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Global endothelial function assessment using pulse wave analysis in hypercholesterolemia

Abstract

To compare global endothelial function assessed by pulse wave analysis (PWA) using the ratio of endothelium dependent vasodilatation (EDV) to endothelium independent vasodilatation (EIV) in patients with hypercholesterolemia and controls. 92 subjects [46 hypercholesterolemics, 46 controls] were studied at standardized conditions. Baseline augmentation index (AIx) was assessed followed by the administration of 0.5 mg sublingual nitroglycerine, an endothelium independent vasodilator. AIx was assessed and the maximum change in AIx after nitroglycerine was recorded as EIV. After a washout period of 30 minutes, 400 µg of inhaled salbutamol, an endothelium dependent vasodilator was administered. AIx was assessed again and the maximum change in AIx after salbutamol was recorded as EDV. Global endothelial function was calculated as EDV:EIV ratio. EDV and EIV in patients with hypercholesterolemia compared to controls were 2.97 ± 3.95 and 6.65 ± 3.80 (p<0.001); and 13.41 ± 4.57 and 15.88 ± 4.78 (p=0.01) respectively. EDV:EIV ratio was significantly reduced in patients with hypercholesterolemia compared to controls; 0.21 ± 0.38 and 0.44 ± 0.24 (p<0.001) respectively. EDV:EIV ratio was significantly reduced in patients with hypercholesterolemia compared to controls. PWA is a potential clinical tool to assess global endothelial function in patients with hypercholesterolemia.

Keywords:
Endothelial function; Endothelium dependent vasodilatation; Endothelium independent vasodilatation; Augmentation index; SphygmoCor

INTRODUCTION

Endothelial dysfunction, which is characterized by a reduction in the bioavailability of vasodilators particularly nitric oxide (NO), and a reciprocal increase in vasoconstrictors has been well implicated in the pathogenesis of atherosclerotic cardiovascular disease (Endemann, Schiffrin, 2004Endemann DH, Schiffrin EL, Endothelial dysfunction. J Am Soc Nephrol. 2004;15(8):1983-1992.; Incalza et al., 2018Incalza MA , D’Oria R, Natalicchio A, Perrini S, Laviola L, Giorgino F. Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases. Vascul Pharmacol. 2018;100:1-19.). It has been shown to precede atherosclerosis and is associated with future cardiovascular events in patients with established coronary disease (Shachinger, Britten, Zeiher, 2000Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000;101(16):1899-906.). Its detection thus may serve as a useful preventive, monitoring as well as prognostic tool in cardiovascular related diseases.

The term global endothelial function refers to the measurement of endothelial function of the conduit and resistant vessels using pulse wave analysis (PWA) (McEniery et al., 2006McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. ). Using the applanation tonometry technique, PWA via its parameter augmentation index (AIx) has been previously tested as a non-invasive and simple tool to assess global endothelial function (Storch et al., 2017Storch AS, Mattos JD, Alves R, Galdino S, Roca HNM. Methods of Endothelial Function Assessment: Description and Applications. Int J Cardiovasc Sci. 2017;30(3):262-273.; Wilkinson et al., 2002Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.). Combining PWA with salbutamol and nitroglycerin (GTN) administration allows the assessments of endothelium-dependent and independent vasodilatations respectively; the ratio of changes in AIx to salbutamol relative to nitroglycerine indicates global endothelial function (McEniery et al., 2006McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. ). A preliminary study in patients with high total serum cholesterol demonstrated a blunted response of AIx to salbutamol (Wilkinson et al., 2002Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.) but the global endothelial function in patients with hypercholesterolemia has not been reported. This study aimed to compare global endothelial function in hypercholesterolemia patients and controls using PWA.

MATERIAL AND METHODS

This was a cross-sectional study involving 46 patients with hypercholesterolemia aged ≥35 years with LDL cholesterol ≥4.1 mmol/l being compared against 46 volunteers with normal LDL cholesterol. Source populations were the patients attending outpatient clinic during the study period. The study protocol was approved by the Human Research Ethics Committee of Universiti Sains Malaysia (USM) and all subjects gave written informed consent according to the Declaration of Helsinki.

Pregnant subjects and subjects who were already on statins or any treatment with vasoactive medications such as ACE inhibitors and Angiotensin Receptor Blockers were excluded from the study. Before endothelial function assessment, baseline heart rate and blood pressure (BP) (Omron, England), AIx, and central arterial pressure (CAP) (PWV Limited, Australia) were assessed.

