Alterations in skin microvascular function in patients with rheumatoid arthritis and ankylosing spondylitis

Abstract

OBJECTIVE: To evaluate the relationship between systemic inflammation and skin microcirculation in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS).

METHODS: We assessed skin microcirculation flux (laser Doppler flowmetry), classical cardiovascular risk factors, inflammatory markers and disease activity (Disease Activity Score 28, Bath Ankylosing Spondylitis Disease Activity Index) in 75 patients with arthritis with a median disease duration of 4 years, and in 26 healthy subjects.

RESULTS: In patients with arthritis inflammatory markers (C-reactive protein, interleukin 6, fibrinogen) were increased, peak flux velocity after the occlusion at the temperature of 36.6°C and maximal heat flux velocity after the heating were significantly lower. These findings were accompanied by the slower increase in the flux rate during local heating. There were positive correlations between inflammatory markers and microcirculation parameters in patients with RA and AS, but only for RA patients between peak flux velocity and disease activity. There were no significant intergroup differences when the classical cardiovascular risk factors were compared except for the lower HDL cholesterol in arthritis patients.

CONCLUSIONS: Patients with chronic systemic inflammatory arthritis presented altered microvascular function and reduced vasodilator capacity of the forearm skin microcirculation.

Keywords

Microcirculation rheumatoid arthritis ankylosing spondylitis laser Doppler flowmetry

1 Introduction

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and atherosclerosis are systemic chronic inflammatory diseases. Inflammation is important in the initiation and propagation of atherosclerosis by the modulation of traditional risk factors and directly by the effect on the vessel wall [40]. It has long been recognized that inflammatory rheumatic diseases are associated with the excess risk of cardiovascular (CV) diseases [10]. Cardiovascular events in RA and AS are the consequence of traditional [9, 22] and many other risk factors, in particular disease activity and its severity measures, including inflammatory markers, seropositivity, corticosteroid and nonsteroidal anti-inflammatory drugs [9, 14].

Skin microcirculation reflects the systemic microvascular function. Sax et al. found that coronary artery disease patients presented an impairment of vasodilator reserve affecting not only coronary circulation but also the peripheral arterial bed [37]. Of note, microvascular dysfunction is linked to CV risk factors such as hypertension and insulin resistance [38, 39].

Patients with RA, AS and systemic sclerosis have impaired microvascular function [3 , 8] and in patients with RA it may precede macrovascular disease [3]. In RA and AS there is an impaired vasodilatory response within forearm skin microcirculation [5 , 44]. Moreover, improvement in the microvascular function in both groups was demonstrated after the anti-inflammatory treatment with tumor necrosis factor-α (TNF-α) inhibitors [36, 44]. Circulating levels of C-reactive protein (CRP) and TNF-α are associated with skin microvascular dysfunction in healthy individualsas well [11, 21].

One of the methods used for the evaluation of skin microcirculation is laser Doppler flowmetry (LDF) evaluating changes in cutaneous perfusion due to post-occlusive and local thermal hyperemia. There are different mechanisms responsible for hyperemia during post-occlusive reaction and local heating, among which myogenic response, flow-induced vasodilation (in response to shear stress), metabolic and neural control have been considered [33]. Post-occlusive reactive hyperemia (PORH) with increase in blood flux after releasing the occlusion is thought to be endothelium-independent and influenced by many different mediators, such as local sensory nerves axon reflexes [26, 28]. On the other hand, thermal hyperemia consists of biphasic rise in skin blood flux with the first increase to a peak within first minutes, with a subsequent brief nadir followed by a progressive rise to a sustained plateau at about 20–30 minutes. The initial peak is mediated mainly by local sensory nerve axon reflexes [28] and the sustained plateau phase is considered to be endothelium-dependent and mediated by a local production of nitric oxide (NO) [28].

