• Open Access

Real-Time RT-PCR Ct Values for Blood GAPDH Correlate with Measures of Vascular Endothelial Function in Humans

Authors

  • Faisel Khan Ph.D.,

    1. Medical Research Institute, Division of Cardiovascular and Diabetic Medicine, Ninewells Hospital & Medical School, University of Dundee, Dundee, United Kingdom
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  • Wen Ling Choong M.Sc.,

    1. Medical Research Institute, Division of Cardiovascular and Diabetic Medicine, Ninewells Hospital & Medical School, University of Dundee, Dundee, United Kingdom
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  • Qingyou Du Ph.D.,

    1. Medical Research Institute, Division of Cardiovascular and Diabetic Medicine, Ninewells Hospital & Medical School, University of Dundee, Dundee, United Kingdom
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  • Aleksandar Jovanovi'c M.D., Ph.D.

    Corresponding author
    1. Medical Research Institute, Division of Cardiovascular and Diabetic Medicine, Ninewells Hospital & Medical School, University of Dundee, Dundee, United Kingdom
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Abstract

Purpose

To date, there is a wide range of methods in use to assess endothelial function, each with its own advantages and limitations. Here, we tested hypothesis that real-time RT-PCR threshold value (Ct), which is reflective of mRNA level, for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from whole blood is indicative of endothelial function in humans.

Materials and Methods

To assess vascular function, we measured baseline skin perfusion, postocclusion reactive hyperemia (PORH), and brachial artery flow-mediated dilatation (FMD) and tested for a possible correlation between vascular responses and blood GAPDH real-time RT-PCR Ct value in 75 healthy volunteers.

Results

Tests known to measure, at least in part, endothelial function such as baseline skin perfusion, the 2-minute recovery PORH, and FMD exhibited significant positive correlations with blood GAPDH Ct values. In contrast, there was no significant correlation between Ct values for blood GAPDH and peak PORH, an endothelium-independent parameter.

Conclusions

Based on these findings, we report that GAPDH mRNA level in the blood correlates with vascular function in healthy subjects. This suggests that GAPDH mRNA level could be a potential biomarker of vascular endothelial function.

Introduction

Early changes in the normal functioning of the endothelium are key initiating factors in the development and progression of atherosclerosis and they are present well before the presentation of clinical symptoms. To date, there is a wide range of methods in use to assess endothelial function, each with its own advantages and limitations.[1] For diagnostic purposes and for indications of progression and outcomes of disease, a methodology measuring a biomarker in the blood would be convenient. Measurement of mRNA levels could provide a good option as: (1) measurement of this parameter requires small amounts of blood and (2) real-time RT-PCR to measure mRNA is relatively straightforward and technically less challenging than techniques used to measure protein levels.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly expressed multifunctional protein with diverse physiological functions and activities involved in glycolysis, transcriptional and posttranscriptional gene regulation, vesicular transport, receptor-mediated cell signaling, chromatin structure, and the maintenance of DNA integrity. GAPDH activity is regulated by many factors, one of them being nitric oxide (NO).[2] NO synthesized and released from vascular endothelium is an important regulator of vascular tone.[3] It has been reported that NO can either inhibit or activate GAPDH in different cell types.[4, 5] As it is known that NO derived from the vascular endothelium targets blood cells,[6] it is possible that GAPDH activity could be regulated by endothelial NO. If so, then GAPDH activity in blood cells could be reflective of vascular endothelial NO production and thus indicative of endothelial function in physiological and pathophysiological conditions.

Although there was no previous work to support our hypothesis, we decided to explore the potential link between vascular endothelial function and GAPDH mRNA level in the blood in healthy volunteers. We found that real-time RT-PCR Ct values of blood GAPDH correlate with vascular function in healthy subjects. This suggests that GAPDH mRNA level could be a potential biomarker of vascular endothelial function.