The method of assessment of endothelial function has previously been described elsewhere (McEniery, 2007McEniery CM. How to measure endothelial function by pulse wave analysis. Artery Res. 2007;1(2):39. ; Wilkinson et al., 2002Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.). Briefly, endothelial independent vasodilatation (EIV) was induced by administering 0.5 mg sublingual nitroglycerine (Myonit Insta, Troikaa Pharmaceuticals Limited, India) for 3 minutes, after which the remaining tablet was discarded. The assessments of AIx after nitroglycerine were recorded at 3, 5, 10, 15 and 20 minutes. After a washout period of 30 minutes, endothelium dependent vasodilatation (EDV) was induced by administering 2 x 200 µg of inhaled salbutamol (Ventolin, GlaxoSmithKline, France) via a spacer device. The assessment of AIx after salbutamol was recorded at 5, 10, 15 and 20 minutes. The maximum changes in AIx after nitroglycerine and salbutamol was were recorded as EIV and EDV respectively. A reduction in EDV: EIV ratio indicates poorer endothelial function. All AIx readings were recorded as the adjusted value at the heart rate of 75 bpm (AIx75), as AIx is known to be affected by heart rate.

Statistical analysis

Global endothelial function is defined by the ratio of AIx changes after salbutamol (EDV) relative to AIx changes after GTN (EIV). The baseline characteristics of the subjects were tested using an independent t-test and Pearson’s chi-square for numerical and categorical variables respectively. The mean difference of EDV: EIV ratio between the hypercholesterolemia group and controls were tested using independent t-test and α value of 0.05 was considered statistically significant. Further analysis was done using analysis of covariance (ANCOVA) which controlled for possible confounders which are age, sex, height, body mass index, smoking status, diabetes mellitus, peripheral and central BP. Data was analyzed using IBM SPSS ((Statistical Packages for Social Science) 22.0 for windows (Inc., Chicago, IL).

RESULTS AND DISCUSSION

The baseline characteristics of the subjects studied are shown in Table I. Global endothelial function represented by EDV: EIV ratio was significantly lower in hypercholesterolemic groups compared to controls; 0.21±0.30 and 0.44±0.24 respectively (p<0.001). Similarly, EDV was also significantly lower in hypercholesterolemic groups compared to normal controls; 2.97%±3.95 and 6.65%±3.80 respectively (p<0.001). Difference still persists after controlling for possible confounders as shown in Table II.

TABLE I
Subjects characteristics
TABLE II
Comparison of global endothelial function between hypercholesterolaemia patients and healthy controls with and without adjustment of possible confounders

EIV that represents AIx response to nitroglycerine was also significantly lower in hypercholesterolemic group compared to controls; 13.41%±4.57 and 15.88%±4.78 respectively (p=0.01).

Our study showed that EDV:EIV ratio which represented global endothelial function was significantly reduced in patients with hypercholesterolemia compared to controls. To our knowledge, this was the first study that assessed EDV:EIV ratio as an index of global endothelial function in patients with hypercholesterolemia using PWA. Our recent finding was consistent with other previous reports on endothelial function in patients with hypercholesterolemia using other methods of detection such as flow-mediated dilatation (FMD) (Ikdahl et al., 2015Ikdahl E, Hisdal J, Rollefstad S, Olsen IC, Kvien TK, Pedersen TR, et al. Rosuvastatin improves endothelial function in patients with inflammatory joint diseases, longitudinal associations with atherosclerosis and arteriosclerosis: results from the RORA-AS statin intervention study. Arthritis Res Ther. 2015;17:279. ; Stapleton et al., 2010Stapleton PA, Goodwill AG, James ME, Brock RW, Frisbee JC. Hypercholesterolemia and microvascular dysfunction: interventional strategies. J Inflamm (Lond). 2010;7:54. ) and PWA (Wilkinson et al., 2002Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.). An earlier study by Wilkinson et al. (2002)Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152. reported a reduced response of AIx to salbutamol in hypercholesterolemia. In our study, this was represented by EDV and similarly it was significantly reduced in patients with hypercholesterolemia compared to controls. Several versions of endothelial function assessments by PWA were described by different studies, but all methods were based on the waveform changes in response to salbutamol and nitroglycerine (Lekakis et al., 2011Lekakis J, Abraham P, Balbarini A, Blann A, Boulanger CM, Cockcroft J, et al. Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation. Eur J Cardiovasc Prev Rehabil. 2011;18(6):775-789. ). Whilst the earlier studies defined global endothelial function as salbutamol-induced changes in AIx as represented by EDV (Hayward et al., 2002Hayward CS, Kraidly M, Webb CM, Collins P. Assessment of endothelial function using peripheral waveform analysis: a clinical application. J Am Coll Cardiol. 2002;40(3):521-528. ; Wilkinson et al., 2002Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.), the later studies described endothelial function as EDV:EIV ratio (McEniery, 2007McEniery CM. How to measure endothelial function by pulse wave analysis. Artery Res. 2007;1(2):39. ; McEniery et al., 2006McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. ). McEniery et al. (2006)McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. described global endothelial function as the ratio of AIx change to salbutamol relative to nitroglycerine whereby a reduction in the ratio indicates worse endothelial function. In their study, global endothelial function in healthy population was described (McEniery et al., 2006McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. ). Studies in patients with risk factors and cardiovascular-related disease are still lacking and ours was the first study to describe the ratio in patients with hypercholesterolemia. Compared to our cohort of hypercholesterolemia, global endothelial function, as described by McEniery et al. (2006)McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. appeared among a healthy population.

