Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis

    Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis E. Butoi, A.M. Gan, M.M. Tucureanu, D. Stan, R.D. Macarie, M. Calin, M. Simionescu, I. Manduteanu PII: DOI: Reference:

S0167-4889(16)30082-9 doi: 10.1016/j.bbamcr.2016.04.001 BBAMCR 17844

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

5 November 2015 30 March 2016 5 April 2016

Please cite this article as: E. Butoi, A.M. Gan, M.M. Tucureanu, D. Stan, R.D. Macarie, M. Calin, M. Simionescu, I. Manduteanu, Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis, BBA - Molecular Cell Research (2016), doi: 10.1016/j.bbamcr.2016.04.001

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ACCEPTED MANUSCRIPT

Cross-talk between macrophages and smooth muscle cells impairs collagen and

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metalloprotease synthesis and promotes angiogenesis

Manduteanu

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E. Butoi, A.M. Gan, M.M. Tucureanu, D. Stan, R. D. Macarie, M. Calin, M. Simionescu, I. Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Biopathology and

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Therapy of Inflammation, Bucharest, Romania

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E-mail address of the authors: Elena Butoi: [email protected] Ana Maria Gan: [email protected]

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Monica Madalina Tucureanu: [email protected]

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Daniela Stan: [email protected] Cristina Constantinescu: [email protected]

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Razvan Daniel Macarie: [email protected] Manuela Calin; [email protected]

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Maya Simionescu: [email protected] Ileana Manduteanu: [email protected]

Corresponding author: Elena Butoi (Dragomir), PhD Institute of Cellular Biology and Pathology "Nicolae Simionescu" 8, B.P. Hasdeu Street, P.O.Box:35-14, Bucharest, ROMANIA Phone: +4021 319 45 18 Fax:

+4021 319 45 19

e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Coronary atherosclerosis complicated by plaque disruption and thrombosis is a critical event in myocardial infarction and stroke, the major causes of cardiovascular death. In

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atherogenesis, macrophages (MAC) and smooth muscle cells (SMC) are key actors; they

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synthesize matrix components and numerous factors involved in the process. Here, we design experiments to investigate whether SMC–MAC communication induces changes in ECM

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protein composition and/or neo-angiogenesis. Cell to cell communication was achieved using trans-well chambers, where SMC were grown in the upper chamber and differentiated MAC in the bottom chamber for 24 or 72h. We found that cross-talk between MAC and SMC

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during co-culture: (i) significantly decreased the expression of ECM proteins (collagen I, III, elastin) in SMC; (ii) increased the expression and activity of metalloprotease MMP-9 and

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expression of collagenase MMP-1, in both MAC and SMC; (iii) augmented the secretion of soluble VEGF in the conditioned media of cell co-culture and VEGF gene expression in both cell types, compared with control cells. Moreover, the conditioned media collected from

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MAC-SMC co-culture promoted endothelial cell tube formation in Matrigel, signifying an

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increased angiogenic effect. In addition, the MAC-SMC communication led to an increase in inflammatory IL-1β and TLR-2, which could be responsible for cellular signaling. In

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conclusion, MAC-SMC communication affects factors and molecules that could alter ECM composition and neo-angiogenesis, features that could directly dictate the progression of atheroma towards the vulnerable plaque. Targeting the MAC-SMC cross-talk may represent a

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novel therapeutic strategy to slow-down or retard the plaque progression.

Keywords: macrophage-SMC cross-talk, angiogenesis, extracellular matrix proteins, matrix metalloproteinases, atherosclerosis

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ACCEPTED MANUSCRIPT Introduction Coronary atherosclerosis complicated by plaque rupture and thrombosis is one of the leading causes of cardiovascular death [1]. One important pathophysiologic process

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implicated in the progression of the plaque towards a vulnerable one is the alteration of the

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extracellular matrix (ECM) composition. Many atherosclerotic lesions which are deficient in collagen fibrils progress towards plaque rupture and myocardial infarction [2]. Therefore, the

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strength of the fibrous cap and its resistance to rupture depend on the relative balance of collagen (particularly collagen I and collagen III) deposition and its degradation [3]. Smooth muscle cells (SMC) both synthesize and degrade the surrounding ECM, and

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type I and III collagens are proteolytic substrates for several metalloproteinases (MMPs) such as MMP-1, -2, -8, -9, and -13. Collagen destruction is a two-step process, with collagenases

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(MMP-1, MMP-8, and MMP-13) initiating the first steps in the degradation of native collagen I and collagen III, resulting in the generation of three-quarter and one-quarter length fragments [4]. In the second step, these cleaved fragments are further degraded by gelatinases

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(MMP-2 and MMP-9) and MMP-3 leading to reduced resistance to mechanical stresses [5].

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These enzymes digest the fibrous cap, particularly at the edges, causing the cap thinning and ultimately rupture. Both, the collagenases and gelatinases are increased in human atheroma

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[6] and are expressed mainly by activated SMC and macrophages (MAC) [4], [7]. Healthy SMC have an important role in stabilizing the vessel wall through the elaboration of collagen fibrils. The presence of an inflammatory process impedes SMC from synthesizing and depositing collagen, and increase the MMPs levels which support matrix destruction [8].

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In addition to its action on extracellular matrix composition, inflammation has been shown to be an essential factor in angiogenesis [9], also. As recently reported, the pathological neo-vascularization is a major contributor to plaque progression and rupture, and is involved in both early and late stages of the atherosclerosis [10]. Neo-angiogenesis is directly responsible for erythrocyte leakage and hemorrhages and participates in leucocyte extravasation into the newly vascularized plaques [11]. It was found that the medial vascular SMC are the main source of vascular endothelial growth factor (VEGF)-A, in response to lipid products generated at the plaque level, revealing an important role of SMC in angiogenesis in the early stages of atheroma [12]. In addition to producing VEGF, the SMC produce or express other crucial players in the angiogenic process such us: (1) MMPs - which degrade extracellular matrix components allowing endothelial cells to penetrate and reshape connective tissue [13]; (2) IL-1β - stimulates cells of endothelial lineage to secrete VEGF [14]; or (3) Toll-like receptor 2 (TLR-2) which mediates the upregulation of angiogenic 3

ACCEPTED MANUSCRIPT factors VEGF and IL-8 [15]. TLRs, the key players in innate immunity, are upregulated in atherosclerotic lesions, but their role in the progression of atherosclerosis is not well known. Monaco’s group demonstrated that TLR-2 produced by human atheroma cell culture signal

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through MyD88 and plays a predominant role in inflammation and matrix degradation in

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human atherosclerosis [16]. Further research is required to establish the exact mechanisms underlying the tightly regulated angiogenic processes and matrix degradation in

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atherosclerosis.

