Alessandra Bitto

Letteria Minutoli

Maria Rosaria Galeano

Domenica Altavilla

Francesca Polito

Tiziana Fiumara

Margherita Calò

Patrizia Lo Cascio

Lorena Zentilin

Mauro Giacca

Francesco Squadrito
email: francesco.squadrito@unime.it

Prof F Squadrito

Angiopoietin-1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression

Keywords: Ang-1, rAAV, diabetes, wound healing, eNOS, angiogenesis, VEGF, VEGFR-2, PECAM-1

INTRODUCTION

Angiogenesis is a multistep process that involves endothelial cell (EC) sprouting from the parent vessel, followed by migration, proliferation, alignment, tube formation, and anastomosis to other vessels [START_REF] Conway | Molecular mechanisms of blood vessel growth[END_REF]. Only under certain physiological and pathological situations, active angiogenesis occurs in the adult life. This process is associated with expression of cytokines, as well as angiogenic factors, such as VEGF [START_REF] Ferrara | The biology of VEGF and its receptors[END_REF]. Members of the VEGF family, including VEGF (also known as VEGF-A), VEGF-B, and placental growth factor, play important roles in angiogenesis. The effects of VEGF are mediated via receptors termed Flt-1 or VEGF-R1 and KDR/Flk-1 or VEGFR-2 [START_REF] Tammela | The biology of vascular endothelial growth factors[END_REF]. VEGF levels are increased during hypoxic conditions, making VEGF-driven angiogenesis a central response to low oxygen tension.

Insufficient angiogenesis is involved in the impaired healing of diabetic skin ulcers [START_REF] Goodson | Studies of wound healing in experimental diabetes[END_REF][START_REF] Bohlen | Adult microvascular disturbances as a result of juvenile-onset diabetes in db/db mice[END_REF]. Indeed, healing impairment in diabetics is characterized by delayed cellular infiltration and granulation tissue formation, decreased collagen organization and, of course, reduced angiogenesis [START_REF] Goodson | Studies of wound healing in experimental diabetes[END_REF][START_REF] Bohlen | Adult microvascular disturbances as a result of juvenile-onset diabetes in db/db mice[END_REF]. As far as angiogenesis is concerned a defect in VEGF regulation, characterized by an altered expression pattern of VEGF mRNA during skin repair has been shown in db/db mice [START_REF] Altavilla | Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse[END_REF]. In addition to confirm the pivotal role of VEGF in skin repair abnormalities of diabetic animals, it has been reported that adeno-associated viral vector (AAV) mediated human VEGF165 gene transfer, stimulates angiogenesis and wound healing in the genetically diabetic mice [START_REF] Galeano | Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse[END_REF].

Besides angiogenesis, vasculogenesis may also play a crucial role in the healing process.

Vasculogenesis, the in situ differentiation of the primitive endothelial progenitors known as angioblasts into endothelial cells that aggregate into a primary capillary plexus, has been shown to be responsible for the development of the vascular system during embryogenesis [START_REF] Peichev | Expression of VEGFR-2 and AC133 by circulating human CD34 + cells identifies a population of functional endothelial precursors[END_REF]. However, vasculogenesis is also present in adults and occurs through the action of circulating or resident bone marrow-derived cells called endothelial progenitor cells (EPCs) [START_REF] Asahara | Isolation of putative progenitor endothelial cells for angiogenesis[END_REF]. Further cell-lineages not bone-marrow derived may be found at different sites and have been demonstrated to differentiate into endothelial cells under hypoxic conditions or during physiologic replenishment of skin and gut [START_REF] Urbich | Endothelial progenitor cells functional characterization[END_REF]. Moreover vasculogenesis appears more present and effective when angiogenesis is failing: this is the case of the healing of diabetic ulcers in which there is S t a g e 2 ( a ) P O S T -P R I N T an impairment of haemostasis, inflammation, matrix deposition and most of all angiogenesis [START_REF] Wetzler | Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair[END_REF].

Angiopoietins are known to work in concert with VEGF in vascular growth, development and maintenance as well as in the regulation of microvascular permeability [12]. Angiopoietin-1 has been also proposed to stimulate angiogenesis via the activation of Akt signalling pathway and finally stimulates endothelial Nitric Oxide Synthase (eNOS) [START_REF] Babaei | Angiogenic actions of angiopoietin-1 require endotheliumderived nitric oxide[END_REF]. This mechanism could be extremely important in the repair of wounds especially during impaired wound healing which may be a consequence of metabolic derangement such as diabetes [START_REF] Stadelmann | Impediments to wound healing[END_REF].

