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Department of Vascular Surgery, Amsterdam University Medical Centres, location VUmc, Amsterdam, the NetherlandsDepartment of Physiology, Amsterdam University Medical Centres, Amsterdam Cardiovascular Sciences, Amsterdam, the NetherlandsDepartment of Surgery, Zaans Medisch Centrum, Zaandam, the Netherlands
Department of Angiology and Vascular Surgery, São João University Hospital Centre, Porto, PortugalDepartment of Surgery and Physiology, Cardiovascular Research Unit, Faculty of Medicine, University of Porto, Portugal
Department of Vascular Surgery, Amsterdam University Medical Centres, location VUmc, Amsterdam, the NetherlandsDepartment of Physiology, Amsterdam University Medical Centres, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands
Department of Vascular Surgery, Amsterdam University Medical Centres, location VUmc, Amsterdam, the NetherlandsDepartment of Physiology, Amsterdam University Medical Centres, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands
Corresponding author. Amsterdam University Medical Centres, location VUmc, Departments of Surgery and Physiology, Amsterdam Cardiovascular Sciences, De Boelelaan 1118, P.O. Box 7057, 1081 HV, Amsterdam, the Netherlands.
Department of Vascular Surgery, Amsterdam University Medical Centres, location VUmc, Amsterdam, the NetherlandsDepartment of Physiology, Amsterdam University Medical Centres, Amsterdam Cardiovascular Sciences, Amsterdam, the NetherlandsDepartment of Vascular Surgery, Amsterdam University Medical Centres, location AMC, Amsterdam, the Netherlands
Perivascular adipose tissue (PVAT) contributes to vascular homeostasis and is increasingly linked to vascular pathology. PVAT density and volume were associated with abdominal aortic aneurysm (AAA) presence and dimensions on imaging. However, mechanisms underlying the role of PVAT in AAA have not been clarified. This study aimed to explore differences in PVAT from AAA using gene expression and functional tests.
Human aortic PVAT and control subcutaneous adipose tissue were collected during open AAA surgery. Gene analyses and functional tests were performed. The control group consisted of healthy aorta from non-living renal transplant donors. Gene expression tests were performed to study genes potentially involved in various inflammatory processes and AAA related genes. Live PVAT and subcutaneous adipose tissue (SAT) from AAA were used for ex vivo co-culture with smooth muscle cells (SMCs) retrieved from non-pathological aortas.
Adipose tissue was harvested from 27 AAA patients (n [gene expression] = 22, n [functional tests] = 5) and five control patients. An increased inflammatory gene expression of PTPRC (p = .008), CXCL8 (p = .033), LCK (p = .003), CCL5 (p = .004) and an increase in extracellular matrix breakdown marker MMP9 (p = .016) were found in AAA compared with controls. Also, there was a decreased anti-inflammatory gene expression of PPARG in AAA compared with controls (p = .040). SMC co-cultures from non-pathological aortas with PVAT from AAA showed increased MMP9 (p = .033) and SMTN (p = .008) expression and SAT increased SMTN expression in these SMC.
The data revealed that PVAT from AAA shows an increased pro-inflammatory and matrix metallopeptidase gene expression and decreased anti-inflammatory gene expression. Furthermore, increased expression of genes involved in aneurysm formation was found in healthy SMC co-culture with PVAT of AAA patients. Therefore, PVAT from AAA might contribute to inflammation of the adjacent aortic wall and thereby plays a possible role in AAA pathophysiology. These proposed pathways of inflammatory induction could reveal new therapeutic targets in AAA treatment.
A multicentre study was conducted to assess inflammatory gene expression in perivascular adipose tissue (PVAT) of patients with abdominal aortic aneurysm (AAA) and healthy controls. PVAT from AAA patients and healthy aortic smooth muscle cells stimulated with this adipose deposit both showed increased pro-inflammatory and decreased anti-inflammatory gene expression. These findings suggest that inflammation of PVAT and thereby the aortic wall contributes to AAA pathophysiology. The pathway of inflammatory induction could reveal new therapeutic targets for both prevention and conservative treatment of AAA.
Recent literature has shown an increased interest in the role of inflammation in abdominal aortic aneurysm (AAA).
The transmural inflammation is a pathological feature of AAA and involves a variety of inflammatory cell types, where macrophages and lymphocytes are the most prominent with mast cells and neutrophils migrating to a lesser extent.
