Pathways of Microcirculatory Endothelial Dysfunction in Obstructive Sleep Apnea: A Comprehensive Ex Vivo Evaluation in Human Tissue

Abstract

Background

The mechanism and markers of cardiovascular disease (CVD) in obstructive sleep apnea (OSA) remain unknown. The microcirculation is the site of early changes in OSA patients who are free of CVD risk.

Methods

Patients with newly diagnosed moderate to severe OSA (n = 7) were studied before and 12 weeks after intensive treatment with continuous positive airway pressure (CPAP), along with weight and age matched controls (n = 7). Microcirculatory vessels were isolated from gluteal biopsies and changes in critical functional genes were measured.

Results

The following genes changed after 12 weeks of intensive CPAP therapy in the microcirculatory vessels: angiotensin receptor type 1 (AGTR-1) (11.6 (3.4) to 6 (0.8); P = 0.019); NADPH oxidase (NOX4) (0.85 (0.02) to 0.79 (0.11); P = 0.016); and dimethylarginine dimethylaminohydrolase (DDAH 1) (1 (0.31) to 0.55 (0.1); P = 0.028). Despite decreased nitric oxide (NO) availability as measured indirectly through brachial artery flow-mediated dilation, endothelial NO synthase (NOS3) did not change with CPAP. Other disease markers of OSA that changed with treatment in the microcirculation were endothelin, hypoxia inducible factor 1a, nuclear factor kappa B, interleukin-8, and interleukin-6.

Conclusions

In this ex vivo evaluation of the microcirculation of patients with OSA and no CVD risk, several pathways of CVD were activated supporting that OSA independently induces microcirculatory endothelial dysfunction and serving as disease-specific markers for future pharmacological targeting of OSA-related CVD risk. The findings support the role of renin–angiotensin activation and endothelial oxidative stress in the decreased microcirculatory NO availability in OSA.

Obstructive sleep apnea (OSA) is present in up to 25% of all middle-aged individuals in the United States.¹ OSA has a causal relationship with hypertension^2–4 and is independently associated with the progression or poor outcome of other cardiovascular disorders including arrhythmia, heart failure, stroke, and coronary artery disease.^5–10 Previously, several studies have supported that the standard therapy of OSA such as continuous positive airway pressure (CPAP) can reverse the immediate cardiovascular effects of OSA such as hypertension, and insulin resistance.^11–13 Recently, however, long-awaited randomized controlled trials evaluating the effect of treating OSA with CPAP on cardiovascular disease (CVD) demonstrated no significant effect on important CVD outcomes including mortality,^14–16 calling into question the role of OSA as a CVD risk factor¹⁷ and/or the role of CPAP in reversing OSA-related CVD.¹⁸ Important questions that remain largely unaddressed include the pathways of OSA-induced CVD; the subpopulations of OSA patients at risk for CVD complications; and the effective therapeutic approaches to reversing CVD risk in OSA.

Microcirculatory vascular endothelial dysfunction (VED) is critical for the pathogenesis of hypertension.^19,20 Therefore, the microcirculation would be an important site for studying the early vascular consequences of OSA. We previously employed an experimental approach in which we study microcirculatory endothelial tissue from younger adults with OSA who are free of CVD risk before and after a short period (12 weeks) of enhanced CPAP therapy (exceeding 70% of sleep time). Using this approach, we found evidence of microcirculatory VED in normotensive patients with OSA).^21–23 We specifically found that decreased nitric oxide (NO) availability in OSA is due to oxidative inhibition in endothelial nitric oxide synthase (eNOS).²¹ However, the pathways involved in increased oxidant overproduction in the microcirculatory endothelium in OSA remained unknown. Furthermore, there are minimal to no established OSA disease-specific markers of CVD risk that can serve as surrogates for mechanistic or therapeutic studies in humans. Therefore, we sought to identify markers of early microcirculatory dysfunction that are specific to OSA and can assist in profiling OSA patients, serve as surrogate targets in clinical trials, and provide molecular signature for in vitro models.