MATERIALS AND METHODS rAAV vector preparation and characterization.

Two recombinant AAV vectors were obtained in this study, expressing the Lac-Z reporter gene (control gene) and the cDNA for mouse Angiopoietin-1 (Ang-1) under the control of the strong and constitutive cytomegalovirus (CMV) immediate early promoter. Both constructs were based on plasmid pFU-5, kindly provided by N.

Muzyczka (University of Florida, Gainesville, Fla.,USA) [START_REF] Zolotukhin | A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells[END_REF]. pAAV-LacZ was obtained by substituting the GFP open reading frame with the Lac-Z gene from plasmid pCH110 (Pharmacia, Uppsala, Sweden). The cDNA for Ang-1 was obtained by RT-PCR amplification of total RNA with appropriate primer pairs and again cloned to replace GFP in the pFU-5 vector digested with Bam HI and Eco RI. Infectious vector stocks were generated in 293 cells, cultured in 150-mm-diameter Petri dishes, by cotransfecting each plate with 15 µg of each vector plasmid, together with 45 µg of the packaging/helper plasmid, pDG (kindly provided by J.A. Kleinschmidt) expressing AAV and adenovirus helper functions [START_REF] Grimm | Novel tools for production and purification of recombinant adenoassociated virus vectors[END_REF]. Twelve hours after transfection, the medium was replaced by fresh medium and 3 days later the medium was collected and the cells harvested by scraping. After three freeze-thaw cycles in dry ice/ethanol bath and 37°C water bath, cell lysates were fractionated using ammonium sulphate precipitation. rAAV particles were then purified by CsCl gradient centrifugation in a SW41Ti rotor at 288,000×g for 36 h. Twelve to sixteen fractions of ten drops each were collected by inserting a G-21 needle below the rAAV band and their refractive index was determined.

The six fractions with index closest to 1.3715 (corresponding to a density of 1.40 g/cm3) were dialiyzed against phosphate buffered saline (PBS) at 4°C overnight and stored at -80°C. rAAV titres were determined by measuring the copy number of viral genomes in pooled, dialyzed gradient fractions. This was achieved by a competitive PCR procedure [START_REF] Diviacco | A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates[END_REF] using primers and competitors mapping in the CMV promoter region common to all vectors. The purified viral preparations used for this work had particle titres of about 1×10 12 viral genomes per ml. The infectious titre in this preparation is usually 100 to 1000 times less, depending on either the type of viral preparation and the target tissue. Each animals received the same dose of rAAV as stated in the experimental plan.

Animals

All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD). Female genetically diabetic C57BL/KsJ Lep db (hereinafter db + /db + ) mice and their normoglycaemic littermates C57BL (hereinafter db +/+ m) were produced by the Jackson Laboratories and obtained from Charles River Laboratories (Pordenone, Italy). Animals were 14 weeks old at the start of the experiments. Diabetic mice were obese and severely insulin resistant as evidenced by hyperglycemia (plasma glucose 560 +/-70 vs. 143 +/-8 mg/dl in db +/+ m, p<0.05) and hyperinsulinemia (plasma insulin 14.2 +/-0.48 vs. 1.43 +/-0.05 ng/ml in db +/+ m, p<0.05) compared to nondiabetic animals. Diabetic mice weighed 30-32 g, whereas nondiabetic littermates weighed 22-24 g. The animals were housed one per cage maintained under controlled environmental conditions (12-h light/dark cycle, temperature 23°C), and provided with standard laboratory food and water ad libitum.

Experimental protocol

After general anesthesia with thiopental sodium (80 mg/kg/i.p.), the hair on the back was shaved and the skin washed with povidone-iodine solution and wiped with sterile water. Two full-thickness longitudinal incisions (4 cm) were made on the dorsum of the mice, and the wound edges were closed with 4-0 silk surgical suture placed at 1-cm intervals. 10 animals for each group were killed after 7 and 14 days, respectively, and the wounds were divided into three segments (0.8 cm wide and 1.2 cm long). The caudal and cranial strip were used for molecular analysis and wound breaking strength, whereas the central one was used for histological and immunohistochemical evaluations. The animals were randomized to receive a single dose of either rAAV encoding for Ang-1 ( 100µl; 1x10 11 viral particles) or rAAV encoding for Lac-Z ( 100µl; 1x10 11 viral particles). Both rAAV were administered in the wound edges immediately after incision through 8 intra-dermal injections with a total volume of 100µl, thus infecting the 100% of wound area.