The infiltration of inflammatory cells in the media and adventitia, along with medial smooth muscle cells (SMCs) and adventitial fibroblasts from the aortic wall, are involved in the production of proteases,
Perivascular adipose tissue (PVAT) surrounding the conduit arteries was until recently thought to function merely as mechanical support. However, there is a growing body of literature that support PVAT as a tissue interacting with arteries in a paracrine fashion, with a pathogenic role in cardiovascular diseases.
This was, however, not previously correlated with histological findings.
Similar to other adipose tissues, PVAT secretes different biologically active substances (adipokines, including cytokines, chemokines, and growth factors) at different anatomical sites, which can prevent, decelerate, induce, or exacerbate atherosclerosis. For example, the protective role of adiponectin (ADIPOQ) in the onset of cardiac atherosclerosis is not found in aortic atherosclerosis.
the computed tomography scan results in that study raised follow up questions regarding pathological alterations and underlying pathways, answers to which might lead to new preventive or conservative treatments in AAA. In the current study, the genetic mechanisms potentially underlying the role of PVAT in the occurrence of AAA were investigated, and the hypothesis that PVAT plays a role in inflammation of the aneurysmal abdominal aortic wall was tested.
Materials and methods
An extensive version of the methods including laboratory protocols is available online as Supplementary Methods.
The recent study was carried out in accordance with the Code of Ethics of the World Medical Association (Helsinki Declaration) of 1975, as revised in 1983 and was approved by the Medical Ethics Committee of the AUMC, location VUmc (Amsterdam, The Netherlands) and local approval was obtained for the remaining participating centres.
Informed consent was retrieved for all patients. This was a prospective translational study in which human aortic PVAT was collected during open surgery from patients suffering from AAA and from post mortem renal donation in patients without image or local signs of aortic atherosclerosis (control tissue). Furthermore, clinical characteristics were collected from electronic health records. The study set up is summarised in Figure 1.
RNA isolation and quantitative polymerase chain reaction (qPCR) of snap frozen PVAT specimens was performed. Relative gene expression of all genes was based on normalisation factor of housekeeping genes Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ), TATA Binding Protein (TBP), and Hypoxanthin Phosphoribosyltransferase (HPRT), based on previous literature. No significant differences in expression were detected between case and control samples for the chosen housekeeping genes. The following primers were designed to study relative gene expression in PVAT: Protein Tyrosine Phosphatase Receptor Type C (PTPRC) or CD45, IL6, CXCL8 (IL8), metalloproteinase (MMP) 2, MMP9, Peroxisome Proliferator Activated Receptor Gamma (PPARG). Internal control for adipose tissue was performed using ADIPOQ and LEP. Based on the primary results, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, ELIXIR, Cambridgeshire, UK) was used to study potential interacting genes. These were assessed within the same cohort. A complete overview of forward and reverse primer sequences can be found in Supplementary Table 1.
Using PVAT and subcutaneous adipose tissue (SAT) of AAA patients, ex vivo stimulation of control aortic SMCs was performed. Transplantation and sectioning of PVAT was performed according to a previously published method to cut live aortic tissue sections.
Live PVAT and SAT sections of AAA patients (n = 5) were added to live control SMC and cultured. Also, control SMCs without stimulation were cultured. After a week, PVAT, SAT, and culture medium were removed and gene expression of live SMCs was performed. The same genes as previously assessed, extended with SMTN (smoothelin), were studied in the SMCs. A ratio of PVAT stimulated SMCs and a ratio of SAT stimulated SMCs (both compared with non-stimulated SMCs) was calculated.
Data were analysed with IBM SPSS Statistics v22.0 (Chicago, IL, USA). Data were presented as median with interquartile range (IQR). Mann–Whitney U tests, Fisher’s exact tests, and Kruskal–Wallis tests with Bonferroni correction were performed. Tests were considered statistically significant at p ≤ .050.
For PVAT gene expression analysis, demographics and PVAT were collected for a total of 27 patients (n [control] = 5, n [elective AAA] = 16, and n [acute AAA] = 6). In the control group, 100% were male and in the AAA group 68.2% were male. The median age of all controls was 63.5 years (IQR 5.8), and median age of all AAA patients was 72.0 years (IQR 8.3). The median maximum AAA diameter of all AAA patients was 69.5 mm (IQR 19.0). A complete overview of the baseline variables can be found in Table 1.