METHODS

Our experimental approach includes the evaluation of relatively younger adults with newly diagnosed OSA who are free of CVD risk factors. We provide intensive CPAP adherence program to achieve ≥5 hours of CPAP use during the 12-week period treatment. Our research design assumes that the changes in microcirculation with short term (12 weeks) of intensive CPAP therapy (>5 hours/night) are an indication of resolution of the OSA effects on the microcirculation. We still compare the change to weight and age matched control for added precision. This rationale and selected duration of follow-up is supported by several previous studies.^22,24,25

Participants

Patients with OSA

Newly diagnosed patients were recruited from an academic Sleep center after their diagnostic polysomnography and prior to the initiation of CPAP therapy. Only patients with an apnea–hypopnea index >15 events/hour of sleep were included. Very low cardiovascular risk status (≤2%) was required in all participants using the published Framingham Heart Study 10-year cardiovascular event calculators.^26,27 Therefore, the exclusion criteria included any diagnosis or ongoing treatment of hypertension, hypercholesterolemia (total cholesterol >200 mg/dl regardless of age), a diagnosis of diabetes, or any current or history of smoking. None of the participants were on any prescribed medications; and any supplements were discontinued at least a week prior to participation. The purpose of the restrictive inclusion criteria is to ensure that the microcirculatory dysfunction in participants is due to OSA only and not to other subclinical CVD risk factors.

Non-OSA controls

We enrolled healthy participants with no OSA to serve as controls. The same inclusion and exclusion criteria were used in selecting controls. Specifically, the same Framingham risk score was required. Attempts were made to frequency-match OSA patients with similar sex, weight, and age controls. All non-OSA participants underwent either a polysomnography or home sleep test to rule out OSA.

Procedures

OSA patients were asked to provide a baseline visit within 2 weeks of initial OSA diagnosis, prior to starting CPAP; and a second conclusion visit after 12 weeks of CPAP therapy. Participants with OSA received an adherence intervention with regular reminder calls and their usage of CPAP was verified with device download during the conclusion visit. Patients who used CPAP more than 5 hours/night proceeded to complete the second visit with the biopsy and endothelial reactivity studies. Non-OSA volunteers underwent both procedures once.

Gluteal subcutaneous biopsy and vessel isolation

Incisional skin biopsy techniques were used to obtain 1–2 cm³ of subcutaneous tissue from the lateral superior gluteal region, as described in a previous publication.²⁸ The tissue from the subcutaneous biopsy was immediately dissected on ice using a dissecting Leica microscope and microsurgical tools to isolate microcirculatory vessels (50–300 µm), which were placed in an Eppendorf tube, immediately frozen in liquid nitrogen, and then kept at −80 °C prior to RNA isolation.

Endothelial reactivity studies

Doppler ultrasound was used to measure flow-mediated dilation (FMD) of the brachial artery. Image acquisition was done with a linear array transducer (7 MHz frequency) and color spectral Doppler (GE Vivid 7). FMD measurements were performed according to published guidelines²⁹ and our previously reported studies.^21,22 Briefly, participants underwent the measurement between 7 and 9 AM prior to the biopsy procedure in a climate-controlled room. All participants were instructed to abstain from caffeine for at least 12 hours prior to the measurements. None of the participants used tobacco, and all discontinued any supplements at least 1 week prior to participation.

Peripheral blood mononuclear cells isolation

Blood was collected in 5 ml heparinized tubes (green top) and spun down at 1,200 g for 10 minutes. Plasma was collected, aliquoted, and stored at −80 °C for cytokine analysis. After plasma collection, equal volume of Ca/Mg free phosphate-buffered saline was added to the blood cells, and peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation at 600 × g for 20 minutes at room temperature. The mononuclear layer was removed and washed twice in RPMI 1640 (BioWhittaker, Walkersville, MD). The cells were either immediately lysed for RNA and protein isolation or placed in a 12-well plate (3 × 10⁶) in RPMI 1640 supplemented with 10% fetal bovine serum (Atlas Biological). The cells were resolved in Trizol reagent (Invitrogen, Live Technologies, Carlsbad, CA) immediately after isolation, and RNA extraction was done according to the manufacturer’s protocol.

