Kurt Farrell

Phillip Simmers

Gautam Mahajan

Ludovic Boytard

Andrew Camardo

Jyotsna Joshi

Anand Ramamurthi

Florence Pinet

Chandrasekhar R Kothapalli
email: c.kothapalli@csuohio.edu

Alterations in phenotype and gene expression of adult human aneurysmal smooth muscle cells by exogenous nitric oxide

Keywords: abdominal aortic aneurysm, elastin, matrix proteins, cell modulus, MMPs, nitric oxide, LILAS study, abdominal aortic aneurysm, elastin, matrix proteins, cell modulus, MMPs, nitric oxide, LILAS study

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

 March 19, 2019To, Prof. Neil Hogg, Editor-in-Chief Medical College of Wisconsin Dear Prof. Hogg, We hereby submit our manuscript titled, "Improvement of altered phenotype and genotype of adult human aneurysmal smooth muscle cells by exogenous nitric oxide" to be considered for publication in the Nitric Oxide journal as an original full-length article. All authors have read and agreed with the submission of the manuscript to this journal. This manuscript hasn't been submitted or published elsewhere, either in part or in full.

Novelty of this work:

It is known that the pathogenesis of aortic aneurysms leads to extracellular matrix degradation in blood vessels and disruption of vascular smooth muscle cell (SMC) interactions with endothelium and medial elastic fibers. In this work, we report for the first time, on the basal amounts of numerous matrix proteins synthesized and released by aortic aneurysmal SMCs derived from LILAS (Lille Aneurysmal Study in France) patients, changes in their phenotype and genotype, the cytokines/ chemokines/ growth factors they release, and their intrinsic biophysical characteristics (cell stiffness and surface roughness). In addition, we compared such outcomes from aneurysmal cells to SMCs derived from healthy humans. Finally, we report on the benefits of delivering exogenous nitric oxide cues to restore some of these lost biophysical and biochemical functionalities in aortic aneurysmal SMCs.

The outcomes from our study are relevant to not only understand the disease etiology but also develop effective NO delivery based pharmacological, tissue engineering and regenerative medicine strategies to repair and regenerate faithful mimics of elastic fibers in aneurysmal blood vessels. The results would be of great interest and utility to researchers working on vascular biology, translational medicine, nitric oxide signaling, cellular mechanics, and clinical studies.

Introduction

Chronic vascular pathological conditions (e.g., hypertension, atherosclerosis, trauma, infection) promote localized release of proteinases and enzymes which break down the intact extracellular matrix (ECM; e.g., elastin, glycosaminoglycans (GAGs)) within the adult abdominal aortic walls [1; 2]. Unfortunately, the major cell types in aorta, i.e., smooth muscle cells (SMCs) and adventitial fibroblasts, are limited in their ability to repair, replace or regenerate the degraded vascular matrix, which often results in progressive weakening and burgeoning of blood vessel diameter (> 50% increase relative to healthy vessel) -a condition clinically termed as abdominal aortic aneurysm (AAA). AAAs are also typically characterized by the presence of innate and adaptive immune cells (e.g., macrophages, T-cells, neutrophils, lymphocytes) which release abnormal levels of cytokines and matrix metalloproteinases (MMPs) to exacerbate the breakdown of elastic fibers and other matrix proteins [3; 4]. This, in turn, disrupts the signaling between elastic fibers and SMCs, promotes SMC apoptosis, and weakens elasticity in blood vessel walls, to permanently alter the mechanical properties and native microenvironment of the vessel wall [5; 6].

Among the major ECM components native to blood vessels, collagen, GAGs and elastin play an important role in regulating cell adhesion, anchoring, proliferation, and migration [4; 7].

They also regulate blood vessel homeostasis by supporting axial and circumferential loadings and conferring anti-thrombogenic properties [START_REF] Wagenseil | Vascular Extracellular Matrix and Arterial Mechanics[END_REF]. These ECM molecules are synthesized, crosslinked, and deposited as mature matrix fibers by SMCs and adventitial fibroblasts within the aorta. While the turnover of collagens and GAGs is maintained even through adolescence, elastic matrix fiber deposition primarily occurs only during developmental stages and preserved throughout the adult life (elastin half-life is ~74 years) [START_REF] Sivaraman | Advances in biomimetic regeneration of elastic matrix structures[END_REF]. Since healthy elastic fibers play critical biochemical and biomechanical roles in maintaining vessel homeostasis, abnormalities in elastin formation due to genetic defects or elastin degradation due to diseases could result in peptides formation, which invalidates the beneficial role of intact elastic fibers [10; 11].

Given the limitations of existing surgical (e.g., endovascular aneurysm repair, open surgery), pharmacological (e.g., anti-inflammation agents, beta-blockers, statins) and preservation (e.g., MMPs inhibition, TIMPs delivery, elastases suppression) interventions for comprehensive AAA treatment [9; 12; 13; 14; 15], ECM (specifically elastin) repair and regeneration approaches focusing on tissue-engineered grafts, stem cells, 3D scaffolds, bimolecular cues, biomechanics, and their combinations, are being explored [16; 17]. We have recently reported on the benefits of delivering exogenous nitric oxide (NO) cues via S-nitrosoglutathione (GSNO), to cell proliferation and ECM deposition by adult human aortic SMCs (Hu-SMCs) within 3D biomimetic cocultures with adult human endothelial cells (Hu-ECs) [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF]. While the phenotype and genotype of ECM proteins synthesized by healthy Hu-SMCs has been reported [19; 20; 21; 22], similar outcomes from their AAA-counterparts are unknown. Specifically, the baseline levels of matrix proteins synthesized and deposited by human AAA-SMCs, changes in their phenotype and genotype, the cytokines and chemokines released, their stiffness levels, and the responses to exogenous biomolecular cues, remains unexplored. Such information is critical for understanding the disease etiology and identify effective pharmacological, tissue engineering and regenerative medicine strategies to repair and regenerate elastin in AAA vessels.

In this study, we first investigated the changes in transcription levels of AAA-SMC genes, involved in the pathological progression of this disease. Next, we assessed the in vitro baseline levels of cell proliferation, phenotypic and genotypic expression, ECM and biomolecular (elastin, sulfated glycosaminoglycans (sGAGs), hyaluronan (HA), LOX, desmosine, inducible nitric oxide synthase -iNOS) synthesis and deposition, release of cytokines/ chemokines/ MMPs/ TIMPs, and biomechanical characteristics of human AAA-SMCs. Similar outcomes from healthy Hu-SMCs were assessed under identical culture conditions for comparison. Finally, the response of AAA-SMCs to exogenously-delivered NO cues was evaluated and compared to their healthy counterparts, to establish the utility of delivering exogenous NO for AAA treatment.

Materials and Methods

Population study

LILAS was a case-control study that enrolled 42 men, some with AAA, who needed a vascular surgery or endovascular treatment at the Lille University Hospital Centre (Lille, France) between September 2003 and July 2005 [START_REF] Lamblin | Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study[END_REF]. Case patients were considered eligible if the AAA diameter, measured by abdominal ultrasound exceeded 50 mm, or if it had increased more than 10 mm during the past six months and required initial surgical treatment. The study conformed to the principles outlined in the Declaration of Helsinki, ethics committee of the Lille University Hospital Centre (France) approved the study, and each patient provided written informed consent. Clinical data items were presented as mean ± SD, and frequencies with proportions as appropriate. Statistical significance was assigned at p < 0.05.

Isolation of human AAA-SMCs

Aortic samples (Fig. 1A) were obtained for 20 of the 24 AAA patients and SMCs were isolated from all the 20 AAA tissue samples. Individual cultures of AAA-SMCs were established for each patient by dissection of a residual segment of abdominal aorta and then cutting out the media into 1-2 cu.mm pieces and culturing them on type 1 collagen coated dishes (rat tailderived, BD Biosciences, France) in medium 231 supplemented with smooth muscle growth supplement (Cascade Biologics, France) and antibiotics (50 µg/mL streptomycin, 50 U/mL penicillin; Sigma-Aldrich). SMCs were characterized by western blot analysis. Twenty microgram fractions of cell lysates were separated by SDS-PAGE (9% polyacrylamide), transferred to Hybond C membranes (GE Healthcare, France) and blotted with antibodies against α-smooth muscle actin (α-SMA) (1A4, Dako, 1:5000), fibronectin (E3A, Sigma-Aldrich, 1:3000), and vinculin/ meta-vinculin (hVIN-1, Sigma-Aldrich, 1:400). The blots were subsequently washed in PBS-Tween 20 and incubated with appropriate secondary antibodies (peroxidase-conjugated anti-mouse IgG, GE Healthcare) at a dilution of 1:5000. The blots were then washed three times in PBS-Tween 20 for 15 min, incubated with enhanced chemiluminescence reagents (GE Healthcare), and exposed to a radiographic film scanned with an Imagescanner® (GE Healthcare). AAA-SMCs were subsequently passaged with Medium 231 (Life Technologies).

Transcriptomic analysis of human AAA-SMCs

Changes in SMCs were validated for selected genes with qRT-PCR. RNA samples isolated from SMCs were converted to cDNA with an in vitro transcription reaction. We used cDNA as templates for Taqman qRT-PCR with ABI Assays-on-Demand on an ABI Prism 7900 sequence detection system. The specific assays were designed by TaqMan® Gene Expression Assays (Applied Biosystems) and used Hs 00199841-m1 (ADAMTS 5), Hs 00199836-m1 (ADAMTS 8), Hs 00166915-m1 (renin), Hs 00174179-m1 (ACE1), and Hs 00377632-m1 (L-cathepsin). All samples were run in triplicate, with GADPH (Hs99999905_m1) and Cyclophilin A (Hs99999904_m1) as internal controls to normalize transcript abundance (2 -dCt method).

Triplicates were averaged to calculate an expression value for each sample.

Human AAA and healthy aortic SMC cultures

AAA-SMCs derived from one patient (details in Table 1) were investigated further using biochemical and biomechanical analysis to establish the baseline. Healthy adult Hu-SMCs isolated from a 16-year old male were obtained from Life Technologies Corp. (Carlsbad, CA, USA) and passaged using Medium 231 (ThermoFisher Scientific, USA). For experiments in this study, cells from passages 3-5 for healthy cells and 2-3 for AAA cells were used. AAA-SMCs were cultured on 2D collagen-coated (50 µg/mL) substrates or within 3D collagen hydrogels (2 mg/mL; pH ~ 7.2). Results from 2D substrates will help establish the baseline values, while cultures in 3D gels will provide more physiological-mimic microenvironment. Cells were cultured for 21 days in SMC media, containing 100 nM GSNO (Sigma, Saint-Louis, MO, USA), a NO donor. Control cultures received no GSNO (termed 0 nM). Healthy and AAA-derived SMCs were imaged at random locations during passaging using a Zeiss Axiovert A1 florescence microscope equipped with Hamamatsu camera and image acquisition software. At least ten images per cell type were processed using ImageJ (NIH) to quantify the perimeter and area of each cell type. humidified incubator (37 °C, 30 min) to polymerize the collagen gels. For 2D cultures, collagencoated 24-well tissue culture plastic plates were seeded with 10 4 cells per well. GSNO dose used in this study was identified from our previous study [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF] where a range of GSNO doses were tested to evaluate their effects on ECM synthesis and deposition. Media was changed once every two days and the spent medium was pooled over the 21-day culture and stored at -20 °C for further biochemical analyses. Similarly, cell matrix layers at the end of 21 days were trypsinized and processed for biochemical assays.

