Endothelial repair capacity and apoptosis are inversely related in obstructive sleep apnea

Abstract

Purpose:

To investigate the impact of obstructive sleep apnea (OSA) on endothelial repair capacity and apoptosis in the absence of potentially confounding factors including obesity.

Patients and methods:

Sixteen patients with a body mass index <30 and newly diagnosed OSA and 16 controls were studied. Circulating levels of endothelial progenitor cells, a marker of endothelial repair capacity, and endothelial microparticles, a marker of endothelial apoptosis, were quantified before and after four-week therapy with continuous positive airway pressure (CPAP). Endothelial cell apoptotic rate was also quantified in freshly harvested venous endothelial cells. Vascular reactivity was measured by flow-mediated dilation.

Results:

Before treatment, endothelial microparticle levels were greater and endothelial progenitor cell levels were lower in patients with OSA than in controls (P < 0.001 for both). Levels of endothelial microparticles and progenitors cells were inversely related (r = −0.67, P < 0.001). Endothelial progenitor cell levels increased after effective treatment (P = 0.036).

Conclusions:

In the absence of any co-morbid conditions including obesity, OSA alone impairs endothelial repair capacity and promotes endothelial apoptosis. These early endothelial alterations may underlie accelerated atherosclerosis and increased cardiovascular risk in OSA.

Keywords:

• sleep apnea
• endothelium
• apoptosis
• endothelial repair capacity

Data supplement

Methods

Immunofluorescence for endothelial cell apoptosis

Endothelial cells were permeabilized in PBS/0.5% Triton X-100. Polyclonal anti-von Willebrand factor antibodies (DAKO, Glostrup, Denmark) were used, followed by Texas red-conjugated secondary antibodies to identify endothelial cells. Nuclei were stained with diaminophenylindole (DAPI) (Molecular Probes, Carlsbad, CA). Apoptotic cell nuclei were identified using in situ apoptosis peroxidase detection kit, the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) according to the manufacturer’s instruction (Intergen). Nucleated endothelial cells were identified by red (von Willebrand factor staining) and blue fluorescence (DAPI DNA staining), and apoptosis was detected by green fluorescence (TUNEL). Apoptotic endothelial cells were detected by co-localized red and green fluorescence. Slides from study participants were stained concurrently with two slides of human umbilical venous endothelial cells (HUVEC) that served as positive and negative control. For negative controls, terminal deoxynucleotidyltransferase was omitted from the labeling mixture. For positive control, HUVEC slides were treated with DNase buffer for five minutes. Endothelial cells were analyzed with a fluorescent microscope under identical conditions (Nikon Eclipse E600, Melville, NY), and were captured by digital camera (Q Imaging Retiga EXi, Surrey, BC, Canada). The reader was blinded to subjects’ identity. Slides were systematically read left to right and top to bottom. The number of TUNEL-positive endothelial cells was scored in 10 randomly chosen high-power fields.

Flow cytometry for circulating endothelial progenitor cells

Twelve mL of venous blood was withdrawn from a forearm vein via the angiocath inserted for the endothelial harvesting. Mononuclear cells were isolated by density-gradient centrifugation with Ficoll (Sigma) and counted using a Coulter Counter (Beckman Coulter, Fullerton, CA) within one hour of blood sample collection. One million mononuclear cells were aliquoted and incubated with 15 μL mouse serum (Sigma) at room temperature to block nonspecific binding of antibodies. Mononuclear cells were incubated for 30 minutes in the dark with monoclonal antibodies against human kinase insert domain receptor (KDR) which was phycoerythrin (PE)-labeled (10 μl; R&D Systems, Minneapolis, MN), and for 10 minutes with CD34 (fluorescein isothiocyanate [FITC]-labeled) (20 μl; Becton Dickinson), and CD133 (APC-labeled) (20 μl; Miltenyi, Auburn, CA). EPC were defined as cells positive for monoclonal antibodies against human KDR, CD34, and CD133.^1–3 These bone-marrow-derived hematopoetic progenitor cells differentiate into mature endothelial cells and contribute to endothelial repair and new vessel formation after ischemic injury.^3–6 Isotype-identical antibodies IgG1-PE-FITC (Becton Dickinson) and IgG2b-APC (eBioscience, San Diego, CA) served as negative controls. Cell fluorescence was measured immediately after staining. FACSCalibur flow cytometer and CellQuest Software (Becton Dickinson) were used for data acquisition. Data were gated on the lymphocyte population, and 20,000 events were collected in the gated region for each sample.¹ The percent of KDR+/CD34+/CD133+ cells was expressed as the percent of the gated events.

