Human Vascular Wall Mesenchymal Stromal Cells Contribute to Abdominal Aortic Aneurysm Pathogenesis Through an Impaired Immunomodulatory Activity and Increased Levels of Matrix Metalloproteinase-9

Abstract

Background: The main histopathological features of abdominal aortic aneurysm (AAA) are tissue proteolysis mediated by matrix metalloproteinases (MMPs) and inflammation. This study aimed at verifying the presence and contribution of mesenchymal stromal cells (MSCs) to aneurysmal tissue remodeling.

Methods and Results: MSCs were successfully isolated from the AAA wall of 12 male patients and were found to express mesenchymal and stemness markers. MMP-2/-9 are involved in AAA progression and their mRNA levels in AAA-MSCs resulted higher than healthy MSCs (cMSCs), especially MMP-9 (400-fold increased). Moreover, MMP-9 protein and activity were pronounced in AAA-MSCs. Immunomodulation was tested in AAA-MSCs after co-culture with activated peripheral blood mononuclear cells (PBMCs) and revealed a weak immunosuppressive action on PBMC proliferation (bromodeoxyuridine incorporation, flow cytometry assay), together with a reduced expression of anti-inflammatory molecules (HLA-G, IL-10) by AAA-MSCs compared to cMSCs. MMP-9 expression in AAA-MSCs was shown to be negatively modulated under the influence of cMSCs and exogenous IL-10.

Conclusions: MSCs with stemness properties are niched in human AAA tissues and display a dysregulation of functional activities; that is, upregulation of MMP-9 and ineffective immunomodulatory capacity, which are crucial in the AAA progression; the possibility to modulate the increased MMP-9 expression by healthy MSCs and IL-10 suggests that novel therapeutic strategies are possible for slowing down AAA progression. (Circ J 2015; 79: 1460–1469)

Abdominal aortic aneurysm (AAA) is a focal dilatation of the arterial wall, most commonly affecting men over 65 years of age, without specific signs or symptoms.^1–4 The progressive weakening of the artery wall can lead to rupture, if left untreated, carrying a high risk of mortality.^4,5 The only treatment currently in use is surgery, through the conventional repair or the endovascular approach, while there is not a pharmacological agent able to arrest aneurysm growth and delay its rupture.⁶ For this purpose, many studies addressing the molecular pathways crucial for AAA development and degeneration are in progress.¹ The mechanisms primarily involved in AAA formation are: congenital malformations, hypertension, atherosclerosis, inflammation, oxidative stress, hypoxia and aging.^1,7 Except for malformations, these processes are associated with the unbalanced activity of proteolytic enzymes; that is, the upregulation of metalloproteinases (MMPs) that degrade extracellular matrix (ECM). MMPs are a large family of zinc-endopeptidases (28 members) classified according to their substrate: collagenases, gelatinases, stromelysins and membrane-bound MMPs. Collagenases include MMP-1, -8, -13, -18 and degrade fibrillar collagen type I-II-III, while Gelatinases A and B are represented by MMP-2, -9 and digest denatured collagen (gelatine).^8,9 MMPs are involved in many physiological processes such as vascular remodeling, angiogenesis and cell migration/proliferation. In physiological conditions, MMPs are tightly regulated through many mechanisms, including transcriptional modifications, activation of pro-forms or zymogens, interaction with ECM components and modulation by endogenous tissue inhibitors (TIMPs).^1,8 An abnormal MMP regulation or an imbalance between MMP and TIMP production may be responsible of pathological vascular remodeling. Many investigators have demonstrated an altered MMP/TIMP balance, together with increased MMPs transcription and activity, in many cardiovascular diseases associated with vascular remodeling such as aneurysm,¹⁰ atherosclerosis,^11,12 hypertension¹³ and arteritis caused by Kawasaki Disease.¹⁴ In this context, collagenases and gelatinases are the MMPs mainly involved in aortic wall weakening.^15–17

Editorial p 1439

Recently, a population of multipotent stromal stem cells have been discovered and isolated from different arterial segments of multiorgan donors.^18,19 This population shares properties of human bone marrow mesenchymal cells with stemness features, as well as with the ability to survive and be enriched under anoxia and cryoinjury post mortem stresses.²⁰ This same cell population also had the ability to modulate immune response through the suppression of immune cell activation and proliferation.^20,21

This study aimed to detect the presence of mesenchymal stromal (stem) cells (MSCs) in AAAs and explore their potential involvement in ECM degradation and inflammation. For this purpose, after MSC isolation from AAA tissues and basal characterization, we analyzed the expression of AAA molecular mediators, MMP-2 and MMP-9; therefore, we investigated the AAA-derived MSC response to an in vitro inflammatory condition, in order to evaluate if these MSCs can behave erratically, suggesting a role into the mechanisms leading to pathological arterial remodeling.

Methods

Study Protocol and Tissue Collection

The study protocol was approved by the Local Ethic Committee (ethic protocol number APP-13-01) and written informed consent was obtained from patients.

