD3100 treated animals. The inserts show a triple positive cell (arrow) in more detail. Please note that the bright white stained dots indicate cells with very strong PCNA staining. Nuclear counterstaining: 4',6-diamidino-2-phenylindole (DAPI). Magnification: 630×. Scale bar: 20 µm. (<b>B-C</b>) Quantification of the number of c-kit⁺, c-kit⁺ vWF⁺ and c-kit⁺ α-SMA⁺, as well as PCNA⁺, c-kit⁺ PCNA⁺, c-kit⁺ vWF⁺ PCNA⁺ and c-kit⁺ α-SMA⁺ PCNA⁺ cells in and around the pulmonary arteries of SuHx + vehicle and SuHx + AMD3100 animals. n = 3 animals per group. * <i>P</i><0.05 and ** <i>P</i><0.01.</p

Accumulation of c-kit⁺ cells in the SU5416/chronic hypoxia (SuHx) model.

<p>(<b>A</b>) Representative images of <i>in situ</i> hybridization images demonstrating <i>Kit</i> mRNA expression. In naïve control animals, occasional low <i>Kit</i> expression was found in cells of vessels and alveolar walls<b>,</b> whereas multiple cells in lumen-obliterating regions and vessel wall/perivascular region expressed <i>Kit</i> (granular staining pattern) in the lung of a SuHx animal. The open arrows indicate cells with strong <i>Kit</i> expression. Counterstaining with Gill’s Hematoxylin. Magnification: 400×. Scale bar: 20 µm. (<b>B</b>) Quantification of the number of c-kit⁺ cells/vessel over time. n = 3 animals per group. * <i>P</i><0.05, ** <i>P</i><0.01. (<b>C</b>) Images demonstrate representative optical sections (confocal microscopy). c-kit⁺, c-kit⁺ von Willebrand Factor⁺ (vWF⁺) and c-kit⁺ α-smooth muscle actin⁺ (α-SMA⁺) cells were occasionally found in alveolar walls and vessel walls of naïve control animals. c-kit⁺ and c-kit⁺ α-SMA⁺ cells accumulated in and around the pulmonary arteries of SuHx animals over time. The number of c-kit⁺ vWF⁺ cells also increased until day 21 in and around the pulmonary arteries of SuHx animals and started to decline thereafter. Arrows indicate c-kit⁺ vWF⁺ or c-kit⁺ α-SMA⁺ cells. Nuclear counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Magnification: 630×. Scale bar: 20 µm. (<b>D-E</b>) Quantification of the number of c-kit⁺ vWF⁺ (D) and c-kit⁺ α-SMA⁺ (E) cells per vessel. n = 3 animals per group. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.0001.</p

Effect of AMD3100 treatment on CXC chemokine receptor 4⁺ (CXCR4⁺) cells.

<p>Quantification of the number of total CXCR4⁺ cells (<b>A</b>), CXCR4⁺ von Willebrand Factor⁺ (vWF⁺) cells (<b>B</b>) and CXCR4⁺ α-smooth muscle actin⁺ (α-SMA⁺) cells (<b>C</b>) per vessel in pulmonary arteries of SuHx animals treated with vehicle or AMD3100. The data indicate that while the number of total CXCR4⁺ cells and CXCR4⁺ α-SMA⁺ cells per vessel was significantly reduced by AMD3100 treatment, there was only a small trend towards decreased number of CXCR4⁺ vWF⁺ cells per vessel in AMD3100 treated SuHx animals. n = 4 animals/group. * <i>P</i><0.05, *** <i>P</i><0.0001.</p