Regular paper

mRNA Expression of thrombospondin 1, 2 and 3 from proximal to distal in human abdominal aortic aneurysm – preliminary report

Aleksandra Augusciak-Duma1✉, Marta Lesiak1, Karolina L. Stepien1, Ewa Gutmajster2 and
Aleksander L. Sieron1

1Department of Molecular Biology, Faculty of Medical Science in Katowice, Medical University of Silesia, Katowice, Poland; 2Department of Medical Genetics, Faculty of Medical Science in Katowice, Medical University of Silesia, Katowice, Poland

Abdominal aortic aneurysm is a process involving the disruption and reconstruction of the extracellular matrix and the apoptosis of smooth muscle cells under the strong influence of the immune system. Thrombospondins are proteins that influence a wide range of cell-matrix interactions. While THBS1 and THBS2 are widely studied, the effects of THBS3 on extracellular matrix and vascular cells are poorly understood. Additionally, it is not known whether expression of these genes’ changes along the aneurysm tissue. Here we analyzed the expression of THBSs mRNA isolated from the harvested tissues along the aneurysm divided into three zones based on their morphology. Total mRNA was isolated from 13 male patients undergoing scheduled open aortic repair, with each aneurysm divided into a proximal part, an aneurysm bag, and a distal part with border tissue as a control. Two step real-time PCR analysis with random hexamers was performed, which allowed the detection of significantly increased expression of all analyzed thrombospondins, especially THBS3, at the control tissue. Overexpression of THBSs may have a destabilizing effect on the structure of the extracellular matrix by affecting both the matrix producing cells and by inhibiting the activity of matrix proteins.

Keywords: abdominal aortic aneurysm; pathology; thrombospondins; gene expression; THBS3

Received: 29 March, 2021; revised: 20 May, 2021; accepted: 14 June, 2021; available on-line: 20 October, 2021

✉e-mail: aaugusciak@sum.edu.pl

Acknowledgements of Financial Support: The work was in part financially supported by the Insti­tutional grant KNW-1-049/N/9/0 (awarded to AAD). The equipment for molecular analyses used in this work was purchased from the Silesian Bio-Farma Center for Biotechnology, Bioengineering and Bioinformatics Project (no POIG.02.01.00-00-166/08 THE OPERATIONAL PROGRAMME INNOVA­TIVE ECONOMY FOR 2007-2013. Priority Axis 2).

Abbreviations: AAA, abdominal aortic aneurysm; ECM, extracellular matrix; MMP, matrix metalloproteinase; THBS, thrombospondin; VEGF, vascular endothelial growth factor

INTRODUCTION

Abdominal aortic aneurysm (AAA) is a heterogeneous asymptomatic disease which, if not detected, can lead to death from aortic rupture (Keisler & Carter, 2015; Sakalihasan et al., 2005). Aortic dilation results from persistent inflammation (Liu et al., 2015a; MA3RS Study Investigators 2017; Sawyer et al., 2016), associated with death of endothelial (EC) and smooth muscle cells (SMC) (Kokot et al., 2013; Siasos et al., 2015) and abnormal remodeling of the extracellular matrix (ECM) (Quintana & Taylor, 2019; Didangelos et al., 2011; Kadoglou & Liapis, 2004). An increased risk of developing AAA is strongly correlated with gender, age, smoking, family history of AAA, atherosclerotic diseases, spinal cord injury, and genetic predispositions (Sakalihasan et al., 2005; Lederle et al., 2000; Li et al., 2013). In the overall European population, the prevalence in 2011–2013 was 4.3%, with 80% mortality resulting from AAA rupture (Li et al., 2013). In the Polish population aged over 65 years, the incidence of AAA is 2.62% and almost 4 times higher in men (4.32%) than in women (1.23%) (Mikołajczyk-Stecyna et al., 2013; Tkaczyk et al., 2019).

Thrombospondins (THBSs) seem to be important inhibitors of AAA, keeping the growth of the aneurysm under control. Thrombospondins are classified in the family of secreted glycoproteins that have very complex and often opposite biological functions. They contain domains for interacting with cell surfaces, growth factors, cytokines, and ECM components (Adams & Lawler, 2011). Trimeric A subfamily consists of THBS1 and THBS2 and has been studied in AAA, pentameric THBS3 is less studied, and its function and importance in angiogenesis and vascularization are poorly understood (Stenina-Adognravi, 2013).