Many cell types are involved in atherogenesis and complex cross-talks take place between different cells within the plaque. Recently we and others found that interaction(s)

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between SMC and monocytes/macrophages results in an inflammatory cascade, leading to monocytic activation and the switch of SMC toward a synthetic phenotype [17], [18], [19].

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We hypothesize that the molecular signals resulting from the cross talk between SMC-MAC could induce modification of extracellular matrix composition and angiogenesis, changes that are known to occur within the atherosclerotic plaque. We provide evidence that

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the communication between SMC-MAC leads to an unbalanced secretion of some matrix

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proteins by SMC (collagens, elastin, MMP-9) and concurrently induces the secretion of VEGF and IL-1β, reputedly known for their angiogenic capacity. These alterations could

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explain the susceptibility and propensity of the plaque for rupture and the ensuing atherothrombosis.

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Material and methods Chemicals

SiRNAs (TLR-2/scrambled) were from Santa Cruz Biotechnology and siRNA

transfection reagent Hiperfect were obtained from Qiagen. RT‐PCR reagents and enzymes were from Invitrogen. Monoclonal antibodies, anti‐human MMP-1 (MAB901) and MyD88 (AF2928) were from R&D Systems, TLR-2 (sc-21759), IgG (sc-66931) and IL-1β (sc-7884) from Santa Cruz Biotechnology, anti MMP-9 (ab76003) from Abcam, anti-collagen I (PA535380) from Thermo Scientific and anti-collagen III (C7805) and anti‐actin (A5060) from Sigma– Aldrich. The angiogenesis assay kit was from Millipore (USA & Canada). The secondary antibodies and all the other reagents were from Sigma Aldrich Chemie GmbH (Germany).

Cell culture 4

ACCEPTED MANUSCRIPT Human aortic SMC were isolated from the media of fetal thoracic aorta and characterized as a pure cell line devoid of any contaminants. The cells exhibited an elongated spindle‐shaped morphology, grow as multilayers with the characteristic hills and valley

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pattern (as assessed by phase‐contrast microscopy), and exhibited bundles of cytoplasmic

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myofilaments and numerous caveolae at the cell periphery (as demonstrated by electron microscopy). In addition, immunoblotting and immuno‐histochemistry experiments revealed

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that they were positive for smooth muscle alpha‐actin, and for vinculin, negative for von Willebrand factor [20], and displayed functional store‐operated channels responsive for capacitative calcium entry [21]. SMC were cultured in DMEM as described [22].

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Monocyte‐like cell line THP-1 were grown in suspension in the RPMI 1640 culture medium containing 10% FCS and were split 1:5, twice a week. The investigation was carried out

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according to the principles outlined in the Declaration of Helsinki [23]. The Ethics Committee of the Institute of Cellular Biology and Pathology “Nicolae Simionescu,”

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Bucharest, approved the protocol.

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Experimental design: the co-culture system The MACs and SMC were placed in Transwell co-cultures chambers separated by a membrane filter at a distance that prevented direct cell-to-cell contact. Briefly, monocytes

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(1x106 cells) were plated in 6 well plates and differentiated towards activated MAC by exposure to 100nM PMA, for 3 days. As previously suggested, PMA activation of monocytes induces an unpolarized M0 macrophage phenotype [24], or a certain degree of classical

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activation (M1) MAC phenotype [25]. SMC (5x104 cells/well) were grown to confluence on the filter inserts (Falcon, 0.4 µm pore diameter). The SMC containing inserts were placed in the upper wells (MAC at the bottom) in RPMI without serum, for 24h or 72h. For every experiment, control differentiated macrophages or SMCs (not co-cultured) were maintained for the same time period (24 or 72h), in the serum free medium, as the co-culture samples. The total volume in each well was 4 ml. In other experiments, the SMC were seeded on the bottom of the culture plate and MAC on the insert filters. At the end of co-culture time, the conditioned media and the cells were isolated and analyzed individually by different techniques (see below).

Western Blot The protein expression of IL-1β, MyD88, MMP-1, MMP-9, TLR-2, collagen I, collagen III and actin were assessed in the total extract of SMC or MAC obtained by 5

ACCEPTED MANUSCRIPT homogenizing the cells in Laemmli electrophoresis buffer as described [22]. The signals were visualized using SuperSignal West Pico chemiluminescent substrate (Pierce) and quantified by densitometry employing gel analyzer system Luminiscent image analyzer LAS 4000

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(Fujifilm) and Image reader LAS 4000 software.

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Transfection of small‐interfering RNA (siRNA)

The human siRNAs (TLR-2/scrambled) were transfected into SMC (at 24h after they were passed to the bottom of the 6 wells culture plates), using siRNA transfection reagent Hiperfect® (Qiagen) according to the manufacturer’s protocol. Twenty‐four hours after

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transfection, the cells were washed, incubated with serum free medium and co-cultured with PMA-differentiated macrophages, for another 72h. Then, the conditioned media were

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collected and the cells were processed for further protocols, as described.

Real-Time PCR

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Total cellular RNA was isolated from cells using GenElute® Mammalian Total RNA

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kit (Sigma). First‐strand cDNA synthesis was performed employing 1µg of total RNA and MMLV reverse transcriptase, according to the manufacturer’s protocol (Invitrogen).

