extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Structural Characterization of a Gcn5-Related N-Acetyltransferase from Staphylococcus aureus

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

Introduction
The Gcn5-related N-acetyltransferases (GNATs) are a very large enzyme superfamily with more than 10,000 members identified across all kingdoms of life [1]. They were first identified as aminoglycoside acetyltransferases from bacteria that developed antibiotic resistance to kanamycin and gentamicin [1]. The GNATs catalyse the transfer of an acetyl group from acetyl CoA to the primary amine of substrates including antibiotics aminoglycosides, glucosamine-6-phosphate, histones, serotonin, spermine, spermidine, and other small molecules [2][3][4][5][6]. In spite of the substrate and functional diversity, the basic structure of GNAT members is highly conserved. Members of the family share a common fold known as the GNAT fold, comprised of 6-7 antiparallel b-strands and 4 a-helices in the topology b1-a1-a2-b2-b3-b4-a3-b5-a4-b6-b7.
The GNAT fold contains four conserved motifs A-D, arranged in the order C, D, A and B in the primary sequence. The most highly conserved motif across the superfamily is motif A, followed by motif B, D, and C. The hallmark of motif A is the ''P-loop'', connecting helix a3 and strand b4, which plays an essential role in binding the b-mercaptoethylamine and pantothenic acid moieties of acetyl-CoA. Motif B spans conserved regions within a-helix 4 and is involved in binding the 39, 59-adenosine diphosphate of acetyl-CoA. Motif D, encompassing b2 and b3 strands is not directly involved in substrate or cofactor binding, but stabilises core structural elements of the protein, while motif C, located at the N-terminus of the protein, is the least conserved with some histone N-acetyltransferases lacking this motif completely. These motifs together comprise the common structural core known as GNAT domain. A signature of the GNAT fold is a splay between b4 and b5 strands, forming a V-shape opening in the central b sheet which is crucial in the transfer of acetyl group and binding of acetyl-CoA [7].
Whilst cell regulation through acetylation has been well characterised in eukaryotes, the role of protein acetylation within prokaryotes has only emerged recently, providing support that acetylation based regulation is an important and universal process. Staphylococcus aureus, an important pathogenic and increasingly multi-drug resistant bacterium, contains 35 putative GNAT enzymes, many of which remain uncharacterised both functionally and structurally. It is also an opportunistic human pathogen and frequent cause of infection ranging from mild to life threatening illnesses including bacteremia, meningitis, osteomyelitis, liver cirrhosis, keratitis, pneumonia, septic phlebitis and endocarditis [8,9]. Moreover, rates of S. aureus infections have increased over past decade as has antibiotic resistance to commonly used antibiotics including rifampicin, vancomycin and methicillin [10,11]. Resistance towards the aminoglycoside antibiotics can occur through a range of mechanisms including aminoglycoside modifying enzymes, ribosomal mutations, or excretion of the aminoglycoside. Aminoglycoside-modifying enzymes can inactivate antibiotics by covalently attaching either a phosphate, nucleotide, or acetyl moiety to either the amine or the alcohol  key functional group (or both groups) of the antibiotic, changing the charge or sterically hindering the antibiotic [12,13]. Thus, characterisation of proteins capable of playing a role in antibiotic resistance and regulatory functions within important pathogenic bacteria provides an important platform for rational drug design, development of new inhibitors, and an enhanced understanding of the putative functional roles.
Here, we describe the structure of an uncharacterised, GNAT family member from S. aureus. Our structure confirms that the protein exhibits many of the classical GNAT motifs, has high structural similarity with the phosphinoacetyl GNAT proteins, and is likely to exist as a dimer in solution based on biophysical and crystallographic properties.

Cloning and expression
The gene encoding the putative N-acetyltransferase from S. aureus subsp. aureus Mu50 NP_373053 (SaGNAT) was PCR amplified from genomic DNA purchased from American Type Cell Culture (ATCC), and cloned into the expression vector pMCSG21. The fidelity of the clone was confirmed by DNA sequencing and the plasmid transformed into E. coli BL21 (DE3) pLysS for recombinant expression. A 5 ml Luria-Bertani (LB) broth starter culture containing 100 mg/ml spectinomycin was used to inoculate 500 ml of auto-induction media [14] containing 100 mg/ml spectinomycin grown at 25uC for 24 h. The cells were harvested by centrifugation and the cell pellet resuspended in 50 ml of His buffer A (50 mM Phosphate buffer pH 8.0, 300 mM NaCl, 20 mM Imidazole) and stored at 220uC.

Protein purification and crystallisation
The E. coli cells were lysed by 2 repetitive freeze-thaw cycles in the presence of 20 mg of lysozyme, and the lysate centrifuged at 15,000 rpm for 30 min. The supernatant was filtered through a 0.45 mm filter and the supernatant loaded onto a 5 ml Ni 2+ column (HisTrap HP, GE Healthcare) in His buffer A. Following extensive washing of the column (.10 column volumes) in His buffer A, the protein was eluted using an increasing gradient of His buffer B (50 mM phosphate buffer pH 8.0, 300 mM NaCl, 500 mM imidazole). Elution fractions were pooled and treated with tobacco etch virus protease (100 mL of 3.3 mg/mL) overnight at 4uC to remove the affinity tag. The cleaved protein was further purified by size exclusion chromatography (Superdex 200 column, GE healthcare) in GST buffer containing 50 mM Tris, pH 8.0, and 125 mM NaCl). The fractions containing protein were pooled and concentrated to 27 mg/ml using an Amicon ultrafiltration device (Millipore). The purity of the protein was assessed by SDS-PAGE and stored at 280uC. Crystallisation screening was undertaking using the hanging-drop vapour-diffusion method and commercially available screens (Hampton Crystal Screen, PEG/Ion, Crystal Screen 2 and PEG/Ion 2). The drops contained 1.5 ml of the protein, to which an equal volume of reservoir solution was mixed, and suspended over 300 ml of reservoir solution at 296 K. Plate shaped diffraction quality crystals were obtained in 1 M sodium acetate trihydrate, 100 mM HEPES pH 7.5, and 50 mM cadmium sulphate hydrate.

Data collection, structure determination and refinement
Crystals were flash-cooled at 100 K in liquid nitrogen with reservoir solution containing 30% glycerol as a cryoprotectant. Diffraction data were collected from a single crystal at the MX2 crystallography beamline at the Australian Synchrotron. Data were indexed and integrated using iMOSFLM [15] and scaled in AIMLESS [16]. Molecular replacement was undertaken using Phaser [17] and chain A of PDB 2JLM (48% sequence identity) as a search model. Model building and refinement was performed in Coot [18] and Phenix respectively [19].

Protein production and structure determination
To determine the x-ray crystallographic structure of SaGNAT, the gene encoding the protein was cloned into bacterial expression vector pMCSG21 [20] and recombinantly expressed as a 6-His tagged fusion protein in E. coli BL21 (DE3) pLysS. The protein was solubly over-expressed using the auto-induction method [14] (see Fig. 1 lanes 1 and 2), and a two-step purification incorporating affinity and size exclusion chromatography resulted in greater than 95% purity (Fig. 1). SaGNAT protein crystals produced in 1 M sodium acetate trihydrate, 100 mM HEPES pH 7.5, and 50 mM cadmium sulphate diffracted to 2.15 Å and were indexed and integrated in the space group C2, with unit cell parameters a = 97.5 Å , b = 78.9 Å , c = 66.0 Å , a = 90u, b = 112.0u, c = 90u. Molecular replacement using Phaser [17] and chain A of PDB model 2JLM was used to place 2 molecules in the asymmetric unit, corresponding to a Matthews coefficient of V M 3.18 Å 3 Da 21 and 61.4% solvent content [21]. Extensive model building and refinement using COOT [18] and Phenix [19] respectively produced a final model with an R cryst and R free 0.18 and 0.22 respectively. All amino acid residues were modelled with the exception of the final C-terminal residue. Coordinate and structure factors have been validated and deposited to Protein Data Bank and assigned the PDB ID code 4MBU. Data-collection and refinement statistics are summarized in Table 1.

