Plasma nitric oxide level is correlated with microvascular functions in the peripheral arterial disease

Abstract

At present there is no widely accepted biomarker for monitoring of vascular functions. The purpose of this prospective study was to investigate the association of some blood biomarkers with vascular reactivity in patients with peripheral arterial diseases (PAD). A prospective evaluation was made of 3 groups comprising a control group of healthy individuals, and patients with PAD caused by either atherosclerosis or Buerger’s disease. Microvascular perfusion was examined using laser Doppler imaging of cutaneous erythrocyte flux after iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP). The correlation of microvascular reactivity with endothelium-related biomarkers was assessed. ACh-induced and SNP-induced vasodilations were significantly diminished in the PAD groups. The plasma nitric oxide (NO) levels of PAD patients were significantly higher than those of the control group, but asymmetric dimethylarginine, total antioxidant capacity and hydrogen sulphide levels were similar. Plasma NO level was negatively correlated with ACh and SNP-stimulated microvascular flow increase, whereas a positive correlation was detected with blood glucose and glycated hemoglobin (HbA1c) levels in all groups. These results indicate that a high plasma level of NO in PAD patients is associated with diminished endothelium-dependent and independent flow increase in the microvascular bed. An excessive amount of NO-induced nitrosative stress in an inflammatory condition that might be a reason for vascular dysfunction should be taken into consideration in the diagnostic and therapeutic approaches to PAD.

Keywords

Peripheral arterial disease atherosclerosis microcirculation endothelial dysfunction nitric oxide

1 Introduction

Peripheral arterial disease (PAD) is an important health problem in all societies and is characterized by impaired microcirculation in the extremities. There are two major pathological conditions, namely atherosclerosis (AS) and Buerger’s disease (BD), causing PAD and both have a different etiology and clinical presentation. The atherosclerotic lesions in AS and non-atherosclerotic inflammation of the arteries in BD cause a progressive reduction of blood flow in the extremities often leading to intermittent claudication, ischemic rest pain and necrosis [20, 26].

Endothelial cells play a pivotal role in the regulation of vascular tone by secreting various active substances [24]. Among these nitric oxide (NO) is involved in various physiological and pathological responses, including smooth muscle relaxation, immune regulation and platelet function. Under physiological condition, a low level of NO is produced by the endothelial NO synthase (eNOS). Endothelial dysfunction has been proposed as a primary reason/result of vascular pathology in PAD [1 , 7] and, endothelial dysfunction has been correlated with a decrease in the synthesis, release or effect of NO. In addition, increased plasma level of endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) was also linked to endothelial dysfunction in PAD patients [3, 14].

On the other hand several immunological agents stimulate the inducible form of NO synthase (iNOS) to generate higher amounts of NO. In the inflammatory condition seen in PAD, endothelium-derived NO production decreases and huge amounts of inflammatory cells-derived NO is produced [16, 19]. Inflammation also causes oxidative stress that is responsible for decreased bioavailability of NO, by inhibiting endothelium-derived NO synthesis and scavenging NO by superoxide anions. It has been suggested that oxidative stress is closely related to the arterial dysfunction in PAD [2 , 27]. Furthermore, reaction of increased production of superoxide anion with excess amount of NO caused nitrosative stress. Another gaseous transmitter is hydrogen sulfide (H₂S) has been reported to interacts with NO metabolism and oxidative status in physiological and pathological situations [23].

Discovery of a correlation between vascular functions and blood markers in PAD patients would be important for evaluating the pathological state, monitoring and determining therapeutic/preventive approaches. In the present study, possible association of endothelium-dependent and -independent microvascular relaxation with some blood biomarkers (NO, ADMA, TAC, H₂S) was investigated in patients with PAD caused by AS and BD.

2 Methods

In this prospective study, 148 individuals were assessed, comprising 34 healthy subjects and consecutive patients with peripheral arterial disease due to AS or BD. Study protocols were approved by the Institutional Ethics Committee and all subjects provided written informed consent. PAD was assessed by the ankle/brachial pressure index (ABPI) measured at intervals, the walking distances on treadmill of patients with claudication, the condition of ischemic lesions, surgical operations, amputation and tissue loss. The lesions of patients with AS or BD were classified according to Rutherfords’s Clinical Chronic Ischemia Category [25].