Real Time PCR for ANG-1 and VEGF mRNA

Total RNA was extracted from skin by using a commercial kit (Trizol Reagent, Invitrogen, USA) according to manufacturer's instruction and spectrophotometrically quantified (Biophotometer, Eppendorf, USA). Total RNA was then treated with DNaseI Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

to digest residual DNA contamination, subsequently 5µg of total RNA was reverse transcripted using High capacity cDNA Archive Kit (Applied Biosystems, USA) and random primers according to manufacture's instruction. 10 ng of total cDNA was used to quantitate the amount of Ang-1 and VEGF-cDNA by Real Time Polimerase Chain Reaction method (Real-Time PCR), as well as β-actin cDNA as endogenous control.

Both reactions were carried out in the same microwell (biplex) with Taq Man Universal PCR master mix and Assays on Demand ready to use primers and probes with different reporter dye (Applied Biosystems).

The progression of PCRs was monitored by 7300 Real Time-PCR System (Applied Biosystems), and the relative quantitation was determined by standard curve method for both target and endogenous reference. After normalization, the median of Ang-1 and VEGF levels in normal skin from control mice was considered as calibrator (1x sample) and the results were expressed as an n-fold difference relative to normal controls (relative expression levels).

The skin from diabetic and non diabetic mice not subjected to rAAV injection were also tested.

Determination of Angiopoietin-1, VEGFR1, VEGFR2 and eNOS by Western blot analysis

Briefly, skin samples were homogenized in 1 ml lysis buffer (20mM Hepes pH7.6, 50mM β-glycerophosphate, 1.0 mM DTT, 2.0 mM EGTA, 1% Triton, 10% glycerol) added with proteases and phosphatases inhibitors (2mM phenyl methyl sulfonyl fluoride, 1mg/ml aprotinin, 2mg/ml pepstatin A, 5mg/ml leupeptin and 100mM Na 3 VO 4 ) with a Ultra Turrax (IKA, Staufen, Germany) homogenizer. The homogenate was subjected to centrifugation at 15.000 g for 15 min at 4°C. The supernatant was collected and used for protein determination using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA, USA).

Protein samples (30 µg) were denatured in Laemmli sample buffer (Bio-Rad) with 5% β-mercaptoethanol and separated by electrophoresis on an SDS (12%) polyacrylamide gel. The separated proteins were transferred on to a nitrocellulose membrane by using the transfer buffer (39 mM glycine, 48 mM Tris pH 8.3, 20% methanol) at 200 mA for 1 h. The membranes were stained with Ponceau' S (0.005% in 1% acetic acid) to confirm equal amounts of protein and were blocked with 5% non fat dry milk in TBS- Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society 0.1% Tween for 1h at room temperature, washed three times for 10 min each in TBS-0.1% Tween, and incubated with a primary antibody for Ang-1, VEGFR1, VEGFR2 and for the constitutive and phosphorilated form of eNOS (Ser1177) (Chemicon, Temecula, CA, USA and Cell Signaling, Danvers, MA, USA) in TBS-0.1% Tween overnight at 4 o C. After being washed three times for 10 min each in TBS-0.1% Tween, the membranes were incubated with a specific secondary antibody peroxidaseconjugated (Pierce, UK) for 1 h at room temperature. After washing, the membranes were analyzed by the enhanced chemiluminescence system according to the manufacture's protocol (Amersham, NJ, USA). The protein signal was quantified by scanning densitometry using a bio-image analysis system (Bio-Profil, Celbio, Milan, Italy). The results from each experimental group were expressed as relative integrated intensity compared with normal skin measured with the same batch. Equal loading of protein was assessed on stripped blots by immunodetection of β-actin with a rabbit monoclonal antibody (Cell Signaling) diluted 1:500 and peroxidase-conjugated goat anti-rabbit immunoglobulin G (Pierce, UK) diluted 1:15000. All antibodies are purified by protein A and peptide affinity chromatography.

Determination of VEGF in wounds

The amount of VEGF in wounds was determined by an enzyme-linked immunoasorbent assay (ELISA). Briefly tissues were homogenized in 1.0 ml of 1 x phosphate-buffered saline containing complete protease inhibitor cocktail (Boehring Mannhein Indianapolis. IN). Homogenates were centrifuged to remove debris and were filtered through a 1.2-µm pore syringe filter. Analysis was performed with a commercially available VEGF-specific ELISA kit (R&D Systems). The amount of VEGF was expressed as picograms per wound.