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Assessment of matrix proteins and other molecules mRNA expression was done by amplification of cDNA using a LightCycler 480 Real Time PCR System (Roche) and SYBR Green I chemistry. The primer sequences are shown in Table I. Optimized amplification

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conditions were 0.2mM of each primer, 2.5mM MgCl2, annealing at 60°C and extension at 72°C for 40 cycles. Actin gene was used as internal control. The relative quantification was done by comparative CT method and expressed as arbitrary units. The beta-actin was used as reporter gene for all the investigated molecules. Table 1 Gene

GenBank® Accession Number

Coll I

NM_000089

Sequences of Oligonucleotide

Predicted size (bp)

Primers

5’-aattggagctgttggtaacgc-3’

125

5’-caccagtaaggccgtttgc-3’ Coll III

NM_000090.3

5’-aggtcctgcgggtaacact-3’

226

5’-actttcacccttgacaccctg-3’ Elastin

NM_001081752.1

5’-cattcctacttacggggttgga-3’ 5’-ctccgacactagggacacc-3’ 6

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ACCEPTED MANUSCRIPT 5’-gaaggcttgaaccaacctacg-3’

Fibronectin NM_014923

96

5’-tgattcagacattcgttcccac-3’ MMP-2

5’-ccgtcgcccatcatcaagtt-3’

NM_004530

169

5’-gtgcgtcttccccttcactttcct-3’

NM_004994

199

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MMP-9

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5’-ctgtctggggcagtccaaag-3’ 5’-ggaatgatctaagcccagcg-3’ 5’-atcctccaatcaggttctct-3’

NM_003264

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TLR-2

118

5’-ggacaggtcaaggctttttaca-3’ IL-1β

5’-tggccctaaacagatgaagtgc-3’

NM_000576

488

VEGF

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5’-tcaacacgcaggacaggtacag-3’ 256

5’-catgtacgttgctatccaggc-3’

250

5’-gctactgccatccaatcgag-3’

NM_001025366

β-actin

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5’-tctttctttggtctgcattcac-3’ NM_001101

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5’-ctccttaatgtcacgcacgat-3’

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VEGF quantification by ELISA

VEGF antigen were quantified in the cell-conditioned media using an ELISA assay

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(R&D systems, UK) which measure VEGF165 levels in cell culture supernatants, serum and plasma, according to the manufacturer's instructions.

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Matrigel assay

The formation of tube-like structures by human vascular endothelial cells (HUVEC EA926 line) was assessed employing a solid gel of basement membrane proteins and growth factors (Millipore, USA & Canada). The bottoms of 96-well culture plates were coated with Matrigel (50 µl per well) and incubated for 1h at 37 °C for gelatination. The gels were overlaid with 100 µl of conditioned media collected from control cells or from co-cultured SMC-MAC and next, 20000 endothelial cells/well were added. The tube formation was monitored using an inverted phase contrast microscope (Zeiss, Axio Vert.A1, Germany) and pictures were taken by AxioCam ERC 5s camera. Tubule branching points, meshes and total length were analyzed using Angiogenesis Analizer soft and Image J program (Gilles Carpentier. Angiogenesis Analyzer. ImageJ News, 2012). For controls, blocking studies were performed. Briefly, the conditioned media from SMCMAC co-culture was pre-incubated with human IL-1β (5 μg/ml, sc-7884, Santa Cruz) for 30

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ACCEPTED MANUSCRIPT min (37 °C), before starting angiogenesis assay. As negative control, a non-specific immunoglobulin IgG (sc-66931, Santa Cruz) was used. Gelatin zymography assay

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The gelatinolytic activity of MMP-2 and MMP-9 was evaluated by gelatin

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zymography, as we previously described [17]. Briefly, cell lysates or conditioned media

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collected from control or co-cultured SMC-MAC was centrifuged at 800g, mixed with nonreducing Laemmli sample buffer and subjected to electrophoresis under nonreducing conditions on 10% polyacrylamide gels containing 1 mg/ml gelatin as substrate. After

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electrophoresis, the gels were re-natured in 2.5% Triton X-100 (2×30 min) and then incubated with 50 mM Tris–HCl, pH 7.4, containing 10 mM CaCl2 and 0.2 mM PMSF (18 h, 37 °C); subsequently the gels were stained with 0.2% Coomassie brilliant blue R-250 and de-

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stained with 10% acetic acid and 25% methanol. The white bands against the blue background were indicative of the presence of gelatinolytic activity. Image acquisition was

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Statistical analysis

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done with Image Master VDS and LisCap software (Amersham Pharmacia Biotech).

The data obtained from experiments were expressed as means standard deviation

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(SD). Statistical evaluation was carried out by one‐way ANOVA -Bonferroni test- and Twosample T-test from Origin-Pro7.5 software. A value of p<0.05 was considered statistically

Results

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significant. The “n” from the figure legends indicate the number of independent experiments.

MAC - SMC co-culture affects differently the expression of several extracellular matrix proteins Types I and III collagen, elastin and fibronectin, warrant the structural strength and elasticity of the atherosclerotic plaques, and are synthetized mainly by SMC. We evaluated the effect of co-culture of SMC and MAC for 24h or 72h, on the gene expression of Col I, III and elastin in each of these cells comparatively with controls, consisting in MAC and SMC grown independently, (as described in the experimental design). The quantitative PCR results showed that communication in co-culture, between SMC and MAC for 24h, significantly decreased the expression of collagen I, III and elastin in SMC (figure 1A); however, fibronectin mRNA was not significantly modified. Interestingly, when the cells were co-

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ACCEPTED MANUSCRIPT cultured for longer time (72h), the expression of all these proteins (including fibronectin) were considerably reduced (figure 1A). For a long time it was believed that MAC do not produce collagen; however, Schnoor

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group reported that MAC express almost all known collagen and collagen-related mRNA

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[26], although some of the collagens (type II and III) are expressed at very low levels. We have also found that MAC express matriceal molecules (Coll I, III, elastin and fibronectin),

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much less compared with SMCs (CT for SMC is around 14 and CT for macrophages is around 27), and these reduced levels were not considerably affected by SMC-MAC cocultures conditions. Actually we detected that MAC, upon co-culture with SMC exhibited a

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slightly increase in elastin mRNA at 24h only, and an increase in fibronectin mRNA at 72h (supplementary figure 1B). When the collagens were investigated for protein expression, we

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also found reduced levels of collagen I and III in SMCs (figure 1B) and undetectable levels of collagen protein in macrophages. Interestingly, in the validation studies where instead of macrophages derived from THP-1 we have used macrophages derived from human freshly

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isolated monocytes (supplementary material), the gene and protein expression of collagens

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were also significantly reduced in SMC (supplementary figure 6 A, C).