Table 1Description of baseline characteristics for control and abdominal aortic aneurysm (AAA) groups included in perivascular adipose tissue gene expression analysis
Additionally, demographics, PVAT and SAT were collected within a total of five additional AAA patients (n [elective AAA] = 5) for live co-culture analyses. This group included five males (100%) that underwent elective AAA surgery, with a median age 71.5 years (IQR 15) and a median maximum AAA diameter of 62.0 mm (IQR 15.3). In this cohort, statin use was 80.0% and 80.0% were currently or formerly smokers. One healthy SMC cell line of the previous control group was used for live SMC and adipose tissue co-culture.
Gene expression of perivascular adipose tissue in AAA and controls
qPCR was used to quantify gene expression (n [control] = 5 and n [AAA] = 22). A significant increase in PTPRC, which encodes the enzyme CD45 (a pan-leucocyte protein), was found in PVAT of AAA compared with controls (1.43 [IQR 0.22] vs. 0.32 [IQR 2.93], p = .008). Furthermore, a significant increase in CXCL8, which encodes the chemokine IL8 (neutrophil chemotactic factor), was found in PVAT of AAA compared with controls (1.58 [IQR 2.69] vs. 0.21 [IQR 0.92], p = .033). Also, MMP9 which encodes the matrix metallopeptidase 9 was higher in PVAT of AAA than in controls (19.59 [IQR 24.91] vs. 9.74 [IQR 10.52], p = .016). Additionally, a significant decrease in PPARG, which encodes the protein peroxisome proliferator activated receptor gamma (glitazone receptor), was found in PVAT of AAA compared with controls (3.23 [IQR 6.94] vs. 7.82 [IQR 7.93], p = .040). Expression of ADIPOQ and LEP did not differ significantly between PVAT of controls and AAA patients (p = 1.0 and p = .69, respectively); however, both adipocyte genes were expressed in both controls and AAA patients.
Next, based on the statistically significant differences found in the primary analysis, these genes were tested in STRING Search Tool for the Retrieval of Interacting Genes/Proteins, which revealed predicted interactions of: Lymphocyte Cell-Specific Protein-Tyrosine Kinase (LCK), CD44, Platelet Activating Factor Receptor (PTAFR), C-X-C Motif Chemokine Receptor 2 (CXCR2), Jun Proto-Oncogene (JUN), C-C Motif Chemokine Ligand 5 (CCL5), and FYN Proto-Oncogene (FYN). For these genes, mRNA expression levels were measured by qPCR as mentioned above. A significant increase in LCK was found in PVAT of AAA compared with controls (0.31 [IQR 0.69] vs. 0.04 [IQR 0.03], p = .003) and a significant increase in CCL5 was found in PVAT of AAA compared with controls (2.48 [IQR 4.39] vs. 0.72 [IQR 0.68], p = .004). Figure 2 shows the strength of interaction between these genes as found in previous literature and error bars of the statistically significant different genes in the current study.
Live perivascular and subcutaneous adipose tissue co-culture changes in gene expression profile of aortic smooth muscle cells
PVAT and SAT of patients suffering from AAA were collected (n = 5). After co-culture of SMCs without known pathology with PVAT or SAT, the cells were microscopically checked for vitality. All wells contained a solid group of live SMCs, and live adipose tissues were observed. Figure 3A–D shows a schematic overview and photographs of the incubation process. qPCR was used to detect the levels of genes. Analysis showed an increase in PVAT co-cultured SMC ratio for MMP9 (2.15, p = .008), which encodes the matrix metallopeptidase 9 enzyme (Fig. 3E). Furthermore, analysis showed an increase in PVAT co-cultured SMC ratio for SMTN (1.06, p = .008), which encodes the protein smoothelin. Furthermore, an increase in the SAT co-cultured SMC ratio was found for SMTN (1.18, p = .016, Fig. 3F). An overview and interpretation of all the results can be found in Figure 4.
The purpose of this study was to elucidate whether there is a role for PVAT in inflammation within AAA patients. Following previous imaging studies that revealed a positive association between PVAT and thoracic and abdominal aortic dimensions, there was a request for clearance in the role of PVAT in inflammation and AAA. Kugo et al.
recently showed in a rat model that removal of PVAT led to a significant decrease in AAA diameter. Adipocytes from the aortic wall and PVAT were shown to release factors that induce expression of interleukins contributing to AAA progression.