RNA isolation and gene studies in the microcirculatory vessels

0.5 ml of Trizol reagent (Invitrogen, Live Technologies, Carlsbad, CA) was added to the tube with the isolated microvessels. Later, the frozen vessels were homogenized in TissueLyser LT (Qiagen) with a prechilled adaptor and mix of beads for 1 minute. The tissue lysate was then collected and 0.5 ml of fresh Trizol was added, and the homogenization step was repeated. Lysates from 2 homogenization steps were combined, and RNA extraction was continued according to the manufacturer’s protocol. After isolation, 1–2 µg of total RNA was reverse transcribed to cDNA by ThermoScript RNase H-Reverse Transcriptase (Invitrogen Life Technologies) and then diluted to 60 µl by RNAse/DNAse free water (ThermoFisher Scientific). The converted cDNA (20–60 ng) was used for quantitative reverse transcriptase polymerase chain reaction (rtPCR) with SYBR Green PCR Master Mix in the StepOne Plus system (Applied Biosystems, Foster City, CA).

Ethics statement

The protocol was approved by the OSU Institutional Review Board (protocol number 2009H0212). The study was registered in the National Clinical Trials database (NCT01027078).

Analysis

Patient characteristics are reported for OSA patients and non-OSA controls as mean and SD for continuous variables and count and percentage for sex. These characteristics were compared between groups using Wilcoxon rank-sum tests for continuous variables. FMD was compared pre-CPAP vs. post-CPAP within patient using paired t-tests.

FMD was measured according to published guidelines and our previously published studies.^21,22,28 Vessel images were obtained at baseline and at 60 seconds postrelease of occlusion. The images were immediately deidentified with dates removed to be measured later by a blinded vascular imaging technologist.

We used relative quantification to evaluate the expression of selected gene markers linked to endothelial cells function. All primers were designed with the following similar criteria: to give an 80- to 200-nt amplicon, GC range of 30%–70%, melting temperature of 59–60 °C, free of secondary structure, and a free-energy change (ΔG) of less than or equal to −5 kcal/mol. Once selected, primer pairs were validated by PCR, melting curve and high-resolution gel electrophoresis to have a single band of the desired size that were free of primer dimers. For the selected genes, PCR products were sequenced. After optimization, only primer pairs that satisfied our criteria were used. Amplification of genomic DNA was used to verify that the PCR conditions did not amplify any contaminating genomic DNA. Gene expression was calculated in relative copy numbers (RCNs) with the following equation:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{R}\mathrm{C}\mathrm{N}=E^{-\mathrm{\Delta }\mathrm{C}\mathrm{t}}\times 100,}\end{array}}$$

where E is the efficiency of PCR, and ΔCt (cycle threshold) is the difference (Ct target − Ct reference). Ct reference is the average of 2 housekeeping genes, GAPDH and CAP-1. PCR efficiency was calculated by the equation $E={10}^{(-\frac{1}{\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}})}$ as previously reported.^30,31

RESULTS

Participant characteristics

Table 1 includes details of patients and control participant characteristics. No significant differences were present at baseline. All participants had a Framingham risk score less than 2%. One patient with OSA provided partial data on the second visit with only FMD but did not wish to proceed with the repeat biopsy. Data from this participant are reported in Table 1 since the participant was part of the BMI and age matching process with the controls. The changes with treatment in the OSA group are presented in Table 2. Notably, significant change in FMD occurred with CPAP in the OSA group.

Table 1.

Characteristics of participants

Characteristics, mean (SD)	Non-OSA (n = 7)	OSA (n = 7)	P valuea
Age (y)	37 (9.6)	40 (10.7)	0.22
Sex (male)	3	4
Race (Black)	2	1
BMI (kg/m2)	37.2 (10.5)	39.2 (7.4)	0.48
SBP (mm Hg)	121 (8.9)	127 (7.6)	0.27
DBP (mm Hg)	78 (3.2)	86 (6.4)	0.03
ESS	7.7 (3.3)	10 (3.1)	0.79
AHI (events/h)	2.2 (1.7)	37 (20.6)	0.003
Desaturation index (4%) (events/h)	2.7 (1.5)	32 (17.4)	0.006
TST below 90% (%)	0.5 (0.75)	16.6 (7.7)	0.01
FMD (%)	8.8 (9.1)	4 (3.9)	0.094