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Effect of SMC-MAC communication on the expression of matrix metalloproteinases

As already mentioned, collagen degradation is a two steps process which involves the activity of collagenases (such as MMP-1) and gelatinases (MMP-2 and MMP-9) [5], and we

i)

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investigated the effect of cell cross-talk on these molecules. Evaluation of MMP-1 protein in macrophages and SMC after their co-culture

One of the collagenases that cleave the fibrillar collagen monomers into smaller fragments at a bond between glycine and isoleucine is MMP-1 [27]. To identify the factors involved in collagen reduction upon cell communication, we investigated the expression of MMP-1 protein in both SMC and MAC grown independently (control) or co-cultivated. The Western blot results showed that MMP-1 protein expression was significantly increased in SMC (P=0.04) and MAC (P=0.008) that were previously in co-culture, compared with the same cells grown independently (figure 2 A and B). ii)

Effect of communication between macrophage - SMC grown in co-culture on gelatinases (MMP-2 and MMP-9) expression and activity The MMP-2 and -9 expression and activity was assessed in cell lysates and in the

collected conditioned media. The experiments showed that the gene expression of MMP-9 9

ACCEPTED MANUSCRIPT was highly upregulated in SMC which were co-cultured with MAC for 24h (~35 fold; p<0.0001) or 72h (p<0.01, Figure 3A). To a lesser extent, in MAC after prior co-culture with SMC, the MMP-9 gene expression was also significantly up-regulated compared with control

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MAC (p<0.05 for 24h and p<0.05 for 3days of co-culture, figure 3B). The MMP-9 protein

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expression was also significantly increased in both cell types, SMCs and macrophages (figure 3E), and in a large extend, when SMC were co-cultured with macrophages derived from

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human freshly isolated monocytes (supplementary figure 6D). For MMP-2 gene expression no significant differences were found between control cells and co-cultured cells. Zymographic analysis of the conditioned media (CM) collected from co-cultured MAC-SMC

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demonstrated an increase of pro-MMP-9, compared with CM collected from control cells (Figure 3C). Although MMP-2 appear to be also increased in CM from cell co-culture

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compared with control MAC, it was not different from control SMC. In the case of cell lysates, the experiments showed that pro- and active MMP-9 was significantly increased in both, MAC (2 fold P<0.05) and SMC (7 fold, P<0.001) and that pro- and active MMP-2 was

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significantly increased only in MAC (P=0.01) after being subjected to co-culture conditions

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(Figure 3D).

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Conditioned medium collected from SMC-MAC co-culture induces formation of endothelial tube-like structures Since the ECM alteration and matrix metalloproteinases were reported to be crucial players in the modulation of the angiogenic process [28], [27], we explored whether the

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observed changes induced by SMC-MAC co-culture on the cell synthesis of ECM components are functionally important. For this, the effect of conditioned media (CM) on endothelial capacity to form tube-like structures was evaluated in vitro. Before starting the angiogenesis experiments, we evaluated the gene expression of VEGF (a potent stimulator of angiogenesis) in SMC and MAC, and the VEGF protein released in the culture media. The Real Time PCR results showed that VEGF mRNA was significantly increased in both, MAC and SMC that were co-cultured as compared with control cells (Figure 4 A and B). Using the VEGF - ELISA kit assay we have found an increased levels of VEGF released in the CM from cell co-culture, compared with VEGF determined in the CM of control cells (MAC or SMC) (Figure 4C). The angiogenic capacity of the factors released upon SMC-MAC communication during co-culture was tested using Matrigel assay. As shown in the figure 5D (d1,d2,d3), the number of meshes, nodes and the total length of tube like structures was significantly increased by the CM from co-cultured cells (MSc), compared with CM from 10

ACCEPTED MANUSCRIPT control (independently cultured) SMC (S) or MAC (M). When endothelial cells were added on the gel in fresh medium (VEGF free medium), the number of tube-like structures was

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reduced (supplementary figure 4).

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IL-1β gene and protein expression is up-regulated in each cell upon SMC-MAC co-culture and is involved in endothelial tube-like formation

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Previously, it was demonstrated the necessity of a microenvironment-derived IL-1β for tumor angiogenesis [29], [30]. Moreover, it was recently found that IL-1β and VEGF induce each other and act in a complementary manner to promote angiogenesis in Matrigel

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plugs supplemented with tumor cells [31]. Here, we evaluated the effect of SMC-MAC coculture on IL-1β expression and its involvement in angiogenesis process. As shown in figures

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5A and B, upon co-culture, the gene expression of IL-1β was significantly increased in MAC (24h - P=0.003; 3days - P=0.04) and SMC (24h - P=0.0004; 3days - P=0.01). Moreover, the protein level of IL-1β was also increased in cells which were in co-culture for 72h, compared

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with control cells (Figure 5C). The possible involvement of IL-1β secreted in the CM to

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affect angiogenesis, was assessed by incubating the CM collected from co-culture of SMCMAC with anti-IL-1β (or normal IgG - as control) for 30 minutes and then added to the

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Matrigel for testing. As shown in figure 5D and in the statistic graphs (d1, d2, d3), addition of IL-1β antibody significantly reduced the number of meshes, nodes or tube total length, indicating the involvement of IL-1β in the angiogenesis process. The values for calculated statistic P are indicated in figure 5. When the anti-IL-1β and irrelevant IgG were added to the