Determination of NO

Immunohistochemistry

Paraffin-embedded tissues were sectioned (5 µm), and antigen retrieval was performed using 0,05M sodium citrate buffer. Tissues were treated with primary antibody against PECAM-1 (Santa Cruz, USA), VEGFR-2 (Cell Signaling) and VEGFR-1 (Abcam, UK). To assess angiogenic response, microvessel density (MVD) was estimated after PECAM-1 staining. Briefly, five areas per section were randomly selected in the dermis just proximal to the wound site and the positive endothelial cells were counted per highpower field (x40 magnification) by three pathologists blinded to the samples. To assess positive VEGFR-1 and VEGFR-2 staining five areas per section were randomly selected in the dermis and the positive endothelial cells were counted per high-power field (x40 magnification) by three pathologists blinded to the samples.

In each reaction a positive control of human angiosarcoma was used. For negative controls, the primary antibodies were replaced by citrate buffer (pH 6.0).

Breaking strength.

The maximum load (breaking strength) tolerated by wounds was measured blindly on coded samples using a calibrated tensometer (Instron Corp, Canton, Mass., USA) as described previously [START_REF] Pierce | In vivo incisional wound healing augmented by plateletderived growth factor and recombinant c-cis gene homodimeric proteins[END_REF]. The ends of the skin strip were pulled at a constant speed (20 cm/min), and breaking strength was expressed as the mean maximum level of tensile strength (g/mm) before separation of wounds.

In vivo vascular permeability

In vivo vascular permeability was evaluated in an additional set of animals. Briefly, Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

nm was measured, and the extracted Evans blue content was then calculated from its calibration curve.

Statistical Analysis

All data are expressed as the mean plus or minus the standard deviation (mean ± SD).

Before analysis all end points were tested for normality by using the Kolmogorov-Smirnov test. The test suggested that data were normally distributed therefore they were analysed using a two-way ANOVA.

In all cases, a probability error of less than 0.05 was selected as criterion for statistical significance.

RESULTS

Efficacy of Ang-1 gene transfer in wounds.

Angiopoietin-1 is normally expressed in the skin during adult life and is not affected by diabetes. The efficacy of rAAV mediated gene transfer was assessed by either Real-Time PCR and western blot analysis at day 7 and 14 after wounding. The unwounded skin from diabetic and non diabetic mice not subjected to rAAV injection, showed levels of Ang-1 mRNA comparable to those obtained from rAAV-Lac-Z injected animals (diabetic= 5.5 ± 1 n-fold vs β-actin; non diabetic= 5.1 ± 0.8 n-fold vs β-actin).

At day 7 after injury the expression of Ang-1 mRNA was slightly increased in the wounds of diabetic animals administered with (5.7 ± 0.9 n-fold vs β-actin); while Ang-1 gene transfer significantly augmented Ang-1 mRNA expression at each time point (day 7= 11.4 ± 1.7 n-fold vs β-actin; p<0.001 vs rAAV for Lac-Z), (Figure 1). Similar results

were obtained in non diabetic animals (Figure 1).

The enhanced mRNA expression for the angiogenic factor resulted in an increased protein expression 7 and 14 days after wounding, as confirmed by western blot analysis, in either normoglycaemic (11.5 ± 1.1 relative units of integrated intensity; p<0.001 vs rAAV-Lac-Z at day 7) and diabetic mice (12.4 ± 1 relative units of integrated intensity; p<0.001 vs rAAV Lac-Z at day 7) administered with rAAV-Ang-1 at each time point (Figure 2).

Expression of eNOS in wounds

Uninjured skin had a very low content of p-eNOS (1.1±0.9 and 0.5±0.08 integrated intensity, in normoglycaemic and diabetic animals respectively). Wounding caused at day 7 an enhanced expression of p-eNOS in both strain of animals (normoglycaemic animals = 7.1 ± 1.6 integrated intensity; diabetic animals = 4 ± 1.7 integrated intensity;

Figure 3A). In wounds treated with rAAV-Ang-1 we found an higher increase in p-eNOS in both normoglycaemic (13.1 ± 1.5 integrated intensity) and diabetic animals (10.2 ± 1.4 integrated intensity; Figure 3A).

NO products in wounds

We determined the concentration of Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society tissue). We observed a stronger increase in wound NO products in mice subjected to the Ang-1 rAAV-mediated gene transfer (Figure 3B).

Histological assessment.