Abbreviations: AHI, apnea–hypopnea index; BMI, body mass index; DBP, diastolic blood pressure; desaturation index (4%) is the number of decreases by 4% or greater in the oxygen saturation per hour of sleep; ESS, Epworth sleepiness scale; FMD: flow-mediated dilation in the brachial artery; OSA, obstructive sleep apnea; SBP, systolic blood pressure; TST below 90%: is the percent of total sleep time spent with oxygen saturation below 90%. All participants had a Framingham risk score <5%.

^aWilcoxon rank-sum tests except for sex and race.

Table 2.

Cardiovascular and respiratory changes in participants with OSA

Mean (SD)	Pre (n = 6)	Post (n = 6)	P value
Treatment AHI (events/h)	37 (10.5)	4.4 (3.4)	0.003
CPAP average nightly usage (h/night)	5.85 (3)
SBP (mm Hg)	124 (10.9)	121.6 (11.4)	0.337
DBP (mm Hg)	80 (5.3)	74 (7.8)	0.067
HR (beats/min)	83 (16)	76 (7.1)	0.14
FMD (%)	4 (3.9)	9.0 (1.8)	0.013

Abbreviations: AHI, apnea–hypopnea index; CPAP, continuous positive airway pressure; DBP, diastolic blood pressure; ESS, Epworth sleepiness scale; FMD, flow-mediated dilation in the brachial artery; HR, heart rate; SBP, systolic blood pressure.

Impact of OSA on the microcirculation

The genes measured in this study were selected to assay established or highly suspected critical pathways of microcirculatory endothelial function based on previous studies done in OSA patients. Comparisons between pre- and post-CPAP as well as between OSA and non-OSA participants are provided. The genes are grouped by function in Table 3 and discussed further below.

Table 3.

Changes in microcirculatory endothelial genes

Gene (relative copy number)	Non-OSA (n = 7)	OSA pre-CPAP (n = 6)	OSA post-CPAP (n = 6)	P value OSA pre-CPAP vs. OSA post-CPAP	P value non-OSA vs. OSA pre-CPAP
EDN1 (endothelin 1)	4.24 (0.68)	10.07 (1.80)	5.38 (0.811)	0.047	0.021
EDNRB	3.12 (2.12)	6.30 (1.05)	2.68 (0.22)	0.024	0.06
CHD5	5.05 (1.16)	15.90 (4.65)	4.80 (1.6)	0.085	0.08
PECAM1	25.56 (5.6)	34.48 (7.22)	11.84 (1.17)	0.032	0.25
AGTR1	11.61 (3.42)	18.86 (3.90)	6.07 (0.78)	0.019	0.13
TGFB1	6.23 (1.61)	8.94 (1.29)	6.40 (2.74)	0.206	0.17
NOS3 (eNOS)	7.23 (1.25)	10.81 (1.97)	7.66 (2.58)	0.254	0.146
DDAH1	1.0 (0.31)	1.44 (0.33)	0.55 (0.1)	0.028	0.199
DDAH2	83.03 (3.69)	64.63 (3.58)	72.26 (5.91)	0.222	0.014
ARG1	0.06 (0.02)	0.49 (0.18)	0.39 (0.16)	0.356	0.044
HIF1A	4.91 (0.81)	7.30 (1.05)	3.22 (0.484)	0.016	0.124
SOD1	215.93 (25)	234.1 (17.4)	197.4 (11.2)	0.094	0.316
NOX4	0.85 (0.19)	1.81 (0.30)	0.79 (0.11)	0.016	0.024
NFKB1 (NFκB p50)	4.43 (0.88)	4.50 (0.90)	1.20 (0.20)	0.013	0.482
RELA (NFκB p65)	2.89 (0.59)	5.24 (0.94)	2.27 (0.345)	0.049	0.093
CCL2 (MCP-1)	19.84 (5.99)	5.69 (1.11)	14.75 (1.77)	0.006	0.060
IL8	0.57 (0.18)	1.09 (0.27)	0.13 (0.001)	0.0033	0.153
IL6	0.33 (0.11)	0.36 (0.08)	0.07 (0.01)	0.0027	0.439
IL1B	0.31 (0.02)	1.19 (0.21)	0.24 (0.04)	0.0021	0.0027