The study inclusion criteria were no history of arterial surgery or endovascular intervention for PAD and no history of prostaglandin treatment. Exclusion criteria were sepsis or major infection, major diseases like cancer, autoimmune diseases and rheumatological diseases (except for cardiovascular diseases), vasculitis, history of major vascular surgical operation, pregnancy, age below 18 years.

2.1 Assessment of microvascular circulation

Peripheral microvascular perfusion was assessed using a validated technique to quantify vasodilator responses to iontophoresis of ACh and SNP, which has been described in detail elsewhere [11]. All subjects fasted overnight and were asked to refrain from drinking any fluids except for water before the measurements, which were taken in a temperature controlled room (23±1°C). During a 20 min acclimatization period before iontophoresis, the volar aspect of the right forearm was gently cleaned with an alcohol wipe and swabbed with deionized water. After acclimatization, two perspex iontophoresis chambers (LI 611 drug delivery electrodes; Perimed, Jarfalla, Sweden) were attached to the right forearm, 5 cm below the medial condyle with ≥10 cm between them, using double-sided adhesive rings, avoiding hair, broken skin and superficial veins. The anodal chamber was filled with 0.25 ml 1% (w/v) ACh (Sigma-Aldrich Chemicals, UK) and the cathodal chamber with 0.25 ml 1% (w/v) SNP (Sigma-AldrichChemicals, UK). Drug delivery from each chamber was controlled by a battery-powered constant-current iontophoresis controller (Perilont 382 power supply, Perimed, Jarfalla, Sweden).

Forearm cutaneous microvascular erythrocyte flux was assessed using a cumulative dose–response protocol which has been described in detail previously [11]. This was followed by drug delivery at incremental duration and fixed current. The technique is based on the fact that a charged molecule migrates across the skin under the influence of an applied electrical field and ionized drug delivery is dependent on the magnitude of the applied current and its duration (current×time-charge, in Coulombs). Therefore 0.1 mA was applied for 5, 10, 20, 40 and 80 seconds which constitutes 0.5, 1, 2, 4, and 8 milliCoulombs (mC). Non-invasive measurement of skin perfusion was performed with a laser Doppler imager (PeriScan PIM II, Perimed, Jarfalla, Sweden) at wavelength 670 nm, power 1 mW, and beam diameter 1 mm. The technique is based on the Doppler shift imparted by moving blood cells in the underlying tissue to the backscattered light. The laser is scanned in a raster fashion over both chambers and through the cover slips. The backscattered light is collected by photo detectors and converted into a signal proportional to perfusion in arbitrary perfusion (flux) units (PU) that is displayed as a color-coded image on a monitor. Perfusion measurements were obtained using the imager manufacturer’s image analysis software (LDPIwin software, Perimed, Jarfalla, Sweden) by outlining a region of interest (ROI) around the internal circumference of the chamber. Before administering the drug, 4 baseline images were recorded taken totally 120 seconds without current, i.e. no drug was iontophoresed. Each iontophoretic drug application was followed by eight laser scans each taking 30 seconds. In other words, each dose application was then scanned for 240 seconds. Statistical analysis of the ROI was subsequently performed offline to yield the median flux value across approximately 700 measurement points. A total of 44 repetitive scans were taken, the first 4 being a control (before current administration), followed by the incremental time protocol described above.

Total perfusion response can be represented by the area under the curve (AUC) for each of the series of 44 scans. All quantities were measured at baseline and after each iontophoretic charge 0.5, 1, 2, 4, and 8 mC. ACh and SNP-stimulated flow increases were calculated using a percentage enhancement formula.

Flow increase = [(mean value of 8 calculations in each charge–basal value)/basal value]*100

After assessment of the vascular responses, the patients were treated as indicated and followed up for complications of PAD and cardiovascular events.

2.2 Biochemical examinations

The plasma nitrite level was measured as a representation of NO production. It was measured using the spectrophotometric method based on the Griess reaction [21]. This method was modified in our laboratories for 96-well plates. The total antioxidant capacity (TAC) of plasma was measured by the method which was described previously [9], based on the reduction of Cu⁺² to Cu⁺¹ by the antioxidants in the plasma. Neocuproine was used as a chromogenic agent and colored complex was detected spectrophotometrically, at 455 nm. H₂S levels of the plasma were measured spectrophotometrically, according to a previously described method based on the measurements of the absorbance of the methylene blue, which produced the chemical reaction between N, N-dimethyl-p-phenylenediamine and FeCl₃, at 670 nm [28]. ADMA levels were measured by ELISA kits (Immunodiagnostic A.G., Germany) according to the manufacturer’s instructions.