In PVAT of AAA patients, an increased inflammatory gene expression of PTPRC, CXCL8, MMP9, LCK, and CCL5, and a decreased anti-inflammatory expression of PPARG compared with PVAT of controls was observed. PTPRC and LCK are present in leucocytes. The enzyme PTPRC (CD45 or leucocyte marker) is involved in cell growth, mitosis, and differentiation. Upregulation of PTPRC in the AAA wall is a non-specific marker of inflammatory processes, as well as the presence of the corresponding enzyme.
This protein (and its gene expression) plays a role in the inflammatory response to antigens and future studies on the current topic might reveal the role of LCK in the differentiation of specific T cells in AAA.
The pro-inflammatory role of PVAT is further underlined by increased expression of chemokines. CXCL8 (IL8) encodes for the eponymous chemokine and promotes inflammation, stimulates protease expression and it has pro-angiogenic effects via neutrophils and leucocytes, which are known for many years already to play a role in AAA pathology.
CCL5 (Chemokine C-C motif ligand 5) is classified as chemotactic chemokine, which functions as chemo-attractant for blood monocytes, memory T helper cells, and eosinophils, and recruits leucocytes into inflammatory sites. Recently, CCL5 was found to be overexpressed in aortic wall tissue and mentioned as a potential therapeutic target,
In parallel and related to inflammation, PVAT may also play a role in the weakening of the aortic wall via increased expression of MMP9. Matrix Metallopeptidase 9 is a matrixin (enzyme) involved in the degradation of the extracellular matrix and is known to play a central role in both progression and rupture of AAA.
suggesting a link between the adipocytes in the vascular wall and the mechanisms leading to rupture. Potentially, PVAT might lead to degradation of the aortic wall through MMP9 via equal pathways. This hypothesis was further emphasised by the effect of live PVAT and SAT on SMC showing an increased expression of MMP9 in healthy SMC co-culture with PVAT of AAA patients. This implicates that PVAT could play an important role in the breakdown of the extracellular matrix via SMCs in the medial layer and consequently in progression and rupture of AAA.
Another interesting finding is the decreased expression of the seemingly protective gene PPARG (Peroxisome Proliferator-Activated Receptor Gamma), also known as glitazone receptor. This is a nuclear receptor mainly present in adipose tissue, colon, and macrophages and regulates fatty acid storage, glucose metabolism, and adipocyte differentiation. Strikingly, PPARG also decreases the inflammatory response of many cardiovascular cells.
So far, a small cohort study over a period of two months and two animal studies were unable to limit the maximum aneurysm size with PPARG agonists; however, expression of inflammatory genes and matrix metallopeptidases was decreased.
leads to macrophage polarisation, which is important for tissue remodelling and repair. PPARG is likely to continue to upregulate expression of anti-inflammatory cytokines, extend the “compensatory stage”, and decelerate the process of AAA development and rupture.
These findings seem to be in line with the current findings of increased PTPRC and increased SMTN expression in the pathological ex vivo situation. Furthermore, most gene expression outcomes showed comparable results for PVAT and SAT, which might indicate a systemic effect of adipose tissue in AAA patients. An alternative explanation for these findings could be by the interaction between SMCs and adipose tissue.
There was a higher use of statins in the AAA group (73%) vs. the control group (0%). It is widely accepted that statins lower inflammation in vascular diseases and van der Meij et al. specifically showed this for AAA patients.
Selective anti-inflammatory therapy focusing on PVAT might provide a new strategy to prevent progression of AAA. Moreover, this might explain the recent interest in the role of not diabetes but metformin use as being protective against AAA progression,
Further work is required to establish the role of metformin on PVAT.
A potential difference between inflammatory reaction in truly atherosclerotic and aneurysmal vessels was not addressed in paper. Atherosclerotic patients seem to share some but not all the inflammatory features of the aneurysm patients. In previous studies from the group, MMP9 was higher in PVAT of atherosclerotic than control patients and in PVAT of atherosclerotic compared with AAA patients, besides being higher in PVAT of AAA compared with controls (data not published). PVAT adipocytes from AAA patients were smaller than those derived from controls and atherosclerotic patients and PVAT from AAA patients presented larger amounts of fibrosis vs. control and atherosclerotic patients (data not published).
A note of caution is due here since this study was limited by the relatively small number of cases that were included. Furthermore, the included patients had a western European background. Therefore, these findings cannot be extrapolated globally to patients from varying geographical backgrounds.