Abbreviations: AGTR1, angiotensin receptor type 1; ARG1, arginase; CH5, cadherin; CPAP, continuous positive airway pressure; DDAH, dimethylarginine dimethylaminohydrolase (DDAH 1 and 2); EDNRB, endothelin receptor type-B; eNOS, endothelial nitric oxide synthase; HIF1A, hypoxia inducible factor 1A; IL-1B, Interleukin 1B; IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; NFκB p50, nuclear factor kappa-B subunit 50; NFκB p65, nuclear factor kappa-B subunit p65; NOX4, NADPH oxidase; OSA, obstructive sleep apnea; PECAM1, platelet and endothelial adhesion molecule 1; SOD1, superoxide dismutase-1; TGFB, tumor growth factor type-B.

P values for significant comparisons are highlighted in bold.

Vasomotion pathways

In the microcirculation of OSA patients, key endothelial genes including: endothelin 1 (EDN1), endothelin receptor type-b (EDNRB), and angiotensin II receptor type1 (AGTR1) genes were upregulated compared with non-OSA participants and decreased after 12 weeks of CPAP in the OSA patients. There were no significant differences in TGFB1 (tumor growth factor type-B) and the surface adhesion molecule cadherin (CDH5). All gene changes measurements are reported in Table 2 and significant changes in related genes as well as platelet and adhesion molecule (PECAM1) are shown in Figure 1.

Figure 1.

Changes in microcirculatory endothelial vasomotion and injury markers with OSA treatment. Abbreviations: AGTR1, angiotensin II receptor type 1; END1, endothelin; EDNRB, endothelin receptor type-B; OSA, obstructive sleep apnea; PECAM1, platelet and endothelial cell adhesion molecule 1. OSA pre: patients with OSA before CPAP; OSA post: OSA patients post-CPAP. *P < 0.05 for OSA pre vs. non-OSA; **P < 0.05 for OSA pre-CPAP vs. OSA post-CPAP.

Hypoxia sensing, oxidative function, and NO synthesis

These groups of genes were selected to assay the pathways of NO production and availability based on previous findings from human studies.^21,28,32 Endothelial nitric oxide (NOS3) expression was not different between OSA and non-OSA participants and there was no significant change with CPAP. Notably, significant changes were present in dimethylarginine dimethylaminohydrolase (DDAH1, DDAH2) and arginase (ARG1). These genes are critical for NO availability. Figure 2 depicts the changes in genes related to these interconnected pathways. Notably, the profile of changes in NADPH oxidase (NOX4) and superoxide overproduction are other contributors to NO availability in the OSA endothelium.

Figure 2.

Changes in selected endothelial genes related to hypoxia, oxidative stress, and nitric oxide production. Abbreviations: ARG1, arginase; DDAH1, dimethylarginine dimethylaminohydrolase; HIF1A, hypoxia inducible factor 1a; NOS3, endothelial nitric oxide synthase; NOX4, NADPH oxidase subunit 4; SOD1, superoxide dismutase. OSA-pre: patients with OSA before CPAP; OSA post: OSA patients post-CPAP. *P < 0.05 for OSA pre vs. non-OSA; **P < 0.05 for OSA pre-CPAP vs. OSA post-CPAP.

Inflammatory genes

Selected genes involved in the endothelial inflammatory response to hypoxia and CVD were measured and are depicted in Table 3 and Figure 3. Expression of NFkB subunits p50 and p65 (NFKB1 and RELA, respectively), along with several interleukins decreased in the microcirculation after 12 weeks of CPAP.

Figure 3.