Delivery of Ang-1 in the wound edge produced a significant improvement in the healing process as shown by the hematoxylin-eosin staining and by Masson's trichrome staining for the assessment of epidermal regeneration and the thickness of granulation tissue (Figure 4 At day 14 wounds of diabetic animals administered with rAAV-Lac-Z showed a still incomplete reepithelialization, the presence of a crust, a very low organized granulation tissue and a marked adipose substitution in the dermis. By contrast, at the same time point rAAV-Ang-1-treated diabetic wounds demonstrated a complete reepithelialization, well-organized dermal layers, well-formed granulation tissue, reduced adipose substitution and mature collagen bundles (Figure 4 and5).

Non diabetic animals injected with rAAV-Lac-Z showed almost complete reepithelialization at day 14, the administration of rAAV-Ang-1 resulted in an improved reepithelialization process as demonstrated by the presence of hair follicles in the dermis (Figure 4 and5).

Evaluation of new blood vessel formation

PECAM-1 immunostaining was investigated 14 days after surgical procedures to confirm neo-vessel formation (Figure 6). In fact, it represents a highly specific marker for endothelial cells. As reported in the figure the positive staining in the granulation tissue of diabetic rAAV-Ang-1 injected animals was mostly expressed by small vessels and capillaries, while in those receiving rAAV-Lac-Z reactivity was found only in preexisting vessels, demonstrating an augmented angiogenesis mediated by Ang-1 administration. Using the images obtained by PECAM-1 staining the tissue samples from each group were assessed for microvessel density as reported in figure 6.

S t a g

Breaking strength.

Angiopoietin-1 gene transfer improved the tensile strength of the injured tissue as shown in Figure 7 which summarizes the wound breaking strength values obtained for each group at day 14. The breaking strength of incisional diabetic wounds of animals treated with recombinant AAV vector encoding Ang-1 was higher than that of diabetic wounds obtained from mice given with rAAV-LacZ. Similar results were observed in wounds obtained from non-diabetic animals (Figure 7).

VEGF mRNA and protein expression

To understand the basis of the angiogenic process we investigated the expression of VEGF in wounds by Real Time PCR at day 7 and 14 after injury. In fact diabetic animals show a distorted VEGF pattern which is responsible of an inadequate angiogenetic process. In the wounds of diabetic mice administered with rAAV-Lac-Z, VEGF mRNA was markedly lower than in normoglycaemic animals at each time point (p<0.001 vs normoglycaemic animals). Treatment with rAAV-Ang-1 did not modified the disturbed VEGF mRNA expression in diabetic mice at day 7 and 14 (p not significant vs rAAV-Lac-Z), (Figure 8A).

Non-diabetic rAAV-Lac-Z treated mice showed a normally marked induction of VEGF mRNA at day 7 that declined to normal values following 14 day after injury and this expression was not affected by Ang-1 gene transfer (p not significant vs rAAV-Lac-Z)

suggesting that Angiopoietin-1 is not able to induce VEGF expression (Figure 8A).

To confirm this result, we also investigated VEGF protein by ELISA. Overlapping data were obtained (Figure 8B). Administration of rAAV-Ang-1 did not modified the disturbed VEGF protein production in diabetic mice at day 7 and 14 (p not significant vs rAAV-Lac-Z), (Figure 8B). In addition rAAV-Lac-Z treated mice showed a normally marked VEGF protein expression at day 7 that declined to normal values following 14 day after injury and this expression was not affected by Ang-1 gene transfer (p not significant vs rAAV-Lac-Z) confirming that Angiopoietin-1 is not able to induce VEGF expression (Figure 8B).

Assessment of VEGFR-2 immunostaining and protein expression.

At day 7 we performed the immunostaining for VEGFR-2, a marker that identifies endothelial precursor cells [START_REF] Urbich | Endothelial progenitor cells functional characterization[END_REF][START_REF] Asahara | Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[END_REF]; these cells are not only circulating and bone marrow-derived, but they are also present in the subcutaneous adipose tissue. Ang-1 rAAV-mediated gene transfer augmented VEGFR-2 expression in endothelial cells in diabetic animals (Figure 9), thus contributing to the healing process. Similar results

were obtained in normoglycaemic animals (results not shown). In contrast a very slight staining for the VEGFR-1 receptor, that is mostly expressed by mature endothelial cells, was observed in both strains of mice administered with rAAV-Ang-1, further suggesting that VEGF is not involved in Angiopoietin-1 driven angiogenesis. However both Angiopoietin-1 and VEGF are able to recruit endothelial precursors [START_REF] Urbich | Endothelial progenitor cells functional characterization[END_REF][START_REF] Asahara | Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[END_REF].