Another weakness of the study was the collection of control tissue. Due to the clinical handling process, procurement times and protocol differed between AAA and control tissues, since AAA tissue was harvested directly in the operating room, while control tissue was dissected for renal transplantation and transported in Custodiol® and harvested after surgery on the kidney receiving patient had started. Nonetheless, it was concluded that RNA expression of ADIPOQ and LEP did not differ significantly between these groups and both markers are generally expressed in adipose tissue. This finding strengthens the differences found between the AAA and control group. The model that was used was limited to SMC culture, which represents a fraction of the resident vascular cells in the aortic wall. The effect of PVAT was not addressed in the mesenchymal cells and adipose deposits of the adventitia itself which have recently been linked to AAA progression and rupture.
Furthermore, the stimulation tests were performed in healthy aortic SMCs since it is difficult to distinguish whether the PVAT leads to inflammation of the wall or AAA leads to PVAT inflammation.
The stimulation experiment did not test the effect of PVAT or SAT from control patients; as such, whether the effect is specific from adipose tissue deposits from AAA patients or a general effect of any adipose tissue could not be addressed. Unfortunately, the kidney donors were limited, and that experiment could not proceed due to lack of samples. Further stimulation experiments using adipose deposits from control patients might clarify the exact role of individual PVAT genes in the pathology of AAA.
Another limitation was that experiments within the same patient cohort could not be performed. This was a direct consequence of the emergence of new live co-culture research techniques that were unavailable at the initiation of the current study. Lastly, as shown in Table 1, there were statistically significant differences between the groups in age and statin use. Due to the relatively small sample size it was not feasible to adjust for these potential confounding factors.
Finally, full clarification of the contribution of the genetic expression that was found in PVAT around the aorta of AAA patients will require protein experiments to ensure that the differences verified really lead to differential expression of the protein coded by the genes involved. Therefore, the current findings need to be interpreted and discussed under these limitations.
Elective and acute surgical treatment of AAA has improved greatly in terms of outcomes over the last decades. The use of laparoscopic and robotic surgery has been abandoned due to procedural complexity, higher risk of death, and adverse events.
The introduction and rapid evolution of endovascular treatment options has led to promising outcomes compared with open repair within six months after surgery. Nonetheless, aneurysm related mortality after eight years is higher in patients that underwent endovascular aneurysm repair compared with open surgery.
Currently available pharmacological treatment strategies are aimed at reducing AAA risk factors, but no prescription medication is yet approved to prevent the incidence or growth of AAA. As stated in the European Society for Vascular Surgery clinical practice guidelines on the management of abdominal aorto-iliac artery aneurysms, there is a global request for further research into medical treatment to slow AAA growth. As mentioned above, metformin might be a potential candidate drug and with the current results, it is proposed that PVAT might be a key player via either metformin or other to be discovered potential therapeutic targets to prevent or slow AAA growth.
In conclusion, the results show that PVAT of AAA patients presents with higher expression of the currently assessed inflammatory genes and increased extracellular matrix degradation. Adipose tissue of patients suffering from AAA can induce inflammation in healthy SMC originating from control patients. It is proposed that PVAT plays an important role in the onset and progression of AAA and further research may clarify underlying pathways through PVAT which might reveal promising therapeutic targets in both prevention and conservative treatment of AAA.
Conflict of interest
Glória Conceição is supported by Universidade do Porto / FMUP and by FSE – Fundo Social Europeu through NORTE2020 – Programa Operacional Regional do Norte, no âmbito da operação NORTE-08-5369-FSE-000024-Programas Doutorais.
We are grateful to all the patients (and their family members) who donated vascular tissue. The authors thank Willem Wisselink, Hillian Nederhoed, Arjan Hoksbergen, Maarten Truijers, Arno Wiersema, Jur Kievit, Vincent Jongkind, and Vincent Scholtes for providing the vascular specimens in the Netherlands during surgery. The authors also would like to thank José Fernando Teixeira, the director of the Department of Angiology and Vascular Surgery, São João University Hospital Centre, on behalf of the surgeons that provided vascular specimens during surgery in that centre. Furthermore, the authors thank Theodorus van Schaik, Harm Ebben, Orkun Doganer, Sabrina Doelare, Stefan Smorenburg, Niels Keekstra, and Jacqueline Hoozemans for collecting tissue and preparing the experiments.
Appendix A. Supplementary data
The following are the Supplementary data to this article:
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