Cross-over study design.

<p>Healthy volunteers underwent phlebotomy in the antecubital fossa and whole blood (15 ml) was collected in the ethylenediaminetetraacetic acid (EDTA) coated tube. The volunteers were exposed to intermittent hypoxia or control air conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144725#sec002" target="_blank">Methods</a>) for 5 hours followed by whole blood collection. In one week, the volunteers underwent phlebotomy again and had an alternative exposure followed by whole blood collection. Peripheral blood mononuclear cells (PBMC, lymphocytes and monocytes) were isolated immediately after blood collection using Ficoll-Hypaque solution. RBC, red blood cells.</p

Intermittent hypoxia increased expression of toll-like receptor 2 in peripheral blood mononuclear cells.

<p>Expression of toll-like receptor two (TLR2) in peripheral blood mononuclear cells of healthy volunteers was measured during daytime exposure to intermittent hypoxia or control conditions for 5 hours and compared to baseline by real time PCR. The results are expressed as ratios to 18s. * denotes p < 0.05 for the difference between baseline and 5 hours data points.</p

Hypoxia accentuates LOX expression via HIF-1, leading to enhanced collagen cross-linking.

<p>(A) Hypoxic exposure increases expression of HIF-1α in the nuclear extract of wild-type mouse hepatocytes. This change is abolished in hepatocytes from <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice. (B) LOX mRNA expression increases in wild-type hepatocytes exposed to hypoxia, but this increase in LOX is not observed in <i>Hif1a</i>^<i>-/-</i><i>hep</i> hepatocytes. (C) When culture media from wild-type hepatocytes is added to collagen, hypoxia causes increased collagen cross-linking and precipitation; this difference is not seen in <i>Hif1a</i>^<i>-/-</i><i>hep</i> hepatocytes. *, p<0.05; ††, p<0.001.</p

Expression of genes involved in fibrosis, hepatic lipid metabolism, and inflammation/apoptosis.

<p>Expression of genes involved in fibrosis, hepatic lipid metabolism, and inflammation/apoptosis.</p

Liver histology and collagen quantification.

<p>(A) Representative liver H&E (top) and Masson’s trichrome stains (bottom) from wild-type (left) and <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice. More fibrosis can be observed in the wild-type mice in the Masson’s trichrome stain. (B) Sirius red stain of collagen in wild-type (left) and <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice (right). (C) Collagen content of all samples by use of hydroxyproline assay. *, p<0.05.</p

Liver hypoxic profile.

<p>(A) Wild-type (left) and <i>Hif1a</i>^<i>-/-</i><i>hep</i> (right) liver tissue was similarly hypoxic after six months on an HTFD, as qualitatively assessed by Hypoxyprobe stain. (B) As expected, HIF-1α protein levels were reduced in <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice. Some HIF-1 activation in <i>Hif1a</i>^<i>-/-</i><i>hep</i> mouse liver tissue likely remains due to incomplete gene knockout, and preserved HIF-1α in non-hepatocyte cell types. †, p<0.005.</p

Metabolic characteristics of wild-type and <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice.

<p>(A) Hepatocyte knockout of HIF-1α was confirmed by quantifying HIF-1α mRNA expression from the nuclear extract of isolated hepatocytes. (B) Body weight (top) and food intake (bottom) over experiment duration. (C) IPGTT (top) and ITT (bottom) at time of sacrifice. †, p<0.005.</p

Mouse Histologic Characteristics.

<p>Mouse Histologic Characteristics.</p

Extent of OSA recurrence as measured by AHI during CPAP withdrawal.

<p>(A) AHI across the night during alternating periods of therapeutic or atmospheric CPAP (*p<0.01 CPAP-on vs. CPAP-off). (B) Comparison of the baseline AHI, and the average AHI during CPAP-on and CPAP-off periods (*p<0.05 CPAP-on vs. CPAP-off; ¥p<0.001 vs. CPAP-on and CPAP-off).</p

Effects of CPAP depressurization on sleep architecture across the night.

<p>Overall, CPAP depressurization reduced sleep time, increased the proportion of time spent in stage wake and N1, and reduced the proportion of time spent in stage REM sleep (p<0.05 for each comparison). More specifically, sleep efficiency was reduced during the fourth period of CPAP depressurization, as shown by both reduction in sleep time and increase in %wake. There was also reduced REM during the third period of CPAP depressurization (*p<0.05) and a trend towards reduced N3 sleep during the first CPAP depressurization. Median, interquartile range (box), and min/max (whiskers) are shown in each panel.</p