Thrombospondin 1 was shown to be involved in the maintenance of vascular structure by affecting cell proliferation, apoptosis, and adhesion (Liu et al., 2015b). Numerous studies revealed the involvement of THBS1 in the development of AAA through acceleration of vascular inflammation, activation of TGF-beta, and activation of the cofilin pathway (Jana et al., 2020; Liu et al., 2015b; Krishna et al., 2015; Adams & Lawler, 2011; Resovi et al., 2014; Crawford et al., 1998; Yamashiro et al., 2018). THBS1 is also one of the proteins involved in the “angiogenic switch” that changes the phenotype of endothelial cells from quiescent to sprouting (Lawler & Lawler, 2011). THBS2 cooperates with THBS1 for most of its functions (Lawler & Lawler, 2011). THBS2 plays an important role in the structural and functional heart integrity (Golledge et al., 2013a). THBS3 promotes sarcolemmal destabilization by reducing integrin function (Schips et al., 2019). Overexpression of THBS3 in the heart of mice uniformly inhibits the expression of integrins α4, 5, 6, 7, 8, 9, 10, and β1D, which leads to the rupture of the cell membrane. The lack of THBS3 expression protects the heart from pressure overload (Schips et al., 2019).

In presented study, we aimed at detection of the expression pattern of thrombospondins along the AAA using the border segment of aneurysm as a control (Ziaja, 2013; Legaki et al., 2020). Bearing in mind that AAA is a multifactorial and heterogeneous disease with great variability between patients, it is difficult to select the ideal control group to compare expression levels. Therefore, the best way to achieve it was to compare the affected tissue with the phenotypically healthy tissue excised next to the abdominal aortic aneurysm.

MATERIALS AND METHODS

Patient characteristics

A total of 13 samples from males were collected following AAA surgery from patients who were scheduled for open aortic repair (OAR). The patients who underwent surgery for AAA included in this study were male and ranged in age from 57 to 82 years old (mean 67.15±6.47 years) (Supplementary Table I at https://ojs.ptbioch.edu.pl/index.php/abp/). The AAA patients excluded from the study were those who fulfilled the following criteria: (a) chronic obstructive pulmonary disease (COPD); (b) diabetes; (c) creatinine level >1.0; (d) reconstruction of coronary vessels and thoracic aorta (CABG); (e) reconstruction of carotid artery (ICA); (f) diagnosed generalized atherosclerosis (AO); (g) family history of AAA or inherited cardiovascular syndromes; and (h) lack of ability to provide informed consent for surgical treatment.

The study plan was approved by the Bioethics Committee of the Medical University of Silesia in Katowice, no. KNW/0022/KB1/55/14 issued on June 17th, 2014, and its further extension no. KNW/0022/KB1/55/1/14/17 issued on June 27th, 2017.

Materials

Fragments of AAA, on average 50 mm in diameter, were collected from the patients upon surgery. When technically feasible, non-aneurysmal aortic samples of the aneurysm neck (unaffected samples, as confirmed by pathologists) were simultaneously collected and used as controls (Fig. 1).

All surgical procedures were performed in the planned mode. Briefly, the material collected for the research was part of the aneurysm excised during an OAR (ICD9 - 38.424). The samples were collected intra-surgically at the General and Vascular Surgery Department in Katowice-Ochojec, Poland, and secured immediately in the surgery room, at the room temperature and placed in sterile 50 mL tubes filled with 25 mL of high glucose (4.5 mg/mL) Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with penicillin (10 000 U/ml), streptomycin (10 mg/ml), and amphotericin B (25 µg/ml) (PAA Laboratories, Pasching, Austria). The described procedures ensured the maintenance of alive cells, thus, prevent RNA degradation. Then, upon arrival at the cell culture facility, aneurysmal tissue was divided into 4 fragments: border and control/border (C); neck – upper/proximal (1); aneurysm bag or middle/central (2); and the end segment – bottom/distal region (3), where the second part was the aneurysm sack of the excised AAA (Ziaja, 2013). The most altered portion of tissue was called the “aneurysm sac,” and the surrounding tissue were marked as proximal and distal parts. The method of tissue fragmentation was based on the research of Ziaja (Ziaja, 2013). The control tissue was marked as control by the surgeon performing the surgery, from this part, histological examinations were performed to confirm removal of the aneurysm in its entirety by the method of margin analysis (Legaki, 2020). From the fragments, specimens of ~4 mm × 4 mm × 2–4 mm were immediately subjected to RNA isolation and purification (Fig. 1).

Methods

Total RNA was isolated in duplicate using Zymogen Quick RNA Mini Prep (Ambion, Austin, Texas, USA) following sample homogenization in TissueLyser II (Qiagen, Venlo, The Netherlands). Quality and quantity evaluation was performed using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.). Total RNA (1 to 2 µg) was transcribed using a cDNA Transcriptor First Strand cDNA Synthesis Kit (Roche, Penzberg, Upper Bavaria, Germany) with random hexamers. Expression analyses with Real Time Custom Panel 384-96 (config. no 100131839; Roche) and LightCycler 480 Probe Master (Roche, Penzberg, Upper Bavaria, Germany) were performed using a LightCycler 480 II (Roche, Penzberg, Upper Bavaria, Germany). The genes analyzed in this report are listed in Table 1.