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fresh media, there was not observed significant modification of tub-like structure numbers (supplementary figure 4). Silencing of TLR-2 hinders the induction of IL-1β expression and endothelial tube-like formation TLRs are well-characterized-pattern-recognition receptors that mediate innate immunity. Among the different TLRs that have been found in humans and mammals, the increased expression and the importance of TLR-2 and 4 have been demonstrated in atherosclerotic plaques [32]. Recently, it was found that TLR-2 signaling stimulates progression of the pro-inflammatory phenotype of mouse aortic SMC by activation of MMP2 and secretion of pro-inflammatory cytokines [33]. Moreover, synthesis of pro-IL-1β (which we found that is increased upon cell co-culture, figure 5) is tightly regulated by a unique twosignal mechanism, dependent on TLR-2 [34]. Therefore, we set up experiments to evaluate 11

ACCEPTED MANUSCRIPT the expression of TLR-2 in MAC and SMC. The Real-Time PCR results showed that consequent to SMC-MAC co-culture, the TLR-2 expression was significantly increased in both, MAC (Mc, P=0.005) and SMC (P=0.004, Figure 6A). It is known that MyD88 plays a

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key role in TLR-2 signaling. Our experiments demonstrated that the protein expression of

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MyD88 was also induced in MAC (P=0.007) and SMC (P=0.0017), following to their coculture (Figure 6B). The involvement of TLR-2 signaling in the induction of angiogenic

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mediators in the CM of co-cultured cells was investigated using siRNA transfection technique, to down-regulate TLR-2. To this purpose, before cell co-culture, SMC were transfected with siRNATLR-2 or negative control siRNA, and then co-cultured with MAC.

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The transfection efficiency was tested by Real-Time PCR and Western Blot experiments (supplementary figure 2). After transfection, the CM was collected and examined for the

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capacity to induce endothelial tube-like structures in Matrigel. As shown in the figure 6C, CM collected from co-culture in which TLR-2 was down-regulated in SMC, exhibited a 40% decreased number of tube like structure, compared with CM obtained from non-transfected

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cells or transfected with negative control. All the calculated parameters (nodes, meshes, total

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length) were reduced in the co-culture of cells in which TRL-2 was silenced (Figure 6 c1, c2, c3). In addition, we evaluated the effect of TLR-2 knockdown on IL-1β and on the matriceal

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proteins. In these experiments we found that silencing of TRL-2 in SMC prior to SMC-MAC interaction significantly reduced the expression of IL-1β (Figure 5D), but did not affected significantly the expression of collagen I, III and MMP-9 (data not shown).

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Discussion

Atherosclerosis is a multi-factorial disease, which involves a complex scheme of direct and indirect communication between immune cells and vascular cells. We have previously reported the existence of one such cell to cell dialogue taking place between smooth muscle cells and monocytes/macrophages, the result of which is an exacerbated inflammatory process [17]. During atherogenesis, inflammatory chemokines produced by monocytes/macrophages stimulate migration of SMC from the vessel’s media and their proliferation within the intima, leading to the neointima development. In later stages, MAC release more pro-inflammatory cytokines and MMPs that affect SMC phenotype, and neoangiogenesis. These major events could lead sequentially to thinning of the fibrous cap, development of vulnerable plaque [3], and ultimately to plaque rupture [35].

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ACCEPTED MANUSCRIPT In this study, we investigated whether the cross-talk between MAC and SMC - two cells with a known critical role in the development of the atheromatous plaque, has an effect on their capability to produce matrix proteins and angiogenic factors. The findings reported

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here and summarized in the figure 7, show that consequent to the cross-talk between SMC-

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MAC, there is: (1) a drastic reduction in the synthesis of collagens I and III, elastin and fibronectin by SMC; (2) a significant increase in MMP-9 production in both, SMC and

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macrophages; (3) an enhanced synthesis and release of angiogenic factors, VEGF and IL-1β in both cells; these factors are functionally active since they induce angiogenesis by a mechanism that involves, at least in part, activation of TLR-2 signaling pathway.

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Degradation of the fibrous cap collagens has been widely implicated in atherosclerotic plaque rupture, and the unbalance between synthesis and degradation of interstitial collagen

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was considered as a central event in this process [3]. Within the atherosclerotic plaques, this balance can shift either toward reduced synthesis of collagens as a result of shear stress or inflammatory cytokines action, or toward increase matrix degradation, since the MAC and

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SMC (with altered phenotype) secrete different proteinases including collagenases and

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gelatinases, [36], [37]. Reportedly, the pro-inflammatory cytokine IFN-γ decreases the collagen synthesis by down-regulating SIRT1 expression in SMC [36]. Our experiments

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indicated that upon co-culture of MAC with SMC, the latter exhibit a drastic reduction in the expression of collagens I and III, fibronectin and elastin, features that may explain the alteration of ECM composition occurring or leading to plaque progression. As mentioned, in addition to the reduced collagen synthesis, collagen degradation is

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enhanced during atherogenesis due, in part, to the increased activity of matrix MMPs [38]. Here we have found that SMC-MAC communication leads, in parallel to the decrease in matrix protein synthesis, to increased expression in both cell types of MMP-1 and MMP-9, the most important metalloproteases involved in collagen degradation. These results are in good agreement and extend the data showing that MMP-1 dictates the collagen content of the atherosclerotic plaques [39] and that inhibition of MMP-9 leads to increased levels of collagen I and collagen III mRNA expression in vascular SMC isolated from carotid plaques and treated with epidermal growth factor [38]. Using apolipoprotein E/MMP-9 double deficient mice it was found that deletion of MMP-9 results in decreased plaque volume, after 25 weeks of diet [40]. Although the majority of works underline the deleterious effects of MMPs in cardiovascular pathologies, some of the MMPs are also involved in fibrous cap formation. By their proteolytic activity, normal levels of some MMPs controls matrix destruction and the availability of active molecules such as growth factors [41], processes that 13