Biochemical assays

At the end of 21 days, the amounts of various protein types released into the pooled media or deposited within cell matrix of 2D and 3D cultures were quantified as detailed earlier [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF].

Individual biochemical assays were performed on AAA-SMC cultures to quantify total protein (BCA Kit Assay; Sigma-Aldrich), elastin (Fastin assay; Accurate Scientific Corp, Westbury, NY), sulfated glycosaminoglycans (sGAGs; Kamiya Biomedical Company, Seattle, WA), and HA (hyaluronan enzyme-linked immunosorbent quantitative assay; Echelon Biosciences Inc., Salt Lake City, UT) amounts. For BCA assay, the protein amounts within cells was not separated from the total protein quantified within cell layers. However, the protein content in 2 mg/mL collagen gels (3D cultures) and collagen-coated 2D cultures was quantified (without cells) using this assay and deducted from the protein amounts reported in test cases. Although other proteins such as collagens might be released and deposited by SMCs, their amounts were not assessed here. SMC proliferation was quantified using a fluorometric assay detailed earlier [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF],

to quantify the amount of DNA synthesized over the 21 days. Similarly, LOX functional activity was quantified using a fluorometric assay (AmplexRed® Assay; Molecular Probes, Eugene, OR).

Desmosine bioassay

The amount of desmosine produced and released by AAA-SMCs and healthy SMCs into the pooled media was quantified using a sandwich enzyme immunoassay technique (Human Desmosine ELISA Kit; MyBioSource, Inc., San Diego, CA). The desmosine bioassay has a detection range of 0.1-10 ng/mL and a sensitivity of 0.039 ng/mL. Briefly, aliquots of samples and standards were incubated in a 48-strip-well pre-coated with desmosine-specific antibody, and the bound desmosine was serially-tagged with biotin-conjugated DES-specific antibody and avidin-conjugated horseradish peroxidase, with intermediate washings after every step. Finally, desmosine amounts in each sample were assessed calorimetrically based on the intensity of color generated within a substrate solution in proportion to the respective DES amounts.

Real-time PCR analysis of SMCs

For RT-PCR analysis, AAA-SMCs and healthy SMCs were cultured in parallel on 2D collagen-coated substrates or within 3D collagen gels and exposed to 100 nM GSNO for 5 days to induce changes in gene expression, and GSNO-free cultures served as controls. Total RNA was extracted from SMCs using RNAqueous®-Micro total RNA isolation kit (Life technologies) according to the manufacturer's instructions. The RNA concentration of each sample was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). The RNA was reverse-transcribed to cDNA using the high-capacity cDNA reverse transcription kit with RNase inhibitor (Life Technologies) according to manufacturer's instructions. Gene expression in SMCs was quantified using TaqMan® gene expression assays (Applied Biosystems), and TaqMan® Gene expression master mix for each target using the ABI 7500 Real-time PCR System (Applied Biosystems, Forest City CA) limited to only 40 cycles.

The following genes were analyzed: lysyl oxidase (LOX, Assay ID: Hs00942480_m1), elastin (ELN, Assay ID: Hs00355783_m1), fibrillin 1 (FBN1, Assay ID: Hs00171191_m1), and inducible nitric oxide synthase 2 (NOS2, Assay ID: Hs01075529_m1). To identify relative differences in the levels of target genes, gene expression in AAA-SMCs was normalized to endogenous reference gene (18S, Assay ID: 4319413E) and further to corresponding expression in healthy SMCs, using the ∆∆Ct method.

Cytokine/ Chemokine assays

Cytokine, chemokine, TIMP and MMP analyses were performed using Discovery Assays® (Eve Technologies, Alberta Canada), from 2D or 3D cultures of AAA-SMCs and healthy SMCs.

Briefly, cell culture supernatants were collected from each well after 24 h, spun down at 3000 g for 5 min, and stored at -20 °C. The supernatants were then processed using multiplexing LASER bead technology and processed on a dual-laser flow-cytometry system (Bio-Plex 200).

The technology works by utilizing different combinations of red and infrared fluorophore beads

Western blot analysis of iNOS

iNOS expression within healthy and AAA-SMCs, in both 2D and 3D cultures, in the presence or absence of GSNO, was semi-quantitatively evaluated using western blot analysis [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF]. To optimize loading sample volumes for SDS/PAGE western blot, cell pellets from respective cultures were assayed using a DC protein assay kit (Bio-Rad Labs). The protein bands were detected using a primary polyclonal antibody for the iNOS protein (Abcam), visualized using chemiluminescence method and quantified using ImageJ.

TIMP-1 assay

TIMP-1 amounts released by healthy and AAA-SMCs into the pooled media, in the presence or absence of GSNO, were quantified using Human TIMP-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). Spent media (supernates) from the cell cultures in 2D or 3D were pooled and stored at -20 C, and loaded onto a 96-well polystyrene microplate coated with a monoclonal antibody specific for human TIMP-1. Post-incubation (2 h, RT) on a horizontal orbital shaker, the bound proteins were incubated with human TIMP-1 specific horseradish peroxidase-linked polyclonal antibody (1 h, RT), exposed to a chromogen solution containing tetramethylbenzidine for (30 min, RT, dark), and the reaction was stopped with 2 N sulfuric acid.

Final solutions were measured using Epoch BioTek microplate spectrophotometer with absorbance at 450 nm. This assay has a range of 0.2-10 ng/mL in supernatant solutions, with a sensitivity of 0.08 ng/mL.

Immunofluorescence labeling of cell layers

AAA-SMCs were cultured on glass chamber slides ( 10 Vector Laboratories, Burlingame, CA). AAA-SMC layers treated with the secondary probe alone (no primary antibody) served as negative controls. Cell layers were imaged with a Leica SP8 confocal microscope (Leica Microsystems Inc, Buffalo Grove, IL).

Atomic force microscopy (AFM) analysis of cell stiffness and roughness

Healthy and AAA-SMCs were cultured in 35-mm laminin-coated petri dishes and maintained at 37 °C throughout live-cell nanoindentation assay, over a 60-min experiment each time, performed using a MFP-3D-Bio AFM (Asylum Research, Santa Barbara, CA) mounted on an inverted optical microscope (Nikon Eclipse Ti). Tip-less AFM cantilevers (model Arrow TL 1, Nano World, nominal spring constant ~ 0.03 N/m) were modified by attaching a 4.5 μm polystyrene bead using epoxy [START_REF] Hama | Characterizing viscoelasticity of unhydrolyzed chicken sternal cartilage extract suspensions: Towards development of injectable therapeutics formulations[END_REF]. The spherical bead indenter reduces the possibility of destructive deformation of cell due to smaller local strains applied [START_REF] Li | AFM indentation study of breast cancer cells[END_REF], compensates for the x-y drift (usually < 1 μm), and yields higher spatially-averaged measurement with minimal variability, relative to conventional sharp-tip probes. The actual spring constant was determined from a force-distance curve and using thermal calibration method in a clean culture dish containing DMEM. Drifting minimization and thermal/mechanical equilibrium of AFM was achieved by submerging probe in cell medium for stabilization. For each experiment, at least 30 cells were randomly selected, indented between nuclei and cell edges, and 150-200 force curves were obtained per cell in force-volume mode, at a rate of 0.25 Hz with approach/ retraction velocity of 2.5 µm/s. Using Hertz's contact model [ , Young's modulus was 𝐹 = (4𝐸 𝑅𝛿 3 ) 3(1 -𝜇 2 )] determined from these force curves, where F is the indentation force, E is the Young's modulus, µ is the Poisson's ratio (0.5 for cells), R is radius of spherical bead attached to cantilever and  is the indentation depth (300-400 nm) [START_REF] Li | AFM indentation study of breast cancer cells[END_REF]. The analysis of force-indentation curves was automated using Igor Pro 6.37 software. The indentation data was analyzed assuming cells behave as linear elastic at small strain regimes. The mean elastic modulus values were calculated for each cell from the elastic modulus heat maps generated over randomly-selected 90  90 m 2 area, and then averaged across multiple cells for each culture condition group.

From this data, the mean surface roughness (Ra) of each cell -characterized as the arithmetic mean of the deviations in height from the line mean value, and the root mean square (Rq), were calculated [START_REF] Kim | AFM-detected apoptotic changes in morphology and biophysical property caused by paclitaxel in Ishikawa and HeLa cells[END_REF]. Both Ra and Rq were obtained from randomly selected areas (30  30 µm 2 ) on the cell membrane. A two-tailed Student's t-test was applied to all the pairs of samples (untreated-treated cells) for statistical analysis and p < 0.05 were considered significant.

Statistical analysis

All biochemical data were obtained from three independent repeat experiments per case (n = 3 wells/ case/ experiment) and analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance and differences deemed significant for p < 0.05.

The data were assumed to follow a near-Gaussian distribution in all the cases, and the mean and standard errors were calculated accordingly.

Results

Patients

Table 1 summarizes the baseline characteristics of the 42 AAA subjects. One-third of the AAA patients had hypertension, half had dyslipidemia, 17% had diabetes mellitus, and 29% had family members who had a history of heart disease (myocardial infarction and/or acute coronary syndrome). The AAA case patients (n=24) had a mean maximal external aortic diameter of 56.1±11.3 mm (range: 46-95 mm). Fewer AAA patients received ACE inhibitor treatment (21% vs. 44%). None of the AAA patients took non-steroidal anti-inflammatory drugs (NSAIDs).

Primary AAA-SMC cultures

SMCs migrated from AAA explants after 10 days and reached confluence in one month. All the cultured AAA-SMCs had the same elongated, spindle-shaped morphology, and at confluence all cultures assumed a hill-and-valley pattern that was maintained throughout subcultures (Fig. 1A). Protein expression profiles determined by western blot analysis (Fig. 1A)

showed that all the cultures from patient groups expressed α-SMA, vinculin and meta-vinculin, indicating the presence of differentiated SMCs. Fibronectin, a component of basement membrane, was also expressed indicating a proliferative and secretory SMC phenotype. RT-PCR analysis (Fig. 1B) indicated that mRNA expression of three genes appeared to be significantly down-regulated in SMCs from AAA patients: ACE (angiotensin converting enzyme), ADAMTS 5 and ADAMTS 8. The plots of individual mRNA expression for these three genes (normalized to house-keeping genes) shows the variability, with ADAMTS8 being the most suppressed.

AAA-SMC proliferation and dimensions

Despite similar morphologies within 2D cultures, the area and perimeter of AAA-SMCs within 2D cultures were significantly higher compared to their healthy counterparts (Fig. 2A; 52 <n<79; p<0.01 in both cases). The phenotype of AAA-SMCs was confirmed by staining for αactin in monolayer cultures, with more than 98% positive-stained cells. In general, AAA-SMC proliferation is significantly lower in 3D gels compared to their 2D counterparts (Fig. 2B).