Changes with OSA treatment in selected inflammatory genes in the microcirculatory endothelium. Abbreviations: CCL2 (MCP-1), monocyte chemoattractant protein-1; IL1B, interleukin-1B; IL6, interleukin-6; IL8, interleukin-8; NFKB1, nuclear factor kappa-B subunit 50; OSA, obstructive sleep apnea; RELA, nuclear factor kappa-B subunit p65. OSA-pre: patients with OSA before CPAP; OSA post: OSA patients post-CPAP. *P < 0.05 for OSA pre vs. non-OSA; **P < 0.05 for OSA pre-CPAP vs. OSA post-CPAP.

Impact of OSA and CPAP on inflammatory response in PBMCs

Selected inflammatory gene transcription was measured in the PBMCs isolated from patients before and after 12 weeks of CPAP, and in non-OSA controls. The profile of change is depicted in Table 4 and Figure 4.

Table 4.

Inflammatory gene changes in PBMC

PBMC gene	Non-OSA (n = 7)	OSA pre-CPAP (n = 6)	OSA post-CPAP (n = 6)	P value OSA pre-CPAP vs. OSA post-CPAP	P value non-OSA vs. OSA pre-CPAP
PECAM1	9.49 (2.15)	11.72 (3.7)	8.83 (0.94)	0.48	0.60
CCL2 (MCP-1)	0.34 (1.10)	1.77 (1.11)	0.19 (0.06)	0.5	0.409
IL6	0.23 (0.08)	0.40 (0.04)	0.10 (0.04)	0.022	0.38
IL1B	1.14 (0.13)	3.82 (0.53)	2.05 (0.23)	0.046	0.015
CAMP (LL37)	3.35 (0.65)	6.38 (3.07)	1.40 (0.25)	0.44	0.53

Abbreviations: CPAP, continuous positive airway pressure; IL-1B, interleukin-1B; IL-6, interleukin-6; IL-8, interleukin-8; LL-37, human cathelicidin antimicrobial peptide LL-37; MCP-1, monocyte chemoattractant protein-1; OSA, obstructive sleep apnea; PBMC, peripheral blood mononuclear cell; PECAM1, platelet and endothelial cell adhesion molecule 1.

P values for significant comparisons are highlighted in bold.

Figure 4.

Significant changes in inflammatory PBMC genes from OSA patients and controls. Abbreviations: CAMP (LL-37), human cathelicidin antimicrobial peptide LL-37; IL-1B, interleukin-1beta; IL-6, interleukin-6; OSA, obstructive sleep apnea; PBMC, peripheral blood mononuclear cell. OSA-pre: patients with OSA before CPAP; OSA post: OSA patients post-CPAP. *P < 0.05 for OSA pre vs. non-OSA; **P < 0.05 for OSA pre-CPAP vs. OSA post-CPAP.

Discussion

In this study, we examined several established and highly suspected pathways of microcirculatory dysfunction in patients with OSA. The study was done in its entirety in freshly procured microcirculatory tissue from OSA patients, before and after adequate elimination of OSA with CPAP, and from weight and age matched controls. The findings of the study have several implications. The changes in the endothelial gene profiles provide a comprehensive view of the preclinical changes OSA produces in the human microcirculation. The profile of changes in the NO pathway along with upregulation of NOX gene provides an explanation for eNOS uncoupling, decreased NO and increased superoxide in the microcirculatory endothelium of OSA patients.^21,22 Furthermore, the significant activation of several inflammatory genes in the microcirculation provides a novel insight into the effect of OSA on vascular inflammation. The ability to simultaneously measure several genes that are highly relevant to CVD risk in patients with OSA before and after treatment, in addition to its important mechanistic insights, provides a new suite of disease-specific biomarkers that can be used in both mechanistic and interventional studies. Despite the relatively, albeit minimally, invasive nature of the subcutaneous biopsies, these techniques can be highly relevant to clinical trials evaluating surrogates of CVD risk in this population. In this regard, the changes reported in the PBMC can even offer a truly noninvasive set of disease-specific biomarkers to stratify disease severity, determine endotypes, or measure treatment effects.