2.3 Statistical analysis

Values were expressed as mean±SEM. Statistical analyses were performed using the SigmaPlot (Systat Software Inc., USA) version 11 for Windows. Repeated-measures of two-way ANOVA was used to test differences among the groups for skin perfusion. One-way ANOVA was used to test differences among the groups for biomarkers. Values were considered statistically significantly different when p < 0.05. When the p value was statistically significant, comparison procedure was applied using the Holm-Sidak test. Pearson Product Moment was used to test correlations.

3 Results

Demographic properties and risk factors of all groups are seen in Table 1. Diabetes and coronary surgery in the AS group and smoking in the BD group are the most common risk factors. As for their clinical condition, patients with BD had higher upper stages of chronic ischemia and more minor/major amputation history than patients with AS and control (Table 2).

Effect of endothelium-dependent relaxation on microvascular perfusion of skin was assessed using iontophoresis of ACh on the forearm. Release of Ach by electrical stimulation induced charge-dependent increase in capillary blood flow in all groups. ACh-induced increase in blood flow was significantly attenuated in the AS and BD groups (8 mC charge stimulated % increase 97.2±6.4 in the control group, 66.1±6.1 in the AS group and 64.7±5.4 in the BD group) (p < 0.05) (Fig. 1). SNP-induced endothelium-independent relaxations were also significantly smaller in the AS and BD groups compared with the control (8 mC current stimulated % increase 90.9±5.8 in the control, 60.8±4.9 in the AS and 53.4±6.5 in the BD groups) (p < 0.05) (Fig. 1).

Plasma NO levels in the AS and BD patients were significantly higher than the control (24.5±2.3 μM in the control, 71.7±5.5 μM in the AS, 58.1±5.7 μM in the BD groups) (p < 0.05) (Fig. 2). However, plasma TAC, ADMA and H₂S levels were similar in all groups (Fig. 2).

Association of both types of vascular relaxations with endothelium-related biomarkers was determined by calculation of the correlation coefficient in all groups. Although, NO level was negatively correlated with ACh.8 and SNP.8-mediated dilation; plasma levels of ADMA, TAC and H₂S were not correlated with either of the vascular relaxations (Figs. 3 and 4).

Routine blood chemistry of patients are shown in Table 3. While blood cell count, levels of glucose, hsCRP, WBC, hemoglobin and hemoglobin-A1c in the AS group were higher than the control; in the BD group only the level of hsCRP was significantly higher than the control.

Plasma NO level was positively correlated with blood glucose and HbA1c levels in all groups (Table 4). Plasma ADMA level was only negatively correlated with blood homocysteine level. TAC levels were positively correlated with total cholesterol, LDL, VLDL, triglyceride, and uric acid levels. Plasma H₂S level was not correlated with any of these markers. ACh.8-mediated endothelium-dependent dilation was correlated positively with total cholesterol level and negatively with blood hsCRP level. SNP.8-mediated dilation was correlated with total cholesterol and HDL levels, negatively with blood HbA1c level (Table 4).

4 Discussion

The results of this study show that in the PAD groups, both ACh and SNP induced vasodilatations in the upper extremities are attenuated significantly compared with the controls and plasma NO levels are significantly above the level in the control group. Elevated blood NO level in the PAD patients is negatively correlated with failure of the microvascular perfusion to rise in response to stimulation by endothelium-dependent and-independent vasodilators.