Aneurysm tissues were collected from 12 patients (males, mean age=69.2±5 years) undergoing surgical repair and were provided from the Vascular Surgery Unit, S.Orsola-Malpighi Hospital, University of Bologna; thoracic aortic wall tissue from 3 healthy donors (female:male=2:1, mean age=25±6.5 years) was provided by the Cardiovascular Tissue Bank, S.Orsola-Malpighi Hospital (Bologna) and were used as healthy controls for in vitro analysis, with the approval of the Local Ethic Committee (ethic protocol number APP-13-01).

Immunohistochemical Staining

Immunohistochemistry was performed on aneurysm and healthy specimens to characterize the MSC population before enzymatic digestion, using a non-biotin amplified method (Novolink, Newcastle, UK). The following primary antibodies were used: CD44 (BD Biosciences), CD90 (BD Biosciences), MMP-2 (Thermo Fischer Scientific) and MMP-9 (Cell Signaling).

Human Mesenchymal Stromal Cell Isolation and Culture

Sections of the aneurysmal wall measuring 2 cm² were enzymatically digested with 0.3 mg/ml Liberase type II (Liberase TM Researche Grade, Roche) in serum-free Dulbecco’s modified Eagle’s medium (DMEM; SIGMA Aldrich) at 37℃ overnight in a rotor apparatus. The tissue homogenate was filtered using different cell strainers (100–70–40 µm) and centrifuged at 300 g. Cell viability was assessed by Trypan Blue exclusion. MSCs isolated from aneurysm samples (AAA-MSCs) were cultured (37℃ incubator, 5% CO₂) in DMEM enriched with 20% Fetal Bovine Serum (FBS; SIGMA Aldrich) and expanded in vitro. Healthy vascular tissue processing and cell recovery followed the same protocol used for pathological samples.

Immunophenotyiping

Flow cytometer analysis was performed on AAA-MSCs at passage 3 to investigate the expression of typical mesenchymal markers. Briefly, cells at passage 3 were fixed by using a Fixation Kit (Beckman-Coulter) and stored at 4℃ until use. Fixed cells were incubated for 20 min using the following extensive conjugated panel of antibodies: anti-CD44-fluorescein isothiocyanate (FITC), anti-CD73-phycoerythrin (PE), antiCD90-phycoerythrin-cyanine 5, anti-CD14-FITC, anti-CD34-FITC, anti-CD45-allophycocyanin, anti-CD146-PE, and anti-platelet-derived growth factor receptor β (PDGFR-β) (R&D Systems, Inc, Minneapolis, MN, USA). In order to evaluate the immunomodulatory activity of AAA-MSCs, HLA-G expression was detected with anti-human leukocyte antigen-G (HLA-G)-FITC antibody (Abcam, Cambridge, UK). Negative controls were performed using appropriate conjugated irrelevant antibodies. Samples were analyzed using a Navios FC equipped with 2 lasers for data acquisition (Beckman-Coulter). Results were analyzed with Kaluza FC Analysis software (Beckman-Coulter).

Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from cultured cells using TRIreagent (TRIzol reagent; Life Technologies) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed in a 20 µl reaction volume using a High Capacity Reverse Transcription Kit (Life Technologies).

Stemness Gene Expression Analysis

RT-polymerase chain reaction (PCR) was performed on AAA-MSCs to evaluate the stemness genes, Nanog, Oct-4 and Sox-2 expression; primers sequences (SIGMA Aldrich) are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to value the cDNA quality. All samples were loaded on a 2% agarose gel with Tris-acetate-EDTA buffer 1X (TAE) and a 100bp DNA ladder was used to allow amplicone size identification. The gel was subjected to electrophoresis at a constant 100V and observed under ultraviolet light, through ethidium bromide incorporation.

Table 1. Primers Used for Stemness Marker Detection in AAA-MSCs

Gene	Primer sequence	Product size (bp)	T (℃)
GAPDH	FWD 5’-ACCACAGTCCATGCCATCAC-3’	452	61
	REV 5’-TCCACCACCCTGTTGCTGTA-3’
NANOG	FWD 5’-AAGGCCTCAGCACCTACCTA-3’	326	58
	REV 5’-ACATTAAGGCCTTCCCCAGC-3’
OCT-4	FWD a 5’-CTCCTGGAGGGCCAGGAATC-3’	380	62
	FWD b 5’-ATGCATGAGTCAGTGAACAG-3’	402
	REV 5’-CCACATCGGCCTGTGTATAT-3’
SOX-2	FWD 5’ACCGGCGGCAACCAGAAGAACAG-3’	208	62
	REV 5’-GCGCCGCGGCCGGTATTTAT-3’

AAA-MSCs, aneurysm-derived mesenchymal stromal cells; FWD, forward; OCT-4, octamer-binding transcription factor-4; REV, reverse; SOX-2 or SRY, sex determining region Y-box 2.