Data on the microvascular function assessed with LDF in patients with rheumatoid arthritis and ankylosing spondylitis are inconsistent. Therefore the aim of our study was to examine the skin microcirculation in disease-modifying anti-rheumatic drugs (DMARDs) naive patients with RA and AS in relation to the degree of systemic inflammatory activation and the disease activity: Disease Activity Score (DAS28) for RA and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) for AS.

2 Materials and methods

2.1 Study population

We studied 75 adult patients aged 20 to 56 years (mean age 36±9 years; 27 women and 48 men) with arthritis including 28 patients with RA and 47 patients with AS referred to the Outpatient Rheumatology Clinic of the Department of Internal Diseases in the University Hospital in Cracow. The diagnosis of RA was established according to the revised 1987 American College of Rheumatology (formerly the American Rheumatism Association) criteria [2] and the diagnosis of AS according to the modified New York criteria [43]. All patients with AS had predominantly axial disease (involvement of the spine and sacroiliac joints) and were HLA-B27 positive. The median duration of AS was 6 years with the range of 4 to 10 years, and the median duration of RA was 0.95 years with a range of 0.33 to 1.75 years. The patients have not been treated with any biologic or non-biologic DMARDs, have been receiving a stable dose of nonsteroidal anti-inflammatory drugs (NSAIDs) and/or glucocorticoids for at least 4 weeks prior to the enrollment in our study, or have been without any anti-inflammatory treatment (neither NSAIDs nor glucocorticoids). Treatment with DMARDs was given to all patients with diagnosis of RA according to current guidelines directly after completion of the study protocol. Disease activity was assessed in both groups. In RA patients we calculated DAS28 (see Appendix 1) based on high-sensitivity C-reactive protein (hsCRP), the tender joint count (28 joints), the swollen joint count (28 joints), and the patient’s assessment of global well-being (100 mm visual analogue scale, VAS) [32]. DAS28 values in the range of >2,6 and ≤3,2 indicate low disease activity; >3,2 and ≤5,1 –moderate disease activity; >5,1 –high disease activity and ≤2,6 remission. In AS patients we calculated BASDAI score (see Appendix 2) [16], based on the severity of main symptoms of AS such as level of fatigue, duration of morning stiffness, its severity, level of neck, back and hip pain, level of pain/swelling in joints other than neck, back, hips and the level of discomfort from any areas tender to touch or pressure. BASDAI values ≥4 indicate active disease. As a control group we studied 26 healthy subjects (mean age 31±7.5 years; 12 women and 14 men). Exclusion criteria were the same for both groups and included: clinical evidence of atherosclerotic CV disease, uncontrolled/untreated hypertension, diabetes, renal failure, chronic or acute inflammatory diseases, a history of neoplastic diseases and current therapy with DMARDs.

2.2 Study protocol

Patients were recruited from July 2009 to June 2011. The study protocol has been described in details previously [24]. The protocol was approved by the Bioethical Committee of Jagiellonian University and written informed consent was obtained from all participants.

2.3 Laboratory tests

Blood samples were taken from the left antecubital vein. Serum lipid profile [total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triglycerides], and glucose levels were measured with the Hitachi 917 analyzer (Roche Diagnostics, Hitachi Ltd., Japan) and complete blood count was analyzed with SYSMEX K 800 analyzer (Sysmex, Kobe, Japan), using standardized laboratory techniques. The erythrocyte sedimentation rate (ESR) was determined in whole blood. High-sensitivity C-reactive protein (hsCRP) was measured with immunonephelometry (Nephelometer BM II, Siemens Healthcare Diagnostics Inc., USA). Plasma fibrinogen was measured with an BCS analyzer (Siemens Healthcare Diagnostics Inc., USA). Plasma for additional biochemical analyses was separated and frozen at –70°C until being assayed. Cytokines were measured using enzyme-linked immunosorbent assays (ELISA) kits: IL-6 (human IL-6 Immunoassay Quantikine HS) and TNF-α (human TNF-α Immunoassay Quantikine HS). In RA patients, rheumatoid factor was determined by immunoturbidimetric assay (APTEC Diagnostics nv., ALLmed Diagnostics) and anti-cyclic citrullinated peptide antibodies (aCCP) with ELISA (QUANTA Lite CCP 3.1 IgG/IgA ELISA, INOVA Diagnostics, Inc., San Diego, USA).