When used as a control, AIx is expected to remain unchanged after nitroglycerine stimulation. However, in our study, hypercholesterolemia group showed a reduced response to nitroglycerine. While a few previous studies reported no difference in AIx after nitroglycerine stimulation in healthy volunteers (Hayward et al., 2002Hayward CS, Kraidly M, Webb CM, Collins P. Assessment of endothelial function using peripheral waveform analysis: a clinical application. J Am Coll Cardiol. 2002;40(3):521-528. ; Ibrahim et al., 2009Ibrahim NN, Rasool AH, Wong AR, Rahman AR. Prevalence of beta-2 adrenergic receptor (beta 2 AR) polymorphisms and its influence on a model used to assess endothelial function using pulse wave analysis (PWA). Clin Chim Acta. 2009;409(1-2):62-66. ; McEniery et al., 2006McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608. ) and hypercholesterolemic cohort (Wilkinson et al., 2002Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.), a few recent studies have also reported a reduced response in AIx to nitroglycerine in different medical conditions. Using the same method of detection of PWA, Covic et al. (2004Covic A., Goldsmith DJ, Gusbeth-Tatomir P, Covic M. Haemodialysis acutely improves endothelium-independent vasomotor function without significantly influencing the endothelium-mediated abnormal response to a beta 2-agonist. Nephrol Dial Transplant. 2004;19(3):637-643. ) reported a reduced response of AIx to nitroglycerine in chronic kidney disease (CKD) patients and the response improved after dialysis. Similarly, Paul et. al (2009Paul B, Hewitson CL, Woodman RJ, Mangoni AA. Analysis of short-term reproducibility of arterial vasoreactivity by pulse-wave analysis after pharmacological challenge. Clin Exp Pharmacol Physiol. 2009;36(1):49-54. ) also reported a reduced AIx response to nitroglycerine in patients with chronic heart failure. Compared to hypercholesterolemia patients with no concomitant disease in the preliminary study reported by Wilkinson et al. (2002)Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152., our cohort of hypercholesterolemia patients were documented to have other concurrent medical conditions which include hypertension (10 patients) and diabetes mellitus (three patients). Other studies assessing endothelial function via other methods such as FMD also reported impaired EIV by nitroglycerine in subjects with cardiovascular risk factors and coronary heart disease (Maruhashi et al., 2013Maruhashi T, Soga J, Fujimura N, Idei N, Mikami S, Iwamoto Y, et al. Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation. Arterioscler Thromb Vasc Biol. 2013;33(6):1401-1408. ; Raitakari et al., 2001Raitakari OT, Seale JP, Celermajer DS. Impaired vascular responses to nitroglycerin in subjects with coronary atherosclerosis. Am J Cardiol. 2001;87(2):217-219. ). It has been reported that in cardiovascular related diseases, an increased presence of superoxide has been detected (Maruhashi et al., 2013Maruhashi T, Soga J, Fujimura N, Idei N, Mikami S, Iwamoto Y, et al. Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation. Arterioscler Thromb Vasc Biol. 2013;33(6):1401-1408. ). Superoxide, other than inhibiting NO production by endothelium has also been shown to inhibit intravascular signaling in vascular smooth muscle cell by inhibiting guanylyl cyclase and cGMP dependent kinase which may result in smooth muscle cell dysfunction. It has also been suggested that a reduced response to nitroglycerine was correlated with increased intima-media thickness (Maruhashi et al., 2013Maruhashi T, Soga J, Fujimura N, Idei N, Mikami S, Iwamoto Y, et al. Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation. Arterioscler Thromb Vasc Biol. 2013;33(6):1401-1408. ) and other possible structural abnormalities such as increased left ventricular mass (Covic et al., 2004Covic A., Goldsmith DJ, Gusbeth-Tatomir P, Covic M. Haemodialysis acutely improves endothelium-independent vasomotor function without significantly influencing the endothelium-mediated abnormal response to a beta 2-agonist. Nephrol Dial Transplant. 2004;19(3):637-643. ). It was thus suggested that global endothelial function should be interpreted as an index of both EDV and EIV, rather than solely by EDV particularly in high risk patients with cardiovascular related disease.