Structure of SaGNAT15
The refined x-ray crystallographic structure revealed SaGNAT to be an a/b protein comprised of 4 a-helices and 7 b-strands, with a topology b1-a1-a2-b2-b3-b4-a3-b5-a4-b6-b7 (Fig. 2). All b-strands are arranged sequentially according to sequence, with the exception of b7, located between strands b5-6. Two central antiparallel b-sheets (b1-4 and b5-8) are splayed between b4 and b5 to create a V-shape in the protein (Fig. 2). The two b-sheets are held together at the V joint by hydrogen bonding located on the N-terminal residues in strands b4-b5, and diverge at Ser 83 and Ala 117 . This signature feature of GNATs is stabilised by hydrogen bond interactions between water molecules and the amide N and carbonyl O atoms from the protein main chain. The N-terminal arm of the protein is comprised of an antiparallel b-sheet (b1-4) flanked by 3 a-helices (a1, a2 on one side, a3 on the other), and the C-terminal arm is comprised of an antiparallel sheet (b5-7) flanked by a4 on the same side as a3.
To assess both the sequence and structural similarities of SaGNAT with other GNAT-proteins, BLAST and DALI searches were undertaken. A sequence homology search of the nonredundant database using BLASTP (2.2.28) revealed the most closely related enzyme to be a phosphinothricin N-acetyltransferase (YP_008780792) from Bacillus cereus, sharing 60% sequence identity. This low sequence identity between the two closest related homologues is not unusual in the GNAT family, with subfamilies well documented to have highly variable amino-acid sequences, yet retaining very high structural homology [1]. In support of this, a structural homology search using DALI revealed 3 proteins with an rmsd of less than 1 Å , all corresponding to phosphinothricin acetyltransferases (PDB 1yr0/rmsd 0.8/40% sequence identity; PDB 2jlm/rmsd 1.0/48% sequence identity; PDB 2bl1/rmsd 0.9/47% sequence identity). The structural overlay and alignment of these proteins is presented in Fig. 3, with the conserved active site and CoA binding site residues Figure 3. Structure based alignment of the SaGNAT (green) with 3 acyl-transferases and RMSD less than 1 Å . 1YR0 (orange), 1BL1 (blue), and 2JLM (magenta) are crystal structure of phosphinothricin acetyltransferase from Agrobacterium tumefaciens, Pseudomonas aeruginosa, and Acinetobacter baylyi respectively. Blue and red boxes depict conserved CoA binding and active site residues respectively, yellow box indicate residues involved in dimer formation, and unfilled boxes represent strictly conserved residues. doi:10.1371/journal.pone.0102348.g003 highlighted based on homology with other GNAT family members.

Quaternary structure of SaGNAT
SaGNAT is likely to exist as a dimer based on the crystal structure, structural similarity with homologous proteins, and elution profiles from size exclusion chromatography. In the asymmetric unit of the crystal, two SaGNAT molecules were present with a buried surface area of 1,397 Å 2 , strongly suggesting that this interaction is biologically relevant. Analysis of the inteferaces within the crystal using PISA (Proteins, Interfaces, Structures and Assemblies) also predicted this dimer configuration is likely to represent the biological unit, with other possible crystallographic contacts displaying less than 200 Å 2 of surface area. Consistent with this result, the structural homology search above confirmed that the proteins with an rmsd of less than 1 Å also exist in the same dimeric configuration. Finally, the elution profile during size exclusion chromatography supports that the protein exists as a dimer in solution (Fig. 1B). The full dimer conformation is presented in Fig. 4, and detailed interactions that mediate the dimer binding are also described. Briefly, the binding interface in comprised Ala75:Tyr28/Tyr146; Tyr30:Gln77; Arg71:Glu81; Thr140:Ala138; Thr114:Thr142/Asn143; Thr79:Val144; Thr142:Glu158; Asp160:Asn143.

Conclusion
Here, we describe the 2.15 Å structure of a GNAT family member within S. aureus. The structure confirms that the protein exhibits the core GNAT fold, and has high structural homology with phosphinothricin acetyltransferases. Consistent with this, the closest homologue identified by BLAST sequence analysis, was also a phosphinothricin acetyltransferase. Putative residues involved in acetyl-CoA and have been identified based on structural homology within the GNAT family.


s2fieldsofstudy: Biology Medicine Engineering

text:
Engineering an Endothelialized Vascular Graft: A Rational Approach to Study Design in a Non-Human Primate Model

After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs) are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon) models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data—correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.