ACCEPTED MANUSCRIPT facilitate the SMC migration and fibrous cap formation [42]. Therefore, using a model of femoral artery injury in MMP9- or MMP2-deficient mice, it was found that TNF-α induced vascular SMC migration in a MMP9-dependent manner, and PDGF induced SMC

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proliferation in a MMP2-dependent manner, both of which contributed to the mechanism of

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neointimal hyperplasia formation [43]. Moreover, it was found that lesion area was larger in brachiocephalic arteries isolated from apoE/MMP-3 and apoE/MMP-9 double knockout mice

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after 8 weeks, suggesting the protective role of MMP-3 and MMP-9, by limiting plaque growth and promoting a stable plaque phenotype [44]. However, although the MMP-9 contributes to SMC migration and neointima formation, there is possibility that depending of

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plaque evolution step, local agonists and/or cell-cell interactions to further exacerbate the MMPs production, TIMPs reduction and thus destruction of neointima matrix, leading to

MA

vulnerable plaque. This can explain the contrary results obtained in the studies motioned above [40], [44], where the lesions were evaluated at different time of plaque evolution, 25 versus 8 weeks. Interestingly, in our work the MMP-2 and MMP-9 appear to be different

D

affected by cell cross talk. Both MMP-2 and MMP-9 are significantly associated with plaque

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rupture [45], but MMP-9 has been highlighted as one of the most important enzyme related with unstable carotid plaques [46]. In the case of MMP-2, the relationship between its

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expression and unstable plaques is controversial, since there are studies which showed that MMP-2 is not related to carotid instability plaques [46] or that MMP-2 activity may be associated with a stable lesion phenotype [47]. Moreover, the MMP-9 but not MMP-2 was associated with an inflammatory process [48], [49]. Our cross-talk system induces an

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increased inflammatory effect that may explain the differences obtained in the MMP-2 and MMP-9 modulation. We observed that MMP2 and MMP-9 are differently affected by crosstalk, with MMP-2 activity increased only in macrophages while MMP-9 expression and activity are highly increased in both SMC and MAC, supporting the conclusion that cell cross-talk induced matrix degradation. The MMPs activity can be inhibited by endogenous tissue inhibitors (TIMPs), which are essential for the regulation of normal connective tissue metabolism. An elevated MMP/TIMP ration is equivalent with the net increase in proteolytic activity of MMPs and subsequently with disease pathology. An increased MMP-9/TIMP2 ration was recently found at patients with thoracic aortic aneurysms [50] and patients with ischemic stroke [51]. Moreover, systemic adenovirus-mediated gene transfer of TIMP-2 improved plaque stability in high fat-fed apolipoprotein E-knockout mice [52]. We evaluated the effect of SMCmacrophage cross-talk on TIMP-1 and TIMP-2 gene expression and obtained that while 14

ACCEPTED MANUSCRIPT TIMP-1 was not affected, TIMP-2 was significantly reduced by cell cross-talk in SMCs (supplementary figure 5). Therefore, because cell cross-talk augments the production of MMP-9 without affecting the synthesis of TIMP1 and down-regulating the TIMP-2, this may

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favor the ECM degradation. By their properties to degrade extracellular matrix components

major role in initiating and sustaining angiogenesis [53].

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MMPs also pave the way for angiogenesis and this is particularly true for MMP-9 that plays a

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An unexpected finding of our experiments is the demonstration, for the first time, that upon communication in culture between MAC and SMC, pro-angiogenic factors are released from the cells in the conditioned media (CM), which have the capacity to induce in vitro

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angiogenesis. We detected a significant augmented level of VEGF in the CM collected from cell co-culture compared with control SMC or MAC that induced an increased number of

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endothelial-derived tube like-structures compared with CM from control cells. Moreover, the expression of IL-1β - inflammatory molecule with angiogenic properties was increased in both cell types after co-culture. The functionality of the released

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IL-1β in angiogenesis was demonstrated by the experiments in which blockage of IL-1β

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reduced the endothelial cell ability to form tube like structures. These results are in good agreement and extend previous data on the angiogenic properties of IL-1β secreted by MAC

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[14].

Regarding the molecular pathways responsible for the regulation of these molecules by cell cross-talk, previous studies, including ours, revealed that the signaling pathways p38MAPK and JNK, and the inflammatory transcription factors AP-1, NF-kB and STAT3 are

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activated in SMC and/or monocytes/macrophages after their interaction [17], [18], [54]. Moreover, it was found that these signaling pathways regulate the expression of collagens, MMPs and VEGF [17], [55], [56]. Thus, in a recent study it was demonstrated that inhibition of p38-MAPK and JNK-MAPK signaling pathways decreased the expression of MMP-1, and MMP-9 and increased the collagen type I and III expression in EGF-treated vascular SMC [55]. In addition, it was demonstrated that STAT-3 promotes the expression of VEGF [56]. Here, using specific inhibitors for p38 MAPK, ERK1/2, but also STAT3 and NF-kB transcription factors, we have found that MMP-9 is significantly reduced by ERK1/2 (PD90059) and STAT3 inhibitor (S3I-201) in SMC. Since ERK1/2 is a signaling molecule that further activate the AP-1 transcription factor, we may conclude that MMP-9 regulation is dependent by AP-1 and STAT3 pathways. Previously, we have reported that the increased expression of MMP-9 in SMC (after their direct interaction with monocytes) was also dependent on AP-1 transcription factor [17]. Taken together these data may indicate that the 15

ACCEPTED MANUSCRIPT MAPKs signaling and inflammatory transcription factors NF-kB, AP-1 and STAT3 induced by cell cross-talk could be responsible for modulation of collagens, MMPs and VEGF. In the case of IL-1β, knowing that the main pathway that triggers its induction is

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dependent on activation of inflammasome by toll-like receptors [57], we evaluated the

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expression of the main TLRs (TLR--2 and TLR-4). Although the TLR-4 was not significantly affected by cell cross-talk (data not show), we have found a significantly increase of TLR-2

