Age-Related Immunophenotypic Characteristics of Perivascular Mesenchymal Stem Cells in Patients with Heart Defects

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INTRODUCTION
Most human blood vessels are surrounded to varying degrees by perivascular adipose tissue (PVAT) [1]. Previously, it was believed that the function of PVAT in the cardiovascular system (CVS) was solely to create a framework, structural support for vessels, but recent studies have shown that PVAT is closely involved in the modulation of vascular homeostasis and vascular dysfunction associated with cardiometabolic diseases [2]. PVAT is structurally and functionally associated with the adventitia of arteries of various diameters, with the exception of cerebral arteries [3]. It is a connective tissue consisting of adipocytes, preadipocytes, mesenchymal stem cells, fibroblasts, immune cells (macrophages, lymphocytes and eosinophils), contains blood vessels and autonomic nerve fibers [4]. It is known that the active contribution of PVAT to the regulation and maintenance of vascular homeostasis is associated mainly with its endocrine functions [5]. However, stromal progenitor cells were also found in PVAT, which are believed to be able to provide vascular wall regeneration. In studies by Crisan M. et al., it was first shown that perivascular MSCs are present in many organs and tissues, such as skeletal muscles, pancreas, adipose tissue, and placenta, including those that lack expression of hematopoietic, endothelial, and myogenic cell markers. They also identified the multilineage differentiation potential of these cells, including osteogenic, chondrogenic, and adipogenic, but the question of the relationship between perivascular MSCs and vascular remodeling remained open [6]. G. Lin et al. also managed to identify perivascular adipose tissue-derived MSCs, where they apparently expressed both CD34 and smooth muscle actin. To date, the existence of perivascular adipose tissue-derived MSCs is beyond doubt, but their functions and properties remain poorly understood. This makes it important to study the characteristics of perivascular adipose tissue-derived MSCs to understand their contribution to the development of cardiovascular diseases (CVD) [7].
As is known, one of the main non-modifiable risk factors for CVD is age [8]. MSCs, like other cells in the body, are subject to age-related changes, which consist of the accumulation of free radicals, genetic damage, and other processes that lead to disruption of intracellular homeostasis, regenerative and proliferative capacity of the cell [9]. Numerous studies show that MSCs of various localizations obtained from elderly donors have a lower proliferation rate, limited potential for multilineage differentiation, and increased expression of apoptosis genes compared to MSCs from young donors [10, 11]. Since PVAT makes a significant contribution to maintaining cardiovascular homeostasis, it is reasonable to assume that age-related changes in PVAT MSCs may affect the development or course of CVD. To date, there are few studies devoted to the study of age-related characteristics of perivascular MSCs, in contrast to subcutaneous adipose tissue, which is probably explained by the limitations associated with exclusively intraoperative collection of material for research.

TARGET

To understand the contribution of PVAT-MSCs to the development of cardiovascular diseases, it is necessary to study their morphological and functional characteristics, so the purpose of this work was to study the morphological features and immunophenotype of PVAT-MSCs in pediatric and elderly patients with heart defects of various etiologies.
MATERIALS AND METHODS
The study included 16 patients, who were divided into 2 groups: 1st - 8 patients aged 3-6 years with congenital heart defects (ventricular septal defect), 2nd - 8 patients with degenerative acquired non-rheumatic heart defects (aortic stenosis/insufficiency) and aged 60 to 75 years. The examined adult patients signed voluntary informed consent, children were included in the study with the written consent of their parents or other legal representatives. The study protocol was approved by the local ethics committee of the Federal State Budgetary Scientific Institution, the Research Institute for Complex Issues of Cardiovascular Diseases. All patients had indications for open cardiac surgery - heart valve surgery. The study did not include patients with clinically significant concomitant pathologies (diabetes mellitus types 1 and 2, myocardial infarction (MI), anemia, renal and hepatic insufficiency, oncological and infectious and inflammatory diseases in the acute stage, autoimmune diseases).