Quantitative Real-Time PCR (qPCR)

qPCR was performed to indagate stemness transcriptional factors, AAA molecular mediators and anti-inflammatory cytokine mRNA in human MSCs isolated from aneurysm and healthy aortas. Fetal membrane-derived MSCs (MF-MSCs) were used as a positive control for stemness gene expression.²² qPCR was carried out in a Gene Amp 7000 Sequence Detection System (Applied Biosystems) using the TaqMan approach for the housekeeping gene, β-glucoronidase (GUS) (Applied Biostystems), and the SYBR green approach, with specific couples of primers (SIGMA Aldrich; Table 2) for the other genes. Each assay was executed in triplicate and target gene expression was normalized to GUS. The final results were determined by the comparative 2^-∆∆Ct method,²³ where ∆∆CT=[CT Target–CT GUS] AAA-MSCs-[CT Target–CT GUS] cMSCs/MF-MSCs. Results were expressed as fold changes relative to cMSCs and MF-MSCs as controls.

Table 2. Primers Used for Real Time PCR Experiments

Gene	Primer sequence
MMP-2	FWD 5’-CCCCAAAACGGACAAAGAG-3’
	REV 5’-CTTCAGCACAAACAGGTTGC-3
MMP-9	FWD 5’-GAACCAATCTCACCGACCAG-3’
	REV 5’-GCCACCCGAGTGTAACCAT-3’
EMMPRIN	FWD 5’-GGGAGAGTACTCCTGCGTCTT-3’
	REV 5’-CGACTTCACGGCCTTCAC-3’
TIMP-1	FWD 5’-GTGGCACTCATTGCTTGTCC-3’
	REV 5’-CAAGGTGACGGGACTGGAAG-3’
TIMP-2	FWD 5’-TCTCGACATCGAGGACCCAT-3’
	REV 5’-TGGACCAGTCGAAACCCTTG-3’
IL-10	FWD 5’-GGGGCTTCCTAACTGCTACA-3’
	REV 5’-TAGGGGAATCCCTCCGAGAC-3’
HLA-G	FWD 5’-GTCCTGGACTCACACGGAAA-3’
	REV 5’-GTCTGGGGAGAATGAGTCCG-3’
NANOG	FWD 5’-ACCTACCCCAGCCTTTACTC-3’
	REV 5’-GGACTGGATGTTCTGGGTCT-3’
OCT-4	FWD 5’-TGAGTAGTCCCTTCGCAAGC-3’
	REV 5’- GAGAAGGCGAAATCCGAAGC-3’
SOX-2	FWD 5’-AGGATAAGTACACGCTGCCC-3’
	REV 5’- TAACTGTCCATGCGCTGGTT-3’

EMMPRIN, extracellular matrix metalloproteinases inducer; HLA-G, human leukocyte antigen-G; IL, interleukin; MMP, metalloproteinase; TIMP, modulation by endogenous tissue inhibitors. Other abbreviations as in Table 1.

Western Blot

Total cellular proteins were extracted by cultured cells using lysis buffer (KH₂PO₄ 0.1 mol/L pH 7.5, NP-40 1%, 0.1 mmol/L β-glycerolphosphate, supplemented with a complete protease inhibitors cocktail; Roche Diagnostics) and quantified spectrophometrically with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hempstead, UK). Thirty microgram proteins were subjected to 8% SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Amersham) at 30 mA for 2.5 h. The membrane was blocked with 5% non-fat dry milk in TBS-tween (TBS-T) for 1 h at room temperature and incubated with primary antibodies against MMP-9 (1:1,000; Cell Signaling Technology, Beverly, MA, USA) and β-actin (clone AC-74; Sigma-Aldrich) at 4℃ overnight. Incubation with secondary antibodies human anti rabbit/mouse horseradish peroxidise-conjugated (GE Healthcare Milan, Italy) was performed at room temperature for 1 h. The protein signal was detected by using a Westar ηC chemiluminescent substrate (Cyanagen) and band intensities were quantified by densitometric analysis using ImageJ software (NIH, USA).

MMP Activity Assay

MMP activity was assayed by using gelatin zymography. MSCs from healthy and AAA tissues at 80% confluence were cultured in serum-free DMEM; after 24 h, MSC-conditioned media were recovered and stored at –80℃ until use. Equal aliquots of each sample were loaded on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis containing 0.1% gelatin, in non-reducing conditions. The human colon cancer cell line (HCT116) was used as a positive control. After electrophoresis, the gel was washed in Triton X-100 at room temperature for 1 h and incubated at 37℃ in zymography buffer (NaCl, CaCl₂ *2H₂ O, Tris-HCl, pH=8) for MMP reactivation. After 18 h, the gel was stained with Coomassie blue and the gelatinase activity was detected in correspondence of clear bands on dark area and quantified by densitometry using ImageJ software (NIH).

