Disruption of endothelial glycocalyx in patients with rheumatoid arthritis

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BACKGROUND

Endothelial dysfunction is a key link in the pathogenesis of rheumatoid arthritis (RA) [7]. Endothelial glycocalyx (EGC), which is a thin dynamic layer of macromolecules (glycoproteins, proteoglycans, and glycosaminoglycans) on the vascular endothelium surface, plays an important role in the endothelium function regulation [6]. Acting as a biological barrier, it is the first to be damaged in the development of autoimmune diseases. The thinning of EGC impairs the barrier function of the vessels, which results in their increased permeability, increased platelet and leukocyte adhesion to the vascular wall, and changed microvasculature perfusion. All this leads to inflammatory process development [2, 4]. Despite its instability, EGC thickness measurement can be used to diagnose and monitor diseases [2]. Dark-field microscopy allows non-invasive and in vivo EGC assessment, with moderate reproducibility, which increases with repeated measurements, as well as insignificant daily variability [3, 5].

This study aimed to establish the relationship between EGC thickness and activity, serological profile, and risk factors for an unfavorable course of RA.

MATERIALS AND METHODS

The study included 76 patients aged 18 to 65 years (13 males and 63 females) with diagnosed RA according to the American College of Rheumatology and the European League Against Rheumatism criteria in 2010 [1]. The median age was 56 years (49–62.5 years). Rheumatoid factor positivity was established in 74.6% of patients and that for cyclic citrullinated peptide in 79.4% of cases. The study was conducted twice, in the first days after the patient’s hospital admission and before discharge on average of after 9 days (8–11 days), to assess the effect of the therapy on the vascular endothelium. All patients received study-independent treatment according to current clinical guidelines and standards of specialized care.

The main indicator for assessing the EGC state was the size of its perfused boundary region (PBR) determined by the depth of erythrocyte immersion into the EGC thickness in microvessels with an internal diameter of 5–25 μm (PBR_5–9, PBR_10–19, PBR_20–25, and PBR_5–25, respectively). An increased PBR reflects a deeper immersion of erythrocytes into the EGC thickness, which is interpreted as to its thinning [3]. A dark-field video microscope with LED illumination in the green region of the spectrum (KK Research Technology Ltd, Great Britain) and GlycoCheck software (Glycocheck BV, the Netherlands) was used to measure the PBR. The video microscope was placed on the sublingual mucosa of the oral cavity. The analyzed indicators were automatically calculated using the software algorithm. The quantitative measurement of circulating syndecan-1 using the Human Syndecan 1 enzyme-linked immunoassay Kit (RayBiotech Inc., USA) was used as a laboratory marker of EGC damage.

Statistical data processing was performed using the International Business Machines Corporation Statistical Package for the Social Sciences version 26 program. The norm of distribution of indicators in the sample was determined using the Kolmogorov–Smirnov test with the Lilliefors correction, as well as the values of kurtosis and skewness, depending on which parametric or nonparametric methods were used.

The paired t-test or the Wilcoxon test was used to compare related populations and the Spearman coefficient for correlation analysis. Discriminant analysis was performed. Statistical significance was determined at p < 0.05. The clinical study was approved by the First Pavlov Saint Petersburg State Medical University (minutes No. 11/2019 dated December 28, 2019). All participants provided informed voluntary consent in advance.

RESULTS

The relationship between the PBR value and the serological profile and risk factors for an unfavorable course of RA was established (Table).

 

Table. Correlation between the size of the perfused boundary region of endothelial glycocalyx and the serological profile and risk factors for an unfavorable course of rheumatoid arthritis / Таблица. Взаимосвязь величины пограничной области перфузии эндотелиального гликокаликса c серологическим профилем и факторами риска неблагоприятного течения ревматоидного артрита

PBR, µm	Indicator analyzed		p level
	absent	present
PBR5–9*	1. Positivity for cyclic citrullinated peptide		p = 0.017
	1.16 ± 0.03	1.07 ± 0.02
PBR5–25*	2. Compromised history of rheumatic diseases		p = 0.014
	2.02 ± 0.06	2.20 ± 0.04
PBR5–25	3. Large joint involvement during rheumatoid arthritis in the first 2 years after the onset		p = 0.037
	2.07 ± 0.06	2.13 ± 0.03
PBR5–9	4. The need for genetically engineered biological therapy due to rheumatoid arthritis history		p = 0.035
	1.11 ± 0.01	1.21 ± 0.04
PBR5–25*	5. Osteopenia or bony rarefication		p = 0.011
	2.05 ± 0.05	2.25 ± 0.06
PBR5–25*	6. Erosive rheumatoid arthritis		p = 0.027
	1.93 ± 0.05	2.11 ± 0.06

Note: PBR — perfused boundary region; * PBR values after hospital treatment at the repeated examination.

 

A statistically significantly direct correlation between the PBR_5–25 value and the level of syndecan-1 (r_xy = 0.487; p = 0.012) and erythrocyte sedimentation rate (r_xy = 0.378; p = 0.002) was established (Fig. 1, 2).