Isolation of adipose tissue mesenchymal stem cells
MTC-AT were isolated from perivascular AT biopsies (3-5 g). PVAT in patients of the first group was taken from the ascending aorta, in patients of the second group - from the right coronary artery. The obtained AT samples were thoroughly washed with sterile phosphate-buffered saline (PBS) (Gibco, USA) to clear the AT surface from thrombi, erythrocytes and local anesthetics. Then the AT was placed in 20 ml of PBS with the addition of penicillin (600 U/ml) (Gibco, USA) and streptomycin (300 mg/ml, Gibco, USA) in a 50 ml test tube for 5-10 min at room temperature to remove residual blood vessels, connective tissue and/or dermis of the AT. After repeated washing and purification, the AT was transferred to a 10 cm diameter culture dish with the addition of 2 ml of PBS and cut with scissors into irregularly shaped fragments of 1–3 mm2 in size, which were pipetted into 25 cm2 culture flasks (Biologix, Germany) and aligned at intervals of 0.3–0.4 cm. The cells were incubated in a CO2 incubator (5% CO2, 95% air, 37ºC), in a medium supporting the growth of MSCs (MesenCult Proliferation Kit, STEMCELL Technologies, Canada) with the addition of antibiotics and antimycotics (100 U/ml penicillin, 100 U/ml streptomycin, 0.4% amphotericin B, Gibco, USA). When the primary cells reached 80–90% confluency, they were treated with 0.25% trypsin solution containing 0.02% EDTA (Tripsin/EDTA, CELL, USA), transferred to 75 cm2 culture flasks (Biologix, Germany) and cultured to 80–90% cell confluency. Then, the cells were counted using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific, Finland) and immunophenotyping of the cells from the 2nd to the 4th passage was performed.

Cell immunophenotyping (flow cytometry)
The cell suspension of AT-MSCs from passages 2–4, collected using 0.25% trypsin/EDTA, was centrifuged at 100×g for 5 minutes. For staining, 1×105 culture cells removed from plastic and washed with PBS were collected in test tubes. A combination of conjugated monoclonal antibodies was used in the work: CD90 FITC (Biolegend, 328108), CD 34 APC (Biolegend, 343520), CD73 APC Cy7 (Biolegend, 344022), CD 105 PE (Biolegend, 323206), APC HLA-DR (Biolegend, 327022). Antibodies were added to the sample in the volume specified by the manufacturer, with subsequent incubation for 30 min at room temperature in a place protected from light. Stained samples were resuspended in PBS and analyzed on a CytoFlex flow laser cytometer (USA) in the CytExpert 2.1 program. To set up the instrument, samples with appropriate isotype controls were used, and all steps were then performed similarly to the main sample. All samples were analyzed using the same instrument settings. The gating strategy consisted of preliminary removal of cell doublets and debris, sequential selection of the gate on the FSC-H/FSC-A histogram with subsequent transfer to the FSC/SSC histogram and gating of the area of ​​interest. In this gate, the cells were divided into populations depending on the presence of CD90 on their membrane (CD90+ and CD90−). In each of these populations, the expression of CD73, CD105, CD34, HLA DR was studied.
Statistical processing
Statistical processing of the data was performed using the standard statistical methods package of the StatSoft STATISTICA 10 program. The data are presented as the median and 25-75 percentile (Me [25; 75%]). The nonparametric Friedman criterion was used to assess the differences in quantitative characteristics when comparing two or more dependent groups with a distribution different from normal. The critical significance level is "p" <0.05.

RESULTS
The stromal-vascular fraction of adipose tissue taken from the perivascular area in patients of different age groups was cultured under standard cell conditions. On the 3rd-5th day of primary culture, many small spotted cells appeared along the edges of adipose tissue fragments in all flasks, which continued to grow and proliferate. By the 10th day, the cells cultured in vitro began to acquire a fibroblast-like and venter-like shape and remained so throughout the entire cultivation period (passage 2-4) (Figure 1).
Then, the immunophenotype of cells isolated from the stromal-vascular fraction of PVAT in patients of different age groups from the 2nd to the 4th passage was assessed (Table 1).

Table 1. Immunophenotypic characteristics of cells isolated from perivascular adipose tissue in patients of different age groups

Cell populations isolated from PVАT	Group 1			Р	Group 2			P
	Passage number				Passage number
	2	3	4		2	3	4
Proportion of cells with phenotype CD90+73+/−105+/−34− HLA DR− (Ме, [25, 75] %)	47,97 (43,2-49,3)	95,87 (90,1-100)	98 (94,2-101)	< 0,01	95,98 (90,1-102)	98,4 (96,1-103,6)	44,59 (39,1-49)	< 0,01
Proportion of cells with phenotype CD90−73+/−105+/−34− HLA DR− (Ме, [25, 75] %)	50,87 (43,6-54,2)	0,83 (0,4-1,6)	1,57 (1-2,3)	< 0,01	4,02 (3,59-4,6)	0,81 (0,2-1,3)	54,9 (50,1-62)	< 0,01