Immunomodulation Assay

Human AAA and healthy MSCs at passage 3 were seeded at 2.5×10⁵ cells in a 6-well plate in DMEM 20% FBS. After 24 h, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. A step of PBMC adhesion to a plastic flask at 37℃ for 1 h was performed to avoid monocyte contamination. PBMCs were plated at a density of 2.5×10⁶ cells/well on a MSC feeder layer in RPMI 1640 (Lonza, Walkersville, MD, USA) and were activated by the addition of 5 μg/ml phytohemagglutin (PHA; Sigma Aldrich). Activated PBMCs cultured in the absence of MSCs were used as controls. After a 72-h co-culture, PBMCs were recovered and 1×10⁵ cells were transferred in a 96-well plate for the 5-bromo-2’-deoxyuridine (BrdU) incorporation assay. BrdU was added in each culture condition and after 4 h, PBMC proliferation was assessed using the BrdU-Assay kit (Roche Applied Science) according to the manufacturer’s protocol. The remaining PBMCs were fixed with 75% ethanol at 4℃, stained with Propidium Iodide (Beckman Coulter) at room temperature for 10 min and analyzed by using a flow cytometer. The AAA-MSCs and cMSCs were trypsinized after co-culture with PBMCs and processed for HLA-G cytofluorimetric analysis and RNA extraction, as described above.

In Vitro Co-Cultures of Human Aneurysm and Healthy MSCs

A co-culture of MSCs obtained from pathological and healthy aorta was assessed and two culture conditions were tested: (1) AAA and healthy MSCs seeded in a ratio of 1:1/well in a 6-well plate; (2) AAA-MSCs cultured in the presence of healthy MSC-conditioned media.

After 72 h, total RNA was extracted from co-cultures and qPCR was performed to quantify MMP-2 and MMP-9 mRNA levels.

MMP-9 Transcription Following AAA-MSC Exposure to Interleukin-10 (IL-10)

IL-10 was added to AAA-MSC cultures to evaluate its effect on MMP-9 transcription. The AAA-MSCs from 3 different samples were seeded at 60,000/well in a 6-well plate and after 24 h, soluble IL-10 (SIGMA Aldrich, Italy) was added to cultures (0–10–20 ng/ml) in serum-free DMEM. After 24 h, total RNA was extracted and qPCR was performed to determine MMP-9 transcription.

Statistical Analysis

Results are expressed as mean±standard deviation and were analyzed by GraphPad Prism 5 statistical software (GraphPad Software Inc). Statistical analysis was performed using a Student’s t-test, one-way and two-way ANOVA tests followed by the Bonferroni post-hoc test. Results were considered statistically significant at the 95% confidence level (P value <0.05).

Results

Study Subjects

Patients’ clinical data are reported in Table 3; the mean age of the AAA group was 69.2±5.0 years (males). Control subjects’ mean age was 25.0±6.5 years (female:male=2:1).

Table 3. Clinical Characteristics of AAA Patients

	Characteristics of AAA patients (n=12)
Years (mean±SD)	69.2±5.0
Sex	Males
DAAA (mean±SD)	73±3 mm
Cholesterol (mean±SD)	158±25 mg/dl
LDL (mean±SD)	90±24 mg/dl
HDL (mean±SD)	49±8 mg/dl
Tryclicerides (mean±SD)	100±31 mg/dl
Smoking (%)	42
Hypertension (%)	100
CAD (%)	25
COPD (%)	25
CVD (%)	17
PAOD (%)	33
CRF (%)	25
Statins (%)	75

AAA, abdominal aortic aneurysm; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; CRF, chronic renal failure; CVD, cerebrovascular chronic disease; DAAA, AAA diameter; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PAOD, peripheral arterial obstructive disease.

MSCs Can Be Successfully Isolated From AAA (AAA-MSCs)

MSCs were detected in the aortic tissue before the enzymatic procedure. Figure 1 demonstrates representative immunohistochemistry that highlights the presence of the CD44+ and CD90+ cell population with a perivascular origin. As observed in Figure 1A, CD44 antibody stained inflammatory cell infiltrate and parietal cells that showed a perivascular localization in the vasa vasorum niche.

Figure 1.

Representative immunohistochemical staining of 4-μm thick sections obtained from aneurysm tissues. CD44+ (A,C) and CD90+ (B,D) cells are seen in abdominal aortic aneurysm (AAA) tissue; apart from the inflammatory mononuclear cells, CD44 is expressed by spindle cells of the perivascular niche; this cell population also expresses the CD90 molecule (Scale bars indicates 50 μm).

After enzymatic digestion, approximately 8×10⁵ cells were recovered from 2 cm² sections of the aneurismal tissue. This cell recovery was successfully obtained from the tissues of all the patients enrolled. Cells were seeded in DMEM 20% FBS and after 24–48 h, they were adherent to the plastic substrate. Cells showed a typical fibroblast-like morphology and at 7 days from seeding, they reached confluence. The cell populations isolated from the aneurysm wall exhibited a growing kinetic comparable to that seen in MSCs isolated from cryopreserved donor arteries,²⁰ and they could be expanded in vitro for at least 12–13 passages. Morphological features of AAA-MSCs are shown in Figures 2A–D.