Addition of GSNO significantly suppressed SMC proliferation in 3D gels with a trend in 2D counterparts. GSNO-free controls) in total protein, elastin, sGAG, and HA, deposited within cell matrix or released into pooled media, by human AAA-SMCs, in 2D and 3D cultures, in the presence of 100 nM GSNO. The total protein amounts were quantified using a BCA protein assay, elastin amounts using a Fastin Assay kit, sGAG amounts using a quantitative dye-binding sGAG assay kit, and HA amounts using a hyaluronan enzyme-linked immunosorbent quantitative assay. Quantitative results were obtained from cell matrix layers or pooled media after 21 days of culture and normalized to cell count within respective cases (n = 6/ condition). Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance and differences deemed significant for p < 0.05 between respective cases. Data shown represents mean ± standard error in respective cases.

Total protein synthesis

Protein synthesis and release by AAA-SMCs has not been reported earlier. The total protein synthesized by AAA-SMCs, in 2D and 3D cultures, in the presence or absence of GSNO, was quantified as two separate components (Fig. 2C): protein deposited in the cell matrix (termed matrix here) and protein released into the pooled media (termed media here). A significant increase in the total protein deposited in cell matrix was observed with GSNO addition, in both 2D (2.58-fold) and 3D (3.1-fold) cultures. Total protein deposited in cell matrix within 3D cultures was significantly higher than that in 2D counterparts (p<0.01), in the presence or absence of GSNO (16.4-fold and 19.8-fold, respectively). GSNO stimulated significant increase in the total protein released into the pooled spent media in 3D cultures (p<0.01 vs. 2D). The amounts of protein in pooled media were at least two-orders of magnitude higher than in matrix, in all conditions. This study established the baseline for the total protein amounts released and deposited by adult human AAA-SMCs in 2D and 3D cultures (actual amounts provided in Table 2), in the absence of any stimulation. However, less than 2% of total protein produced by AAA-SMCs was deposited as matrix within cell layers, suggesting that additional approaches are required to coacervate the protein into matrix form. Compared to healthy SMCs (Table 2; Supplementary Fig. 1), the total protein deposited within 3D cell layers or released into the spent media by AAA-SMCs was significantly higher (i.e., 2-fold and 19-fold, respectively) even in the absence of GSNO. GSNO stimulated a 7-fold increase in protein deposition within matrix layers and a 17-fold increase in protein release within pooled media by AAA-SMCs within 3D cultures, compared to Hu-SMCs (Supplementary Fig. 1). Table 2. Amounts of various proteins deposited in cell matrix or released into pooled media by healthy and AAA human aortic SMCs, on a per cell basis, over a 21-day period, within 2D or 3D cultures, in the presence or absence of 100 nM GSNO. Data shown here represents the average of at least n = 6/ case. Data for the healthy Hu-SMCs were from our previous publication [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF]. ND: Not detectable within assay limits (i.e., < 1 pg/cell amounts).

tropoelastin). The benefits of exogenous NO to elastin deposition in cell matrix appeared to be dependent on the substrate type. Compared to GSNO-free controls, significantly higher matrix elastin was deposited within 3D cultures with GSNO addition, although matrix elastin deposition decreased in 2D cultures. GSNO stimulated tropoelastin release only within 3D cultures, although the amounts were two-orders of magnitude higher than matrix elastin (Table 2).

The matrix elastin yields (ratio of matrix elastin to the total elastin produced) within 2D AAA-SMCs cultures were 0.4% and 0.08% with 0 and 100 nM GSNO additive-cultures, respectively.

However, 3D cultures modestly enhanced matrix elastin yields in the absence and presence of GSNO (0.5% and 0.8%, respectively). Thus, elastic matrix yields improved by 10-fold in 3D

cultures with the addition of GSNO, similar in trend to that observed for the total protein amounts.

sGAG and HA synthesis

Adding GSNO to AAA-SMC cultures increased sGAG deposition in matrix layers (2.73-and 1.52-fold, respectively) and in pooled media (2.17-and 1.72-fold, respectively) within 2D and 3D cultures, on a per cell basis (Fig. 2C), although the amounts were two orders of magnitude higher in pooled media. Compared to 2D cultures, 3D cultures appeared to promote sGAG release into pooled media or deposition within cell layers, on a per cell basis (Table 2). sGAG yield (ratio of matrix sGAG to the total sGAG produced) within 2D AAA-SMC cultures indicated that matrix yields were 0.9% and 2.3% in controls and GSNO-additive cultures, respectively.

However, 3D cultures did not show any enhancement of matrix sGAG yields, in the presence or absence of GSNO.

GSNO addition modestly increased HA deposition in cell matrix on a per cell basis in both 2D and 3D cultures (1.41-and 1.35-fold, respectively; Fig. 2C), with similar increases in HA release into the pooled media. When AAA-SMCs were cultured in 2D, the matrix HA yields (ratio of HA deposited in matrix to the total HA produced) were 1.6% and 1.7% within controls and GSNO additive-cultures, respectively. However, 3D cultures modestly increased matrix HA yields, in the presence or absence of GSNO (2% and 1.9%, respectively). Representative bands are shown in the insert for comparison. iNOS protein expression by AAA-SMC cultures (white histograms) was normalized to that in 2D GSNO-free cultures of healthy SMCs (black histograms). (D) Similarly, the release of TIMP-1 into pooled media was quantified using a TIMP-1 ELISA assay. GSNO (0 or 100 nM) was supplemented to AAA-SMC cultures in 2D (black histograms) or 3D (white histograms) cultures over 21 days. Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance and differences deemed significant for p < 0.05. Data shown represent mean ± standard error of quantified data and normalized to cell count within respective cases (n = 6 per condition).

LOX enzyme activity

While GSNO addition to 2D cultures did not induce a significant change in LOX activity within cell matrix, it induced a 2.55-fold increase within 3D cultures (Fig. 3A; p<0.01). LOX activity in matrix within 3D cultures was significantly higher (2.2-fold; p<0.01) than that in 2D cultures. LOX activity within pooled media from 3D cultures was 13.5-fold higher than that in 2D cultures in the absence of GSNO. Although adding GSNO to 3D cultures did not enhance LOX activity compared to controls, it significantly increased LOX activity within 2D cultures (14.9-fold; p<0.01).

Desmosine crosslink density

In healthy SMC cultures (Fig. 3B), desmosine levels were higher in 2D cultures than in 3D cultures (p<0.01), and GSNO offered no additional benefit in both 2D and 3D cultures. However, in AAA-SMC cultures, desmosine levels were significantly higher in controls compared to GSNO-receiving cultures (p<0.01), in both 2D and 3D. The basal levels of desmosine by AAA-SMCs remained unaltered in both 3D and 2D cultures. Finally, significant difference in desmosine levels were noted between healthy and AAA derived SMCs, in 3D cultures receiving no GSNO (p<0.001).

iNOS expression and TIMP-1 release

To test if exogenously-delivered NO influenced iNOS protein expression in AAA-SMC cultures, western blot analysis was performed (Fig. 3C; bands in insert). The basal level of iNOS expression (2D control cultures) was significantly higher in AAA-SMCs compared to healthy counterparts (p<0.05). It was found that GSNO addition to 2D cultures significantly promoted iNOS protein expression in healthy SMCs, but not in 3D cultures. Expression of iNOS by AAA-SMCs, on the other hand, was not modified by 2D or 3D cultures, and/or by GSNO addition.

No significant difference in TIMP-1 release was noted between 2D and 3D AAA-SMC cultures in controls (Fig. 3D). GSNO induced a 2.4-fold increase in TIMP-1 release in 3D cultures. While the basal levels of TIMP-1 release was significantly lower in AAA-SMCs compared to adult healthy aortic SMCs in 3D cultures [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF], the levels were similar in the presence of 100 nM GSNO.

Elastin, iNOS, LOX and fibrillin gene expression

Elastin (ELN) gene expression in AAA-SMCs, both in 2D and 3D cultures, in the presence or absence of NO cues, was significantly lower compared to healthy SMCs (Fig. 4A). Such inhibited elastin gene expression levels in AAA-SMCs, compared to healthy SMCs, was in agreement with their respective protein expression levels of matrix elastin (Table 2). Similarly, within AAA-SMCs, elastin gene expression was significantly lower in 2D compared to that in 3D, in agreement with their protein expression levels. Surprisingly, elastin gene expression was significantly lower after GSNO addition, in contrast to tropoelastin and matrix elastin produced by these cells (Fig. 2, C-D).

The iNOS gene expression of healthy and AAA-SMCs were similar in 2D and significantly increased in 3D cultures for AAA-SMCs (Fig. 4A). Adding GSNO had no significant effect on iNOS gene expression in AAA-SMC cultures, suggesting that AAA-SMCs are sensitive to signaling from exogenous NO cues, similar to their healthy counterparts. LOX gene expression was significantly higher (6 to 10 -fold in 2D and 3D cultures, respectively) in AAA-SMCs compared to healthy SMCs, although adding GSNO suppressed such elevated levels (Fig. 4A), in contrast with the LOX functional activity (Fig. 3A). The baseline levels of fibrillin-1 (FBN) gene in AAA-SMCs was significantly higher compared to their healthy counterparts, although such expression levels were significantly downregulated in 2D AAA-SMC cultures with GSNO addition (Fig. 4A).

Figure 4.

(A) Lavik method (ΔΔCt method) was used for relative quantification of the fold changes in ELN, iNOS, LOX, and FBN genes in AAA-SMCs, after addition of GSNO (0 or 100 nM) within 2D (black histograms) or 3D (white histograms) cultures for 21 days. The gene expressions obtained from qRT-PCR analysis within AAA-SMCs were first normalized to housekeeping gene (18S) expression within respective cultures, and further normalized to respective gene expressions within healthy Hu-SMCs cultured under similar conditions. Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance and differences deemed significant for p < 0.05. Data shown represent mean ± standard error (n = 6 independent data points per condition). (B) Heat map showing the amounts of cytokines, chemokines, and MMPs/TIMPs released by healthy and AAA SMCs, in 2D and 3D cultures, in the presence or absence of 100 nM GSNO, as measured using laser bead immunoassay.

Release of cytokines, chemokines, MMPs, and TIMPs

We quantified the baseline levels (2D -0 nM) of cytokines, chemokines, MMPs and TIMPs released by healthy Hu-SMCs [Supplementary Fig. 2 (A: < 10 pg/mL; C: < 250 pg/mL; E: < 40 ng/mL)] and adult human AAA-SMCs [Supplementary Fig. 2 (B: < 10 pg/mL; D: < 250 pg/mL;

F: < 40 ng/mL)].
Their levels in 3D cultures, and in the presence of 100 nM GSNO were also shown in respective panels. The analytes released by SMCs were plotted on a log-scale using a heatmap function (Fig. 4B). MMPs and TIMPs were the most highly-expressed analytes compared to the cytokines (e.g., ILs). Benefits of GSNO delivery in healthy SMC cultures (both 2D and 3D), appeared marginal with no significant modulation in ILs and MMPs release but significant increase in TIMPs release. However, in 3D AAA-SMC cultures, GSNO suppressed the release of several analytes (e.g., IFN-γ, MMPs-8, 9, 12, IL-8) compared to all other conditions, and some analytes were undetectable in most conditions (IL-1 and TNF-). A tenfold increase in IL-6 release by AAA-SMCs was found compared to healthy counterparts.

Variations in analyte concentrations were observed across samples cultured in 2D vs. 3D environments; for instance, SMCs in 2D cultures had higher concentrations of MMP-2, MMP-12, TIMP-2, TIMP-3, and MCP-1, in both healthy and AAA-SMC cultures. In general, cytokine and MMPs production in both cell types appeared to be regulated by GSNO presence as well as culture substrate (2D vs. 3D).