Flow cytometry for circulating endothelial apoptotic microparticles

Blood was collected in citrate tubes and processed within one hour of collection. Plasma derived from 10 mL of blood was centrifuged at 1000g for six minutes to generate platelet-poor plasma. 50 μL of plasma was incubated with 4 μL of PE-labeled monoclonal antibody against CD31 (Becton Dickinson) and 4 μL of FITC-labeled CD42b (Becton Dickinson), then diluted with 1 mL of PBS. PE-labeled IgG1 and FITC-labeled IgG 2a (Pharmingen) served as a negative control. Particle detection was set to trigger by a fluorescent signal greater than background threshold. Data were gated using sizing of microparticles (forward light scatter) in the presence of calibrator beads. EMP were defined as particles ≤1.5 μm in size positive for monoclonal antibodies against CD31 and negative for CD42b.⁷ FACSCalibur flow cytometer (BD Biosciences) and CellQuest Software were used for data acquisition. Population of CD31+/CD42b-microparticles smaller than 1.5 μm is expressed as the number of EMP per μL of platelet poor plasma. Possible contamination with leukocytes microparticles (CD31+/CD45+) was assessed. CD31+/CD45+ particles accounted for a negligible percentage of all CD31+ microparticles in samples from both patients with OSA and controls.

Brachial artery flow-mediated dilation

Vascular response in the brachial artery was assessed by FMD according to the guidelines of the International Brachial Artery Reactivity Task Force.⁸ Brachial artery diameter was measured in the contralateral arm to the endothelial harvesting site. Subjects were evaluated in a quiet, temperature-controlled room. After a 30-minute rest in a supine position, the brachial artery diameter was measured 6 cm proximal to the antecubital fossa using a 7–15 MHz linear array transducer (Philips 5500, Andover, MA). Occlusion blood pressure cuff was placed over the proximal forearm just below the antecubital fossa. FMD was measured as the dilator response to reactive hyperemia induced by a five-minute blood pressure cuff occlusion of the upper arm. The cuff was inflated to 50 mm Hg above systolic blood pressure if the systolic blood pressure was greater than 150 mm Hg, or to 200 mm Hg if the systolic blood pressure was less than 150 mm Hg. Systolic blood pressure was lower than 150 mm Hg in all study participants. Brachial artery diameter (expressed in millimeters up to one decimal place) was measured at rest and during peak hyperemia for one minute after a five-minute occlusion of arterial flow. A blinded reader analyzed brachial artery diameters off-line using analysis software. The percent diameter change for FMD was calculated as follows: $\begin{array}{c}\text{FMD}\hspace{0.17em}(\%)\hspace{0.17em}=[(\text{brachial}\hspace{0.17em}\text{artery}\hspace{0.17em}\text{diameter}\hspace{0.17em}\text{at}\hspace{0.17em}\text{peak}\hspace{0.17em}\text{hyperemia}\\ -\text{diameter}\hspace{0.17em}\text{at}\hspace{0.17em}\text{rest})/\text{diameter}\hspace{0.17em}\text{at}\hspace{0.17em}\text{rest}]\times 100.\end{array}$

Figure S1 Representative histograms of the flow cytometry analysis for endothelial apoptotic microparticles (EMP) from a control subject (Figure 1A) and a patient with OSA (Figure 1B). Particles were gated based on their size (forward light scatter) in the presence of calibrator beads [region 2 (R2)] A). EMP were defined as particles ≤ 1.5 μm in size positive for monoclonal antibodies against CD31 and negative for CD42b B). Isotype-identical antibodies served as negative controls C).

Figure S2 Representative histograms of the flow cytometry analysis for endothelial progenitor cells (EPC) from a control subject (Figure 2A) and a patient with OSA (Figure 2B). Mononuclear lymphocytic cell population was first gated from a plot of forward vs side scatter width [region 1(R1)].A) Those cells were then gated for positive staining for kinase insert domain receptor (KDR) (R2). B) cells that stained positive for KDR were than gated for double positive staining for CD34 and CD133 (R3). C) The percent of KDR+/CD34+/CD133+ cells was expressed as the percent of the gated events, D) Isotype-identical antibodies IgG1-PE, E) and IgG1-FITC and IgG2b-APC F) served as negative controls. Reproducibility of the measurements was assessed by obtaining two separate blood samples from eight control subjects on days 0 and 28. The overall coefficient of variation between the two measurements was 10%.

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Disclosures

The authors were supported by grants from the Irving Center for Clinical Research RR-0645, and American Lung Association CU-52259701 (SJ). The authors report no conflicts of interest in this work.