Figure 2.

Characterization of aneurysm-derived mesenchymal stromal cells (AAA-MSCs). Primary cultures of AAA-MSCs exhibited a typical fibroblast-like morphology (A, 100× magnification; B, 200× magnification); AAA-MSCs nearly confluence at passage 2 and showed a homogenous aspect (C, 100× magnification; D, 200× magnification). Representative flow cytometry analysis on AAA-MSCs at the third culture passage: AAA-MSCs expressed mesenchymal markers (CD44, CD73, CD90, CD146, PDGF-rβ) and were negative for hematopoietic and endothelial markers (CD14, CD34, CD45) (E). Nanog, Oct-4 and Sox-2 expression in AAA-MSCs as found by polymerase chain reaction (PCR) (F) and quantitative PCR (qPCR) in healthy MSCs (cMSCs) and AAA-MSCs compared to fetal membrane (MF)-MSCs (G). Results are expressed as fold change relative to the control group and are representative of MSCs isolated from all the aneurysm tissues enrolled. Values are expressed as mean±standard deviation and are representative of at least 3 independent experiments (two-way ANOVA test, followed by Bonferroni post-hoc test).

The AAA-MSCs at passage 3 were processed for phenotypic and molecular characterization. Flow cytometry analysis showed that AAA-MSCs expressed mesenchymal molecules; that is, CD44, CD73, CD90, CD146 and PDGF-rβ. The AAA-MSCs were negative to CD14, CD34 and CD45, suggesting a non-hematopoietic and non-endothelial cell origin (Figure 2E).

AAA-MSCs Express Stem Cell Markers

Aneurysm-derived MSCs expressed Nanog, Oct-4 (2 isoforms, 308–380bp) and Sox-2 (Figure 2F), typical embryonic stem cell transcriptional factors involved in regulation of pluripotency and self-renewal programs.²⁴ In addition, gene expression was measured through the semi-quantitative approach by qPCR on healthy and AAA-MSCs and compared to MF-MSCs, as control. Both cMSCs and AAA-MSCs showed a reduced stemness gene expression in comparison to the MF-MSCs; meanwhile, no significant differences were observed between healthy and pathological MSCs (Figure 2G).

Transcriptional Levels of Major Aneurysm Determinants Are Altered in AAA-MSCs

The MMP-2, MMP-9 and their respective tissue inhibitors, TIMP-2 and TIMP-1, were analyzed in AAA-MSCs and compared to cMSCs (Figure 3A). qPCR revealed exceedingly high transcription levels of MMP-9 in AAA-MSCs (449.2-fold higher, compared to healthy controls, P=0.0155, unpaired t-test) and a less evident increase of MMP-2 (2.4-fold higher, compared to cMSCs; P=0.0458, unpaired t-test). Regarding tissue inhibitors of MMPs, we observed an increased, but not significant mRNA production of TIMP-1 in AAA-MSCs (3.4-fold higher in AAA-MSCs than cMSCs) and a decrease of TIMP-2 mRNA (0.55-fold lower in AAA-MSCs than cMSCs, P=0.02, unpaired t-test). In addition, the MMP/TIMP ratios resulted higher in AAA-MSCs compared with cMSCs (Figure 3B). We completed the analysis of AAA molecular mediators with EMMPRIN (Extracellular Matrix Metalloproteinases Inducer), which has been recently associated with cardiovascular diseases;^25,26 quantitative analysis showed a 2.6-fold increase of EMMPRIN mRNA in AAA-MSCs compared to cMSCs (Figure 3A; P=0.035, unpaired t-test).

Figure 3.

Expression of abdominal aortic aneurysm (AAA) molecular mediators in aneurysm-derived mesenchymal stromal cells (AAA-MSCs). Relative mRNA expression of metalloproteinases (MMP)-2, MMP-9, extracellular matrix MMPs inducer (EMMPRIN), modulation by endogenous tissue inhibitors (TIMP)-1 and TIMP-2 in AAA-MSCs compared to healthy MSCs (cMSCs) (A). MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios (B). Results are expressed as fold change relative to the control group. Values are represented as mean±standard deviation and are representative of at least 3 independent experiments carried out in triplicate (*P<0.05, unpaired t-test). Immunoblot on AAA-MSCs and cMSCs cell lysates (C) and band intensity quantification (D). Lanes 1–3: AAA-MSCs; lanes 4–6: cMSCs. Gelatin zymography on AAA-MSCs and cMSCs (E) and band intensity quantification (F). Lanes 1–4: AAA-MSCs; lanes 5–7: cMSC; lane 8: HCT116. Results are shown as a representative example of at least 3 independent experiments (**P<0.01; two way-ANOVA test, followed by Bonferroni post-hoc test).