2.4 Laser Doppler flowmetry (LDF)

Skin microcirculatory blood flux was measured according to the procedure that we have described in details previously [18], using PeriFlux laser Doppler flowmetry (LDF) (Periflux System 5000; Perimed, Jarfalla/Stockholm, Sweden) with the probe attached to the palmar surface of the right forearm at the distance of 10 cm from the elbow flexion. The study was performed in the morning in a quiet room at the stable temperature of 22–24°C. The patients were asked to refrain from eating, drinking coffee and smoking prior to the test and they were remaining in a steady, supine position during the whole study. All results were recorded, stored and analyzed afterwards with signal processing software (Perisoft 2.5.5, Perimed). The recording was preceded by 10 minutes period of stabilization of the record. The study protocol included post-occlusive reactive hyperemia (PORH) and local thermal hyperemia (LTH). The day-to-day reproducibility of PORH and LTH has been demonstrated previously [17]. The values of blood flux were expressed as absolute values [arbitrary units –AU] and as an index of cutaneous vascular conductance (CVC) evaluated as flux divided by mean arterial pressure (MAP) [AU/mmHg], calculated from blood pressure measurements in a sitting position. We included CVC considering the influence of MAP on the blood flux in the microcirculation. It is difficult to estimate MAP on the level of microcirculation, therefore we used the values of MAP calculated from the blood pressure measurements on the level of brachial artery, although those values may be higher than on the capillary level. Blood pressure was measured 3 times on the left arm after 5 minutes of a rest in a sitting position; values from 2 last readings were averaged [30]. MAP was calculated using the equation: [diastolic blood pressure +1/3 (systolic blood pressure – diastolic blood pressure)].

2.4.1 Post-occlusive reactive hyperemia (PORH)

We analyzed following parameters: resting flux (RF), area under the curve of the occlusion phase (AO), peak flux (PF), time of rise to peak flux (TM), time of return to resting flux (TR), the area under the curve of the post-occlusive phase (AH). Hyperemia was expressed as peak flux (PF) and as a relative change between peak flux and resting flux expressed as a percentage, calculated according to the formula [PF% RF = (peak flux – resting flux/resting flux)×100% ] and as an area under the curve (AH). We also calculated AH/AO ratio as a marker of response intensity. Reactive hyperemia index (RHI) was calculated according to the formula RHI = (peak flux/resting flux)×100% [% ]. Peak flux velocity (PFV) – the velocity of achieving peak flux after the occlusion at the basic temperature (36,6°C), was calculated according to the formula PFV = peak flux(PF)/time to peak(TM) [AU/s]. The recordings of LDF were analyzed semi-automatically with the Perisoft software.

2.4.2 Local thermal hyperemia (LTH)

The temperature of heating was 44°C. This phase was prolonged to twenty minutes in comparison to the procedure described previously [18], because it allowed the calculation of the area under the curve of maximal hyperemia. We analyzed parameters such as: average heat flux (HF), maximal heat flux (MHF) – the initial peak within the first five minutes, time of rise to maximal heat flux (TM_HF), heat slope (HS) and area under the curve of maximal hyperemia phase. Thermal stimulation ratio (TSR) was calculated according to the formula TSR = HF/RF×100% [% ]. Maximal heat flux velocity (MHFV), which is the velocity of achieving maximal flux after the heating, was calculated according to the formula MHFV = maximal heat flux(MHF)/time to maximal heat flux (TM_HF) [AU/min].(Fig. 1).