Immunofluorescence labeling of matrix proteins

AAA-SMCs deposited detectable matrix elastin and fibulins-4 and 5 within 2D cultures over the 21-day period within control cultures (Fig. 5). However, fibrillin-1, critical for matrix elastin coacervation and crosslinking, was undetectable around the periphery of most cells. GSNO significantly promoted deposition of matrix elastin, fibrillin-1 and fibulins-4, 5, compared to controls.

Figure 5.

Representative immunofluorescence images of AAA-SMC layers in 2D cultures, with or without GSNO, stained with respective primary antibodies for elastin, fibrillin-1, fibulin-4 and fibulin-5, and counterstained with DAPI for cell nuclei. A significant increase in the expression of all the proteins was noted in the presence of 100 nM GSNO. Scale bar: 100 m. For each treatment condition, at least three independent cell layers were imaged, with six random regions captured in each cell layer.

Cell mechanics using AFM

Single cell nanoindentation measurements were obtained to quantify the elastic modulus (E) of live SMCs under different culture conditions (Fig. 6; Supplementary Fig. 3). Representative phase-contrast images (Supplementary Fig. 3), heat maps of elastic moduli (Fig. 6A), and force-indentation curves (Fig. 6B) were shown for healthy and AAA SMCs, in the presence or absence of GSNO. The average modulus of elasticity for AAA-SMCs (17.41±0.34 kPa) was significantly higher (1.6-fold; p<0.001) than for healthy SMCs (10.42±0.29 kPa), indicating an increase of stiffness in AAA-SMCs (Fig. 6C). Upon exposure to GSNO, the average modulus of elasticity for healthy SMCs decreased by 1.3-fold (healthy SMCs: 8.67±0.25 kPa, p<0.05; AAA-SMCs: 13.03±0.35 kPa, p<0.05). Cell roughness measurements showed that AAA-SMCs were smoother than Hu-SMCs (Fig. 6D). GSNO addition significantly increased cell surface roughness in Hu-SMCs (p<0.01), and nearly doubled the roughness in AAA-SMCs, compared to respective controls. In GSNO presence, Hu-SMCs and AAA-SMCs had no significant differences in their surface roughness. Prior studies reported that adult human AAA-SMCs proliferated 4-fold over 16 days [START_REF] Crowther | Increased matrix metalloproteinase 2 expression in vascular smooth muscle cells cultured from abdominal aortic aneurysms[END_REF], 5fold over 21 days [START_REF] Gacchina | Evaluating smooth muscle cells from CaCl2induced rat aortal expansions as a surrogate culture model for study of elastogenic induction of human aneurysmal cells[END_REF], or doubled every 5.3 days [START_REF] Patel | Smooth muscle cell migration and proliferation is enhanced in abdominal aortic aneurysms[END_REF] in 2D cultures, which is significantly higher than that noted in our study. AAA-SMCs proliferated significantly slower compared to their healthy counterparts (Table 2), possibly due to diseased nature and/ or differences in the age of the donors. AAA-SMCs, derived from a relatively older patient, synthesized and deposited more proteins in 3D cultures compared to the healthy SMCs derived from a younger adult. Our results highlight the increased ability of aortic SMCs to proliferate, migrate and deposit ECM under diseased vascular conditions [START_REF] Owens | Molecular regulation of vascular smooth muscle cell differentiation in development and disease[END_REF].

Despite the prominent role of elastin in aortic tissue homeostasis maintenance, quantification of elastin synthesized by human AAA-SMCs has received less attention.

Tropoelastin, the precursor molecule to elastin, is encoded by a single copy of ELN gene in SMCs and fibroblasts. Tropoelastin amounts in human AAA extracts was reported to be 5-fold higher than that within normal human aortic tissues [START_REF] Krettek | Elastogenesis in human arterial disease: a role for macrophages in disordered elastin synthesis[END_REF]. Our current study suggests that healthy Hu-SMCs produce higher amounts of tropoelastin and matrix elastin compared to their AAA counterparts. Adult human AAA-SMCs were shown to release ~10 pg of tropoelastin and deposited 0.02 pg of matrix elastin on a per cell basis in 2D cultures, in the absence of any external cues [START_REF] Gacchina | Evaluating smooth muscle cells from CaCl2induced rat aortal expansions as a surrogate culture model for study of elastogenic induction of human aneurysmal cells[END_REF]. These amounts were significantly lower than what we observed, despite similarities in donor age, disease phenotype and culture duration. Numerous studies have shown that the significant changes in elastin protein within AAA ECM might be due to a related change in the gene expression levels [31; 32]. Similar to our observations here, a downregulation in elastin gene in AAA tissues was reported in a porcine model of induced AAA, compared to healthy counterparts [START_REF] Sadek | Gene Expression Analysis of a Porcine Native Abdominal Aortic Aneurysm Model[END_REF].

Adult human AAA-SMCs in 2D cultures release significantly higher levels of GAGs (e.g., versican, perlecan, heparan sulfate, biglycans) compared to healthy aortic SMCs [START_REF] Melrose | Pathogenesis of abdominal aortic aneurysms: possible role of differential production of proteoglycans by smooth muscle cells[END_REF]. Although the various types of sGAGs released by AAA-SMCs were not individually identified in our study, we quantified the absolute amount of overall GAGs production by AAA-SMCs and found multifold increases in HA release into pooled media and deposition into matrix within AAA-SMC cultures. The combined amount of elastin and GAGs, in both pooled media and cell matrix, constitute a small fraction of the total matrix, suggesting that in addition to elastin and GAGs, numerous other proteins such as collagens and proteoglycans, might have been released and deposited by AAA-SMCs within 2D and 3D cultures.

Such deposited elastin precursors are crosslinked in the presence of LOX enzyme, resulting in the formation of desmosine and isodesmosine linkages between lysine molecules on elastin molecules [START_REF] Schmelzer | Assembly and Properties of Elastic Fibers, Elastic Fiber Matrices: Biomimetic Approaches to Regeneration and Repair[END_REF]. Thus, the final crosslinked elastic fibers typically have a microfibril sheath surrounding the core elastin molecules. Additional stimulation such as alternate scaffold design, elastogenic scaffold material, exogenous LOX, and mechanical stimuli might be necessary to coax the AAA-SMCs to enhance matrix elastin yields. Although it is interesting to note that AAA-SMCs produce LOX which is functionally active, the levels of LOX functional activity within AAA-SMC cultures are at least two orders of magnitude lower than that quantified within healthy SMC cultures (Table 2), on a per cell basis. This difference might be due to the age difference in the donor sources of these cell lines, as well as the phenotypic differences between healthy versus AAA cells. Studies have shown that in addition to regulating vascular SMC phenotype (proliferation, migration), LOX also plays a primary role in ECM crosslinking such as collagens, elastin and GAGs [START_REF] Maki | Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues[END_REF]. However, under diseased conditions such as aneurysms, characterized by remarkable degradation and ECM remodeling, LOX activity and expression is significantly reduced. While it is not clear whether reduction in LOX levels contribute to the onset of the pathogenesis of these diseases or vice versa, numerous AAA-induced animal models demonstrate strong correlation between LOX expression reduction and disease progression [START_REF] Rodríguez | Regulation of lysyl oxidase in vascular cells: lysyl oxidase as a new player in cardiovascular diseases[END_REF].

Desmosine levels in healthy human arterial tissues was noted to be at least 9-fold higher than that in AAA segments [START_REF] Krettek | Elastogenesis in human arterial disease: a role for macrophages in disordered elastin synthesis[END_REF], and reduced desmosine levels were reported in dissecting AAAs [START_REF] Watanabe | Alteration of cross-linking amino acids of elastin in human aorta in association with dissecting aneurysm: analysis using high performance liquid chromatography[END_REF], suggesting diminished levels of mature, crosslinked elastin in AAA tissues. We here measured desmosine levels in the supernatants to determine if sufficient levels of desmosine is being produced that could crosslink tropoelastin in vitro within SMC cultures.

Although vascular NO plays an important role in regulating SMC functions such as vasodilation, survival, gene regulation, and quiescence phenotype, the harmful role of higher NO levels in the pathogenesis of aneurysms has been documented [39; 40; 41]. Specifically, iNOS-stimulated NO release from vascular SMCs was shown to participate in elastin degradation and disruption of vascular ECM [START_REF] Paik | The nitrite/elastin reaction: implications for in vivo degenerative effects[END_REF]. Our observations agree with previous reports that experimental or human AAA-derived tissues had higher iNOS levels compared to control tissues where iNOS could barely be detected [40; 43]. TIMPs-1, 2 regulate ECM remodeling in vasculature by controlling the levels and activity of MMPs. In agreement with our results, no significant differences in TIMP-1 levels (Western blotting) within tissue sections isolated from AAA patients and their healthy counterparts were observed [START_REF] Saito | Matrix metalloproteinase expressions in arteriosclerotic aneurysmal disease[END_REF].

Localized inflammation contributes to weakening of aorta due to imbalances in ECM levels [START_REF] Choke | A Review of Biological Factors Implicated in Abdominal Aortic Aneurysm Rupture[END_REF]. This has prompted research focused on regulating the production of inflammatory molecules as a therapeutic intervention in lieu of invasive surgical approaches [START_REF] Aziz | Role of matrix metalloproteinase inhibitors in preventing abdominal aortic aneurysm[END_REF]. The concentrations noted for MMPs/ TIMPs in our study broadly falls in the range reported by others and our previous work [18; 27]. Given the apparent differences in cytokine production between AAA and healthy SMCs, our data supports the prevailing notion that cytokine stimulation via paracrine or autocrine signaling may in turn modulate MMP activation [START_REF] Galis | Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion[END_REF]. Although it has been suggested that NO can influence the production of some cytokines and chemokines [START_REF] Peshkova | Atherosclerosis and aortic aneurysm -is inflammation a common denominator?[END_REF], we did not see any differences. In summary, our data is consistent with the inflammatory patterns seen in AAA subjects and suggests that NO presence modulates MMP production in a potentially therapeutic manner.

In addition to alterations in composition and functionality of vascular endothelium and ECM, changes in cell stiffness might also contribute to vascular tissue stiffness in AAAs, atherosclerosis and hypertensive conditions. The stiffness (~30 kPa) of hypertensive aorta-derived SMCs, as measured by AFM nano-indentation, was reported to be two-fold higher than that of normal aorta (~14 kPa), and such stiffness was mediated by cytoskeletal proteins [START_REF] Sehgel | Increased vascular smooth muscle cell stiffness: a novel mechanism for aortic stiffness in hypertension[END_REF].

Vascular SMCs from old male monkeys exhibited much higher stiffness than their younger counterparts (42 kPa vs. 13 kPa) [START_REF] Qiu | Short communication: vascular smooth muscle cell stiffness as a mechanism for increased aortic stiffness with aging[END_REF]. The elastic modulus of primary adult male rat arteriolederived SMCs (~13.5 kPa) decreased by 38% upon exposure to NO donor PANOate, most likely due to remodeling of actin cytoskeleton network [START_REF] Hong | Vascular smooth muscle cell stiffness and adhesion to collagen I modified by vasoactive agonists[END_REF]. Marfan vascular SMCs were two-fold stiffer than healthy human SMCs, as measured by AFM, most likely due to higher focal adhesion kinase protein and actin stress fibers expression [START_REF] Crosas-Molist | Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome[END_REF]. Here, we report for the first time on the stiffness and surface roughness of human aortic SMCs from healthy and AAA tissues, using AFM nano-indentation of live cells. Our results show that (i) elastic modulus of healthy Hu-SMCs is lower than that in rats and monkeys; (ii) human AAA-SMCs have higher stiffness and lower surface roughness compared to healthy Hu-SMCs; and (iii) human AAA-SMCs had significantly lower stiffness compared to hypertensive rat SMCs or aged monkey SMCs.