Note: group 1 (age from 3-6 years), group 2 (age from 60-75 years), PVAT-perivascular adipose tissue, p – level of significance when comparing dependent variables (<0.05)

When assessing the immunophenotype of the second passage cell cultures, it was found that in the PVAT of patients in the first age group there are two cell populations: CD90+ CD73+ CD105+ CD34− HLA DR− and CD90− CD73+/− CD105+/− CD34−, HLA DR−. The first population, characteristic of MSCs, accounted for 47.97% of the cells, the second population CD90− 73+ CD105+ CD34− HLA DR− - 50.87%. With an increase in the number of passages, the number of cells with a phenotype characteristic of MSCs increased (p <0.01). Thus, in the third passage, the percentage of cells carrying the CD90+ CD73+ 105+ CD34− HLA DR− phenotype was 95.87%, and by the fourth passage this population constituted 98% of the cells. Accordingly, the population of cells with the CD90−CD73+/−CD105+/− CD34− HLA DR− phenotype decreased as the number of passages increased and by the fourth was represented by 1.57% of the cells (p <0.01) (Figure 2). The expression level of CD markers in the MSC population also varied in different passages. Thus, CD 90+CD73+ was present in only 19.45% of cells in the second passage, and increased as the passage progressed, and by the fourth passage it was already expressed in 80.24% of cells. CD90+CD105+ transcripts were expressed in 88.19% of cells in the 2nd passage, in 92.38% in the third passage, and in 82.43% in the fourth passage.

The opposite pattern was found in the cell culture obtained from the PVAT of patients in the second group. Two cell populations were also identified - CD90+ CD73+ CD105+, CD34−, HLA DR− and CD90−, CD73+/−, CD105+/−, CD34−, HLA DR−. Already at the second passage it was found that the main part of the cell culture (95.98%) is a population characteristic of MSCs - CD90+, CD73+, CD105+, CD34−, HLA DR−. Only 4.02% of the cells had the phenotype CD90−, CD73+/−, CD105+/−, CD34−, HLA DR−. At the third passage, the ratio of the populations remained unchanged. By the fourth passage, the immunophenotypic structure of the culture had changed – cells with the phenotype CD90+, CD73+, CD105+, CD34 - and HLA DR - constituted 44.59%, while a small population of CD90−, CD73+/−, CD105+/−, CD34−, HLA DR− became predominant – 54.9% (Figure 3) (p <0.01).
When studying the expression levels of CD105+ within the population of CD 90 positive cells, it was found that over 3 passages, more than 79% of the cells had this surface marker. With respect to CD 73, the opposite picture was obtained: only 43.57% of cells had this antigen at the 2nd and 3rd passages, and by the 4th passage only 22.94% of cells were positive for CD90 and CD73.