2.5 Statistics

Data are reported as means (SD) or medians (interquartile range, IQR) unless otherwise indicated. Proportions were compared using chi-square test or Fisher’s exact test. The accordance with a normal distribution was tested by Shapiro-Wilk and Kolmogorov-Smirnov tests. For intergroup comparisons between patients with arthritis and control subjects, Student’s t -test or Mann-Whitney test was used where appropriate.

General Linear Models (type III sum of square) were used for age adjusted comparisons between the controls and subjects with arthritis (RA and AS as a one group). Homogeneity of variances was verified by Levene’s test. The Bonferroni test was performed in post-hoc comparisons. In case of the non normal distribution Kruskal– Wallis test was used.

When patients with RA and AS were analyzed separately also General Linear Models (GLM) procedure was used both for comparisons of three groups (RA patients, AS patients, control subjects) and age adjusted comparisons between the controls and subjects with arthritis (type III sum of square).

Spearman rank correlations were applied to test associations between systemic inflammatory markers (ESR, hsCRP, TNF-α, and IL-6) and parameters of skin microcirculation (both PORH and LTH) in patients with arthritis, as well as between skin microcirculation and disease activity based on DAS28 and BASDAI, in patients with RA and AS respectively.

3 Results

The characteristics of the patients with arthritis (rheumatoid arthritis and ankylosing spondylitis) and control subjects are presented in Table 1. Patients with arthritis in comparison to control subjects were older and the results were adjusted for age. We did not find any significant differences in most of the traditional cardiovascular risk factors (i.e. sex, smoking habit, blood pressure, BMI, glucose, TC, LDL-C and triglycerides levels) between the groups, with the exception of HDL, which was lower in patients with arthritis than in the control group. In addition patients with arthritis had significantly higher white blood and platelet counts and lower hemoglobin level. Regarding inflammatory markers, patients with arthritis had elevated levels of fibrinogen (4.26±1.4 vs 2.98±0.7 g/L; p < 0.001), ESR (29.97±24.76 vs 6.42±4.81 mm/h; p < 0.001), hsCRP (12.98±18.59 vs 0.86±0.75 mg/L; p = 0.007) and IL-6 (6.34±6.69 vs 0.86±0.42 pg/mL; p < 0.001). TNF-α was comparable between patients with arthritis and the control subjects (2.42±2.50 vs 1.81±1.11 pg/mL; p = 0.166). RA patients had moderate disease activity (DAS28 : 4.5±1.54). AS patients had active disease (BASDAI = 4.05±2.0). Rheumatoid factor was present in 89% and aCCP in 85% RA patient. Three patients had controlled hypertension. 57 patients (83%) were receiving NSAIDs and 14 patients (20%) – glucocorticoids.

We noted lower peak flux velocity (PFV) during post-occlusive reactive hyperemia (PORH) (Table 2), lower maximal heat flux velocity (MHFV) and heat slope (HS) during local thermal hyperemia (LTH) (Table 3) in patients with arthritis. The cutaneous vascular conductance of maximal heat flux (MHF_CVC) was also decreased in this group. Other analyzed parameters of flux during PORH and LTH were comparable in the study and in the control subjects.

The analysis of microcirculation parameters and inflammatory markers performed separately in RA and AS patients showed similar results (Table 4). In comparison with healthy subjects, patients with both types of arthritis demonstrated lower peak flux velocity (PFV) during post-occlusive reactive hyperemia (PORH) and lower heat slope (HS) during local thermal hyperemia (LTH). Additionally, patients with arthritis (both RA and AS) had higher levels of inflammatory markers (ESR, hsCRP, IL-6, fibrinogen) compared to control subjects except for TNF-α. Patients with RA had elevated ESR and IL-6 in comparison to patients with AS.

We found positive significant (p < 0.05) correlations between parameters of microcirculation flux and markers of inflammation in patients with arthritis. ESR was correlated with changes of peak flux expressed as a percentage from baseline (PF% RF) (r = 0.247) and reactive hyperemia index (RHI) (r = 0.247). Furthermore, IL-6 was correlated with cutaneous vascular conductance of peak flux (PF_CVC) (r = 0.256) and there was a positive correlation between PFV and disease activity (DAS28) in patients with RA (r = 0.484).