NO availability is a critical determinant of endothelial function. The pathways leading to NO deficiency in OSA remain unknown. In 1 study, expression of microcirculatory eNOS protein did not change, while phosphorylated eNOS increased with CPAP.²¹ Certain types of NOS phosphorylation cause dysfunction and O₂^−· Overproduction (uncoupling).³³ Furthermore, overexpression of eNOS by gene transfer can increase NO activity in the vessel wall, while constitutive overexpression of eNOS paradoxically accelerated atherosclerosis in association with increased oxidative stress.^34,35 The findings of unchanged eNOS transcription with CPAP in this study further support that eNOS dysfunction rather than decreased expression is responsible for the decrease in NO availability. We have previously found that microcirculatory eNOS in otherwise health OSA patients demonstrated “uncoupling” ^36,37 with decreased NO production and overproduction of superoxide.²¹ We further confirmed that eNOS uncoupling was dependent, at least partially, on depletion of the critical cofactor tetrahydrobiopterin (BH4) by oxidation.²¹ To this effect, the novel finding in this study of NADPH upregulation provides a source of superoxide overproduction in the OSA microcirculation and explains the oxidative inactivation of BH4 and possibly of eNOS protein modification³⁸ in OSA. Furthermore, this study demonstrated upregulation of AGTR1 in OSA pretreatment patients. This finding, along with previous findings of increased angiotensin receptors (both types 1 and 2) in the OSA microcirculatory endothelium²⁸ confirms the role of renin–angiotensin system (RAS) activation in OSA-related VED. The typical effect of angiotensin II includes activation of NOX-dependent oxidant production.^39,40 In an animal model of intermittent hypoxia, AT-1 overexpression mediated VED.⁴¹ In experimental intermittent hypoxia in healthy adults, the oxidative response was abolished with angiotensin receptor blockade.⁴² We also found that angiotensin receptor blockade reversed eNOS uncoupling ex vivo in OSA patients.²³ Other studies have examined the RAS pathway in the pathogenesis of vascular disease in OSA patients and yielded mixed results.^43–45 Our findings represent the first simultaneous evaluation of these pathways in humans with OSA and provide evidence of early RAS activation resulting in NOX activation and superoxide overproduction in the microcirculation of OSA patients who are otherwise free of CVD risk factors.

This study should not be viewed as an evaluation of the effect of CPAP on reversing CVD risk. Rather, the use of CPAP in this study was designed to counteract the effects of OSA over a set period that has been shown to be long enough to reverse OSA- and intermittent hypoxia-related CVD changes.

The use of CPAP for a short period of time allows for the elimination for the obstructive apnea conditions while the period of 12 weeks sufficiently long to reverse these changes based on previously published studies, this duration is not long enough to allow for significant changes in lifestyle and healthful behavior. Therefore, CPAP use in this study represents an experimental intervention that was made possible by the study design. Nevertheless, the lack of untreated OSA arm is a limitation in the study. This limitation is somewhat addressed by the enrollment of body mass index and age and sex matched controls. Furthermore, studies of lifestyle and behavioral changes do not produce these robust changes in gene expressions in several highly specific pathways for intermittent hypoxia. Finally, the small sample size represents a limitation of this study. This is somewhat mitigated by the finding of consistent changes in several genes representing each tested pathway.

This study demonstrates activation of inflammatory pathways in both PBMC’s and endothelial cells. This large-scale activation of NFkB pathway and upregulation of interleukin-6 supports that OSA is also an inflammatory disorder. The presence and extent of the inflammatory activation in OSA may be a disease severity indicator. In this regard, it is possible that certain individuals with OSA may be exposed to more severe levels of hypoxia in some tissues or organs beyond that is reflected by their apnea–hypopnea index or desaturation index. Also, inflammatory activation may be marker of the impact of OSA on sleep continuity and quality.⁴⁶

In this study, we performed an extensive examination of the microcirculatory tissue of OSA patients who are free of CVD risk and found that several pathways of VED were activated and were reversible with elimination of OSA. These pathways are critical for the development of vascular disease and provide disease-specific biomarkers as well as therapeutic targets for future treatments addressing the vascular risk of OSA. Further, the technical approaches used in this study provide a platform for clinical studies as well as the option to guide bench studies by providing disease-specific molecular signature of OSA.

Funding

This research was supported by a grant from NIH-NHLBI (R56HL127079).

DISCLOSURE

The authors declared no conflict of interest.