Introduction
Vascular grafts are an essential technology for treating cardiovascular disease, the dominant cause of death in our society. An estimated 416,000 bypass grafts procedures occurred in 2009 in the United States alone [1]. Bypass grafts represent an important intervention to treat vessel blockage, with autologous saphenous vein or mammary artery tissues remaining the standard treatment for the past 30 years. Artificial materials are used when native tissues are unavailable; while these materials have the mechanical properties to withstand suturing and arterial blood forces, their compliance mismatch encourages intimal hyperplasia and leads to graft failure. Many of these materials also lack the biologic capacity, predominantly due to the lack of endothelial-dependent regulatory functions, to integrate with the surrounding vessels leading to thrombosis. At small diameters (,5 mm), tissue thickening leads to a decrease in the graft lumen resulting in occlusive failure of the graft. As reviewed elsewhere, various biologic materials are being used or developed for vascular grafts, but current clinical products still have considerable limitations in mechanical integrity, patient wait time, immune rejection, and ultimately patency [2].
Given the limited ability of an artificial graft to fully endothelialize in humans [3], a tissue-engineered graft or endothelialized biomaterial may be a more desirable implant when a native graft is unavailable. Endothelial cells tightly control vascular homeostasis. As reviewed previously, endothelial cells produce a multitude of transmembrane glycoproteins and released factors which regulate coagulation, thrombin generation, and platelet activity [4][5][6]. Of particular interest to this work are activated factor X (FXa), which converts prothrombin to thrombin, and activated protein C (APC), which leads to a downregulation of thrombin. The activation of APC is enhanced by endothelial transmembrane glycoproteins, thrombomodulin (TM) and endothelial protein C receptor (EPCR). Factor Xa results from the complex of transmembrane tissue factor (TF) with factor VIIa, while endothelial cell release of tissue factor pathway inhibitor (TFPI) inhibits both FXa and the TF-factor VIIa complex. Endothelial cells regulate platelet activation through the production of the enzyme endothelial nitric oxide synthase (eNOS) resulting in nitric oxide release and the expression of transmembrane CD39 [5]. In addition to thrombosis and hemostasis, endothelial cells are central regulators of inflammation [7,8]. Intercellular adhesion molecule-1 (ICAM), vascular cell adhesion molecule-1 (VCAM), and platelet endothelial cell adhesion molecule (PECAM) are endothelial transmembrane proteins that facilitate leukocyte adhesion and migration in inflammation. Combined, these endothelial functions cooperatively regulate the processes of hemostasis, inflammation, and intimal hyperplasia. Endothelialization of vascular grafts has been demonstrated to improve vascular graft patency by properly regulating these processes and facilitating integration with the surrounding tissue [9][10][11][12]. Biochemical or mechanical, particularly fluid shear stress, stimulation can be used to alter and improve endothelial cell phenotype. Steady, laminar fluid flow with physiological shear stress leads to endothelial cell elongation and alignment in the direction of flow, decreased cell proliferation, decreased monocyte adhesion, and downregulation of endothelial regulated immunogenicity. Alternatively, low or disturbed flow leads to a lack of cell alignment and an increase in immunogenicity [6]. The phenotype of the elongated endothelial cell improves thromboprotective function, suggesting that preconditioning an endothelialized graft may result in superior in vivo performance.
To obtain autologous endothelial cells for graft endothelialization would require an invasive harvest, while implantation of endothelial cells from allogenic or xenogenic sources would require continuous immune suppression. Given the limited supply of attainable mature vascular endothelial cells, endothelial outgrowth cells (EOCs), which are obtained from a simple blood draw and expand rapidly in culture, are an attractive autologous cell source for an engineered endothelium. This descendent of endothelial progenitor cells has seen widespread use in the past decade. Recent work has begun to characterize EOCs under various shear conditions and found similar results to the well-established shear responses seen with mature endothelial cells [13][14][15][16]. EOCs exhibited the most thromboprotective and immunoprotective phenotype after stimulation with steady fluid shear stress and the most thromboprone phenotype after stimulation with oscillatory shear stress [14]. EOCs were also responsive to tumor necrosis factor (TNF), a potent proinflammatory cytokine [13,15,17]. Stimulation with these mechanical or biochemical stimuli should elicit a large range of EOC phenotypes.
A critical limitation for all research on vascular graft development is the lack of testing modalities that translate into improved clinical function. In vivo testing in relevant animal models will be an essential final step in determining the performance of any graft, but it is neither cost-effective nor feasible for initial studies. Therefore, many researchers test a panel of coagulation or inflammation markers on their cell source and/or biomaterial of choice. As reviewed previously [18], many studies have attempted to mimic in vivo graft performance by examining the response of a single blood component (e.g., platelets or leukocytes), but these simplified models are unlikely to accurately represent the in vivo performance of the graft. Even the majority of implant models lack the strength to generate clinical translatability. Small animal models, which often employ a short (1-2cm length) end-to-end graft, spontaneously endothelialize and do not model human vascular graft responses [19].
To address these limitations we used two, well-established baboon models: an ex vivo shunt model and an in vivo end-to-side graft. The shunt model uses whole blood at physiologic arterial flow conditions to recapitulate coagulation factor transport dynamics. Furthermore, this model characterizes vascular graft thrombogenicity without anticoagulants to more accurately reflect how grafts will perform in patients who will not be given anticoagulants long-term [20][21][22][23][24][25]. The baboon aorto-iliac implant model of an end-to-side 4mm diameter vascular graft is designed to most closely relate to the clinical use of vascular grafts in humans, thereby maximizing the translatability of this research [19,26]. This model does not spontaneously endothelialize, but rather shows an ingrowth of smooth muscle cells and endothelial cells at a rate of 0.2 mm/day [26]. For both these models, we examined EOCs on a clinically-relevant graft material, expanded polytetrafluoroethylene (ePTFE). While this material did not address the compliance mismatch plaguing artificial vascular grafts, the grafts represent the current gold-standard artificial material. By performing in vitro analyses of thrombosis and inflammation in parallel with ex vivo or in vivo responses in these non-human primate models, we identified the in vitro metrics that best correlate to platelet accumulation and performance in the body. Using both biochemical and mechanical methods to alter EOC phenotype we aimed to generate correlations with broad applicability. Specifically, we tested the EOCs in vascular grafts under static culture conditions, steady and oscillatory fluid shear stress preconditioning, and TNF stimulation. These stimuli generated a large range of phenotypic responses which facilitated the development of a linear regression model correlating in vitro EOC responses to platelet or tissue ingrowth in vascular grafts. We hypothesized that 1) steady fluid shear stress would lead to a more anticoagulant and anti-inflammatory EOC phenotype in vitro, reduced platelet accumulation ex vivo, and reduced intimal hyperplasia in vivo, and 2) in vitro metrics of the EOC functions would correlate with ex vivo platelet accumulation. Identification of these metrics now provides an essential starting point for all vascular graft research to more rapidly improve clinical vascular grafts. This multifaceted research determined not only the performance of this clinicallyrelevant, cell-based therapy in vivo, but also the ability to predict in vivo and ex vivo responses based on in vitro analyses.

Experimental Design
These studies were designed to examine EOC coagulation and inflammatory markers in vitro, platelet and fibrinogen accumulation ex vivo, and endothelialized graft performance in vivo on EOC-seeded ePTFE grafts. Oscillatory or steady fluid flow preconditioning, as well as TNF treatment, was performed on EOC-seeded grafts to elicit a range of EOC phenotypes. In vitro analyses were performed on EOC-seeded grafts prepared and treated in parallel with samples used for the ex vivo shunt studies and the in vivo implant studies to determine correlations between in vitro analyses and ex vivo or in vivo performance. Samples were tested in vitro for cell coverage using fluorescent microscopy and DNA quantity, for FXa and APC production using biochemical assays, and for gene expression using qRT-PCR. In parallel, a baboon, femoral, arteriovenous shunt model, which utilizes whole blood lacking anticoagulation with radiolabeled platelets and fibrinogen, was used to quantify platelet and fibrin accumulation on the cellseeded grafts. Alternatively, intimal hyperplasia was quantified on end-to-side aorto-iliac bypasses using a blind analysis on cell-seeded grafts implanted contralaterally to untreated controls in a baboon implant. These parallel analyses of preconditioning EOC-seeded vascular grafts were performed to determine correlations resulting from in vitro metrics of EOC phenotype. Raw data can be found in the supporting files (S1 File).

EOC isolation, culture, and flow pretreatment
Late-outgrowth, baboon EOCs were derived from the mononuclear cells of peripheral blood as described previously [27]. Cell phenotype was confirmed after passage 1 by selecting for PECAM positive cells using Dynabeads (Invitrogen) according to the manufacturer's protocol. EOCs were maintained in endothelial growth media-2 (Lonza) with 10% fetal bovine serum on collagen-coated tissue culture plastic and used at passage 5 in these studies. Graft lengths of 50 or 70 mm of ePTFE with a 4 mm internal diameter (Gore-Tex, W.L. Gore and Associates, Inc.) were connected to silicone tubing, maintaining a smooth lumen. Grafts were then coated with 4 mg/mL bovine collagen I (MP Biomedicals), and seeded with EOCs in a custom biochamber, as described previously [28]. After 24hrs, the media was switched to a higher viscosity flow media to mimic blood viscosity: 15% solution of 100 g/L dextrose and 320 g/L dextran from Leuconostoc mesentreroides (Sigma, molecular weight548,000-90,000) in endothelial basal media (Lonza) with 5% fetal bovine serum, 1% Penicillin-Streptomycin, 1% Lglutamine, and 0.1% epidermal growth factor. Samples were either stimulated with 10 dyn/cm 2 steady shear flow, 0¡10 dyn/cm 2 oscillatory shear flow at 1 Hz, or kept as static cultures for an additional 24hrs.

In vitro assessments
Grafts for in vitro analyses were prepared in parallel with grafts used for in vivo or ex vivo studies. All analyses were performed on all in vitro samples.

Fluorescent staining
EOC-seeded graft segments (about 10 mm in length) were stained for F-actin with AlexaFluor 568 phalloidin and nuclear co-stained with DAPI, as described previously [28]. For PECAM staining, graft sections were incubated with mouse anti-human PECAM (Dako) overnight at 4˚C, washed in PBS with 0.1% BSA, and stained with AlexaFluor 568-tagged goat anti-mouse IgG (Invitrogen). Stained sections were mounted with ProLong Gold antifade reagent (Invitrogen) and imaged using an Olympus FV1000 confocal microscope and Fluoview software.

