Hyperbaric oxygen induces a cytoprotective and angiogenic response in human microvascular endothelial cells

Abstract

A genome-wide microarray analysis of gene expression was carried out on human microvascular endothelial cells (HMEC-1) exposed to hyperbaric oxygen treatment (HBOT) under conditions that approximated clinical settings. Highly up-regulated genes included immediate early transcription factors (FOS, FOSB, and JUNB) and metallothioneins. Six molecular chaperones were also up-regulated immediately following HBOT, and all of these have been implicated in protein damage control. Pathway analysis programs identified the Nrf-2-mediated oxidative stress response as one of the primary responders to HBOT. Several of the microarray changes in the Nrf2 pathway and a molecular chaperone were validated using quantitative PCR. For all of the genes tested (Nrf2, HMOX1, HSPA1A, M1A, ACTC1, and FOS), HBOT elicited large responses, whereas changes were minimal following treatment with 100% O(2) in the absence of elevated pressure. The increased expression of immediate early and cytoprotective genes corresponded with an HBOT-induced increase in cell proliferation and oxidative stress resistance. In addition, HBOT treatment enhanced endothelial tube formation on Matrigel plates, with particularly dramatic effects observed following two daily HBO treatments. Understanding how HBOT influences gene expression changes in endothelial cells may be beneficial for improving current HBOT-based wound-healing protocols. These data also point to other potential HBOT applications where stimulating protection and repair of the endothelium would be beneficial, such as patient preconditioning prior to major surgery.

Figures

Fig. 1

Scatterplots of normalized microarray data. These plots show the pairwise comparison of all 12 samples. Graphs represent the comparison of the normalized intensity data for every probe represented on the array between any two samples. The biological triplicates exhibit very tight correlations serving as a quality control mechanism. Comparisons between HBOT-treated and HBOT-untreated samples show increased and decreased gene expression

Fig. 2

Microarray data analysis. a Differentially regulated genes were selected based on a significant difference in at least one treatment level compared to control. Following statistical analysis, a total of 8,101 (21%) significantly regulated genes were identified. Of that list, about 695 increased and 901 decreased immediately following HBOT, whereas 3,280 increased and 3,968 decreased following the 24-h recovery. b Genes regulated ±5-fold were selected as top-responding genes. White bars indicate up-regulated genes; gray bars indicate down-regulation

Fig. 3

HBOT affects gene expression in the Nrf2 Pathway. Ingenuity Pathway Analysis predicted the Nrf2-mediated oxidative stress response pathway as having significantly changed gene expression for both time points. Molecules are divided into right (HBOT-0 h) and left (HBOT-24 h) halves. Red color indicates up-regulation, green indicates down-regulation, and white indicates no change in comparison to control

Fig. 4

Validation of microarray data. HMEC-1 cells were treated with either HBOT or 100% O2 and RNA extracted at indicated times following treatment. RNA was converted to cDNA and subjected to qPCR for the indicated genes. Genes were selected from pathways of interest including the Nrf2 signaling pathway, cytoprotective genes, and the top-responding genes. HMOX1 heme-oxygenase 1, HSPA1A human heat shock protein 70, Mt1A metallothionein 1 A, ACTC1 actin, alpha cardiac muscle 1, FOS FBJ murine osteosarcoma viral oncogene homolog

Fig. 5

Functional effects of HBOT and 100% O2 on HMEC-1 cells. Cells were plated and incubated for 48 h before being subjected to one round of HBOT or 100% O2. Following recovery for 16 h, cell proliferation was assessed with the MTT assay. a HBOT protected against oxidative stress. Following recovery from HBOT or 100% O2, cells were treated with t-butylhydroperoxide (t-butyl OOH) for 4 h and then analyzed for cell viability using the MTT assay. b HBOT increased cell viability. c HBOT increased vascular tube formation. HMEC-1 cells received either one (1X) or two (2X) treatments separated by a 24-h recovery period of HBOT and were immediately plated on Matrigel coated plates. Representative images are shown for control samples (c1X and C 2X) as well as HBOT cells treated once or twice (HBOT 1X and HBOT 2X)