Gene expression profiling

Gene expression was analyzed using GenEx ver6 software (MultiD Analyses AB; Sweden). Analyses were performed using the Ct(2-ΔΔCt) comparative method as follows: raw data were normalized to sample amount followed by normalization to the reference genes GAPDH, GusB, PPIA, and RPL13a (Table 1). Relative expression of target genes was calculated with the comparison against the control/border samples.

Statistical analyses

The Kolmogorov–Smirnov test was employed to determine if the data from the expression analysis showed a normal distribution. As the data were not normally distributed, a nonparametric test (Mann-Whitney 1-tailed test) was used for analysis of the data (Weissgerber et al., 2015). The threshold for the p-value was set to less than 0.05. For the determination of the differential expression of genes, scatter plot analysis was used with a significance area equal to 1. Spearman correlation coefficients (rS) were calculated to determine the correlation between genes.

RESULTS and DISCUSSION

The expression of all analyzed genes in majority samples from all sections of the aneurysm as well as from the border section were detected at mRNA level. Only one proximal sample revealed negative results (Supplementary Table II at https://ojs.ptbioch.edu.pl/index.php/abp/).

To the best of our knowledge, this is the first report on THBS1, THBS2, and THBS3 expression in human samples along the aneurysm tissue and its border at the mRNA level. On the basis of the relative expression of the samples, we observed that the expression of thrombospondins mRNA is different in the same patient in all four sections of the aneurysm analyzed, but unfortunately too few samples (7 patients with all sections) did not allow statistical results to be obtained, and so we could only observe the trends (Supplementary Fig. I at https://ojs.ptbioch.edu.pl/index.php/abp/).

When combined, significant differences between expression of the three thrombospondins in different parts of surgically removed aneurysm were found (Fig. 2, Table 2). Previous reports from animal models revealed that THBS1 overexpression in a mouse model of AAA largely contributed to its development by inhibiting the expression of TIMP1 (tissue inhibitor of metalloproteinase 1), which allows inflammatory macrophages to infiltrate vascular tissues. Moreover, mice without THBS1 did not reduce the levels of Mmp9, Mmp3, Mmp10, and Mmp12 (Yang et al., 2020). In our work, the highest expression of THBS1 was found in the border tissue and the difference between the border part and the next proximal part was significant. There was also a significant increase in mRNA expression in the aneurysm sac compared with the proximal segment. The same relation was detected between the proximal and distal segments. However, THBS1 expression in the affected segments did not reach levels of that found in control tissues (Supplementary Fig. II at https://ojs.ptbioch.edu.pl/index.php/abp/). High levels of THBS expression in the control tissue could mark an extensive inflammation in these tissues, which may contribute to AAA development.

In the mouse model, 70% of THBS1 was expressed by macrophages, which mainly invade the adventitious layer (composed of endothelial cells) (Liu et al., 2015a; Yang et al., 2020). Therefore, the conclusion could be made that in an already developed aneurysm, the highest macrophage infiltration occurs not in the tissue already phenotypically changed but in the tissue that is still considered as healthy.

The highest change fold ratio between border tissue and the proximal part was for the THBS2 (4.89±0.003) (Table 2). The expression pattern was similar to that of THBS1. This result was not surprising as in numerous studies these proteins showed a similar function under physiological and pathological conditions (Lawler & Lawler; Zhang et al., 2009; Colombo et al., 2010; Yamauchi et al., 2007; Oganesian et al., 2008; Dawson et al., 1997; Jiménez et al., 2000; Sun et al., 2009). Additionally, in the correlation analysis using the Spearman correlation coefficient, strong positive correlations between THBS2 and THBS1 (rS=0.76 ±0) and between THBS2 and THBS3 (rS=0.74 ±0) were found. The analysis in silico of gene expression omnibus set (GEO) (Wan et al., 2018) revealed enrichment of differentially expressed genes (DEG) for THBS2 in human abdominal aneurysm, but not as a potential biomarker or candidate gene for drug therapy. In 2007 only dataset addressing AAA – GDS2838 (http://www.ncbi.nlm.nih. gov/geo/) was published, but based on the information of its profiles for THBS1, THBS2 and THBS3 there is no final conclusion as to theirs rank, they are too heterogenous. Most researchers either take a sample of the AAA center or extract mRNA from the entire aneurysm and compare it with samples taken from a “healthy” aorta during another procedure or after death. We propose a different approach, similar to that used in the analysis of cancer tissues. Thus, the affected tissues are similarly divided with the marginal/border tissue treated as a control sample, thus eliminating individual differences between samples collected from different individuals and focusing only on the differences between different sections of the aneurysm. As shown in the results, these differences are universal and are not due to individual differences.