This study has demonstrated that global endothelial function assessed by PWA combined with pharmacological challenge was reduced in hypercholesterolemia patients. Global endothelial function should be interpreted as the ratio of EDV:EIV particularly in high risk patients with cardiovascular related disease. Further studies should include testing its application in monitoring treatment before it could be widely used as a tool to assess endothelial function.

ACKNOWLEDGEMENT

This study was funded by grants from Universiti Sains Malaysia Short Term Grant (304/PPSP/61312103) and Research University Grant (1001/PPSP/812211).

We also express our gratitude to the Director of the Hospital Universiti Sains Malaysia (USM), Kubang Kerian, Kelantan for granting the permission to the investigators to use patients’ medical record; space and assets belonging to the hospital during the process of conducting the research.

REFERENCES

  • Covic A., Goldsmith DJ, Gusbeth-Tatomir P, Covic M. Haemodialysis acutely improves endothelium-independent vasomotor function without significantly influencing the endothelium-mediated abnormal response to a beta 2-agonist. Nephrol Dial Transplant. 2004;19(3):637-643.
  • Endemann DH, Schiffrin EL, Endothelial dysfunction. J Am Soc Nephrol. 2004;15(8):1983-1992.
  • Hayward CS, Kraidly M, Webb CM, Collins P. Assessment of endothelial function using peripheral waveform analysis: a clinical application. J Am Coll Cardiol. 2002;40(3):521-528.
  • Ibrahim NN, Rasool AH, Wong AR, Rahman AR. Prevalence of beta-2 adrenergic receptor (beta 2 AR) polymorphisms and its influence on a model used to assess endothelial function using pulse wave analysis (PWA). Clin Chim Acta. 2009;409(1-2):62-66.
  • Ikdahl E, Hisdal J, Rollefstad S, Olsen IC, Kvien TK, Pedersen TR, et al. Rosuvastatin improves endothelial function in patients with inflammatory joint diseases, longitudinal associations with atherosclerosis and arteriosclerosis: results from the RORA-AS statin intervention study. Arthritis Res Ther. 2015;17:279.
  • Incalza MA , D’Oria R, Natalicchio A, Perrini S, Laviola L, Giorgino F. Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases. Vascul Pharmacol. 2018;100:1-19.
  • Lekakis J, Abraham P, Balbarini A, Blann A, Boulanger CM, Cockcroft J, et al. Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation. Eur J Cardiovasc Prev Rehabil. 2011;18(6):775-789.
  • Maruhashi T, Soga J, Fujimura N, Idei N, Mikami S, Iwamoto Y, et al. Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation. Arterioscler Thromb Vasc Biol. 2013;33(6):1401-1408.
  • McEniery CM. How to measure endothelial function by pulse wave analysis. Artery Res. 2007;1(2):39.
  • McEniery CM, Wallace S, Mackenzie IS, McDonnell B, Yasmin, Newby DE, et al. Endothelial function is associated with pulse pressure, pulse wave velocity, and augmentation index in healthy humans. Hypertension. 2006;48(4):602-608.
  • Paul B, Hewitson CL, Woodman RJ, Mangoni AA. Analysis of short-term reproducibility of arterial vasoreactivity by pulse-wave analysis after pharmacological challenge. Clin Exp Pharmacol Physiol. 2009;36(1):49-54.
  • Raitakari OT, Seale JP, Celermajer DS. Impaired vascular responses to nitroglycerin in subjects with coronary atherosclerosis. Am J Cardiol. 2001;87(2):217-219.
  • Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000;101(16):1899-906.
  • Stapleton PA, Goodwill AG, James ME, Brock RW, Frisbee JC. Hypercholesterolemia and microvascular dysfunction: interventional strategies. J Inflamm (Lond). 2010;7:54.
  • Storch AS, Mattos JD, Alves R, Galdino S, Roca HNM. Methods of Endothelial Function Assessment: Description and Applications. Int J Cardiovasc Sci. 2017;30(3):262-273.
  • Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol . 2002;22(1):147-152.
  • COMPLIANCE WITH ETHICAL STANDARDS

    All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

Publication Dates

  • Publication in this collection
    13 July 2022
  • Date of issue
    2022

History

  • Received
    05 Dec 2019
  • Accepted
    16 Mar 2020
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br