Total DNA
The DNA content of EOC-seeded graft segments was measured with the PicoGreen dsDNA assay according to the manufacturer's protocol (Invitrogen) and normalized by graft surface area after measurements with digital calipers.

qRT-PCR
Cells were lysed from a 25-35 mm long section of ePTFE, and RNA was isolated from the lysate using an RNeasy mini isolation kit (Qiagen). Samples were reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturers' protocols. Gene expression was quantified using Platinum SYBR Green and ROX reference dye (Invitrogen) with in-house designed primers at 250 nM concentration (S1 Table). All in vitro samples were tested for a panel of coagulation and inflammation genes, specifically, CD39, EPCR, TM, eNOS, ICAM, VCAM, PECAM, TF, and TFPI. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene and ddct values were determined by comparing treated samples to static controls. Data were transformed by subtracting 1 from any positive 2 -ddct values and taking the negative inverse of any negative 2 -ddct values and adding 1. This resulted in a continuous, linear data set and is referred to as fold change, where the static values are 0. Positive values show an increase from paired static samples, while negative values indicate a decrease from the static control.
Functional assays EOC-seeded graft segments approximately 3 mm in length were incubated with 5 nM thrombin (Haematologic Technologies) and 100 nM Protein C (Haematologic Technologies) for 60 min at 37˚C to generate APC. Refludan (Berlex) or hirudin was used to inactivate the thrombin, and a chromogenic substrate (1 mM S-2366, Chromogenix) was used to quantify APC generation by comparing it to an APC standard (Haematologic Technologies). Graft segments for FXa quantification approximately 3 mm in length were incubated with 20 nM factor VIIa (Enzyme Research Laboratories) and 200 nM factor X (Enzyme Research Laboratories) for 60 min at 37˚C. Cold EDTA (5 nM) was used to stop the reaction, and 0.67 mM Spectrozyme FXa (American Diagnostica) was used to measure FXa generation compared to a FXa standard. Absorbance at 405 nm was measured every 20 s for 20 min and the maximum slope over 10 points used to quantify APC or FXa. Data were normalized by the graft segment area.  3,1996). The ONPRC is a Category I facility. The Laboratory Animal Care and Use Program at the ONPRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC), and has an approved Assurance (#A3304-01) for the care and use of animals on file with the Office for Protection from Research Risks at the National Institutes of Health (NIH). The Division of Animal Resources (DAR) is composed of highly trained veterinarians, clinicians, and medical technologists. All nonhuman primates are housed in indoor or indoor/outdoor facilities. Nonhuman primates are routinely fed commercially prepared primate feed milled within the past 90days. This feed is supplemented daily with fruit and special diets prepared in the ONPRC diet kitchen. Fresh, potable water is provided by the municipal water district via automatic water systems. The nonhuman primates are provided with daily enrichment including (1) toys (in cage at all times and rotated every 2 weeks) and (2) devices (foraging manipulanda) on the outside of the cage, also changed every 2 weeks. The devices are filled with trail mix, grain, and/or produce at least 3 days per week. The nonhuman primates receive produce on days the devices are not filled, (3) TV/radio on a weekly rotation schedule, (4) pair housing/grooming contact (as much as possible), (5) positive reinforcement training, and (6) cognitive enrichment (computer tablets, such as Kindles). Steps are taken to ameliorate suffering according to the Weatherall Report ''The use of non-human primates in research.'' During experimentation, the animals are monitored for behavior, appetite, and stool quantity. The technician reports any abnormalities on a daily observation sheet, which is checked daily by a member of the clinical services unit and the behavioral services unit. Any notations by the technician about abnormalities with the animal are addressed by both the clinical services unit and the behavioral services unit.

Animal care and ethics
For all surgical procedures, animals were anesthetized with 1-3% isofluorane delivered at 1-2 L/min in 100% oxygen after induction by ketamine (10-20 mg/ kg intramuscularly) and Telazol (3-5 mg/kg intramuscularly). During the procedure the DAR staff monitored pulse rate, body temperature, blood pressure, ECG, oxygen saturation, and exhaled CO 2 . Adequacy of anesthesia was additionally assessed by observable spontaneous movements, and physical, circulatory, and respiratory responses to surgical stimuli. Animals received a short acting opioid pain medication 3 times/day for 3days post-surgery and a long acting pain medication once/day for 48-72hrs. If it was determined by the veterinary staff that pain medication was needed after the prescribed regime, permission is given by the research staff for it to be prescribed to the animal as needed. After surgery the nonhuman primates were monitored for visible signs of infection or fever, mobility or swelling of the lower limbs, lameness, cold toes, plate limbs, as well as bleeding from surgical sites (anemia, reduced hematocrit); however, this was not observed during these studies, and no animals were terminated prior to study endpoint. At the implant study endpoint, to remove the implanted vascular grafts and analyze the results, the animals were euthanized according to the AVMA Guidelines on Euthanasia June 2007. For euthanasia, the animals were given ketamine 20 mg/kg followed by phenobarbitol (25 mg/kg) and exsanguination.
baboon. An ex vivo femoral arteriovenous shunt loop was established in the absence of anti-coagulant and anti-platelet therapies. EOC-seeded ePTFE grafts within custom chambers were connected to the shunt loop with blood flow controlled at 100 ml/min using a clamp downstream of the graft. This flow rate corresponds to an average of 10 dyn/cm 2 wall shear stress. Platelet deposition was measured for 1 hr using a gamma camera with 5 min exposure increments (static n526, steady n511, oscillatory n59). Fibrinogen accumulation was quantified at the study endpoint after complete decay (.10 half-lives) of the indium-111 (static n524, steady n59, oscillatory n59).

Implant studies
Aorto-iliac bypass ePTFE grafts were implanted bilaterally in juvenile male baboons, weighing 12-16 kg under anesthesia described above. After a midline incision, heparin (100 units/kg) was given and the aorta occluded with atraumatic microvascular clamps. Grafts were positioned between the distal aorta and common iliac artery, at the iliac bifurcation. Static (n57) or steady flowpretreated (n54) EOC-seeded samples were implanted contralaterally to an untreated ePTFE control sample. Grafts were seeded using autologous EOCs. Anastomoses were constructed (end-to-side) using continuous 7-0 polypropylene sutures. This procedure is similar to the standard surgical placement of such grafts in humans and produces progressive proliferative lesion formation at anastomotic sites. After 4wks, grafts were pressure fixed in 10% formalin and explanted. After overnight fixation in 10% formalin, the grafts were sectioned, dehydrated, embedded in paraffin, and stained with hematoxylin and eosin using standard histological procedures. The mid-point of the each anastomoses was analyzed for tissue ingrowth on the ePTFE grafts. The area of intimal hyperplasia was measured from the edge of the anastamosed artery to a 2 mm length into the graft using Image J. Trained viewers quantified these regions while blinded to which samples were controls or treated. The area of ingrowth was averaged across each 2 mm ingrowth section of the anastomosis. Intimal hyperplasia was only analyzed for patent anastomoses. While other groups have found differences between tissue ingrowth in the proximal and distal anatomoses [29,30], the historic data with our animal model for both bare and cell-seeded end-to-side vascular grafts have shown no difference between the two anastomoses [31][32][33][34]. Therefore, data from this work were averaged across proximal and distal locations and analyzed to evaluate the hypothesis of endothelial cell flow preconditioning.