Subjects and Methods

Subjects

Seventy-five young healthy volunteers (41 males, 34 females) were recruited for the study. None of the subjects were smokers, used any medication, or had a history of any symptomatic vascular disease(s). Subject characteristics are shown in Table 1. The study was approved by the Tayside Committee on Medical Research Ethics and written informed consent was obtained from each subject before participation in the study. All subjects attended for one single visit lasting up to 3 hours during which a blood sample was taken and vascular function tests performed (see below). Vascular assessments were conducted in a blood flow laboratory at a temperature of 23°C after 10 minutes of acclimatization. Subjects were asked to refrain from food and drink for at least 2 hours beforehand and also to refrain from physical activity for 1 day before their visit.

Table 1. Subject characteristics. Data are presented means ±SEM
Age (year)22.1 ± 0.3
Sex (male/female)41/34
Weight (kg)65.1 ± 1.1
Height (m)1.70 ± 0.01
Body mass index (kg/m2)22.4 ± 0.3
Heart rate (beats/min)63.2 ± 1.0
Systolic blood pressure (mmHg)115.0 ± 1.2
Diastolic blood pressure (mmHg)68.5 ± 0.9

RNA preparation and real-time RT-PCR

Five milliliters of venous blood was collected from a vein in the upper arm of each subject into a heparinized vacutainer. The vacutainer was placed in a sealed transport plastic bag containing ice and sent immediately to the laboratory for determination of mRNA levels by real-time RT-PCR test. Real-time RT-PCR was performed using primers and controls as we previously described on other human samples.[7, 8] Threshold cycle values (Ct) were directly obtained by this methodology.[7, 8]

Assessment of microvascular and macrovascular functions by postocclusion reactive hyperemia (PORH) and brachial artery flow-mediated dilatation (FMD)

PORH was tested in 56 subjects (30 males, 26 females) and vascular function was assessed with the subjects lying supine on a bed. The forearm was rested at heart level and the skin microcirculation was measured at the volar aspect using a full field laser perfusion imager (moorFLPI, Moor Instruments Ltd., Axminster, United Kingdom). Superficial microvascular perfusion was measured continuously from five individual regions of interest over an area of approximately 30 cm2. Data collected from the five regions of interest were averaged to provide an overall response in arbitrary perfusion units. A blood pressure cuff was placed over the upper arm and a baseline measurement of skin perfusion was obtained for 2 minutes. The cuff was then inflated to a suprasystolic pressure (200 mmHg), thus, occluding any blood perfusion distal to the cuff for 5 minutes. After 5 minutes of ischemia, the cuff was deflated resulting in PORH. The peak perfusion postocclusion was measured and in addition, the average perfusion over 2 minutes following the release of the cuff was determined.

Brachial artery FMD was tested in 37 subjects (20 males, 17 females). Vascular function was assessed with the subjects lying supine on a bed according to standard guidelines.[9] The arm was rested at heart level and the brachial artery was measured above the antecubital fossa in the longitudinal plane at the volar aspect using high-resolution ultrasound imaging (Acuson Sequoia 512, Siemens Medical Solutions, Berkshire, United Kingdom). The ultrasound system was equipped with the vascular software for 2D imaging, a high-frequency vascular transducer (FMD8L5), color and spectral Doppler, and an internal ECG (to provide a trigger for the R wave). A snake arm clamp was used to hold the ultrasound probe in place allowing a stable image of the brachial artery to be obtained throughout the study. The ischemic stimulus was produced by placing a blood pressure cuff above the antecubital fossa and inflating to a suprasystolic pressure of around 200 mmHg for 5 minutes. After cuff was released, a transient increase in blood flow through the brachial artery was produced by reactive hyperemia, which resulted in dilatation of the brachial artery.[10] The 2D images of the brachial artery were acquired for 1 minute at baseline and for 2 minutes postcuff release. FMD was calculated as the maximum percentage change in diameter postreactive hyperemia relative to the baseline diameter.

Statistical analysis

The relationship between Ct GAPDH value in blood and PORH and brachial FMD data was assessed using Pearson's correlation coefficient. Group differences were analyzed using unpaired sample t-tests. Statistical analyses were carried out using SPSS for Windows version 14.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ±SEM and a probability value <0.05 was considered statistically significant.