Our results support our hypothesis that AAA-SMCs do respond to exogenous NO cues to modulate cell proliferation and protein synthesis in 3D cultures. We have recently shown that healthy Hu-SMCs produced significantly higher amounts of tropoelastin and matrix elastin in the presence of 100 nM GSNO, and that an optimal 100 nM GSNO promoted sGAG deposition in matrix layers but not their release into the pooled media, within 3D Hu-SMC cultures [START_REF] Simmers | Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within threedimensional cocultures[END_REF].

An increase in LOX functional activity within 3D AAA-SMC cultures in GSNO presence (Fig. 3), and significantly higher deposition of fibrillar forms of elastic matrix-associated proteins such as fibrillin-1, and fibulins-4, 5 (Fig. 5), is highly encouraging for therapeutic tissue engineering and in situ elastin regeneration. Increased levels of LOX functional activity by exogenous GSNO might partially explain the increases in matrix elastin, GAGs, and tropoelastin in 3D AAA-SMC cultures. Similar increases in tropoelastin but not desmosine levels by human and rat AA-SMCs were reported by others [22; 53]. Higher amounts of GSNO (> 100 nM) could possibly elevate desmosine levels in 3D AAA-SMC cultures, which further enhances elastin crosslinking and maturation.

The increased levels of TIMP-1 in AAA-SMC 3D cultures in the presence of GSNO is encouraging in modulating further degradation of vascular ECM by MMPs under diseased conditions. GSNO induced significant increases in matrix elastin deposition in 3D cultures and reduced MMP-9 concentration by over 50% in all cases (2D/3D AAA-SMC cultures), compared to healthy controls. Given that the upregulation of MMP-9 is linked to the degradation of the aortic ECM, our results are encouraging as they suggest a therapeutic benefit in the supplementation of exogenous NO [START_REF] Wilson | Matrix metalloproteinase-8 and -9 are increased at the site of abdominal aortic aneurysm rupture[END_REF]. Similar trend was noted for MMP-2 in nearly all cases (excluding 3D AAA-SMC cultures). Similar patterns in MMP downregulation were observed in eNOS gene-transfected cells [START_REF] Gurjar | eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity[END_REF], and it has been suggested this pathway is regulated by guanylyl-cyclase dependent and independent pathways [START_REF] Ridnour | Nitric oxide regulates matrix metalloproteinase-9 activity by guanylyl-cyclase-dependent and -independent pathways[END_REF]. NO appeared to have little effect on TIMPs release across most cases. In agreement with literature, we noticed significantly higher IL-6 levels by AAA-SMCs. And finally, delivering exogenous NO significantly lessened AAA-SMC stiffness and enhanced surface roughness, possibly via modulation of cytoskeletal and surface proteins, respectively. We postulate that changes in stiffness beginning at single cell level might contribute to changes in vascular tone and functionality of aneurysmal vessels and delivering GSNO could be explored as a therapeutic strategy. Taken together, results confirm our hypothesis that exogenous NO delivery to AAA-SMCs might help to promote protein synthesis, release and deposition within 3D cultures, in addition to regulating their proliferation.

In conclusion, we established the baseline differences between adult human AAA and healthy SMCs, in terms of their size, proliferation, matrix and associated proteins synthesis and deposition, release of cytokines/ chemokines/ MMPs/ TIMPs, changes in genotype of elastogenic markers, and stiffness and roughness. We demonstrated that exogenously delivering NO via GSNO offers an effective strategy to enhance AAA-SMC functionality. These outcomes might help in further designing tissue engineering and regenerative medicine-based

  conjugated to specific antibodies targeted to the cytokine or chemokine of choice and read using a flow-cytometry based system. The quantity of the specific analyte generated was based off a series of standards set forth by the company. The assays performed include human focused 11-plex, human MMP 9-plex and TIMP 4-plex, to quantify the following analytes: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 (p70), MCP-1, TNF-α, MMPs-1, 2, 3, 7, 8, 9, 10, 12, 13, and TIMPs-2, 3, 4.

  4 cells/well) for 21 days in the presence or absence of 100 nM GSNO, and the cell layers were fixed in cold methanol, washed with PBS, blocked with PBS containing 5% v/v goat serum (ThermoFisher Scientific) and immunolabeled with rabbit anti-human polyclonal antibodies against elastin (1:100 v/v dilution, Abcam), fibrillin-1 (1:100 v/v dilution, Bioss Antibodies), fibulin-4 (1:100 v/v dilution, Santa Cruz Biotechnology), and fibulin-5 (1:100 v/v dilution, Santa Cruz Biotechnology). Cell layers were washed with PBS and labeled with Alexafluor 633 goat anti-rabbit secondary antibody; 1:1000 dilution, ThermoFisher Scientific). The cell layers were washed with PBS and mounted with Vectashield containing the nuclear stain 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI;

Figure 1 .

 1 Figure 1. (A) A sample AAA tissue (n = 20 patients) used for SMC cultures. Representative phase-contrast microscopy of SMCs obtained by explant from human AAA biopsies. Western blot analysis of SMC for fibronectin, meta-vinculin (160 kDa), vinculin (116 kDa) and α-smooth muscle actin (α-SMA). Scale bar: 100 m. (B) mRNA expression ratio of genes (2 -dCt ; normalized to house-keeping genes) in SMCs derived from AAA (n = 12) patients, potentially involved in matrix degrading processes in AAA, as determined by RT-PCR.

Figure 2 .

 2 Figure 2. (A) Representative phase-contrast images of adult healthy Hu-SMCs and AAA-SMCs cultured in this study. Significant differences in average cell area and perimeter were noted between the two cell types. Data shown represents mean ± standard error in respective cases. Scale bar: 200 m. (B) Fold increase in AAA-SMC density within 2D (black histograms) and 3D (white histograms) cultures, in the presence or absence of exogenous 100 nM GSNO. Data shown represents mean ± standard error of cell count after 21 days of culture, normalized to initial seeding density (n = 3/ condition). Cell densities were quantified using a fluorometric assay detailed earlier. (C) Fold-changes (vs.GSNO-free controls) in total protein, elastin, sGAG, and HA, deposited within cell matrix or released into pooled media, by human AAA-SMCs, in 2D and 3D cultures, in the presence of 100 nM GSNO. The total protein amounts were quantified using a BCA protein assay, elastin amounts using a Fastin Assay kit, sGAG amounts using a quantitative dye-binding sGAG assay kit, and HA amounts using a hyaluronan enzyme-linked immunosorbent quantitative assay. Quantitative results were obtained from cell matrix layers or pooled media after 21 days of culture and normalized to cell count within respective cases (n = 6/ condition). Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance and differences deemed significant for p < 0.05 between respective cases. Data shown represents mean ± standard error in respective cases.

Figure 3 .

 3 Figure 3. (A) Fold-changes in LOX enzyme functional activity within cell matrix and pooled media of AAA-SMC cultures, quantified using an Amplex Red® fluorometric assay and quantified on a per cell basis. Cells were exposed to 100 nM GSNO while controls received no GSNO. (B) Desmosine levels in pooled media collected from 2D or 3D Hu-SMCs (black histograms) or AAA-SMC cultures (black histograms), over the 21-day period, in the presence or absence of GSNO. (C) iNOS protein expression was quantified from western blot analysis.Representative bands are shown in the insert for comparison. iNOS protein expression by AAA-SMC cultures (white histograms) was normalized to that in 2D GSNO-free cultures of healthy SMCs (black histograms). (D) Similarly, the release of TIMP-1 into pooled media was quantified using a TIMP-1 ELISA assay. GSNO (0 or 100 nM) was supplemented to AAA-SMC cultures in 2D (black histograms) or 3D (white histograms) cultures over 21 days. Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance

Figure 6 .

 6 Figure 6. (A) Representative force maps of healthy Hu-SMCs (left panels) and AAA-SMCs (right panels) treated with 100 nM GSNO (lower panels). Background from petri dish was subtracted in these images. Representative force-indentation curves (B) and average Young's Modulus (C) of these cells cultured on 2D substrates, in the presence or absence of 100 nM GSNO. The black dotted lines in (C) represent the arithmetic averages in respective cases, i.e., 10.33 ± 0.2624 KPa for untreated Hu-SMCs (n = 605), 8.674 ± 0.2491 KPa for Hu-SMCs

Table 1 .

 1 Baseline characteristics of the study population in AAA group. AAA: aortic abdominal aneurysm; CAD: coronary artery disease; MI: myocardial infarction; ACS: acute coronary syndrome; PTCA: percutaneous transluminal coronary angioplasty. Data expressed as mean  SD, where appropriate.

	AAA
	(n = 24)

Acknowledgements

The authors acknowledge help from Dr. Ali Borazjani (Cleveland Clinic) with RT-PCR analysis, Sarah Carney with confocal imaging, and Arsela Gishto with biochemical assays.

Sources of Funding

This work was partially supported by the Cleveland State University Cellular and Molecular Medicine Fellowships and the Dissertation Research Awards to J.J. and K.F., the Choose Ohio First Scholarship to P.S., the National Science Foundation (CBET, Award # 1337859) to C.K., the National Institutes of Health (R01ES025779) to C.K., the European Fighting Aneurysmal disease grant (Health-F2-2008-200647) to F.P., and the National Institutes of Health (HL132856 and HL119810-05; Project Number: NCAI-17-7-APP-CCF-Ramamurthi) to A.R.

Supplementary Figure 1. Normalizes protein synthesis by human AAA-SMCs in the presence or absence of GSNO, in 2D and 3D cultures. (A) Basal amounts of HA, sGAG, elastin, and total protein deposited within cell matrix or released into pooled media, by human AA-SMCs, in 2D (black histograms) or 3D (white histograms) cultures. (B) Similar proteins and GAGs deposited by AAA-SMCs in the presence of 100 nM GSNO. Data represents mean ± standard error of quantitative results obtained from cell matrix layers or pooled media, after 21 days of culture, and normalized to cell count within respective cases (n = 6/ condition). * indicates p < 0.01 compared to GSNO-free cultures; # indicates p < 0.01 for 2D vs. 3D, at a given concentration of GSNO.

