Greater CD34⁺CD133⁺ progenitors in PAH. (A-D) CD34⁺CD133⁺ progenitors in PAH determined by flow cytometry. Box plots indicate median values, upper and lower quartiles. There are increased hemangioblasts in PAH bone marrow, and higher levels of CD34⁺CD133⁺ progenitors in circulation and within the pulmonary artery endothelium than in controls. (E-F) STAT3 and STAT5 activation and localization in bone marrow biopsies. Cellular localization of phosphoSTAT3 (pSTAT3) by immunohistochemical staining in bone marrow biopsy (arrowheads) from PAH patient and healthy control (E). Immunohistochemical staining for phosphoSTAT5 (pSTAT5) in bone marrow biopsy (arrowheads) from PAH patient and healthy control (F). (G) Reticulin increase in bone marrow of PAH patients. Reticulin staining as a measure of myelofibrosis, was increased in PAH bone marrows. Arrowheads identify reticulin staining in bone marrow biopsies from PAH subject. The reticulin stain around a blood vessel is normally present, and is shown as internal positive in the control tissue. (H) EpoR expression by flow cytometry. Dashed lined histogram indicates background staining with isotype control antibodies, solid histogram indicates EpoR staining. CFU-EC have low levels of EpoR, while lymphocytes are shown as negative control have no EpoR. (I) STAT5 activation in CFU-EC. Electrophoretic mobility shift assay for STAT5 DNA binding activation in whole cell extract of CFU-EC from PAH patient (lane 4) and healthy control (lane 3) is shown. STAT5 DNA-binding activation is similar among PAH and control CFU-EC. A549 cells stimulated with Epidermal Growth Factor (EGF) is a positive control, and the supershift using antibody to STAT5 identifies the STAT5-DNA complex (arrow). Scale bars: 6 μm (E); 10 μm (F); 40 μm (G).