Correlations and linear regression model development
Single linear regression models were developed to correlate all in vitro data from each individual data point (FXa generation, APC generation, DNA quantity, CD39 gene expression, EPCR gene expression, TM gene expression, TF gene expression, TFPI gene expression, eNOS gene expression, ICAM gene expression, VCAM gene expression, and PECAM gene expression) to the parallel ex vivo data point (platelet accumulation at 60 min). The R 2 coefficient/value and p-value were used to examine the strength and significance of these single variable regressions. To create a predictive model of platelet accumulation, additional samples were added to challenge the data set and create a more robust model. In addition to the oscillatory flow, steady flow, and static samples described previously, a group of samples of EOC-coated grafts were stimulated with 100 U/ mL TNF (R&D Systems) for 4hrs prior to testing in vitro and ex vivo. This potent cytokine elicited a proinflammatory EOC phenotype, increasing the range of cell responses and potentially broadening the applicability of the developed regression model. When using gene data in the correlation analysis, values were converted to a linear scale using 2 -dCt . When a single factor correlation's p-value was less than 0.25, that factor was considered potentially important to develop the regression model. Factors that were strongly correlated to one another were not included in the same regression model. Models which resulted in strong correlations (R 2 .0.6) were tested using the in vitro control data collected parallel to implanted grafts to determine the applicability of the model to predict in vivo healing responses.

Statistical analysis
All data are presented as mean ¡ standard deviation. Sample numbers varied throughout this experiment, partially because static control samples were always run parallel to flow-or TNF-treated samples; however, n$3 for all treatment groups. Outlier analysis, defined as greater than 3 standard deviations away from the mean, was performed, but points were only removed when data collection indicated an error may have occurred. For example, samples were excluded if actin staining on parallel in vitro control samples indicated insufficient cell coverage on the graft. Unless otherwise stated, data were analyzed with a 1-way analysis of variance (ANOVA) to determine changes between treatment groups. Significance was indicated when p,0.05, and a Tukey's post hoc test was performed. A 2-way ANOVA was performed on the platelet accumulation data with factors of treatment and time. Data were checked for assumptions of ANOVA. Specifically, a Levene's test was performed to check for unequal variances, since sample sizes were not equal. Occasionally the data showed a slight skewedness (non-normality) in the distribution, and in these cases, a Box-Cox transformation was performed [35]. The same statistical analysis (ANOVA with a Tukey's post hoc) was run on the transformed data. Since there were no changes in the statistically-significant results, these transformations are not presented.
In the linear regression analysis, significance was determined using a main effects test for the overall regression and a 2-tailed t-test for the individual coefficients of the model. The standardized coefficients (beta) were determined in SPSS (IBM corp.) and indicate the scaled variables to compare the dominance of each of the variables compared to the others.

Hemodynamic treatments altered the phenotype of EOCs
EOC phenotype in response to no flow (static), mean arterial (steady) flow, and disturbed (oscillatory) flow was characterized. In the absence of flow, the morphology of EOCs on ePTFE vascular grafts was typical of mature endothelial cells with elongation occurring in somewhat random orientation ( Fig. 1A and  S1A Fig.), but with the majority of the alignment seen in the circumferential direction (S2 Fig.). Steady shear-treated EOCs (Fig. 1B and S1B Fig.) consistently showed elongation of the cells and alignment of the EOCs' actin cytoskeleton to the direction of fluid shear stress. Oscillatory shear-stimulated EOCs (Fig. 1C and S1C Fig.) exhibited a rounded morphology. Despite these large morphological changes, cells were retained under all testing conditions and the DNA quantity (Fig. 1D) was unchanged between all treatment types. The hemodynamic conditions altered the expression of genes related to thrombin generation, platelet attachment, and inflammation relative to no-flow controls (Table 1). Steady shear stress treatment caused a significant increase in eNOS and CD39 gene expression by the EOCs compared to the oscillatory shear stress treatment. Additionally, the hemodynamic conditions altered production of the two key components by which endothelial cells regulate thrombin generation, APC and FXa. Steady flow pretreatment of the EOCs significantly increased APC production ( Fig. 2A) compared to oscillatory shear stress pretreatment. Oscillatory shear-treated EOCs produced significantly more FXa (Fig. 2B) compared to the EOCs which remained under static conditions. Combined, these data show the prothrombotic phenotype elicited in the EOCs from oscillatory flow preconditioning.

Hemodynamic treatment of EOCs altered platelet and fibrin accumulation
To determine the effect of EOC phenotype on the antithrombotic properties of the endothelialized vascular grafts, flow preconditioned endothelialized grafts were placed in a well-established non-human primate model of vascular thrombosis. Platelet accumulation in the absence of anticoagulants was significantly higher in the oscillatory flow-stimulated sample compared to both the steady flow and static samples (Fig. 3A). Steady flow conditioning of EOCcoated samples did not alter the amount of platelet accumulation compared to the static conditioned grafts. Fibrinogen incorporation was not significantly different between the three treatment groups (Fig. 3B); however, the average amount of fibrinogen in the oscillatory treatment samples was higher compared to static and steady flow samples.

Steady flow treatment did not alter vascular healing response on ePTFE grafts
To test whether steady flow treatment of EOC endothelialized vascular grafts would improve long term vascular responses, autologous EOC-coated vascular grafts with contralateral control ePTFE grafts (without cells) were implanted in a non-human primate, end-to-side aorto-illiac bypass model. All animals survived this study; however, in one implant, all four anastomoses (control and static cellseeded) were closed at explant. Two additional anastomoses from different animals both from cell-seeded distal sections without flow pretreatment were also not patent. Representative histology images from the distal anastomoses of each treatment group are shown in Fig. 4. To account for the animal to animal  Engineering an Endothelialized Vascular Graft variation, the amount of intimal hyperplasia on the endothelialized vascular grafts was normalized to the contralateral ePTFE control graft. The flow pre-treatment of the EOCs did trend toward lower intimal hyperplasia, although the difference was not statistically different (p50.162, Fig. 4D).

Linear regression model results
To determine the potential for in vitro assessments of EOC function on vascular grafts to predict platelet accumulation, we developed a linear regression model. The linear regression analysis was performed on single in vitro variables for all individual data points against the corresponding total platelet accumulation at 60 min using the mechanically-and biochemically-modified EOC phenotypes ( Fig. 5 and S2 Table). APC generation, FXa generation, DNA per surface area, and eNOS gene expression showed significant correlations (p,0.05); however, their R 2 values were still quite low, suggesting the need for multiple factors in Engineering an Endothelialized Vascular Graft generating a regression model. Variables that resulted in highly not significant (p.0.5) correlations were ICAM, VCAM, and TF gene expression. Nineteen models, having between 2 and 4 variables, were tested based on the significance of the individual correlations when the TNF data were included. The R 2 values of these combined models ranged from 0.187 to 0.746 (S3 Table). The highest R 2 value resulted from combining the factors FXa, eNOS, and CD39 ( Fig. 6A and S4  To determine the utility of the linear regression model predicting in vivo outcomes of implanted grafts, the model was applied to the data set from the in vitro grafts constructed in parallel to the implant grafts. From the regression Fig. 3. Ex vivo platelet and fibrin data. EOC-seeded grafts in the ex vivo shunt loop had significantly more platelet deposition over 60 min when pretreated with oscillatory shear stress compared to either steady shear stress or no flow preconditioning (A) (*p50.01). Fibrinogen incorporation (B) also trended higher in oscillatory treated samples, but was not statistically different (p50.13). In samples that occluded before 60 min, platelet count at the time of occlusion was included through 60 min for the purposes of illustration. This occurred in three of the oscillatory samples. ANOVA, N$9. doi:10.1371/journal.pone.0115163.g003 Engineering an Endothelialized Vascular Graft model, the platelet accumulation was predicted and compared to the in vivo outcome of anastomotic intimal hyperplasia. The FXa/eNOS/CD39 model correlated to intimal hyperplasia measurements at 4wks after graft implant with an R 2 value of 0.626 and a significance of 0.019 (Fig. 6B).