4 Discussion

Our results indicate altered function and reduced vasodilator capacity of the forearm skin microcirculation in patients with chronic arthritis and its positive correlation with inflammatory parameters. We found significantly lower velocity of achieving maximal flux after the occlusion at the basic temperature (PFV) and after the heating (MHFV) accompanied by diminished heat slope (HS) indicating slower increase in flux rate over the time during local heating.

Traditional risk factors were comparable in patients with arthritis and healthy controls with the exception of decreased level of HDL in arthritis patients. The lower level of HDL may be associated with increase in disease activity as described previously in patients with AS [46] and furthermore, as a cardiovascular risk factor it may have the influence on vascular system. Low HDL cholesterol may be associated with diminished endothelium-dependent vasodilation and capillary recruitment [20]. However, other studies indicate that there is no relationship between nitric oxide (NO)-dependent vasodilation during local thermal hyperemia and HDL concentration [23]. Patients with arthritis were smoking more frequently in comparison to control subjects (33% vs 15% respectively), and though the difference was not significant it could have had the influence on microvascular function. Cigarette smoke may induce a significant reduction in capillary blood flux velocity and elongation of time to peak [19].

We analyzed all patients with arthritis as a one group, although the inclusion of patients with RA and AS in one group may be questionable due to the differences in the pathogenesis of the two diseases. However, chronic inflammation is present in both AS and RA. There was an increase in inflammatory markers in both groups (ESR, hsCRP, IL-6, fibrinogen). The results were similar when these two groups were analyzed separately, and as a one group of arthritis patients. The duration of symptoms in AS was longer (median 4 years) but the early diagnosis of AS is difficult due to the insidiously progressive nature of AS and long delay between onset of symptoms and diagnosis [12]. Although patients with RA were older than AS subjects, the symptoms duration in RA was shorter than in AS. While the evidence for higher CV risk is less evident for AS than RA, European League Against Rheumatism (EULAR) applies comparable recommendations for CV risk management for RA andAS patients.

The results of the studies on skin microcirculation in patients with AS or RA are inconsistent, probably due to different methods used for the evaluation of microcirculation and diversity of studied groups. Endothelial microvascular function may be estimated during LDF using reactivity tests such as mechanical stimuli during occlusion of the artery, thermal provocation or local administration of pharmacological agents. After iontophoresis of acetylcholine and sodium nitroprusside, endothelial microvascular dysfunction was observed in AS patients with active disease, elevated inflammatory markers and long disease duration [44]. Endothelial microvascular dysfunction was also present in RA patients with shorter disease duration and moderate disease activity [44] and in patients with chronic RA [13]. On the contrary, patients with newly diagnosed, moderately active, DMARDs-naive RA with low systemic inflammatory activity [45] and those with longer disease duration but with low disease activity and low levels of systemic inflammatory markers had preserved skin microvascular function [35]. These data indicate that the intensity of systemic inflammation, disease duration and activity are probably important determinants of skin microcirculation function in patients with inflammatory rheumatologic diseases.

Our results showing positive correlations between the inflammatory markers and blood flux during PORH, and for the RA – between disease activity and blood flux during PORH are in accordance with the previously published data. Galarraga et al. showed lower vasodilator responses to acetylocholine in RA patients with higher CRP levels compared to those with lower CRP [15]. Van Eijk et al. demonstrated impaired microvascular function in patients with AS, and its improvement after TNF-α inhibitors [44]. Circulating levels of inflammatory markers (CRP and TNF-α) are associated with skin microvascular dysfunction even in healthy individuals [11, 21]. Nevertheless, in another study endothelium-independent microvascular dysfunction correlated negatively with markers of inflammation, and it was not improved by short-term anti-inflammatory therapy [13]. Furthermore, there was no relationship between markers of inflammation, disease activity or disease duration and microvascular endothelium-dependent or endothelium-independent function in RA patients [36]. The results of previous studies on the inflammation and microvascular function are inconsistent, presumably due to multiple factors contributing to the alterations in the skin microcirculationin arthritis.