Results

Out of 75 subjects, GAPDH mRNA was detected in whole blood of 59 subjects. The average Ct value for GAPDH was 19.28 ± 0.64 (n = 59; Figure 1). To determine markers of vascular function, we measured baseline skin perfusion, PORH, and FMD and assessed possible correlations between vascular responses and blood GAPDH Ct. Baseline skin perfusion, the 2-minute recovery PORH, and FMD exhibited significant positive correlations with Ct values (baseline skin perfusion: r = 0.406, p = 0.001, n = 59; PORH: r = 0.402, p = 0.002, n = 58; FMD: r = 0.356, p = 0.030, n = 37; Figure 1). High Ct values equate to lower levels of GAPDH mRNA, thus a positive correlation means that higher values of basal skin perfusion, 2-minute recovery PORH, and FMD are associated with lower expression of GAPDH in the blood. In contrast, there was no significant correlation between Ct values for GAPDH and peak PORH (r = 0.132, p = 0.323, n = 58; Figure 1).

Figure 1.

Ct GAPDH value from blood correlate with baseline skin perfusion, recovery hyperemic response (PORH), and flow-mediated dilatation (FMD) percentage increase, but not with peak PORH. (A) Original progress curves done in duplicate for the real-time PCR amplification of GAPDH cDNA. (B) Scatter plot and linear regression of GAPDH threshold cycles (Ct) versus baseline skin perfusion, PORH, peak PORH, and FMD percentage increase. Each point represents a single subject (n = 37–59).

Discussion

In this study, we have examined whether a relationship exists between markers of vascular function and Ct for blood GAPDH in humans. Although Ct value is affected by many factors, it is recognized that this value is mostly an indicator of a measured gene mRNA level.[7, 8] Basal perfusion, recovery PORH, and FMD are all parameters of vascular function that are known to be dependent on the integrity of the vascular endothelium. In contrast, peak PORH is a parameter that seems to be predominantly independent from the endothelium and can be largely accounted for by myogenic mechanisms.[1, 10] We have found that three parameters of vascular function that, at least in part, measure endothelial function correlate significantly with the GAPDH Ct in the whole blood. Alternatively, peak PORH, which is endothelium-independent, did not show any correlation with blood GAPDH Ct. Taken all together, this suggests that blood GAPDH Ct correlates with measures of endothelial function.

At present, it is not possible to conclude about the nature of observed association between vascular endothelial function and Ct value, i.e., GAPDH mRNA level. One possibility is that NO produced by vascular endothelium regulates the level of expression in blood cells (most likely leukocytes as they would have required machinery to respond to NO by changes in the mRNA level) and data we have obtained allow such notion. Whether this is indeed a mechanism underlying a connection between GAPDH and endothelial function is yet to be fully tested, but, regardless of the mechanism, it seems that there is an exciting prospect of exploiting Ct blood GAPDH value as a biomarker for endothelial function and early development of atherosclerosis. The fact that differences in blood Ct GAPDH values were successful to describe differences in vascular function within the physiological range of vascular responses in healthy individuals suggests that this parameter could be very accurate to detect conditions associated with damaged endothelium. As far as we know, this would be the first potential biomarker of an endothelial condition based on mRNA levels that can be found in the whole blood. A particular strength of this methodology is that it is technically straightforward, minimally invasive, and requires small amounts of blood. It remains to establish whether a relationship exists between blood GAPDH mRNA and endothelial function in patients with vascular diseases. Based on current findings, it is plausible that such a relationship would be found. In turn, measuring GAPDH mRNA in the blood could be used in the future as a biomarker for endothelial function and early detection of vascular diseases, in particular atherosclerosis.

Conclusion

Based on these findings, we report that GAPDH mRNA level in the blood correlates with vascular function in healthy subjects. This suggests that GAPDH mRNA level could be a potential biomarker of vascular endothelial function.

Acknowledgment

This research was supported by grants from the British Heart Foundation, TENOVUS-Scotland and Anonymous Trust.

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