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expression in both MAC and SMC after their co-culture. This result arises an interesting question, namely how cell cross-talk could induce stimulation of TLR-2. An explanation came from previous data which found that different component of matrix (biglycan, decorin,

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versican) act as endogenous ligands for TLR-2 and TLR-4 [58]. These proteoglycans are usually stationary components of ECM, but when they are released during inflammation or

MA

tissue injury, they become available in soluble form and are endogenous ligand for TLR-4 and TLR-2. Further investigations are necessary to follow the effect of macrophage-SMC cross-talk on these proteoglycans synthesis and release. In addition to TLR-2, the adaptor

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molecule MyD88 expression was also up-regulated by cell co-culture, in both, MAC and

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SMC. In experiments in which TLR-2 was knockdown, IL-1β mRNA was significantly reduced, indicating the involvement of TLR-2 signaling pathway in IL-1β regulation.

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Consequently, we tested the direct involvement of TLR-2 in the angiogenic process by silencing its expression in SMC before co-culture with MAC. Interesting, the number of endothelial tube-like structures were significantly reduced as a result of TLR-2 knockdown, providing evidence for its direct involvement in the angiogenic process. This assumption is

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also supported by results showing that VEGF mRNA produced by SMC and soluble VEGF released in the CM are reduced by TLR-2 silencing (supplementary figure 3). Interestingly, in a recent study it was also found that TLR system participates in the angiogenic response of the vessel wall, and was suggested that TLRs may play important role in the regulation of angiogenesis in pathologic processes [59]. An interesting aspect of our results is that all the modification obtained are mediated by

soluble

factors.

As

the

lesion

develops,

activated

SMC

and

lesional

monocytes/macrophages secrete many pro-inflammatory factors as well as additional factors, and engage in paracrine cross talk and autocrine signaling. Previously we have shown that interaction of monocytes to SMC in direct co-culture resulted in increased levels of soluble TNF-α and IL-6 [17], and here we found the IL-1β is also increased in indirect co-culture between MAC-SMC. Moreover, our recent data reveal increased levels of MCP-1 and fractalkine secreted in the culture medium following MAC-SMC indirect interaction 16

ACCEPTED MANUSCRIPT (unpublished data). All these inflammatory cytokines/chemokines released extracellular might be predicted to participate in subsequent paracrine effects on both macrophages and SMCs. Therefore, the influence of TNF, IL-6 and IL-1β on the plaque evolution is widely

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recognized, and recently evidence shown that MCP-1, RANTES, and fractalkine may

IP

promote instability of coronary atherosclerotic plaque [60]. It is possible that increased MMP-9 expression observed in human plaques may be an effect of a paracrine effects of

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these cytokines/chemokines, released as an effect of cell cross-talk. In addition, the soluble cytokines may participate to the phenotypic modulation (or polarization) of macrophages. These polarized macrophages may be then able to signal back to the local SMC to either

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exacerbate or maintain their activation state. This feed-forward signaling would be expected to ultimately result in worsened neointima formation in response to injury.

MA

Study limitation: In this study, for co-culture studies, we have used a human aortic SMC line and the macrophages differentiated from human monocytic cell line THP-1. Further studies employing human atherosclerotic plaques biopsies will be necessary to

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strengthen the significance of our novel observations.

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Taken together, the data indicate that macrophages and smooth muscle cells talk to one-another and that this crosstalk induces changes in both cells: decrease synthesis of

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extracellular matrix components by SMC, increased production of MMPs by both cells and facilitated production of angiogenic factors (VEGF and IL-1β) by mechanism that include activation of TLR signaling pathway. These novel findings explain the significant contribution of SMC–MAC cross-talk to the development of vulnerable plaque, and suggest

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that disruption of this cellular cross talk in the atherosclerotic plaque could represent a possible therapeutic strategy to slow down the plaque evolution.

Acknowledgements We are thankful to Gabriela Mesca for skillful assistance. This work was supported by two grants of the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-0965”, project number PN-II-IDPCE-2011-3-0928 and Romanian Academy.

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[56] A. Albasanz-Puig, J. Murray, M. Namekata, and E. S. Wijelath, “Opposing roles of STAT-1 and STAT-3 in regulating vascular endothelial growth factor expression in vascular smooth muscle cells,” Biochem. Biophys. Res. Commun., vol. 428, no. 1, pp. 179–184, Nov. 2012. [57] R. G. Snodgrass, S. Huang, I.-W. Choi, J. C. Rutledge, and D. H. Hwang, “Inflammasome-mediated secretion of IL-1β in human monocytes through TLR2 activation; modulation by dietary fatty acids,” J. Immunol. Baltim. Md 1950, vol. 191, no. 8, pp. 4337–4347, Oct. 2013. [58] R. V. Iozzo and L. Schaefer, “Proteoglycans in health and disease: novel regulatory signaling mechanisms evoked by the small leucine-rich proteoglycans,” FEBS J., vol. 277, no. 19, pp. 3864–3875, Oct. 2010. [59] A. C. Aplin, G. Ligresti, E. Fogel, P. Zorzi, K. Smith, and R. F. Nicosia, “Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors,” Angiogenesis, vol. 17, no. 1, pp. 147–161, Jan. 2014. [60] Z. X. Zhong, B. Li, C. R. Li, Q. F. Zhang, Z. D. Liu, P. F. Zhang, X. F. Gu, H. Luo, M. J. Li, H. S. Luo, G. H. Ye, and F. L. Wen, “Role of chemokines in promoting instability of coronary atherosclerotic plaques and the underlying molecular mechanism,” Braz. J. Med. Biol. Res. Rev. Bras. Pesqui. Médicas E Biológicas Soc. Bras. Biofísica Al, vol. 48, no. 2, pp. 161–166, Feb. 2015.