The changes in skin microcirculation might as well be related to other factors, such as endothelium dysfunction and medications. We have previously shown that some markers of endothelial activation were elevated in RA patients [24]. Post-occlusive reactive hyperemia might be associated with endothelial dysfunction via cyclooxygenase-dependent pathways and EDHF (endothelium-derived hyperpolarizing factor) effects [27, 34]. About 83% of the patients were taking NSAIDs, which may alter skin microvascular function. It has been shown previously that forearm muscle and skin reactive hyperemia may be reduced by pharmacological inhibition of cyclooxygenase [1]. Indomethacin was reported to decrease the total blood flux response to occlusion during post-occlusive reactive hyperemia in humans [26]. The impaired recruitment of non-perfused capillaries during stimulation as a result of endothelial dysfunction may be another factor contributing to the diminished peak flux velocity during post-occlusive hyperemia and maximal heat flux velocity during local thermal hyperemia [29]. Reduced postischaemic capillary recruitment have been already observed in patients with longstanding AS [44]. On the other hand, in patients with newly diagnosed RA with low systemic inflammatory activity capillary density and capillary recruitment are similar to control subjects [45], which may suggest their association with disease duration.

We did not evaluate the influence of endothelial dysfunction on skin microcirculation by recording the flux during the sustained phase of thermal hyperemia, because of relatively short time of the recording of thermal hyperemia in the study. We measured the area under the curve during local heating, that has been used previously by Kruger et al. as an indicator of endothelial dysfunction [25], and we found no significant differences between the groups. Assuming comparable endothelial function in arthritis and controls, observed changes might suggest involvement of other mechanisms in the impairment of skin microcirculation in patients with arthritis, such as neural control. Both reactivity tests used in the study (post-occlusive reaction and the initial peak of thermal hyperemia, expressed in our study as a maximal heat flux) are also dependent on axon reflexes. The changes in the skin microcirculation observed in our study may have hypothetically resulted from autonomic neuropathy, that has been reported previously in rheumatoid arthritis [4, 41] and ankylosing spondylitis [6, 42]. However, the presence of autonomic neuropathy was not tested in our study.

5 Limitations of the study

Our study has a few limitations. First of all, our conclusions are constrained by low number of participants and a cross-sectional design of the study that makes it impossible to follow changes in markers of inflammation and skin microvascular function in the course of disease and after starting DMARDs. Second, NSAIDs could have had the influence on skin microvascular function in the patients with arthritis. Finally, we used values of blood pressure measured in a sitting position to estimate of CVC in microcirculation and the position affects blood pressure. However, the differences of mean blood pressure values in supine and sitting position are rather small, since diastolic pressure is lower and systolic pressure is higher when measured in a supine position [30, 31].

6 Conclusions

In conclusion, patients with arthritis present altered skin microvascular function related to the intensity of systemic inflammation. These changes might be the result of different factors including impaired neural vascular control mechanisms, capillary rarefaction or endothelial dysfunction.

7 Perspectives

The alterations in skin microvascular function in patients with early phase of inflammatory arthritis (RA and AS) suggest impaired vasodilator capacity of the forearm skin microcirculation and may result from disturbances in neural control. Whether these changes might predict the autonomic dysfunction, that may be responsible for increased mortality due to cardiovascular diseases in patients with inflammatory arthritis, remains to be elucidated. Therefore, the assessment of skin microcirculation seems to be promising method for evaluating patients with early phase of inflammatory diseases.

Footnotes

Appendix 1.

Appendix 2. Acknowledgments

This study was supported by a Research Grant from the Ministry of Science and Higher Education, Warsaw, Poland (no. NN402267636).

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