Discussion
Endothelialization is expected to be a critical component for improving the patency of small diameter, artificial, vascular grafts. The past decade has seen a substantial increase in the interest in EOCs, and endothelial progenitor cells in general, for multiple therapeutic applications; however, considerable work is still needed to determine the applicability of these cells for vascular devices. The objectives of these studies were (1) to determine the ability of flow preconditioning to alter hemostatic regulation of EOCs in a clinically-relevant graft large animal model and (2) to correlate in vitro thrombogenic and inflammatory markers of EOCs with ex vivo and in vivo outcomes. Completing these objectives improves our understanding of EOCs, a clinically-feasible source for autologous endothelial cells, and suggests predictive metrics for vascular graft success in future implant designs.
There is a considerable body of scientific work that has examined the response of mature endothelial cells to fluid shear stress, but only a handful of recent publications have explored the response of EOCs to these same stresses. Based on previous work with endothelial cells and EOCs [13][14][15][36][37][38][39], it was expected that the application of steady flow would lead to a more anticoagulant and antiinflammatory EOC phenotype in vitro, reduced platelet accumulation ex vivo, and reduced intimal hyperplasia in vivo. When compared to oscillatory shear stress, this hypothesis was largely true. EOC gene expression of the anti-platelet molecules CD39 and eNOS were significantly increased after steady shear compared to oscillatory shear. EOCs produced significantly more APC in vitro when preconditioned with 10 dyn/cm 2 steady fluid shear stress compared to oscillatory, while oscillatory shear increased FXa production compared to static controls. Oscillatory flow preconditioning also increased platelet accumulation when compared to static or steady flow samples. These results corresponded well to previous work, which used the same conditioning on EOCs seeded on tissue culture plastic. Ankeny et al. [14] measured increases in EOC gene expression of thromboprotective genes (including eNOS and CD39) but saw no change in Engineering an Endothelialized Vascular Graft inflammatory, TF, or TFPI genes for oscillatory stimulation compared to steady shear. Oscillatory flow consistently produced a procoagulant phenotype in the EOCs, confirming EOC sensitivity to disturbed hemodynamic conditions. An unexpected observation was the lack of differences between EOCs under steady fluid shear stress compared to static conditions. There were no significant differences between steady flow and static preconditioning for APC or FXa generation. The platelet accumulation on these samples was nearly identical over 60 min, and no differences were seen in fibrinogen incorporation. Ahmann et al. [13] observed no change in ICAM or VCAM gene expression between static and steady shear stress, but most previous work observed changes in thrombosis markers with steady flow. Previous work on EOCs demonstrated an increase in TM protein [36] and gene expression [36,37,40], an increase in TFPI gene expression [36], and an increase in APC production [36,40] with steady shear stress compared to static. NO production [13], eNOS gene expression Engineering an Endothelialized Vascular Graft [13,15,36,37,39], and eNOS protein production [15] by EOCs have all been observed to increase under steady shear stress compared to no flow conditions. Platelet binding also decreased with steady flow preconditioning of EOCs compared to static samples; however, both these groups used heparized blood over a shorter period of time than the work presented here [13,39]. The TF results due to steady flow preconditioning on EOCs were not consistent with one group observing a decrease in TF gene expression [15], another observing an increase in TF gene expression and protein [40], and a third observing no significant change in TF gene expression [38]; however, as with mature endothelial cells, the application of steady fluid shear stress has generally led to a more anticoagulant phenotype in previous studies of EOCs. While the differences seen with this work could be attributed to minor differences in experimental design such as species, matrix coating, or magnitude of the fluid stress, such profound differences suggest a more fundamental change has occurred.
The largest difference between the previous work and the work presented here is likely the material on which the cells were adhered and the way this material impacted cell morphology, altering the static EOC phenotype. Most of the previous work comparing steady shear to static preconditioning was completed on flat surfaces of matrix-coated tissue culture plastic or glass [14,15,36,37]. The collagen-coated ePTFE grafts used here have a distinct topography with a fiber structure that encourages elongation of the EOCs in the circumferential direction. While EOCs seeded on a truly flat surface will exhibit a primarily cobblestone morphology in the absence of flow, the EOCs in this study were frequently observed to be elongated on the ePTFE without fluid shear stimulation. The significance of this morphological change is the corresponding phenotypic change that accompanies endothelial cell elongation. As reviewed elsewhere [41], endothelial cells that are compelled into an elongated morphology without flow exhibit many similarities to endothelial cells elongated with steady fluid shear stress. Multiple researchers have shown through a direct comparison of endothelial cells elongated with flow or with cell patterning that the extent of elongation and the orientation of the cytoskeleton are very similar between flow and patterned endothelial cells. The transcription factor krüppel-like factor 2, which was previously thought to be shear stress-dependent, has also been shown to increase in patterned endothelial cells, illustrating the importance of elongation on endothelial cell phenotype [42]. Elongation due to either patterning or flow decreased primary endothelial cell expression of both TFPI and von Willebrand factor genes [43], which likely contributes to the similarities seen here in FXa production and platelet accumulation between static and steady flow conditioned EOC samples. Additionally, previous work showed that both methods of elongation lead to a decrease in the expression of inflammation markers such as VCAM and E-selectin [42], offering a potential explanation to the lack of statistical difference in intimal hyperplasia between static and steady shear preconditioned EOC grafts when implanted in an aorto-illiac bypass graft for 1mth. The ePTFE topography contributed to the elongation of the EOCs and the resulting elongated morphology may provide a thromboprotective phenotype similar to that previously observed with steady fluid shear stress. Future work toward altering EOC phenotype through either fluid shear stress or physical cell alignment will be important toward improving antithrombotic function.
In this study, the elongation imparted on the EOCs though the ePTFE ultrastructure resulted in equivalent EOC phenotype for the no flow and steady flow preconditioned cells. However, there was a trend that steady shear stress decreased intimal hyperplasia, when normalized to the contralateral acellular control graft. Additional research should further examine the potential benefit of flow preconditioning of EOC-seeded grafts. Work that seeks to improve biological integration of vascular grafts by harnessing the regulatory function of the native endothelium should be pursued to permit small diameter graft patency. Future work should also identify methods to improve the durability of an intact endothelial layer on graft materials.
Much hope has been placed in the potential to tissue engineer a vascular graft to improve clinical outcomes at small diameters, but constructs that have been created and characterized in vitro and subsequently tested in vivo with small animal models have yet to translate into a viable graft replacement. For example, work by Stroncek et al. [29]. observed no significant change in intimal hyperplasia when EOCs were transfected with TM compared to unmodified EOCs even though their previous work saw a dramatic increase in APC production with the transfection in vitro [44]. While their work did support EOC endothelialization of vascular grafts compared to bare ePTFE grafts, it also motivates a better understanding of the cell phenotype necessary for improving in vivo outcomes. Determining the in vitro properties that correlate to positive in vivo outcomes is essential for the development of successful bioengineered grafts. Combining all of the in vitro assessments performed here in parallel to ex vivo assessments allowed for a thorough examination of the extent to which these factors directly correlate to platelet accumulation. The second hypothesis of this work was that in vitro measures of the EOC functions would correlate with ex vivo platelet accumulation. Individually, a variety of in vitro metrics of EOC-seeded vascular grafts significantly correlated to platelet accumulation: APC generation, FXa generation, DNA per surface area, and eNOS gene expression. However, none of these individual correlations was very strong. This was not surprising given the multiple regulatory pathways of coagulation, further motivating the combination of factors in a regression model. A multifactor linear regression analysis determined that a model combining FXa generation, eNOS gene expression, and CD39 gene expression correlated well to platelet data. Interestingly, FXa generation was consistently the most predominant of these factors, as was indicated in the statistical analysis by larger beta values, larger t-values, and lower p-values, but expression of genes relating to FXa generation, TF and TFPI, were not significant factors. This suggests the strength of this functional assay compared to gene expression data, which does not reflect post-translational regulation. It was surprising that many of the variables did not significantly correlate to the platelet accumulation, further emphasizing the importance of determining predictive in vitro metrics. While many researchers test inflammatory markers, most often using gene expression, the lack of correlation with ICAM and VCAM observed here suggests that inflammation-induced gene expression data may not translate well into clinical performance. This is the first work of this type, which attempts to determine predictive metrics for in vivo vascular graft performance from in vitro endothelial markers. The model was created from ex vivo platelet data; however, the model of predicted platelet attachment when applied to in vitro data from parallel implant samples correlated strongly to intimal hyperplasia. This is a very encouraging result, although not unexpected given the ability of platelets to release growth factors like TGF and PDGF, which encourage smooth muscle cell migration and proliferation-critical to the tissue ingrowth characteristic of intimal hyperplasia [45][46][47]. The strength of this correlation is intriguing and suggests the true potential of these specific in vitro tests to predict in vivo performance. However, more work is needed to confirm this potential. The study of the ability of EOCs to alter vascular responses in vivo is currently being studied with sheep EOCs on tissue engineered grafts [12,48]. Future work should examine the applicability of our regression model to other tissue engineered graft conditions, including other sources of endothelial cells, graft surfaces, or preconditioning methods. Genetic manipulations to increase single factors within a thromboprotective and thromboprone EC phenotype could identify key regulators of platelet attachment and tissue ingrowth. Having strong predictors for thrombogenicity and ultimately in vivo remodeling could drastically decrease the resources necessary to translate a tissue engineered graft into a successful clinical product.
This work presents comprehensive in vitro, ex vivo, and in vivo results on flow preconditioning of EOCs on vascular grafts using steady and oscillatory flow conditions. Additionally, this work addresses an important gap in knowledge by beginning to determine the predictive factors important in graft implant success. Gaining a better understanding of these factors should improve future work on vascular grafts for the long-term, clinical improvement of small diameter vascular graft products.