To confirm this result we quantified VEGR receptors protein expression by Western Blot analysis in skin wounds. A low expression of VEGFR-1 protein was present in normoglycaemic and diabetic animals injected with rAAV-Ang-1 (Figure 10A). By contrast Ang-1 rAAV-mediated gene transfer enhanced VEGFR-2 protein (Figure 10A).

In vivo vascular permeability

At day 14 we evaluated in vivo vascular permeability by administering compound 48/80 which causes Evans blue leakage. As shown in Figure 10B

DISCUSSION

Blood vessels form through two distinct processes, vasculogenesis and angiogenesis [START_REF] Patan | Vasculogenesis and angiogenesis[END_REF]. In vasculogenesis, endothelial cells differentiate de novo from mesodermal precursors, whereas in angiogenesis new vessels are generated from pre-existing ones.

Angiogenesis and neovascularization can be unwanted processes in certain diseases, including cancer and diabetic retinopathy. However, formation of the new blood vessels can also help alleviate some states, as in the formation of collateral circulation in ischemic myocardium, limbs as well as during the healing of wounds.

VEGF and Ang-1 have strong effect on angiogenesis and vasculogenesis respectively.

VEGF specific activities include endothelial cell survival, proliferation, migration and tube formation; while Ang-1 has no proliferative effect, but it is a strong inducer of vasculogenesis recruiting and differentiating mesodermal resident precursors as well as bone marrow-derived EPCs. These cells present the surface marker VEGFR-2 (or flk-1) specific for the committed as well as for the young endothelial cells and, driven by growth factors, migrate from the lateral plate mesoderm toward the dorsal aorta to the site of neovasculogenesis [START_REF] Asahara | Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[END_REF][START_REF] Patan | Vasculogenesis and angiogenesis[END_REF][START_REF] Visconti | Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF)[END_REF][23].

In our experiment the rAAV mediated gene transfer of Ang-1 improved the altered healing process, enhanced the impaired angiogenesis and increased wound breaking strength in diabetic animals. However Ang-1 gene transfer did not modify the disturbed pattern of VEGF expression. In contrast Ang-1 stimulated the expression of VEGFR-2 in new vessels close to the wound site in genetically diabetic mice.

In diabetes the need of neovascularization arises from the inadequate VEGF production and release in wounds: thus, the classic angiogenetic process is extremely delayed and poor and, as a consequence, therapeutically valuable approaches have been addressed to the aim of stimulating the expression of the impaired angiopoietic factor [24]. However our results clearly suggest that active angiogenesis may also occur in diabetes without the involvement and intervention of VEGF. Under these specific circumstances, vasculogenesis may represents an alternative pathway to induce new blood vessel formation. More specifically our data strongly suggest that Ang-1 causes, at least in diabetes, angiogenesis in a VEGF independent manner and likely through the endothelial progenitor cells. The enhanced PECAM-1 staining in endothelial cell at the wound site confirms an active angiogenesis. It could be hypothesized that the slightly positive staining for VEGFR-1 is reliant to the low but still present VEGF protein presence in the skin of diabetic animals after the surgical injury. On the other hand the marked staining for VEGFR-2 after Ang-1 administration could be related to the presence of endothelial precursors, that usually present this marker [START_REF] Ferrara | The biology of VEGF and its receptors[END_REF][START_REF] Urbich | Endothelial progenitor cells functional characterization[END_REF].

To date, it is well established that NO substantially regulates wound matrix deposition

[25], as inhibition of wound NO synthesis was paralleled by decreased wound collagen accumulation and wound breaking strength. Interestingly we found active eNOS protein to be low expressed at the wound site in diabetes impaired healing in db/db mice. This observation clearly suggests that in the diabetic wound healing process there is also a defect in nitric oxide pathway that might also contribute to impair the healing process. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250

