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Phenotypic abnormalities in microvascular endothelial cells isolated from the Sugen/Hypoxia rat
Author(s) -
Suresh Karthik,
Servinsky Laura,
Huetsch John,
Shimoda Larissa
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.696.3
Subject(s) - cd31 , hypoxia (environmental) , microbiology and biotechnology , endothelial stem cell , chemistry , calcium , mitochondrion , biology , biochemistry , oxygen , in vitro , organic chemistry
The mechanisms behind microvascular endothelial dysfunction in PAH are unclear, although mitochondrial dysfunction, increases in reactive oxygen species (ROS) and increased intracellular calcium concentration ([Ca 2+ ] i ) have all been implicated in endothelial cell samples from humans with PAH. To better understand the mechanisms behind endothelial dysfunction in PAH, we recently isolated lung microvascular endothelial cells (LMVEC) from normoxic rats (N‐LMVEC) and rats undergoing Sugen/Hypoxia (SuHx‐LMVEC), an experimental model of PAH. To induce PH, rats were injected with SU5416 (Sugen), a VEGFR inhibitor, and then placed in hypoxia for 3 weeks, followed by return to normoxia for 2 weeks. Following hemodynamic measurements, LMVEC were isolated from rats at 5 weeks using a magnetic bead approach, with anti‐CD31 and Griffonia simplicifolia (GS‐II) lectin conjugated beads. Calcium measurements were made in Fura 2‐AM loaded cells placed in a heated flow chamber perfused with Krebs buffer. Immunofluorescence was performed using primary and secondary antibodies and DAPI nuclear stain on rat LMVEC. To measure ROS, LMVEC were infected with a baculovirus vector containing a plasmid encoding roGFP, a ratiometric ROS dye. Cells were imaged 48 h after infection. Mitochondrial respiration was measured using the Seahorse metabolic analyzer system. N‐LMVEC rats appeared phenotypically similar to mouse and human LMVEC, while SuHx‐LMVEC appeared more spindle‐shaped. N‐ and SuHx‐LMVEC stained positive for GS‐II and CD31. Unlike N‐LMVEC, SuHx‐LMVEC additionally stained positive for smooth muscle actin and myosin heavy chain. Mitochondrial respiration was decreased in SuHx‐LMVEC compared to N‐LMVEC, whereas ROS, basal [Ca 2+ ] I , migration and proliferation were all increased. However, basal [Ca 2+ ] i , migration and proliferation in SuHx‐LMVEC were attenuated with treatment with TEMPOL, a ROS scavenger. We conclude that LMVEC isolated from SuHx rats exhibit metabolic and phenotypic differences from control cells that result from elevations in ROS, possibly due to mitochondrial dysfunction. Support or Funding Information F32HL124930