Recently, first reports of increased expression levels of THBS2 have been described in protein studies in human AAA tissue samples, (Qi et al., 2020). Elevated THBS2 concentration in serum plasma was associated with the risk of cardiac mortality in patients with AAA (Golledge et al., 2013b) and aortic dissection (AD) (Qi et al., 2020). Furthermore, its polymorphism was linked to hypertension susceptibility (Yamada, 2009). THBS2 protein was detected in smooth muscle cells and at a lower level in endothelial cells where it positively correlated with TNF-α and IL-6 suggesting that THBS2 may regulate the inflammatory response (Qi et al., 2020). THBS2 also reduced the presence of active MMP2. THBS2-deficient mouse fibroblasts have been reported to produce pro-MMP2 twice compared with wild-type cells (Yang et al., 2000).

Here, too, we present the analysis of THBS3 expression in human AAA for the first time. There was a significant expression decrease between the border tissue and the proximal part of the aneurysm (change fold=4.01±0.02) and between the proximal part and the aneurysm sac (change fold=1.2±0.02). Additionally, a strong positive correlation of expression was found between THBS3 and THBS1 (rS=0.76 ±0), and between THBS3 and THBS2 (rS=0.74 ±0). THBS3 is the least known member of the thrombospondin family. Particularly, its function in AAA is poorly understood. In an aging mouse model THBS3 expression was increased in the myocardial ECM of elder mice, which may lead to Smad2 activation in epithelial cells and age-related cardiac inflammation (Toba et al., 2016). In contrast, the expression level of THBS3 was not associated with pulmonary arterial hypertension (PAH), although THBS1 was directly involved in the activation of TGF-β in the mouse PAH model and was both required and sufficient for the development of PAH (Kumar et al., 2017). In osteosarcomas, THBS3 expression is correlated as a prognostic factor of worse overall survival and as a stimulator of tumor progression due to its high ability to promote angiogenesis (Dalla-Torre et al., 2006). Multi-omics analysis of vascular calcification also highlighted the unknown role of THBS3 in this process, possibly by imposing sarcolemmal instability (Qian et al., 2021; Schips et al., 2019). Overexpression of THBS3 is the result of a response to an inflammatory process. Improved remodeling, expansion, and fibrosis of the ventricles in experimental mice was a result of inhibition of integrin expression, which led to rupture of the cell membrane (Schips et al., 2019).

Vascular smooth muscle cells, which are an essential component of the aorta (they constitute >90% cells in there) during AAA development, undergo excessive apoptosis followed by destruction of the dynamic balance of ECM (Quintana & Taylor, 2019). THBS1 and 2 enhance apoptosis by promoting the expression of inflammatory factors and activating apoptotic pathways, as well as by inhibiting angiogenesis (Armstrong & Bornstein, 2003; Lawler, 2000). Since they play a protective role for healthy tissue under severe macrophage attack at the onset of inflammation in the aorta, thrombospondins respond by eliminating the potential source of inflammation. In chronic conditions, their overexpression leads to disruption of homeostasis in the ECM and then to a positive feedback loop when overexpression of MMPs and its inhibitors, remodel the matrix. In addition, the hemodynamic load pressure contributes to further imbalance, and with THBS3 overexpression, it additionally disturbs the stability and the presence of integrins on the cell membrane.

CONCLUSION

We found that there was a significantly variable expression pattern in the aneurysm tissue taking into account the segmentation of the AAA. The most prominent feature found was the sudden increase in expression in the marginal tissue compared to the aneurysm segments. Based on our and previous studies, we hypothesize that THBS3 downregulates the expression of integrins, which can affect cell membranes and destabilize the complex junction of the ECM structure in the aortic vessel. The destabilization of the ECM and the connection of cells to it may lead to ease the access of THBS1-expressing macrophages to the ECM of the abdominal aorta. The further overexpression of THBS1 might induce structural changes in the ECM by, for example, inhibition of the TIMP1 at protein level, while overexpression of THBS2 in the endothelial and smooth muscle cells inhibits the MMP2 protein further destabilizing the correct balance in ECM homeostasis.

Our research has its limitations. First, the sample size in the experiment was small and a larger number of tissue samples are needed to confirm our findings. Second, mRNA expression is not a precise indicator of the level of protein in tissues and its specific location. More research is needed to understand the molecular mechanisms by which thrombospondins work in AAA.

Acknowledgements

The authors would like to acknowledge Prof. Krzysztof Ziaja and Prof. Wacław Kuczmik from the Department of General and Vascular Surgery, Medical University of Silesia, Katowice, Poland, for constructive medical consultation of the manuscript and granting the tissues of AAA.

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