2 - /NO 3 -- 2 -

 232 Wound samples were frozen in liquid nitrogen until use. NO products, were determined in wound lysates using the Griess reaction. Skin wounds samples were homogenized in 2 x lysis buffer (1 x lysis buffer: 1% Triton x-100, 20 mM Tris/HCL, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml leupeptin). The tissue extract was cleared by centrifugation and the supernatant was diluted 1:1 with water. Lysate (1 ml) were cleared by a centrifugation step at 100.00 x g for 30 mins. Cleared wound lysates (200 µl) were adjusted to 4° C and mixed with 20 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society µl sulfanilamide (dissolved in 1.2M HCL) and 20 µl N-naphtylethylendiamine dihydrochloride. After 5 min at room temperature, the absorbance was measured at 540 nm with a reference wave-length at 690 nm. Combined NO 2 reductase in the presence of reduced nicotinamide adenine dinucleotide phosphate. For this, 80µl of samples were incubated with 10µL of Aspergillus nitrate reductase (1 unit/ml, Sigma, St. Louis MO) and 10 µL of NDPh 1 mmol/L for 1 hr at 27°C before the addition of the Griess reagent. All samples and standards, were assayed in triplicate. Data were expressed as mean ± SD of NO dihydrochloride, were obtained from Sigma Biochemicals (St. Louis, MO).HistologyAll tissue specimens were fixed in 10% neutral buffered formalin for at least 24h at room temperature. After fixation, perpendicular sections to the anterior-posterior axis of the wounds were dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Five-micron-thick sections were mounted on glass slides, dewaxed, rehydrated with distilled water, and stained with hematoxylin and eosin or with Masson's trichrome according to routine procedures for light microscopy. For the amount of granulation tissue the areas proximal to the incision were evaluated by three blinded pathologists in the all section of each slide. As part of the histological evaluation, all slides were examined by a pathologist without knowledge of the previous treatment by means of an eyepiece grid under the microscope from x5 to x100 magnification. The following parameters were evaluated and scored: reepithelialization, dermal matrix deposition and regeneration, granulation tissue formation and remodelling and angiogenesis. The edges of the wound in each of the sections as well as normal control wounds were used as comparison for scoring.

  Secondary antibody was provided by Pierce, and the location of the reaction was S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society visualized in brown with 3,3'-diaminobenzidine tetra-hydrochloride (Sigma) and nuclear fast red counter stain was used. Slides were then mounted with coverslips.

Female

  genetically diabetic C57BL/KsJ Lep db (db + /db + ) mice and their normoglycaemic littermates C57BL (db +/+ m) treated either with rAAV encoding for Ang-1 ( 100µl; 1x10 11 viral particles) or rAAV encoding for Lac-Z ( 100µl; 1x10 11 viral particles). On the day of trial (day 14) the mice were intradermally injected with 5 µg of compound 48/80 (Sigma, St. Louis MO) dissolved in 50 µl of physiological saline into the wound edges. Compound 48/80 administration is followed by increased leukocyte rolling, increased leukocyte adhesion and increased vascular permeability in postcapillary venules. Immediately after an intravenous injection of a 0.5% Evans blue solution (5 ml/kg/mouse). Animals were sacrificed 30 minutes after the compound 48/80 injection and the skin of the reaction locus was removed for a quantitative determination of the extravasated dye. Evans blue was extracted from each piece of dissected skin with 1 ml of 6M KOH at 45°C for 6 h. This extract was neutralized by 1 ml of 6M HCL, and then 2 ml of acetone was added. The extract was clarified by filtration, its absorbance at 595 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

NO 2 - /NO 3 -

 23 in wound lysates from rAAV-Lac-Z or with rAAV-Ang-1 injected normoglycaemic and diabetic mice. Wounding caused at day 7 a marked increase in NO 2 -/NO 3 -content compared to control uninjured skin (normoglycaemic animals= 3.3±0.6 nmol g tissue; diabetic animals= 1.1±0.08 nmol/g S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250

  e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

and intradermal stimulation of 5

 5 µg of compound 48/80 caused a significantly leakage of Evans Blue that has been intravenously injected before compound 48/80. No significant difference were observed among the several groups treated either with rAAV-Lac-Z or rAAV-Ang-1. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Dec 2007 as manuscript CS20070250 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Ang- 1 Angiopoietin 2 S

 12 Figure 2 S t a g e 2 ( a ) P O S T -P R I N T

Figure 3 S

 3 Figure 3 S t a g e 2 ( a ) P O S T -P R I N T

Figure 6 SFigure 7 S 10 S

 6710 Figure 4

Figure 1 .

 1 Figure 1. Ang-1 mRNA levels evaluated by Real Time PCR in skin wound samples collected from either normoglycaemic (db +/+ m) and diabetic mice (db + /db + ) given either rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles). Each bar represents the mean ± S.D. of seven experiments. *p<0.001 vs rAAV-Lac-Z.

Figure 2 .