Elastin synthesis and deposition

The total elastin synthesized by AAA-SMCs (Fig. 2C; Table 2) was quantified as that deposited in the cell layers (i.e., matrix elastin) or released into the pooled media (i.e., Supplementary Figure 2. Amounts of various cytokines and chemokines released by human healthy aortic SMCs (A, C, E) or aneurysmal SMCs (B, D, F) over 21 days, within 2D or 3D cultures, in the presence or absence of exogenous GSNO (100 nM). Data was obtained from multiplexing laser bead assays using respective supernatants pooled over the culture duration. Panels A and B depict the analytes detected within 0 -10 pg/mL levels (low amounts), C and D depict analytes detected within 10 -250 pg/mL levels (moderate amounts), and E and F depict analytes detected within 0.2 -35 ng/mL levels (high amounts).

receiving GSNO (n = 550), 17.41 ± 0.3405 KPa for untreated AAA-SMCs (n = 524), and 13.03 ± 0.3498 KPa for AAA-SMCs receiving GSNO (n = 469). (D) The surface roughness parameters Rq and Ra in Hu-SMCs and AAA-SMCs, in the presence of absence of GSNO. Data were analyzed using one-way ANOVA followed by Tukey's HSD post-hoc testing, assuming unequal variance and differences deemed significant for p < 0.05. Data shown represent mean ± standard error (n = 6 independent data points per condition). Supplementary Figure 3. Elastic modulus of cells acquired from AFM analysis. Histograms of elastic modulus data and corresponding representative phase contrast images of healthy Hu-SMCs (A, A-1), healthy Hu-SMCs treated with 100 nM GSNO (B, B-1), AAA-SMCs (C, C-1), and AAA-SMCs treated with 100 nM GSNO (D, D-1). Representative force-indentation curves (Hertz model fit) and corresponding elastic moduli measured from those curves for healthy Hu-SMCs (A-2), healthy Hu-SMCs treated with 100 nM GSNO (B-2), AAA-SMCs (C-2), and AAA-SMCs treated with 100 nM GSNO (D-2).

Discussion

In this study, we compared the differences and behaviors of human aneurysmal SMCs and their healthy counterpart with regard to cell proliferation, phenotypic and genotypic expression, transcriptomic analyses, immunofluorescence imaging, biomechanical characteristics, and their response to exogenously-delivered NO cues, as a therapeutic AAA treatment. approaches, consisting of 3D scaffolds and localized drug delivery options, specifically for in situ elastin regeneration and remodeling to arrest aneurysm progression.

Conflicts of Interest: None declared.


Diakaridja Nikiema

Zéphirin Mouloungui

Koua Oi Koua

Muriel Cerny

Éric Lacroux

Romain Valentin

Adjou Ané

Effect of dehulling method on the chemical composition of the lipid constituents of the kemels and oils of Ricinodendron heudelotii seeds

Keywor ds

Introduction

Ricinodendron or Jatropha heudelotii is a tree belonging to the Euphorbiaoeae family. lt is known under various common names in Africa: djansan in Cameroon, akpi in Côte d1voire and wama in Ghana. lt can grow to a height of almost 30 m, and it produces drupes, each of which contains no more than four seeds. lt grows in humid tropical zones and its distribution range in Africa extends from Senegal to Kenya. The fruits of Ricinodendron heudelotii reach maturity between June and September. They are harvested periodically, under the trees, by women.

Sinoe the United Nations Conferenoe on Environment and Development (UNCED) in Rio in 1992, the ecological and socio economic importance of non ligneous forest products (NLFPs) has been recognized in the management of forest ecosystems. In Cameroon, an economic and social mode! based on NLFPs was reoently implemented, in order to structu re the NLFP sector s, including the Ricinodendron

• Corresponding author.

E-mail address: zephirin.mouloungui@ensiacet.fr (Z. Mouloungui).

heudelotii sector. This sector was included in the mode! based on the properties of the lipids this tree produces, its cultural importance, its dietary value and its potential profitability [START_REF] Roques | Un modèle économique et social pour la conservation de la biodiversité: des filières oléagineuses durables issues de produits forestiers non ligneux (PFNL) pour préserver la réserve de faune du dja (RFD) au Cameroun[END_REF]. The seeds are extracted from the fruits and boiled to extract the kernels, which are used as aroma providing condiments in traditional African cooking. The oil has an agreeable flavor and is very popular for cooking. In the last few years, Riànodendron heudelotii has been used by the National Center for Agronomie Research (CNRA) in Côte d1voire as part of an agroforestry system based on cocoa plantations, and in ex perimental fields, with a view to its domestication. There are two main reasons for wanting to domesticate Ricinodendron heudelotii: (i) the fruits, the kernels of which are rich in oil and sold on most of the markets of West Africa for use as a seasoning and thickening agent in various African dishes [START_REF] Coulibaly | Effect of Post-harvest traditional technologies on the nutrient content and antioxidant compounds of defatted flours from Ricinodendron heudeloti (baill. Pierre ex Pax) Seed Kernels[END_REF] and Ciü Ricinodendron heudelotii is accepted by foresters in Côte d1voire as a canopy tree in cocoa plantations. This tree has been the subject of several scientific studies in recent https://doi.org/10.1016/j.indcrop.2019.111614

years. Studies of its kernels have reported an oil content of 42 to 60%, depending on the extraction technique used [START_REF] Dandjouma | Enzymeassisted hexane extraction of Ricinodendron heudelotii (bail.) Pierre ex pax seeds oil[END_REF]. This is a potentially important oleaginous natural resource, due to the high oil content of its seeds. According to studies performed by César Kapseu in 1995 and confirmed by Tchiégang et al. in 1996, the oil from kernels isolated by traditional heat treatment has α eleostearic acid (51%) and linoleic acid (28%) as its major fatty acids [START_REF] Kapseu | Chemical properties of Ricinodendron heudelotii (bail.) Seed oil[END_REF][START_REF] Tchiegang | Ricinodendron heudelotii (bail) kernels: a novel ingredient for tropical food agro-industries[END_REF]. By contrast, Samuel and coworkers found, in 2011, that the major fatty acid of the oil obtained from kernels isolated in the same conditions was linoleic acid (58.7%) [START_REF] Yeboah | Compositional and structural studies of the Major and minor components in Three Cameroonian seed oils by GC-MS, ESI-FTICR-MS and HPLC[END_REF]. [START_REF] Leudeu | Ricinodendron heutelotii (bail.) Or tetracarpidium conophorum müll. Oils fed to male rats lower blood lipids[END_REF] study on oil from kernels isolated by traditional heat treatment, found that the major fatty acid was linoleic acid, which accounted for 36% of the fatty acids present [START_REF] Leudeu | Ricinodendron heutelotii (bail.) Or tetracarpidium conophorum müll. Oils fed to male rats lower blood lipids[END_REF]. By contrast, according to [START_REF] Sompila | Identification des triacylglycérols en HPLC[END_REF], the major fatty acid in Ricinodendron heudelotii oil is α eleos tearic acid, which accounts for 35% of the fatty acids present [START_REF] Sompila | Identification des triacylglycérols en HPLC[END_REF]. The α eleostearic acid (C18:3 n 5) is a fatty acid containing 18 carbon atoms and three conjugated double bonds (9c, 11 t, 13 t) with the following physico chemical data : molecular weight 278.42 g/mol, melting point 49 °C, boiling point 235/12 (°C/mm Hg), density 0.902 g/ ml (at 50 °C), iodine value 274. This polyunsaturated fatty acid and its isomers (punicic acid, catalpic acid, β eleostearic acid and jacaric acid) have demonstrated many health benefits. Studies have shown that these bioactive molecules may be used as dietary supplements for treatment of diseases such as obesity, cancer and diabetes [START_REF] Białek | Conjugated linolenic acid (CLnA) isomers as new bioactive lipid compounds in ruminant-derived food products. A review[END_REF][START_REF] Yuan | Conjugated linolenic acids and their bioactivities: a review[END_REF].

Ricinodendron heudelotii oil is a siccative oil, it can be used as a re newable raw material for the formulation of surface coating systems, paints and adhesives, as demonstrated by the work of Assanvo et al. [START_REF] Assanvo | Synthesis, characterization, and[END_REF]. α eleostearic acid, β eleostearic acid and cat alpic acid from this oil are polymerizable fatty acids. They can be used in the polymer industry for the synthesis of biodegradable polymers (biopolymers) like tung oil. The importance of CLnA in Phytomedicine may promote the use of Ricinodendron heudelotii oil in the pharmaceu tical industry.

This overview of the state of the art reveals a variability of fatty acid profiles for oils extracted from kernels isolated by traditional heat treatment. Certain factors, such as heat, humidity, ultraviolet radiation, and storage time and conditions, can change oil fatty acid composition.

In addition to contributing to the structuring of NLFP sectors, the study performed in Cameroon by [START_REF] Roques | Un modèle économique et social pour la conservation de la biodiversité: des filières oléagineuses durables issues de produits forestiers non ligneux (PFNL) pour préserver la réserve de faune du dja (RFD) au Cameroun[END_REF] highlighted the need to improve knowledge of these products and the procedures for their transformation, to target and optimize the parameters likely to influence product quality, including, in particular, oxidation factors. We performed a survery to obtain reliable data concerning the sites at which Ricinodendron heudelotii is grown and fruits are harvested in 23 regions of Côte d'Ivoire. We present here a comparative study of the chemical (protein content, oil content, fatty acid profiles, minor com pounds) and physicochemical (kinematic viscosity, iodine value, acid value) properties of oils and kernels obtained by traditional heat treatment or by manual mechanical dehulling.

Materials and methods

Materials

The seeds of Ricinodendron heudelotii used in this study came from Sinfra (6°36′29.4″N 6°01′18.2″W), a community in the Marahoué re gion of Côte d'Ivoire, in West Africa. The fruits were collected from under the trees, during the month of August 2014. The seeds were extracted from the pulp of the fruit by hand and dried in a ventilated shelter for five days. The seeds were then frozen and stored loose in freezer bags at +4 °C until their transport to the agro industrial chemistry laboratory in Toulouse, France. They were then stored in a cold room at +4 °C.

Methods of kernel isolation

Two methods were used to isolate the kernels from the seeds during this study. The first was the traditional heat treatment, performed in a ventilated shelter, and the second was manual mechanical dehulling, performed in the agro industrial chemistry laboratory in Toulouse, France.

Traditional heat treatment

The seeds of Ricinodendron heudelotii consist of a kernel surrounded by a husk (Fig. 1).

The most widely used technique for separating the kernel from the husk is the traditional three step heat treatment. In the first step, soaking, 5 kg of seeds are immersed in tepid water in a round stainless steel 20 liter cuvette for 24 h. In the second stage, the seeds are boiled in water over a wood fire for 12 h. This process generates sufficient heat to fissure the husk. The final step is the release of the kernels with the aid of a nail or small knife. For the traditional heat treatment in this study, we dehulled the seeds in this way, then dried the kernels in the sun for about 10 days, after which they were frozen and stored in freezer bags at +4 °C.

Manual mechanical dehulling

In this technique, the kernels are released from the seeds without prior treatment. The seeds (Fig. 1) of Ricinodendron heudelotii fruits were directly dehulled by hand, one by one. A standard pincer type nutcracker was used, with the seed placed where the walnut would usually be inserted. The two arms of the pincer were closed, exerting sufficient pressure on the seed to break the husk without shattering the kernel inside it. The kernel was then removed from the husk with a small stainless steel spatula.

Extraction of oil from the Ricinodendron heudelotii kernels by two methods

We used two methods of oil extraction, depending on the amount and quality of oil desired for the various analyses. We extracted lipids in solvent with a Soxhlet extractor for measurements of oil content, and

Determination of standard values for the oils

Acid value, iodine value, and acidity were determined by volu metric methods, according to the protocols of standards, NF EN ISO 3961 for iodine value and NF EN ISO 660 for acid value and acidity/ eleostearic acid equivalent. Acidity was assessed by a theoretical method based on the fatty acid profile of Ricinodendron heudelotii oil.

