Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Hypoxia-induced mitogenic factor (FIZZ1/RELMα) induces endothelial cell apoptosis and subsequent interleukin-4-dependent pulmonary hypertension

doi: 10.1152/ajplung.00279.2013

Figure Lengend Snippet: Human resistin (RETN) and IL-4 induce EC apoptosis and activation, and the conditioned media from activated ECs enhances human pulmonary artery smooth muscle cell (HPSMC) proliferation. A: both RETN (200 ng/ml, 6 h) and IL-4 (50 ng/ml, 6 h) caused TUNEL-positive reactions in human PMVECs (×630 magnification). B: quantification of mean fluorescence intensity of RETN- and IL-4-induced TUNEL-positive cells is shown as a ratio of DAPI staining [n = 8 low-magnification (×100) images from each group]. C: colocalization of RETN- or IL-4-induced Ang2 and vWF in PMVECs. Primary human PMVECs were treated with or without rhRETN (100 ng/ml) or rmIL-4 (50 ng/ml) for 24 h. Images are shown at ×630 magnification. D: quantification of mean fluorescence intensity of RETN- and IL-4-induced Ang2 expression is shown as a ratio of DAPI staining [n = 8 low-magnification (×200) images from each group]. E: PMVECs were treated with 100 ng/ml rhRETN or 50 ng/ml rhIL-4 for 24 h, and the conditioned medium was added to serum- and growth factor-starved HPSMCs for 24 h. Serum-free smooth muscle basal medium (SmBM) was used as a negative control. rhRETN (100 ng/ml) or rhIL-4 (50 ng/ml) was added directly to HPSMCs for comparison. F: PMVECs were treated with 100 ng/ml rhRETN for 24 h, and ET-1 level in the conditioned medium was measured by ELISA. Values are means ± SE (n = 4 separate culture cells/group, Student's t-test). Results are shown as means ± SE. ****P < 0.0001 compared with serum-free SmBM medium; ####P < 0.0001 compared with EC medium control (VEM-2 media with 1% FBS); n = 6/group. These experiments were repeated at least three times with similar results.

Article Snippet: Human pulmonary artery smooth muscle cells (HPSMCs; Lonza) were cultured in smooth muscle basal medium (SmBM; Lonza) supplemented with 5% FBS, human epidermal growth factor, human fibroblast growth factor-B, insulin, gentamicin, and amphotericin B (Bulletkit CC-4149; Lonza).

Techniques: Activation Assay, TUNEL Assay, Fluorescence, Staining, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Hypoxia-induced mitogenic factor (FIZZ1/RELMα) induces endothelial cell apoptosis and subsequent interleukin-4-dependent pulmonary hypertension

doi: 10.1152/ajplung.00279.2013

Figure Lengend Snippet: HIMF-induced pulmonary vascular remodeling is significantly suppressed in IL-4 KO mice. A: hematoxylin- and eosin-stained lung tissue from WT and IL-4 KO mice 30 days after iv injection with HIMF or vehicle. Scale bars: 100 μm. B: bar graph showing the percentage of small pulmonary arteries in WT and IL-4 KO mice that are nonmuscular (NM), partially muscular (PM), or fully muscular (FM). **P < 0.01 and ****P < 0.0001 vs. WT control; ##P < 0.01 and ####P < 0.0001 vs. WT HIMF. More than 500 vessels were counted in each group (3–5 animals/group). C: ELISA was used to measure IL-4 level in bronchoalveolar lavage fluid (BALF) from WT mice 30 days after treatment with or without HIMF. Values are means ± SE (n = 6/group, Student's t-test). D: systemic injection of WT mice with HIMF increased smooth muscle actin (SMA)-positive cells (identifying vascular media) and thrombomodulin-positive cells (identifying vascular intima). These increases were strongly suppressed in IL-4 KO mice. Scale bars: 50 μm (×1,000 magnification).

Article Snippet: Human pulmonary artery smooth muscle cells (HPSMCs; Lonza) were cultured in smooth muscle basal medium (SmBM; Lonza) supplemented with 5% FBS, human epidermal growth factor, human fibroblast growth factor-B, insulin, gentamicin, and amphotericin B (Bulletkit CC-4149; Lonza).

Techniques: Staining, IV Injection, Enzyme-linked Immunosorbent Assay, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Hypoxia-induced mitogenic factor (FIZZ1/RELMα) induces endothelial cell apoptosis and subsequent interleukin-4-dependent pulmonary hypertension

doi: 10.1152/ajplung.00279.2013

Figure Lengend Snippet: Proposed pathway by which HIMF mediates EC apoptosis, pulmonary vascular inflammation, and PH development. Schematic diagram showing our conceptualization of the pathway by which HIMF activates pulmonary EC apoptosis (IL-4 independent) and subsequent vascular inflammation that leads to PH development (IL-4 dependent). Augmented HIMF expression in the pulmonary vasculature causes EC apoptosis via the caspase 3-stress-activated kinase pathway and IL-4-dependent vascular inflammation in the early phase (day 7). These changes lead to subsequent PH (day 30). Human RETN, a human homolog of HIMF, also causes pulmonary EC apoptosis and promotes EC-mediated alteration of pulmonary vascular smooth muscle cells to a proliferative phenotype. Results from previous studies are shown in the dash-lined box (60, 62), and results from the current study are shown in solid-lined boxes.

Article Snippet: Human pulmonary artery smooth muscle cells (HPSMCs; Lonza) were cultured in smooth muscle basal medium (SmBM; Lonza) supplemented with 5% FBS, human epidermal growth factor, human fibroblast growth factor-B, insulin, gentamicin, and amphotericin B (Bulletkit CC-4149; Lonza).

Techniques: Expressing