DISCUSSION
Studying the immunophenotype of perivascular MSCs may shed light on their role in the pathogenesis of CVD development and make these cells therapeutic targets in the treatment of cardiac patients. The immunophenotype of MSCs is highly variable; to date, there is no generally accepted surface marker for identifying these cells. However, the International Society for Cellular Therapy has established immunophenotypic criteria for identifying MSCs, namely, expression of CD105, CD73, and CD90, absence of CD45, CD34, CD14 or CD11b, CD79alpha or CD19, and HLA-DR surface molecules [12]. The intensity of expression of these antigens characterizes the ability of cells to proliferate, differentiate, have an immunomodulatory effect, secrete soluble factors, etc. [13]. This paper presents the results of immunophenotyping of MSC cultures of PVAT obtained from patients with heart defects of different etiologies, by the main positive MSC markers - CD73, CD90, CD105, as well as by the negative antigens CD34 and HLA DR. Our study showed that in PVAT of patients with PPS there are both CD90 - positive and CD90 - negative cell populations. Cells bearing the CD90 antigen in the 2nd passage prevailed in the group of patients aged 60-75 years, but by the 4th passage their proportion significantly decreased, while in the group of pediatric patients the opposite pattern was observed. CD90 (Thy-1) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein with a molecular weight of 25-37 kDa. CD 90 is expressed in large quantities by MSCs, which is associated with their multipotent (undifferentiated) state [14]. Knockdown of CD 90 expression in MSCs enhances their differentiation into osteoblasts and adipocytes [15]. In addition to maintaining multipotency (stemness), this antigen provides intercellular adhesion, migration and homing of MSCs by enhancing the expression of CD 44 (hyaluronan receptor). [16]. Perhaps, MSCs from PVAT of older patients, due to the presence of CVD, had lower viability when cultured in vitro, which may explain the loss of CD 90 expression during passaging. Our results are consistent with the study of Bithiah G. et al., who showed that in elderly patients with hematological diseases, CD90 expression in BM MSCs was lower compared to young people [17]. When studying the expression of CD 105 within the population of CD 90-positive cells, no significant differences in the level of this antigen were found between the groups of elderly and children. Apparently, the age characteristics of the donor do not affect the level of expression of this antigen, which is consistent with the work of Massaro et al., who found that more than 98% of MSCs obtained from the bone marrow of young (9+-1 years) and elderly (61+-1 years) patients express CD105 [18] CD105 (endoglin) is a type I membrane glycoprotein with a molecular weight of 90 kDa. B. Levi et al. showed that CD 105 functions as a co-receptor for TGF-β1, which is a known inhibitor of osteogenic differentiation of MSCs, accordingly, a decrease in the level of CD 105 expression leads to a weakening of the effect of TGF-β1 and, as a consequence, an increase in osteogenesis in multipotent cells. The same group of authors established an inverse correlation between the CD 105 level and the expression of osteogenic genes in MSCs [19]. This allows us to conclude that endoglin is an important regulator of multipotent cell differentiation. In addition, existing studies indicate the importance of CD 105 in providing the immunomodulatory function of MSCs. Petinati N et al. showed that when MSCs are cultivated in the presence of proinflammatory cytokines, the expression of CD 105 on their surface increases significantly, which contributes to an increase in the adhesive capacity of leukocytes. [20] Perhaps this explains the equally high expression of endoglin in both elderly and pediatric MSCs, since the presence of cardiac defects, accompanied by hemodynamic disturbances, undoubtedly leads to an inflammatory pathological process in adipose tissue and, as a consequence, in the microenvironment of MSCs [21].

When studying the level of CD73 expression, it was found that the proportion of MSCs positive for this antigen, obtained from the PVAT of patients aged 60-75 years, was less than 50%, and by the 4th passage it decreased to 23%. In the group of pediatric patients, a low level of CD 73 was also detected, but only at the beginning of cultivation, the 3rd and 4th passages included 75-80% of cells positive for this antigen. CD73 is an ecto-5'-nucleotidase, a glycoprotein with a molecular weight of 69 kDa [22]. It is one of the recognized markers for identifying MSCs, but there is evidence that the level of its expression may depend on the tissue source and passaging time [23]. Kimura K. et al. showed that CD 73 - positive MSCs demonstrate pronounced proliferative potential and high ability for multilinear differentiation in vitro, compared with CD 73 - negative cells [24]. Based on these data, it can be assumed that PVAT-MSCs from elderly patients had a low capacity for proliferation and differentiation, since CD 73 was weakly expressed on them, compared to MSCs from patients aged 3-6 years. Thus, the immunophenotype of PVAT-MSCs obtained from elderly patients with heart defects was characterized by variable expression of CD 90, consistently high expression of CD 105 and low expression of CD 73 during passages. Based on the existing knowledge about the function of these antigens, it can be concluded that these MSCs may have a reduced capacity for proliferation and differentiation, and also possess immunogenic activity. In the pediatric group, an increase in the expression of the studied antigens was found during passaging. This means that the viability and proliferative potential of these MSCs is preserved and enhanced during cultivation. Our findings are consistent with the results of other studies. Thus, Stenderup K. et al. reported that BM MSCs from young donors (18-29 years) demonstrate greater viability and slow aging compared to elderly donors (66-81 years) [25]. Mareschi K. et al. found that children's MSCs (3-6 years) exhibit significantly greater proliferative activity than adult donors 25-35 years old [26]. The presence of hemodynamic disorders present in the patients under study may affect the ability of PVAT MSCs to express the studied markers, since existing studies show that almost 100% of MSCs obtained from different tissue sources of healthy donors express CD 90, CD105 and CD73 [27]. It is known that hypoxia, the main pathological process accompanying heart defects, leads to remodeling of adipose tissue and stimulates the secretion of many proinflammatory adipokines [21]. It is possible that the inflammatory environment created in the PVAT of patients with heart defects contributed to the change in the immunophenotypic characteristics of MSCs and affected the stability of the expression of all the studied markers.
CONCLUSION
It should be noted that this study is the first to present the results of immunophenotyping of cells included in the PVAT for the main markers of MSCs. It was found that the age of the donor in combination with comorbidity affects the immunophenotypic features of MSCs in the PVAT, which is manifested in a decrease in the expression of surface markers on cells obtained from elderly patients as they are passaged.