s2fieldsofstudy: Biology Medicine

text:
Effect of Dietary Ethanolic Extract of Lavandula officinalis on Serum Lipids Profile in Rats

Antioxidants are effective in prevention and treatment of cardiovascular diseases. Lavandula officinalis possesses antioxidant activity, therefore, in this study; the effects of Lavandula officinalis extract were investigated on serum lipids levels of rats. Experimental mature male Wistar rats were treated with 100, 200 or 400 mg/Kg/day of lavender ethanolic extract or distilled water for 25 days via gastric gavage (n=8 each group). At the end of 25th day, the serum cholesterol, triglyceride, HDL, LDL and VLDL levels, as well as atherogenic indices were determined in rats’ serum. The ethanolic extract of lavender decreased serum cholesterol, triglyceride, LDL and VLDL levels in 100 mg/Kg group (p=0.03, p=0.001, p=0.001, p=0.001, respectively). Serum HDL level increased in 100 mg/Kg/day group (p=0.01). Lavender extract decreased LDL/HDL level at doses of 100 and 200 mg/Kg/day (p=0.001, p=0.001, respectively). The TG/HDL levels decreased in experimental groups with doses of 100 and 200 mg/Kg/day (p=0.001, p=0.001, respectively). Lavandula officinalis extract exerts hypolipidemic effect in rats and might be beneficial in hyperlipidemic patients.

Introduction
Cardiovascular diseases are the leading causes of death in the United States of America. High levels of plasma cholesterol, especially the LDL (low density lipoprotein) is known as important risk factors for coronary heart disease (CHD). Whereas high levels of HDL reduces the risk factors of CHD (1). It is now generally accepted that oxidation of low density lipoprotein (LDL) plays a significant pathogenic role in atherosclerosis (2). Atherosclerosis is a chronic disease of large and medium sized arteries with hardening and loss of elasticity of the arterial walls and narrowing of the arterial lumen. Atherosclerosis is the principal cause of cardiovascular and cerebrovascular diseases leading to angina pectoris, myocardial infarction and ischemic stroke and is the principal cause of death in Western countries (5). Fatty acid composition of dietary influences on blood lipids and blood lipoproteins are associated with the development of atherosclerosis and ischemic heart disease (1). The process of atherogenesis has been considered by many to consist largely of the accumulation of lipids within the artery wall. High plasma concentrations of lipids, in particular those of low-density lipoprotein (LDL) cholesterol, are the principal risk factors for atherosclerosis (6). There are several groups of drugs for hyperlipidemia, however, most of them possess high adverse effects (7). Nowadays, there has been a great increase in the use of complementary treatments such as herbal remedies in the treatment of diseases (8).
was then shacked, filtered and evaporated in a rotary evaporator under reduced pressure until dryness (15).

Total phenolic compounds determination
The amount of total phenolic compounds in the Lavandula officinalis extract was determined colorimetrically with Folin-Ciocalteu reagent. Mixed with the Folin-Ciocalteu reagent (1:10 diluted with distilled water) and aqueous Na2CO3 (0.4 mL, 7.5%) was added 0.5 mL of the extract. The mixture was allowed to stand for 30 min and the total phenols were determined by spectrophotometer (Unico UV-2100, USA) at 765 nm. A standard curve was prepared using 12.5, 25, 50, 62.5, 100 and 125 mg/L solutions of gallic acid in methanol and water (60:40, v/v). Total phenol values were expressed in terms of gallic acid equivalent (mg/g), which is a common reference compound. The experiment was repeated 3 times (16).

Total flavonoid and flavonol determination
The amount of total flavonoids in the Lavandula officinalis extract was determined using the colorimetric method. Mixed with 1.5 mL of methanol (60%), 1 mL of 2% aluminum chloride, and 6 mL of 5% potassium acetate was added 1 mL of the Lavender extract or rutin. The mixture was left at room temperature for 40 min. The absorbance of the reaction mixture was then measured at 415 nm. The calibration curve was prepared using rutin solutions at concentrations of 25, 50, 100, 250 and 500 ppm in methanol. The aluminum chloride colorimetric method was employed for flavonol determination, but the incubation period was 150 min and the absorbance of the reaction mixture was determined at 440 nm. The experiments were repeated 3 times. Total flavonoids and flavonols were expressed in terms of rutin equivalent (mg/g), which is a common reference compound (17).

Measurement of the antioxidant activity by Beta-carotene-linoleate method
Beta-carotene-linoleate method was used for measuring antioxidant capacity. Two milligrams of betacarotene was solved in 2.0 mL of chloroform. 20 mg linoleic acid and 200 mg tween 40 were added to the emulsion. 40 mL of Lavandula officinalis (family: Lamiaceae, Synonyms: Lavandula officinalis Chaix ex Vill), commonly known in Iran as "stoechas", is one of these plants which has been traditionally used for different diseases (9). Phytochemical studies revealed that linalool, linalyl acetate and some other monoterpene and sesquiterpenes, flavonoids like luteolin, triterpenoids like ursolic acid and coumarins like umbelliferone and coumarin were the main components of the aerial parts and flowers of Lavandula officinalis which might be effective on serum lipids levels (10). Following oral administration of this plant extract at the doses of 500, 1000, 1500, 2000, 3000, and 5000 mg/Kg no toxicity and no significant changes in the body weight have been reported in control and treated groups (11). Treatment with Lavender oil significantly decreased neurological deficit scores, infarct size, the levels of Malondialdehyde (MDA), carbonyl and reactive oxygen species (ROS), and attenuated neuronal damage, upregulated superoxide dismutase (SOD), catalase (CAT), GSH-Px activities and GSH/GSSG ratio. It has also shown to have neuroprotective activity against cerebral ischemia/reperfusion injury which might be attributed to its antioxidant effects (12). This plant possesses high levels of polyphenol compounds having antioxidant properties. Antioxidants are effective in prevention and treatment of cardiovascular diseases, particularly, atherosclerosis (13).
Therefore, this study was aimed to determine the composition of polyphenols, phenols and antioxidant properties of Lavender extract, other than evaluating its effect on blood lipids profile.