MMP-9 Protein Detection in AAA-MSCs

Immunohistochemical staining of MMP-2 and MMP-9 was performed on healthy (Figure S1A) and aneurysm aortic tissues (Figures S1B–E), revealing an evident MMP-9 expression only in aneurysm-affected walls. MMP-9 was localized at both the inflammatory infiltrate and at the perivascular level of vasa vasorum (Figures S1B,C). As shown in Figures S1D,E, MMP-9 also labeled spindle stromal cells. Meanwhile, the MMP-2 protein was mainly detectable on inflammatory cells (Figure S1F). Representative Western blot analysis on whole cell lysates of mesenchymal cells isolated from the aortic wall demonstrated a pronounced MMP-9 expression in AAA-MSCs (92–84kDa); conversely, a weak signal was detected in cMSCs (Figure 3C), thus confirming our observation on healthy tissue. Band intensities quantified by densitometry were normalized to cMSCs (Figure 3D).

MMP-2 and MMP-9 Secretion in AAA-MSC Conditioned Media

The MMP-2 and MMP-9 activity was assayed by gelatin zymography on cMSCs and AAA-MSCs conditioned media; as shown in Figure 3E, MMP-9 secretion was detected only in AAA-MSCs (lanes 1–4), displaying a lower signal in lane 4. Pro-MMP-2 (72 kDa) and active MMP-2 (62 kDa) band expression pattern was similar between AAA-MSCs and cMSCs (lanes 5–7), exhibiting a major secretion of the protein latent form. Band intensities quantified by densitometry were normalized to cMSCs (Figure 3F).

HLA-G and IL-10 Expression Is Reduced in AAA-MSCs Co-Cultured With Activated PBMCs

Real-time PCR on AAA-MSCs cultured together with activated PBMCs showed a decreased mRNA production of anti-inflammatory cytokines, IL-10 (3-fold decreased in AAA-MSCs; P=0.0386, unpaired t-test) and HLA-G (4-fold decreased in AAA-MSCs; no significant differences were observed), compared with cMSCs in the same inflammatory condition (Figure 4A). Therefore, flow cytometer analysis confirmed that HLA-G was not detected on the AAA-MSC surface after co-culture with activated PBMCs, whereas cMSCs were positive to HLA-G, as expected (Figures 4B,C).

Figure 4.

Immunomodulatory properties of aneurysm-derived mesenchymal stromal cells (AAA-MSCs) compared to healthy MSCs (cMSCs). mRNA levels of interleukin (IL)-10 and human leukocyte antigen-G (HLA-G) in AAA-MSCs co-cultured with activated peripheral blood mononuclear cells (PBMCs) (A). Results are expressed as fold change relative to controls (cMSCs co-cultured with activated PBMCs). Values are represented as mean±standard deviation and are representative of at least 3 independent experiments (*P<0.05; unpaired t-test). HLA-G expression on AAA-MSCs and cMSCs, cultured without (B) or with (C) activated PBMCs. Black histograms refer to AAA-MSCs, while white histograms are referred to cMSCs. Results are shown as a representative example of at least 3 independent experiments. Evaluation of PBMC proliferation was conducted after co-culture with AAA and healthy control MSCs. A 5-bromo-2’-deoxyuridine (BrdU) incorporation assay in activated PBMCs cultured with cMSCs and AAA-MSCs for 72 h was also conducted. Data are expressed as mean of optical density units (D). Cell cycle analysis of PBMCs activated with phytohemagglutin (PHA) in the culture conditions described above, revealed a weak immunosuppressive role for AAA-MSCs (E). Results are shown as mean±standard deviation and are representative of at least 3 independent experiments (*P<0.05, **P<0.01, ***P<0.001; one way-ANOVA and two way-ANOVA test, followed by Bonferroni post-hoc test).

AAA-MSCs Exert a Weak Immunosuppressive Effect on PBMC Proliferation

After 72 h, PHA-stimulated PBMCs cultured on a cMSC feeder layer showed a lower proliferation rate (P<0.05, one-way ANOVA test), confirming our recent results about cMSC immunomodulation.¹⁷ Conversely, activated PBMCs grown on AAA-MSCs exhibited a higher BrdU incorporation rate (P<0.05, one-way ANOVA test) compared with cMSCs, whereas there were not significant differences with activated PBMCs cultured alone (Figure 4D).

The effect exerted by pathological and healthy MSCs on PBMC proliferation was confirmed by cell cycle analysis on PBMCs (Figure 4E). PBMCs cultured in the absence of PHA were quiescent in the G0/G1 phase (100%), while activated PBMCs were in a proliferative state (65.9±1.6% in G0–G1; 15.2±0.9% in S; 18.9±4.8% in G2/M). The presence of cMSCs reduced the percentage of activated PBMCs during synthesis and mitosis (S: 8.6±0.2% and G2/M: 1.3±0.2%), increasing their concentration during the latent phase (G0-G1: 91.2±1.4%). PBMCs cultured on AAA-MSCs were mainly concentrated during the G2/M phase (14.2±1.5%) than the G0/G1 phase (74.4±5.4%), while the amount in the S phase was similar to PBMCs grown with cMSCs (10.5±0.9%).