About the authors

Tamara A. Slesareva

Research Institute For Complex Issues Of Cardiovascular Diseases
Kemerovo State Medical University

Author for correspondence.
Email: soloveva081296@mail.ru
ORCID iD: 0000-0003-0749-4093

Postgraduate student of the Department of Pathological Physiology, Clinical Laboratory Diagnostics Doctor

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6; 650000, Russia, Kemerovo, st. Voroshilova 22а

Evgenia G. Uchasova

Research Institute For Complex Issues Of Cardiovascular Diseases

Email: evg.uchasova@yandex.ru
ORCID iD: 0000-0003-4321-8977

Cand.Sc. (Med),Senior Researcher, Laboratory of Homeostasis Research 

650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha,6

Yulia A. Dyleva

Research Institute for Complex Issues of Cardiovascular Diseases

Email: dyleva87@yandex.ru
ORCID iD: 0000-0002-6890-3287

Cand.Sc. (Med), Senior Researcher, Laboratory of Homeostasis Research

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha, 6

Ekaterina V. Belik

Research Institute For Complex Issues Of Cardiovascular Diseases

Email: sionina.ev@mail.ru
ORCID iD: 0000-0003-3996-3325

Cand.Sc. (Med), Senior Researcher, Laboratory of Homeostasis Research

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6

Vera G. Matveeva

Scientific Research Institute of Complex Problems of Cardiovascular Diseases

Email: matveeva_vg@mail.ru
ORCID iD: 0000-0002-4146-3373
ResearcherId: I-9475-2017

Cand.Sc. (Med), Senior Researcher of the Cell Technology Laboratory of the Experimental Medicine Departmen

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6

Evgeniya A. Torgunakova

Research Institute For Complex Issues Of Cardiovascular Diseases

Email: tevgeniyatorgunakova@mail.ru
ORCID iD: 0009-0005-0683-991X

Junior Researcher, Laboratory of Cell Technologies

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6

Ivan V. Dvadtsatov

Research Institute For Complex Issues Of Cardiovascular Diseases

Email: dvadiv@kemcardio.ru
ORCID iD: 0000-0003-2243-1621

Cand.Sc. (Med), cardiovascular surgeon

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6

Ivan K. Khalipulo

Research Institute For Complex Issues Of Cardiovascular Diseases

Email: halivopulo@mail.ru
ORCID iD: 0000-0002-0661-4076

Cand.Sc. (Med), cardiovascular surgeon

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha

Olga L. Tarasova

Kemerovo State Medical University

Email: pathophysiology_kaf@mail.ru
ORCID iD: 0000-0002-7992-645X

Cand.Sc. (Med), associate professor, Head of the Department of Pathological Physiology

Russian Federation, 650000, Russia, Kemerovo, st. Voroshilova 22а

Evgeniya E. Gorbatovskaya

Research Institute For Complex Issues Of Cardiovascular Diseases; Kemerovo State Medical University

Email: eugenia.tarasowa@yandex.ru
ORCID iD: 0000-0002-0500-2449

Cand.Sc. (Med), junior Researcher, Laboratory of Homeostasis Research, аssistant at the Department of Medical Biochemistry

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6; 650000, Russia, Kemerovo, st. Voroshilova 22а

Olga V. Gruzdeva

Research Institute for Complex Issues of Cardiovascular Diseases; Kemerovo State Medical University

Email: o_gruzdeva@mail.ru
ORCID iD: 0000-0002-7780-829X

Doc.Sc. (Med), associate professor, professor of the Russian Academy of Sciences, Head of the Homeostasis Research Laboratory, Head of the Department of Medical Biochemistry 

Russian Federation, 650000, Russia, Kemerovo, boulevard named after academician L.S. Barbarasha 6; 650000, Russia, Kemerovo, st. Voroshilova 22а

References

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