PAD is characterized by impaired perfusion of the extremities caused by arterial stiffness that originates from atherosclerotic lesions in AS and non-atherosclerotic inflammation in BD [20, 26]. Reduced arterial perfusion causes poor quality of life due to limited functional performance, ischemic pain and ulceration of extremities. In the present study, besides the sufferings of a painful life, some of the PAD patients had ulcer, gangrene and amputation history. Various methods have been described for in vivo examination of arterial responses, one of which is the measurement of cutaneous microvascular perfusion during vasodilator iontophoresis [11]. Using this method, decreased microvascular perfusion has been observed in affected legs of PAD patients [13], and symptoms of PAD were reported to be correlated with vascular dysfunction of lower extremities [6]. In the present study, arterial function in the upper extremities of individuals was assessed by the same method. ACh-induced endothelium-dependent and SNP-induced endothelium-independent relaxations of the brachial microvascular bed in the PAD patients were lower than that of the control group in our study. These results have revealed impaired microvascular perfusion in the healthy upper extremity in PAD suggesting a systemic microvascular pathology. In addition, blood levels of hsCRP, HbA1c, cholesterol and HDL were correlated with both relaxation responses. These correlations are better taken into consideration for the diagnostic and therapeutic approaches to PAD.

Activity of vascular smooth muscle cells and blood cells is regulated by the endothelium via release of potent bioactive substances such as NO [24]. Under physiological conditions endothelial NO causes relaxation of smooth muscle, inhibition of platelet aggregation and regulation of the immune response. Bioavailability of endothelium-derived NO is associated with functional integrity of the endothelium. It has been hypothesized that vascular dysfunction in PAD originates from failure of endothelial functions [1 , 7]. Decreased synthesis or release of NO derived from the endothelial cell is correlated with endothelial dysfunction in PAD. It had been observed that reduced endothelium-dependent skin blood flow was related to failure of NO production in limb of PAD patients [12]. According to them,ACh-induced NO-mediated increases in forearm skin microcirculation were impaired in PAD patient in the present study.

On the other hand, great amounts of NO derived from inflammatory cells is hazardous and known to cause nitrosative stress under pathological conditions [16, 19]. Increased blood nitrite level and its negative correlation with flow-mediated dilation of brachial artery had been observed in PAD patients [8]. However, the correlation of blood NO level with ACh and SNP-induced microvascular flow increase had not been determined previously. The present study showed that elevated blood NO level in PAD patients was correlated with decreased ACh and SNP-induced microvascular perfusion of skin in the upper extremity. In addition, our results revealed correlation of NO level with glucose and hemoglobin A1c in plasma. Thus, we suggest that plasma NO level may give an idea about both the functional integrity of the microvascular bed and diabetic condition.

In addition, oxidative stress induced by elevated reactive oxygen species has been reported to be associated with arterial dysfunction in PAD [2 , 27]. Oxidative stress is not only detrimental on vascular smooth muscle cells, but also reduces NO bioavailability due to both removal by superoxide radicals and decreased NO synthesis/release from the endothelium. In the current study, plasma TAC levels in the PAD patients were not different from the controls, but were correlated with blood lipid profile, and uric acid level. An association of blood LDL level with oxidant status has been previously reported in PAD patients [10]. The relation of TAC to all lipids and uric acid was firstly obtained in the current study.

Another substance related to endothelial dysfunction is ADMA, an endogenous competitive inhibitor of NO synthase. Blood level of ADMA has previously been reported to be high in PAD [3, 14]. In the present study, the ADMA level in the PAD patients was not different from the controls, but was negatively correlated with homocysteine levels. ADMA level has previously been reported to be positively related to homocysteine in diabetic patients in PAD [17]. These discrepancies probably depend on differentiation of disease status and heterogeneity of our groups. Further studies with bigger homogenous subgroups are needed to better understand the relation of ADMA and relatedparameters.

H₂S is a newly reported gaseous signaling molecule associated with NO metabolism and oxidative stress in cardiovascular diseases [23]. In the present study, plasma H₂S level in the PAD patients was not different than the control group and there was no correlation with other markers. In a previous study increased levels of H₂S has been measured in PAD [22]. Because of the use of different experimental methods and different disease status of individuals, it is hard to compare our study with that data. Further studies are needed for the blood H₂S levels in PAD by using same method and standardized subgroup populations.

The results of this study indicate that systemic impairment of microvascular functions in PAD is correlated with increased plasma level of NO. Various biomarkers have been evaluated to aid in the diagnosis of PAD grade, progress, monitoring and therapeutic approach. Unfortunately, no biomarker to date has obtained sufficient value for the assessment of disease status. These findings may have important implications for consideration of predictive, preventive, diagnostic and therapeutic approaches to PAD.

Grants

This study was supported by a grant from The Ankara University Research Foundation (20030809171).

Disclosures

The authors declared no conflict of interest.

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