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Figure legends

Figure 1. Macrophage–SMC cross-talk decrease expression of matriceal proteins in SMC. S -

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control smooth muscle cells; Sc- SMC which were co-cultured with MAC. A. The gene expression of collagen I and III, fibronectin and elastin mRNAs in SMC after their co-culture with macrophages for 24h or 72h. The mRNA of matriceal proteins was normalized to actin

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mRNA. Note that the co-culture condition reduces significantly the collagen I, III and elastin mRNAs in SMC at 24h (Sc 24h). This decrease is even more prominent at 72 hours (Sc 72h), when compared to control SMC (S). n=5, *p<0.01, **p<0.001 co-cultured (Sc) vs. control (S, individually cultured) cells. B. Protein expression of collagen I and III in control SMC versus co-cultured SMC (Sc), detected by Western Blot, n=3, *p<0.05.

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Figure 2. Communication between macrophages and SMC grown in co-culture for 72h increases the MMP-1 collagenase expression. A,B: Evaluation of protein expression of

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MMP-1 (Western blot) in SMC (A) and macrophages (B) grown independently, for control

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(S, M) or after subjected to co-culture conditions (Sc, Mc) cells. n=3, *p <0.05.

Figure 3. Effect of macrophage–SMC co-culture on cellular MMP-9 and MMP-2 expression and enzymatic activity. Gene expression of MMP-9 and MMP-2 was evaluated in SMC (A) and macrophages (B) that were previously co-cultured for 24h and 72h. The mRNA of MMP23

ACCEPTED MANUSCRIPT 2 and MMP-9 was normalized to actin mRNA Note that upon co-culture, MMP-9 mRNA increases in both cell types (Sc, Mc) compared with control cells (S, M). There is no effect on MMP-2 mRNA expression. n=4. C, D. Enzymatic activity of MMP-2 and MMP-9 (SDS-

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PAGE zymography) as assessed in the conditioned media (C) and cell lysate (D) isolated

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from co-cultured macrophage-SMC and from control cells. M, S - control macrophages and smooth muscle cells; Mc, Sc- macrophages and SMC which were co-cultured, M+S Note that while MMP-9 protein is

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conditioned medium from SMC-MAC co-culture.

significantly increased in both, the conditioned media collected from co-cultured cells (M+S) and in co-cultured MAC and SMC in cell lysates (Mc, Sc), MMP-2 is induced only in co-

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cultured macrophage Mc, in cell lysates. n=4, *p<0.05; **p<0.001. E. Evaluation of protein expression of MMP-9 (Western blot) in SMC and MAC grown independently (control, S, M)

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or after subjected to co-culture conditions (Sc, Mc) cells. n=4, *p <0.05

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Figure 4. Expression and secreted VEGF are increased in SMC and MAC consequent to their co-culture and the conditioned media collected after co-culture has angiogenic effect. A, B. Evaluation of VEGF mRNA expression in macrophages and SMC as a result of their crosstalk. Note that consequent to the co-culture, VEFG is significantly induced in both, 25

ACCEPTED MANUSCRIPT macrophages (A) and SMC (B), at both time used (24h and 72h). n=4, *p<0.05. C. VEGF protein released into the conditioned medium (CM) of control SMC (S), control macrophages (M), and SMC-MAC co-culture (MSc) for 72h. Note that the level of soluble VEGF released

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in the CM is also increased by cell-to cell cross-talk. n=4, *p <0.05.

Figure 5. IL-1β evaluation in SMC and MAC independently cultured (control) or consequent

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to their co-culture; angiogenic properties of IL-1β. A, B. The IL-1β mRNA is significantly

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induced in macrophages (A) and SMC (B) consequent to cell to cell cross talk in culture for both 24h and 72h. n=4, *p<0.05, **p<0.001. C. Protein expression of IL-1β determined by

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Western Blot in independently cultured MAC and SMC used as control (M, S) and in SMC and MAC after co-culture (Mc, Sc). n=3, * P<0.05. D. Angiogenic properties of IL-1β: the CM from co-cultured cells (M+S) was pre-incubated with anti- IL-1β and then endothelial

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tube formation was tested in Matrigel. Note that blocking IL-1β leads to a reduced number of nodes, meshes and the length of tube-like structures (statistical graphs d1, d2, d3). No significant effect is detected in control, in which the CM from co-culture cells was supplemented with an irrelevant IgG (M+S IgG). n=3, the p values are specified in the graphs.

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Figure 6. Macrophage –SMC cross-talk affect molecules of TLR-2 pathway. A. TLR-2 gene expression is increased in co-cultured macrophages (Mc) and SMC (Sc). n=4, * p<0.05. B.

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Protein expression of MyD88 determined by Western blot in control cultured macrophages and SMC (M, S, respectively) or upon their co-culture (Mc, Sc). n=3, *p <0.05. C. Effect of TLR-2 silencing on endothelial tube-like formation. SMC were transfected with TLR-2 siRNA before SMC-MAC co-culture and then the conditioned medium (CM) was collected and tested for angiogenic properties. As compared with CM from co-cultured cells (M+S), CM obtained from co-culture of MAC with transfected SMC (M+S siTLR-2) reduces the number of nodes, meshes and total length of tube-like structures (c1, c2, c3). n=3, the p values are specified in the graphs. D. Role of TLR-2 in the regulation of IL-1β gene expression. SMC were transfected with TLR-2 siRNA or control siRNA (NC) and cocultured with macrophages. Note that TLR-2 knockdown significantly decreases the IL-1β mRNA. n=3, *p<0.05.

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Figure 7. Summary figure. The interaction between

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muscle cells (SMC) release different soluble factors that future act in a paracrine mode and produced important effects: decrease the collagens expression, increase the metalloproteases (MMP-1 and MMP-9) expression and activity and release angiogenic factors with functional capacity to produce tube-like structures. EC- endothelial cells.

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MAC and SMC cross-talk decrease the expression of extracellular matrix proteins MMP-1 and MMP-9 are increased by cell-to-cell cross-talk Soluble factors released by cell-to-cell cross-talk have angiogenic properties IL-1β and TLR-2 mediate endothelial tube-like formation.

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Highlights

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