Fatty acid composition determination by gas chromatography (GC)

The fatty acid profile of the oil was determined by the trimethyl sulfonium hydroxide (TMSH) method (standard NF EN ISO 12966 3, 2016). The oil (15 mg) was solubilized in 1 ml of TBME (tert butyl methyl ether), methylated with trimethyl sulfonium hydroxide (TMSH, 0.2 ml/l in methanol) and the resulting fatty acid methyl esters (FAMEs) were analyzed with a gas chromatograph (Varian 3900) equipped with a CP Select CB column (50 m, 0.25 mm i.d., 0.25 μm film thickness). We injected 1 μl, using the following parameters: injector in split mode, ratio of 1/100, 250 °C (55 min). The carrier gas was helium, at a flow rate of 1.2 ml/min. The oven temperature was initially set at 185 °C for 40 min, then increased to 250 °C at a rate of 15 °C/minute and maintained at 250 °C for 11 min. The temperature of the flame ionisation detector was set at 360 °C. FAMEs were identified by com paring the retention times obtained with those for a mixture of com mercial standards, Mix 37 (Sigma Aldrich), tung oil and oil from Catalpa bignonioides.

Determination of tocopherol content by HPLC

The tocopherol content of the oil was determined by HPLC fluori metry, according to the analytical method described in the AFNOR NF ISO 9936 standard. We used a liquid phase chromatograph coupled to a Dionex fluorescence detector (λex290 nm; λem 317 nm) and an oven heated to a fixed temperature of 22 °C. We injected 20 μl of a 10 mg/ml solution of oil in cyclohexane into the HPLC apparatus. The mobile phase was a mixture of isooctane and isopropanol (99.5/0.5 v/v), at a flow rate of 1.1 ml/minute. Quantification was based on external cali bration with standards (α tocopherol, γ tocopherol, and δ tocopherol) from Sigma.

Determination of sterol content by GC

The determination of sterol content began with the preparation of a 2 mg/ml solution of cholestanol (internal standard). We added precisely 50 μl of this solution (corresponding to 100 μg of cholestanol) to a 15 ml screw top tube. After chloroform evaporation, 100 mg of oil was added. Saponification was carred out through addition of 2 ml of 1 M KOH in ethanol. The mixture was vortexed and heated for 20 min in a water bath at 75 °C. The reaction mixture was allowed to cool, 1 ml of de mineralized water plus 6 ml of cyclohexane were added and the con tents of the tube were mixed by vortexing. We allowed the mixture to separate and recovered the organic phase containing the unsaponifiable matter. We mixed 160 μl of this solution with 40 μl of silylation reagent (99% N,O bis[trimethylsilyltrifluoroacetamide] plus 1% trimethyl chlorosilane: BSTFA/TMCS, 99/1). Sterol determinations were per formed by GPC with a Perkin Elmer Auto System XL equipped with a CPSil 8CB (Varian) 30 m x0.25 mm column, with a film thickness of 0.25 μm. The carrier gas was helium, at a column head pressure of 100 kPa. We injected 1 μl of solution in the on column mode. The oven temperature was maintained at 160 °C for 0.5 min and was then in creased gradually to 260 °C at a rate of 20 °C/minute. Oven temperature was then increased to 300 °C at a rate of 2 °C/minute, and finally to 350 °C at a rate of 45 °C/minute. The total analysis time was 50 min. The temperature of the flame ionisation detector (FID) was set at 360 °C [START_REF] Roche | Management of environmental crop conditions to produce useful sunflower oil components[END_REF]. we used the cold extraction of lipids in cyclohexane to obtain oil for chemical and physicochemical characterization. The different solvent mediated lipid extractions were performed at least three times.

Extraction of lipids with solvent in a soxhlet extractor

The kernels were ground with a bladed grinder. A mass of 15 g of ground kernels was placed in the extraction cartridge and inserted into the Soxhlet extractor. With the aid of 150 ml of cyclohexane in a 200 ml balloon flask, the lipid matter within the ground kernels was extracted by solvent reflux (at about 80 °C) for six hours. The oil was obtained by evaporating off the solvent in a rotary evaporator at 50 °C, under va cuum, at 200 mbars. The amount of oil extracted was then determined relative to the mass of ground material.

Cold extraction in cyclohexane

A mass of 2.5 g of ground kernels (ground in a bladed grinder, as described above) was placed in a 30 ml centrifuge tube with 25 ml of cyclohexane. The tube was vortexed for 30 s and then centrifuged for 15 min at 10,000 x g. The supernatant was decanted off and the organic phase (lipid matter) was recovered. This process was repeated twice. All of the organic phases collected were combined and filtered through a funnel containing a glass fiber filter paper on which a few grams of anhydrous sodium sulfate were placed. The oil was recovered by eva porating off the cyclohexane in a rotary evaporator at 50 °C, under vacuum, at 200 mbars. The amount of oil extracted was then de termined relative to the mass of ground material.

Physicochemical characterization of the kernels by the ADF NDF method

We used the revised version (March 23, 2011) of the Van Soest three part sequential analysis procedure to determine the lig nocellulosic composition of the Ricinodendron heudelotii kernels. The first step was the determination of the insoluble fiber content of 1 g of ground Ricinodendron heudelotii kernels from which the lipids had been removed by Soxhlet extraction (cyclohexane), by solubilizing and eliminating the proteins and water soluble material with a neutral de tergent solution (sodium lauryl sulfate) at 95 °C. The residue obtained is known as NDF (neutral detergent fiber). It consists of hemicelluloses, celluloses, lignins and ash. It was filtered and washed with deminer alized water heated to 90 °C. Calcination at 450 °C for 6 h was per formed to determine the ash content.

The second stage of the procedure was the solubilization of hemi cellulose in an acid detergent solution consisting of acetyl trimethyl ammonium bromide in 0.5 M sulfuric acid. The residue obtained by this procedure is called the ADF (acid detergent fiber) and consists of cel lulose and lignin. This residue was filtered, washed in hot water and dried.

Finally, the ADF residue was treated with potassium permanganate to oxidize and eliminate lignins, for the determination of cellulose content.

Determination of protein content

We used an indirect method based on the determination of the percent total nitrogen (% N) by the Kjeldahl method, in accordance with the NF V 18 199 standard. Kjeldahl's method involves miner alization of the organic nitrogen present in the sample to generate ammoniacal nitrogen, which is then determined by titration against acid. For mineralization, a mass of 0.8 g of dried kernels delipidated by the Soxhlet method was placed in a glass tube, to which 12.5 ml of 95% sulfuric acid was added. The mixture was placed in a fume hood for 16 h. It was then mineralized by heating at 400 °C for 1 h. Finally, the analysis was performed with a KjeltecTM 8400 analyzer.

Results and discussion

Mapping Ricinodendron heudelotii in côte d'ivoire

The localization of Ricinodendron heudelotii trees and fruit har vesting was studied at 23 sites: Abidjan, Yamoussoukro, Sinfra, Gagnoa, Tiassalé, Toumodi, Oumé, Divo, Danané, Man, Lakota, San Pedro, Soubré, Adiaké, Adzopé, Kotobi, Bouaké, Arrah, Akoupé, Daloa, Sikensi, Agboville, and Rubino. With the aim of collecting as much information as possible, three villages were selected per site for the collection of information on the presence of Ricinodendron heudelotii and kernel production. According to this survey, this plant was present at all the sites evaluated. Ricinodendron heudelotii seed harvest and kernel production were strongly linked to the dietary habits of the local population. We identified two large production areas in the south east and south west of the country. Overall, 82.5% of the sites studied produced Ricinodendron heudelotii kernels, but there were no harvesting or transformation activities at the remaining 17.5% of the sites at which this plant was found.

In Côte d'Ivoire, the annual harvest of Ricinodendron heudelotii seeds generates about 100 tonnes of kernels. The fruits are harvested by the women in certain parts of the country. The kernels are almost ex clusively destined for human consumption, by local populations. Much of this production is destined for the supply of large towns, such as Abidjan, Yamoussoukro and Bouaké, for reasons of profitability. The buyers in these towns are solvent and the kernels are sold at 8000 Francs CFA (equivalent to 12 Euros) per kilogram.

Composition of Ricinodendron heudelotii kernels

Table 1 shows a breakdown of kernel contents for kernels obtained by the two dehulling methods. Similar kernel content results were ob tained for the two dehulling methods.

Water and dry matter contents were 7% and 93%, respectively, cellulose content was 6%, hemicellulose content was 7.5% and lignin content was 3%. These values indicate that Ricinodendron heudelotii kernels have low contents of cellulose, hemicellulose and lignin. By contrast, they have a high protein content, at 21.3 ± 1.9%. This pro tein content is consistent with the protein contents of 21% reported by Saki et al. (2005), and 24% reported by [START_REF] Coulibaly | Effect of Post-harvest traditional technologies on the nutrient content and antioxidant compounds of defatted flours from Ricinodendron heudeloti (baill. Pierre ex Pax) Seed Kernels[END_REF]. Ac cording to these results, regardless of the method of kernel isolation used, the same amount of lipid is extracted, corresponding to 47.4 ± 0.2% of the kernel contents. Similar results were obtained by [START_REF] Dandjouma | Enzymeassisted hexane extraction of Ricinodendron heudelotii (bail.) Pierre ex pax seeds oil[END_REF]Saki et al. in 2005, with these authors reporting rates of oil extraction by the Soxhlet method of 50% and 48.9% for kernels isolated by the traditional heating method. The re sults obtained for protein content and lipid extraction rate indicate that Ricinodendron heudelotii seeds can be considered as oleoproteaginous. Such seeds are a good source of both lipids and proteins.

Physicochemical characterization of Ricinodendron heudelotii oil

Table 2 summarizes the results for kinematic viscosity, iodine value, acid value and acidity for the oils extracted from kernels by the tradi tional heating method and by manual mechanical dehulling.

The densities of the oil samples obtained following the two dehul ling methods were almost identical, at 0.92 g/ml and 0.94 g/ml. This demonstrates the reproducibility of the mass of oil obtained from ker nels isolated by the two methods of dehulling. The oil obtained from kernels isolated by traditional heat treatment had a higher viscosity (169 mPa.s), a higher acid value (5.7 ± 0.3) a higher acidity (2.8 ± 0.2) and a lower iodine value (179.6 ± 1.1) than the oil ob tained from kernels isolated by manual hulling. This reflects a slight deterioration of the oil during the traditional heat treatment. Indeed, the traditional method for isolating kernels from Ricinodendron heude lotii seeds involves uncontrolled techniques, such as a long period of heating in boiling water and the drying of the kernels in the sun, both of which are factors favoring oxidation of the oil. These results demon strate that the mode of seed treatment for kernel isolation has a sig nificant influence on the iodine value of the extracted oil. Iodine value is lower following the traditional heat treatment of the seeds to isolate the kernels. The iodine values obtained here are different from those reported in previous studies. According to the studies performed by the groups of Tchiégang and Assanvo, the iodine value of this oil is 150 [START_REF] Tchiegang | Conditions for the extraction of oil by pressing of Ricinodendron hendelotii (bail.) Pierre ex pax[END_REF][START_REF] Assanvo | Synthesis, characterization, and[END_REF]. In this study, we ob tained iodine values of 179.6 ± 1.1 and 195.2 ± 1.7 for the oils ex tracted from kernels isolated by the two dehulling methods. These va lues were thus high, for both methods. They indicate that this oil from Ricinodendron heudelotii is highly siccative, even more so than tung oil, which has an iodine value of 170 according to Samadzadeh et al. (2011).