Plant material and preparation of extracts
The plant was purchased from a local provider in Shahrekord, and authenticated in Medical Plants Research Center, Shahrekord University of Medical Sciences. A voucher specimen was deposited in Herbarium unit of this university (No. 421). For preparation of ethanolic extract, air-dried and powdered flowering branches of the plant were macerated with ethanol (96%, Ghadir Industries, Iran) for 48 h. The macerated powder water saturated with oxygen was added to the above materials. Lavandula officinalis extract was prepared at a concentration of 2.0 milligrams per liter in pure ethanol. 4 mL of the prepared solution was added to 2 mL of the prepared extract and control (ethanol). Antioxidant activity of the extract based on Beta-carotene bleaching rate at a wavelength of 470 nm for a period of 180 minutes (15 min intervals) was calculated using the following formula. The mean of obtained values was considered as the percent of antioxidant activity of the extract (18).
AA: antioxidant activity A 0 : sample absorbance at time zero A t : control absorbance at different times during 180 minutes A0 0 : sample absorbance at time zero A0 t : control absorbance at different times during 180 minutes Animals All experimental animal procedures were conducted with the approval of the Ethics Committee of Shahrekord University of Medical Science, Iran. Male Wister rats (body weight 200-250 g) were housed under conditions of controlled temperature (22 ± 2 • C) and constant humidity, with 12 h light/dark cycle (light on 07:00-19:00). Food and water were available ad libitum. The rats were divided into 4 groups (8 animals per group). Control group received gastric gavage of distilled water and three treatment groups received Lavender ethanolic extract with doses of 100, 200, 400 mg/Kg/day for 25 days. On the 25 th day of the experiment the blood lipid profile was determined. Doses were chosen based on previous studies (11).
Lipid profiles At the end of 25 th day, all animals were anesthetized with chloral hydrate (400 mg/ Kg i.p.) and a venous blood sample was drawn and centrifuged at 7000 g for 10 min. The supernatant (serum) was frozen at −20 • C until assay for HDL, LDL, VLDL, triglyceride, and cholesterol content (Pars Azmun, Iran according to manufacturer›s instructions) using autoanalyzer (Liasys, Roma, Italy) (19).
Statistical analysis Cholesterol, triglyceride, LDL, HDL, and VLDL were analyzed using one-way ANOVA test (post hoc LSD). Data were expressed as means ± SEM. P < 0.05 was considered significant.

Analysis of Lavandula officinalis extract
The amount of phenolic compounds was 79.1 mg/g of dry extract equivalent to gallic acid. The amounts of flavonol and flavonoid compounds were 45 mg/g and 21.8 mg/g dry extract, equivalent to Rutin. The antioxidant activity of Lavender extract was 40% as compared to Betacaroten.

Effects of ethanolic extract of Lavandula officinalis on serum lipids profile
Serum cholesterol level was lower in the 100 and 200 mg/Kg/day experimental groups when compared with control group (p =0.03, p = 0.007, respectively). Lavandula officinalis extract significantly decreased serum triglyceride level in the doses of 100, 200 and 400 mg/Kg/ day groups when compared with control group (p=0.001, p=0.001, p=0.01, respectively). Serum HDL level was similar in the control and 200, 400 mg/Kg extract groups while higher in 100 mg/Kg group (p=0.01). The serum LDL level was reduced in the 100, 200 and 400 mg/Kg/ day extract groups when compared with control group (p=0.001, p=0.001, p=0.007, respectively). Lavandula officinalis extract with doses of 100, 200 and 400 mg/Kg/day significantly decreased serum VLDL level when compared with control group (p=0.001, p=0.001, p=0.01, respectively). Lavender extract decreased LDL/HDL level at doses of 100 and 200 mg/Kg/day (p=0.001, p=0.001, respectively). The TG/HDL level decreased in experimental groups with doses of 100 and 200 mg/Kg/day (p=0.001, p=0.000, respectively) (Figures 1 and 2).

Discussion
In our study lavender extract decreased the serum cholesterol, triglycereide, LDL and VLDL levels in experimental groups. Also Lavender extract improved the LDL/HDL ratio. Lavender extract decreased the LDL/HDL and TG/HDL in experimental groups. Lavender extract increased serum HDL level in dose of 100 mg/Kg/day. It has been identified that high plasma LDL levels are major risk factor for vascular diseases (VD), whereas high levels of HDL are considered a negative risk factor for VD (20). The concentration, the size and the chemical modification of LDL are important for atherogenesis. For more than two decades, it has been known that lipid oxidation plays a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways (21). Flavonoids are polyphenolic antioxidants naturally present in vegetables, fruits, and beverages. Flavonoid intake is inversely associated with mortality from coronary heart disease and show an inverse relation with incidence of myocardial infarction (22). Antioxidants such as vitamin E can also reduce free-radical formation by modified LDL (22). Our study indicated that ethanolic extract of Lavandula officinalis had high antioxidant property and may reduce free radical formation by decreasing the serum LDL levels. Flavonoids, a class of water-soluble antioxidants, are useful in reducing the risk of coronary heart disease. Flavonoids inhibit oxidation of low-density lipoprotein and reduce thrombotic tendency (22). Total extracts from traditional natural plants, rich in phenolic compounds, have been shown to inhibit the development of atherosclerosis in animal models (23). However, data from several studies suggest that water-soluble antioxidants, such as vitamin C, are effective in inhibiting LDL oxidation via the preservation of endogenous antioxidants in LDL (24). Lavender extract may reduce endothelial damage and the severity of atherosclerosis. Oxidative stress via increased production of ROS and LDL oxidation induces inflammatory response (25). Our result showed that Lavender plant had high levels of polyphenols such as phenols, flavonoids and flavonols and high antioxidant capacity that may inhibit LDL oxidation.
The essential oil of lavender plant had potent anti-inflammatory activity against carrageenan in mouse (10). Previous studies showed that lavender oil (100 mg/Kg/day for 5 days) significantly reduced thrombotic events without inducing prohemorrhagic complications at variance with acetylsalicylic acid used as reference drug in mice (26).
In our study lavender extract decreased the serum cholesterol, triglycereide, LDL and VLDL levels in experimental groups. This is also an effective way to increase the ability to slow down the lipid peroxidation process and to enhance antioxidant enzymes activity (12). Flavonoids, which are mostly hydrophilic and do not bind LDL molecule, either scavenge free radicals or act as chelating agents, or protect Vitamin E, β-carotene and lycopene in the LDL particle or preserve serum paraoxonase activity, that itself promotes hydrolysis of lipid peroxides. Several in-vitro studies strongly suggest that phenolics protect LDL from oxidation. Flavonoids, significantly increased LDL lag time and significantly decreased the LDL oxidation rate in healthy adults (27). Evidence linking dietary antioxidants to atherosclerosis in humans is still circumstantial and although in some studies the association of antioxidant intake and low risk for atherosclerosis is perceptible, in others this association cannot be established.
In the present study, the higher dose of the Lavender extract had lower effect. The exact mechanism is not clear. However, although initial studies suggested that antioxidant supplements might promote health, however, large clinical trials with a limited number of antioxidants detected that excess supplementation with certain putative antioxidants may be harmful (28). Antioxidants in fruits and vegetable, work as a continuous chain to combat the (3) (4) damaging effects of oxidative stress. However, if an antioxidant is not restored by the following antioxidant in the chain, it may become a prooxidant. In this situation, the final effect of such supplementations would be no effect or a damaging effect. Furthermore, sometimes high levels of antioxidants change to prooxidants and act opposite of antioxidants (29,30), which might explain the low effect of higher dose of Lavender in this study.
It should be noted that in the present study, the effect of lavender extract on serum lipids levels in rats with hyperlipidemia was not measured and could be the next target. Also further phytochemical and biological tests are suggested to determine the active chemical constituent responsible for these activities.  (29)