Taken together, the results obtained from immunomodulatory assays revealed that AAA-MSCs are not able to suppress mononuclear cell proliferation effectively as performed by control cMSCs, suggesting that resident MSCs have lost the ability to negatively regulate the inflammatory state that occurs in aneurysm development (associated with the hyper-production of MMPs).

cMSCs Can Negatively Modulate MMP-9 mRNA Expression of AAA-MSCs Both by Cell Contact and in a Paracrine Manner

The MMP-9 transcription by AAA-MSCs was significantly reduced after a 72-h co-culture with cMSCs (a 80% reduction was recorded, in comparison to AAA-MSC control; P=0.004, paired t-test), and a 20% MMP-9 mRNA decrease was observed when AAA-MSCs were grown in cMSC-conditioned media (0.8-fold lower than AAA-MSCs grown in conventional DMEM; P=0.0043, paired t-test; Figure 5B). Conversely, the MMP-2 mRNA levels were not influenced by cMSCs (Figure 5A), confirming that the MMP-2 transcriptional levels are steady and constitutively expressed by cells of mesenchymal origin. Moreover, a decreased MMP-9 transcription was observed in AAA-MSCs exposed to IL-10 (20 ng/ml) for 24 h (a 0.2-fold decrease relative to the unexposed control, P=0.04, paired t-test; Figure 5C).

Figure 5.

Metalloproteinase (MMP)-2 and MMP-9 mRNA expression in aneurysm-derived mesenchymal stromal cells (AAA-MSCs) co-cultured with healthy MSCs (cMSCs). (A) MMP-2 and (B) MMP-9 mRNA expression in AAA-MSCs after co-culture with healthy MSCs at the ratio density of 1:1 and after culture with cMSC-conditioned media. The MMP-9 mRNA expression in AAA-MSCs was observed after 24 h exposure to interleukin (IL)-10 (C). Results are expressed as fold change relative to untreated AAA-MSCs. Values are represented as mean±standard deviation and are representative of 3 independent experiments (*P<0.05, **P<0.01, ***P<0.001; paired t-test).

Discussion

Inflammation and tissue remodeling are responsible of the structural alterations that characterize AAA disease. These mechanisms involve many molecular mediators such as MMPs, exerting a key role in aneurysm pathogenesis, as supported by several studies that demonstrate an increased MMP production in aneurysm tissue, as well as an imbalanced ratio between MMPs and their endogenous inhibitors (MMPs/TIMPs).^15–17 The pathological pathways occurring in AAA formation involve both resident and recruited vascular wall cell populations; in the present study, we have investigated the potential contribution exerted by MSCs resident in the perivascular niche. Human MSCs are of great interest thanks to their well-established survival/self renewal property and their ability to negatively modulate immune response through the inhibition of T lymphocytes and NK cell proliferation. These properties, together with a transcriptional profile typical of embryonic stem cells; that is, expression of the transcriptional factors, Nanog, Oct-4, Sox-2, are well-expressed by a population of multipotent stromal cells recently isolated from the vascular wall of multiorgan donors.^19,20

Pathological vascular tissues were collected from 12 patients undergoing vascular repair for AAA disease; we succeeded in obtaining a cell population that in vitro had growth characteristics; that is, fibroblast morphology and growing kinetic, similar to those seen in multipotent stromal (stem) cells we recently isolated from cryopreserved donors.²⁰ In accordance with our previous observations, AAA-MSCs expressed surface antigens typical of human MSCs (CD90, CD44, CD73, CD146 and PDGF-rβ) along with an embryonic stem cell genetic profile, as demonstrated by mRNA expression of genes involved in stem cell pluripotency/self-renewal. These data demonstrate for the first time the presence of a consistent number of MSCs also in pathological conditions, indicating that the pathogenetic pathway involved in aneurysm formation does not influence the survival of MSCs resident in the vascular niche, and also does not affect their stemness property, especially with regards to their self-renewal ability. As shown by flow cytometry analysis, the cell population that we isolated from the aneurysm wall did not express the hematopoietic surface markers, CD34 and CD45. These data suggest a parietal origin of the cells we are studying, even if we cannot definitely exclude that circulating cells derived from hematopoietic stem cell lineage (monocyte-derived multipotential cells) can also be involved because they can be mobilized, under physiologic and pathologic conditions, and transferred to the vessel wall through the mural thrombus.²⁷ Meanwhile, immunostaining on AAA wall, before enzymatic digestion, confirmed the presence of MSCs, CD44+ and CD90+, niched in the perivascular area of the vasa vasorum district. In addition, cells belonging to the perivascular niche as well as stromal cells resulted positive to MMP-9 labeling.