Fatty acid composition of the oils

Ricinodendron heudelotii is one of the rare highly polyunsaturated plant oils, with a CLnA content of at least 84%, depending on the method used to dehull the kernels. An analysis of the CLnA content of the oils obtained revealed that α eleostearic acid and linoleic acid were the two principal fatty acids present. Nevertheless, their contents varied with the method used to obtain the kernels. Oil from kernels isolated by manual mechanical hulling without prior treatment contained 60.1 ± 0.2% α eleostearic acid and 22.8 ± 0.1% linoleic acid, whereas oil extracted from kernels obtained by traditional heat treat ment contained 50.5 ± 0.1% α eleostearic acid and 24 ± 0.0% lino leic acid. These two oils also contained CLnA known to be isomers of α eleostearic acid: β eleostearic acid (1.1 ± 0.1% in oil from kernels isolated by manual mechanical hulling and 8.4 ± 0.0% in oil from kernels isolated by traditional heat treatment) and catalpic acid, which was present only in oil from kernels isolated by traditional heat treat ment (0.40 ± 0.1%). Published results for the CLnA content of Ricinodendron oils are shown in Table 3.

Oils extracted from kernels obtained by traditional heat treatment have α eleostearic acid contents of 32.5% to 51.1% and linoleic acid contents of 28.1% to 36.0%. This composition is similar to the CLnA profile of our oil extracted from kernels obtained by traditional heat treatment: 50.5 ± 0.1% α eleostearic acid and 24.0 ± 0.0% linoleic acid. Two parameters must be factored into this comparative study: the location and type of kernel production. Even with the same method of oil extraction in solvent (Soxhlet extraction), CLnA content varied considerably, ranging from 35% to 60% for α eleostearic acid and 22% to 36% for linoleic acid. Furthermore, another study have reported the presence of β eleostearic acid and catalpic acid [START_REF] Sompila | Identification des triacylglycérols en HPLC[END_REF]. An examination of our data and those of Sompila and al revealed a clear tendency towards a decrease in α eleostearic acid content in favor of linoleic acid and β eleostearic acid. For an oil obtained by the tradi tional dehulling method, an 8.4% β eleostearic acid content was de termined in our study, and 9.7% were found by Sompila and al. The traditional dehulling method involves some isomerization of α eleostearic acid. This acid is isomerized into β eleostearic acid and catalpic acid, in the form of triglycerides in native oil.

The oil enriched in β eleostearic acid have beneficial effects on human health. According to some studies, this fatty acid may has cy totoxic effects on cancer cells [START_REF] Shinohara | Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo[END_REF][START_REF] Sun | Beta-eleostearic acid induce apoptosis in T24 human bladder cancer cells through reactive oxygen species (ROS)-mediated pathway[END_REF].

Alpha eleostearic acid, β eleostearic acid and catalpic acid in Ricinodendron heudelotii oil are isomers of conjugated linolenic acid (CLnA). There are other isomeric fatty acids of CLnA, namely punicic acid (C18:3 9c, 11 t, 13c) in Punica granatum oil (79.3%), jacaric acid (C18:3 8 t, 10 t, 12 t) in Jacaranda mimosifolia seed oil (62%) [START_REF] Hernandez | Fatty acid composition and total lipid content of seed oil from three commercial pomegranate cultivars. Production, Processing and Marketing of Pomegranate in the Mediterranean Region: Advances in Research and Technology[END_REF][START_REF] Takagi | Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: analysis by open-tubular gas liquid chromatography and high performance liquid chromatography[END_REF] Recent research shows that these fatty acids from CLnA family are used in phytomedicine. They have a beneficial effect on health. They have favourable physiological effects, such as anti arteriosclerosis, anti obesity, anti tumour, anti hypertension and anti cancer [START_REF] Gasmi | Jacaric acid and its octadecatrienoic acid geoisomers induce apoptosis selectively in cancerous human prostate cells: a mechanistic and 3-D structure-activity study[END_REF][START_REF] Yuan | Conjugated linolenic acids and their bioactivities: a review[END_REF].

α eleostearic acid in Momordica charantia seed oil is a potential therapeutic agent in the treatment of breast cancer and leads to self destruction of colon cancer cells (Caco 2) [START_REF] Shinohara | Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo[END_REF].

Punicic acid in Punica granatum improve insulin secretion without altering fasting blood sugar [START_REF] Nekooeian | Effects of pomegranate seed oil on insulin release in rats with type 2 diabetes[END_REF]. Catalpic acid in Catalpa bignonioides seed oil causes a decrease in fasting blood sugar and insulin [START_REF] Hontecillas | Catalpic acid decreases abdominal fat deposition, improves glucose homeostasis and upregulates PPAR α expression in adipose tissue[END_REF].

Characterization of the minor constituents of Ricinodendron heudelotii oils

Table 4 summarizes the tocopherol content of the oils. Total tocopherol content was 178.3 mg/100 g for oil extracted from kernels isolated by manual mechanical dehulling and 135 mg/ 100 g for oil extracted from kernels isolated by traditional heat treatment. This value was influenced by the treatment of the seeds before oil extraction from the kernels. This tocopherol content is similar to that of poly unsaturated oils such as linseed oil, which contain 110 280 mg/100 g. Regardless of the pretreatment method during dehulling, the major constituent was γ tocopherol, at 92 mg/100 g. However, these results show that the traditional heat treatment degrades the tocopherols present in the oil within the seeds.

An analysis of the sterol composition of Ricinodendron heudelotii oil showed that β sitosterol was the major component, accounting for 83% of the sterols present, and that total sterol content stays the same be tween seed treatments, ranging from 402.5 ± 8.0 to 412.0 ± 8.0 mg/ 100 g. Ricinodendron heudelotii oil is, thus, moderately rich in sterols, including β sitosterol in particular.

Conclusion

This study focused on the characterization of two sorts of oil from Ricinodendron heudelotii kernels: oil from kernels isolated by the tradi tional heat treatment and oil from kernels obtained by manual me chanical dehulling. The two dehulling methods tested resulted in a different chemical composition readily identifiable in terms of CLnA contents. Oil from kernels isolated by manual mechanical dehulling contained 60.1% ± 0.2 α eleostearic acid, 22.8% ± 0.1 linoleic acid, and 1.1% ± 0.1 β eleostearic acid, whereas oil from kernels isolated by the traditional method contained 50.5% ± 0.1 α eleostearic acid, 24% ± 0.0 linoleic acid, 8.4% ± 0.0 β eleostearic acid and 0.40% ± 0.1 catalpic acid. Thus, oils from mechanically dehulled ker nels were very rich in α eleostearic acid, whereas oils from kernels dehulled by the traditional method displayed enrichment in β eleos tearic acid and catalpic acid, due to the isomerization of α eleostearic acid. Oils are rich in CLnA (84%), with high iodine values (180 and 195, respectively). The minor constituents, including γ tocopherol and 3.1 ± 0.1 2.9 ± 0.2 β-sitosterol(%)

83.6 ± 0.5 83.7 ± 0.3 Stigmastanol (%) 1.7 ± 0.01 1.5 ± 0.9 5-avenasterol (%) 4.3 ± 0.11 4.6 ± 0.4 Total sterols (mg/100 g) 412.0 ± 8.0 402.5 ± 8.0

Table 3 Published results concerning the chemical composition of Ricinodendron heudelotii oil.

Fig. 1 .

 1 Fig. 1. Ricinodendron heudelotii (seeds, kernels, husks).

Table 1

 1 Characterization of kernel components.

	Kernel samples isolated:
	Constituent contents (%)	By traditional heat	By manual mechanical
		treatment	dehulling
	Water	7.3 ± 0.2	7.5 ± 0.1
	Dry matter	93.1 ± 0.2	92.5 ± 0.1
	Ash	7.3 ± 0.2	7.5 ± 0.1
	Cellulose	6.5 ± 0.2	6.1 ± 0.3
	Hemicellulose	7.8 ± 0.3	7.5 ± 0.2
	Lignin	3.0 ± 0.2	3.2 ± 0.2
	Protein	21.3 ± 1.9	21.1 ± 0.2
	Oil (Soxhlet extraction)	47.4 ± 0.2	47.3 ± 0.6

Table 2

 2 Physicochemical characteristics of the oils.

	Samples of oil from kernels isolated:
	Physical characteristics of the	By traditional heat	By manual mechanical
	oil	treatment	dehulling
	Density (g /ml at 25 °C)	0.92	0.94
	Viscosity (mPa.s)	169 ± 0.5	128.0 ± 0.1
	Acid value	5.7 ± 0.3	3.7 ± 0.3
	Acidity (% by mass)	2.8 ± 0.2	1.8 ± 0.1
	Iodine value	179.6 ± 1.1	195.2 ± 1.7

  kernels bought on the market. a kernels isolated by manual mechanical dehulling. b kernels isolated by traditional heat treatment. Chemical characteristics of the minor components of the oil.

	Fatty acid	Kapseu 1995 *	Dandjouma 2007 *	Sompila 2014 *	Leudeu 2009 *	This study
						Kernel a	Kernel b
	Palmitic acid C16:0	5.5	6.4	5.4	9.4	3.8 ± 0.0	4.1 ± 0.0
	Stearic acid C18:0	6.4	7.5	8.0	10.6	5.1 ± 0.0	5.3 ± 0.0
	Oleic acid C18 :1n-9	7.4	7.18	11.2	10.3	6.7 ± 0.0	7.1 ± 0.0
	Linoleic acid C18:2n-6	28.3	30.7	28.1	36.0	22.8 ± 0.1	24.0 ± 0.0
	α-eleostearic acidC18:3n-5(c,t,t)	51.1	48.4	35	32.5	60.1 ± 0.2	50.5 ± 0.1
	β-eleostearic acid C18:3n-5(t,t,t)	--	--	9.7	--	1.1 ± 0.1	8.4 ± 0.0
	Catalpic acid C18 :3n-5 (t,t,c)	--	--	1.3	--	0.0	0.40 ± 0.1
	Saturated fatty acids (SFAs)	11.9	13.9	13.4	20.0	8.9 ± 0.1	9.4 ± 0.0
	Monounsaturated fatty acids (MUFAs)	7.4	7.18	11.2	10.3	6.7 ± 0.0	7.0 ± 0.0
	Polyunsaturated fatty acids (CLnA)	79.4	79.07	74.1	68.5	84.4 ± 0.4	83.3 ± 0.1
				Table 4
				Minor components	Samples of oil from kernels isolated:
				Tocopherols (%)	By traditional heat	By manual mechanical
					treatment	dehulling
				α-tocopherol	4.0 ± 0.01	7.9 ± 0.02
				γ-tocopherol	92.9 ± 0.02	89.9 ± 0.18
				δ-Tocopherol	3.1 ± 0.02	2.5 ± 0.46
				Total (mg/100 g)	135.0 ± 0.4	178.3 ± 0.4
				Sterols %
				Campesterol (%)	7.2 ± 0.11	7.2 ± 0.19
				Stigmasterol (%)

*

Acknowledgments

This work was funded by the Ministry of Higher Education and Scientific Research of Côte d'Ivoire and the French government by the program entilted AMURGE CI C2D.