Pulmonary Vascular Disease |

Hypoxia Modulates the Cellular Signaling in Cultured Rat Lung Microvascular Endothelial Cells FREE TO VIEW

Badri Giri; Shekhar Ghamande; David Zawieja; Mohammad Uddin
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Department of Pulmonary and Critical Care, Scott and White Hospital, Temple, TX

Chest. 2014;146(4_MeetingAbstracts):857A. doi:10.1378/chest.1990585
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SESSION TITLE: DVT/PE/Pulmonary Hypertension

SESSION TYPE: Original Investigation Slide

PRESENTED ON: Wednesday, October 29, 2014 at 07:30 AM - 08:30 AM

PURPOSE: The origin of several vascular pathologies including pulmonary hypertension involves pulmonary endothelial dysfunction. Acute and chronic oxidative stress to lung endothelial cells is a causative factor for endothelial dysfunction. The underlying mechanism of hypoxia induced lung endothelial cell dysfunction has not yet been determined. We hereby examined the cellular signaling following exposure to hypoxia in rat lung microvascular endothelial cells (RLMEC).

METHODS: The RLMEC were maintained at 37°C, 5% CO2, 10% O2 and 99% humidity. RLMEC were initially incubated in in hypoxia (2% O2) for 24 h and 48 h, hyperoxia (21% O2) and normal condition (10% O2). The hypoxic cells were then incubated in normal condition (10% O2) for 24 h and 48 h. The expression of pro-apoptotic proteins; Bcl-2-associated X protein (Bax), anti-apoptotic Bcl-2 protein and pro-inflammatory cyclooxygenase-2 (Cox-2) expression were assayed by western blot (WB). The expressions of endothelial Nitric Oxide Synthetase (eNOS), angiotensin II type 1 receptor (AT1), NFkappaB, and vascular endothelial growth factor receptor 1 (VEGFR1) and p38 MAPK phosphorylation were evaluated by WB. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test.

RESULTS: Hypoxia induced the up-regulation (p<0.05) of stress signaling (p38 MAPK,1.5 fold change; Cox-2, 1.4 fold change), apoptotic signaling (BAX/Bcl-2, 1.4 fold change), and vasoconstrictor receptors AT1 (1.3 fold change) in RLMEC compared to normoxia. However, the expression of eNOS (p<0.05) and NFkappaB (p<0.05) was down-regulated and VEGFR1 remains unchanged in hypoxic condition. The 48 h hypoxia induced higher expression of apoptotic and stress signaling proteins compared to 24 h. The cells that were exposed to normoxia for 24h following the 24h hypoxic condition showed a reversal of the signaling. However, those were not reversed by 24h or 48h exposure to normoxia after 48 h hypoxia. In contrast eNOS was found to be reversible to normal by 48 h exposure to normoxia after 48 h hypoxic condition.

CONCLUSIONS: Hypoxia induces the stress and apoptotic signaling in cultured rat lung microvascular endothelial cells, thus indicating the endothelial dysfunction. Hypoxia for certain duration seems to be reversible in vitro testing. However hypoxia of more prolonged duration produces irreversible changes in apoptotic and stress signaling.

CLINICAL IMPLICATIONS: This in vitro system can be used as a tool to study hypoxia-induced endothelial dysfunction.

DISCLOSURE: The following authors have nothing to disclose: Badri Giri, Shekhar Ghamande, David Zawieja, Mohammad Uddin

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