 

Fig. 1. Correlation between PBR_5–25 and erythrocyte sedimentation rate. PBR — perfused boundary region / Рис. 1. Корреляционная связь PBR_5–25 и скорости оседания эритроцитов. PBR — пограничная область перфузии

 

Fig. 2. Correlation between PBR_5–25 and syndecan-1. PBR — perfused boundary region / Рис. 2. Корреляционная связь PBR_5–25 и уровня синдекана-1. PBR — пограничная область перфузии

 

The dependence of PBR_5–25 on the treatment response, estimated by the DAS28 C-reactive protein index (p = 0.019), was revealed. The mean PBR_5–25 value was 2.27 ± 0.23 in the absence of treatment response, whereas 1.99 ± 0.32 µm in cases of a satisfactory response. Differences between groups with satisfactory and good treatment responses were not statistically significant (p = 0.469).

The relationship between the PBR value and the need to use methylprednisolone pulse therapy in a hospital was analyzed. The PBR_5–25 value in the group with subsequent pulse therapy was higher than those without it (p = 0.001) (Fig. 3).

 

Fig. 3. Differences in PBR5-25 depending on subsequent methylprednisolon pulse therapy. PBR — perfused boundary region / Рис. 3. Различия PBR_5-25 в зависимости от последующего проведения пульс-терапии метилпреднизолоном. PBR — пограничная область перфузии

 

A discriminant analysis was performed to clarify the effect of baseline PBR on the probability of pulse therapy. The following model was obtained:

y = –9.107 + 3.831x,

wherein y is a discriminant function that characterizes the probability of pulse therapy and x is the PBR_10–19 value. The discrimination constant was 0.156. Wilk’s λ coefficient was used to establish statistically significant differences (p = 0.020). The model sensitivity was 68.2% and the specificity was 69%.

DISCUSSION OF RESULTS

As of September 2021, any original publications on the study of EGC in RA using dark-field microscopy were not found.

Our study results revealed the relative EGC thinning in females and patients with risk factors for an unfavorable course of RA. The relationship of this indicator with the serological profile of the disease was not revealed.

The inverse correlation between EGC thickness and syndecan-1 levels and erythrocyte sedimentation rate confirms the involvement of EGC in the inflammatory process in RA. The EGC evaluation using dark-field microscopy predicted the subsequent prescription of methylprednisolone pulse therapy to patients in the hospital.

Upon repeated examination, EGC thinning was noted in the presence of risk factors for an unfavorable course of RA, negativity for cyclic citrullinated peptide, or lack of treatment response.

CONCLUSION

Dark-field microscopy enables in vivo and non-invasive EGC thickness assessment, thus providing important information both from a fundamental point of view, advancing in knowledge about the pathological disease physiology, and from a clinical point of view, which enables the consideration of EGC thinning as a marker of an unfavorable course of RA.

ADDITIONAL INFORMATION

Funding. The study had no external funding.

Conflict of interest. The authors declare no conflict of interest.

All authors made a significant contribution to the study and article preparation, read and approved the final version before its publication.

About the authors

Daniel A. Shimanski

First Pavlov State Medical University of Saint Petersburg

Author for correspondence.
Email: shimanskidaniel@gmail.com
ORCID iD: 0000-0002-6903-2217
SPIN-code: 2022-5223
ResearcherId: AAW-1441-2020

post-graduate student of the Department
of Hospital Therapy with a course of Allergology and Immunology named after acad. M. V. Chernorutsky with a clinic

Russian Federation, 6/8 Lva Tolstogo St., Saint Petersburg, 197022

Irina I. Nesterovich

First Pavlov State Medical University of Saint Petersburg

Email: nester788@gmail.com
SPIN-code: 8921-1751

MD, Dr. Sci. (Med.)

Russian Federation, 6/8 Lva Tolstogo St., Saint Petersburg, 197022

Oksana V. Inamova

Clinical Rheumatology Hospital No. 25

Email: b25@zdrav.spb.ru
ORCID iD: 0000-0001-9126-3639
SPIN-code: 8841-5496

MD, Cand. Sci. (Med.)

Russian Federation, Saint Petersburg

Vasilii I. Trophimov

First Pavlov State Medical University of Saint Petersburg

Email: trofvi@mail.ru
SPIN-code: 1306-5645

MD, Dr. Sci. (Med.), Professor

Russian Federation, 6/8 Lva Tolstogo St., Saint Petersburg, 197022

Olga V. Galkina

First Pavlov State Medical University of Saint Petersburg

Email: nephrolog1985@gmail.com
ORCID iD: 0000-0001-7265-7392
SPIN-code: 4251-6056

Cand. Sci. (Biol.), Assistant Professor

Russian Federation, 6/8 Lva Tolstogo St., Saint Petersburg, 197022

Elena N. Levykina

First Pavlov State Medical University of Saint Petersburg

Email: nephrolog1985@gmail.com
ORCID iD: 0000-0001-8024-2904
SPIN-code: 9550-6270

a researcher of biochemical homeostasis laboratory of Nephrology Research Institute

Russian Federation, 6/8 Lva Tolstogo St., Saint Petersburg, 197022

Timur D. Vlasov

First Pavlov State Medical University of Saint Petersburg

Email: tvlasov@yandex.ru
ORCID iD: 0000-0002-6951-7599
SPIN-code: 8367-1246

MD, Dr. Sci. (Med.), Professor

Russian Federation, 6/8 Lva Tolstogo St., Saint Petersburg, 197022

References

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