We therefore hypothesized that, in the diseased arterial wall, the surviving resident MSCs could undergo an alteration of their physiologic function, loosing their innate capacity to neutralize the inflammatory infiltrate in AAA tissue and acquiring a “pathologic” phenotype that makes them potential mediators of aneurysm pathogenesis. AAA-MSCs were characterized for MMP and TIMP production, revealing a remarkable mRNA and protein production of MMP-9; however, we did not observe significant differences in MMP-2 transcript levels between the healthy and AAA-MSCs. We also noticed a higher expression of TIMP-1 transcript in AAA-MSCs, as a compensatory but insufficient response to the MMP-9 hyper-production, as revealed by the high MMP-9/TIMP-1 ratio. The TIMP-2 mRNA resulted decreased in pathological MSCs, while the MMP-2/TIMP-2 ratio remained higher in aneurysm samples, thus demonstrating an altered balance between MMPs and TIMPs, shifted toward a degradative behaviour. MMP activity assay confirmed transcript results and revealed that, unlike MMP-2, MMP-9 is mainly released by AAA-MSCs in their growing media. Data on MMP-2 transcript and activity observed in AAA-MSCs are consistent with the constitutive nature of MMP-2 expression. Indeed, MMP-2, together with MT-MMP-1 and TIMP-2, is constitutively expressed by bone-marrow-MSCs, contributing to their invasive capacity, while MMP-9 is released under specific stimuli and diseased conditions.²⁸

We next investigated the role of the MMP inducer, EMMPRIN, also known as CD147, in our cell model, in relation to aneurysm progression. EMMPRIN is a cell-surface glycoprotein, member of the immunoglobulin family, and it is expressed on tumor cells where stimulates surrounding stromal fibroblasts to produce MMPs, facilitating the stromal invasion process.^25,26 Recent studies on murine models of angiotensin II-induced aneurysm have reported an increased expression of EMMPRIN in aortic aneurysm tissue.²⁹ We found an increased EMMPRIN transcription (4-fold higher) in AAA-MSCs, suggesting a potential regulatory role of vascular MSCs activities.

In order to explore the critical step responsible for vascular wall MSC alterations, we supposed that vascular MSCs could have lost their innate ability to modulate an immune response. As showed by BrdU incorporation and cell cycle assay on PHA-stimulated PBMCs after co-culture with MSCs from healthy and aneurysm aorta, AAA-MSCs have a weak suppressive effect on lymphocyte proliferation, compared with cMSCs. This reduced immunosuppressive activity could be explained with the loss of IL-10 and HLA-G cytokine expression, as demonstrated by molecular analysis on AAA-MSCs after co-culture with activated PBMCs. The loss of vascular MSC immunomodulation could be of critical importance in AAA pathogenesis, and this was confirmed by co-culture experiments of healthy MSCs and pathological MSCs; a significant reduction of MMP-9 transcription following direct contact between healthy and AAA-MSCs was detected; a less evident decrease was observed in AAA-MSCs cultured in cMSC conditioned media, suggesting that cMSCs may regulate pathological mediators at the mRNA level, also in a paracrine manner. Moreover, preliminary data showed that MMP-9 could be modulated at the transcriptional level following AAA-MSCs exposure to IL-10, indicating a key strategy of MMP-9 regulation, in addition to the ability to restore MSC immunomodulation.³⁰

Our study demonstrates that the molecular mechanisms involved in arterial remodeling, as occurs in AAA development, do not affect the survival/self-renewal properties of the vascular MSC reservoir. Meanwhile, the MMP-9 hyperexpression and the weak immunosuppressive property observed in AAA-MSCs suggest that the pathological context could alter MSC behavior. Exploring the mechanisms and the molecular targets able to restore and improve MSC immunomodulation could lead to novel promising strategies to monitor aneurysm progression.

Study Limitations

Limitations of our study reside in the selection of the control subjects, because they are limited (3 controls vs. 12 AAA patients) and not sex- and age-matched with patients. Regarding the age gap, we cannot exclude that our data on MMP-9 mRNA hyperexpression and activity could be a result of the physiologic vascular aging process. Preliminary data on total RNA extracted from aortic tissue of 2 male subjects, aged between 56 and 70 years, age-matched with AAA patients collected, showed an increased MMP-9 mRNA production (9-fold increase), in comparison to healthy aortic tissues used in our study (data not shown). Conversely, MSCs isolated through enzymatic digestion behaved like cMSCs from young donors, as revealed by results on MMP-9 mRNA and immunomodulatory ability (data not shown). They were not used to extend the control group in our study because their clinical data (ie, cause of death associated with cerebral hemorrhage, presence of vascular calcifications) were not in accordance with the inclusion criteria. Thus, the age of the control subjects does not significantly influence the differences between our study subject groups, as the aortic dilation does. Our controls can be considered as healthy subjects with no cardiovascular risk and as negative controls for AAA molecular mediator expression in human perivascular MSCs.

Supplementary Files

Supplementary File 1

Methods

Figure S1. Representative metalloproteinase (MMP)-9 and MMP-2 immunostaining on healthy and aneurysm-affected aorta.

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-14-0857

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