 2 Figure 2. Representative western blot analysis and a quantitative assessment of Ang-1 protein expression in skin wound specimens obtained from either normoglycaemic (db +/+ m) and diabetic mice (db + /db + ) given rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles). Each bar represents the mean ± S.D. of seven experiments. *p<0.001 vs rAAV-Lac-Z.

Figure 3 eNOS

 3 Figure 3 eNOS expression (constitutive and phosphorilated form) (A) and NO products (B) in skin wound samples collected from either normoglycaemic (db +/+ m) and diabetic mice (db + /db + ) given either rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles). Each bar represents the mean ± S.D. of seven experiments. *p<0.001 vs rAAV-Lac-Z; §p<0.05 vs db +/+ m .

Figure 4 .

 4 Figure 4. Histological photomicrographs, haematoxylin-eosin, (original magnification ×10.) A: rAAV-LacZ-treated nondiabetic (db +/+ m) wound at day 14. Re-epithelialization is almost complete. Granulation tissue is well-formed without inflammatory infiltrates. B: rAAV-Ang-1-treated nondiabetic (db +/+ m) wound at day 14. Complete reepithelialization with presence of hair and restoration of normal architecture in dermis. C: rAAV-LacZ-treated diabetic (db + /db + ) wound at day 14. Thin and immature epithelial layer, with presence of crusting and poorly-formed granulation tissue. S t a g e 2 ( a ) P O S T -P R I N T

Figure 5 .

 5 Figure 5.Masson's trichrome staining of skin wound from either diabetic mice (db + /db + ) administered with rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1( 100µl; 1x1011 viral particles) at day 14 and quantification of collagen bundles.

Figure 6 .

 6 Figure 6. Representative immunohistochemical PECAM-1 staining of skin wounds samples from diabetic mice (db + /db + ) given with rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles) at day 14, (original magnification x40) and comparison of semiquantitative assessment of microvascular density (MVD) between each group. Each bar represents the mean ± S.D. of seven experiments. *p<0.05 vs rAAV-Lac-Z.

Figure 7 .

 7 Figure 7. Tensile strength of wounds obtained at day 14 from non diabetic (db +/+ m) and diabetic mice (db + /db + ) administered with either rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles). Each bar represents the mean ± S.D. of seven experiments. *p<0.001 vs rAAV-Lac-Z.

Figure 8 .

 8 Figure 8. VEGF mRNA levels evaluated by Real Time PCR (A) and VEGF protein studied by ELISA (B) in skin wound samples collected from either normoglycaemic (db +/+ m) and diabetic mice (db + /db + ) given either rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles). Each bar represents the mean ± S.D. of seven experiments. *p<0.001 vs rAAV-Lac-Z and diabetic animals.

Figure 9 .

 9 Figure 9. A and B: Representative immunohistochemical VEGFR-2 staining of skin wound samples from diabetic mice (db + /db + ) given with rAAV-Lac-Z ( 100µl; 1x10 11 viral particles) or rAAV-Ang-1 ( 100µl; 1x10 11 viral particles) at day 7. (Original magnification x40).

  and 5). Complete skin normalization in untreated mice occurred at day 22±1 after wounding in non-diabetics and at day 35±2 in diabetics. No side effect, nor signs of inflammation were noted in either diabetic and normoglycaemic animals injected with the viral vectors. Diabetic animals usually have a thicker epithelial layer than normal mice, in addition the different layers are less differentiated and adipose infiltrates are present in dermis, impairing the normal elasticity of the skin that in consequence is more prone to a delayed healing.

THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070250 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

db + + rAVV LacZ db + /db + +


Marie C Guldstrand

Caroline L Simberg

High-fat diets: healthy or unhealthy ?

In current dietary recommendations for the treatment and prevention of Type 2 diabetes and related complications there is flexibility in the proportion of energy derived from monounsaturated fat and carbohydrate as a replacement for saturated fat. Over the past few years, several population studies have shown that subjects eating a lot of refined grains and processed foods gain a much larger increase in waist circumference than those following a diet higher in monounsaturated fat, protein and carbohydrates rich in fibres and whole grains.

In this issue of Clinical Science, Sinitskaya and co-workers have demonstrated that, in normal-weight rodents categorized into three groups of high-fat, medium-carbohydrate (53/30 ratio of fat/CHO % of energy, 4.7 kcal/g), high-fat, low-carbohydrate (67/9 ratio of fat/CHO % of energy, 5.2 kcal/g) and high-fat, carbohydrate-free (75/0 ratio of fat/CHO % of energy, 5.9 kcal/g) diets, the high-fat diets that also contained carbohydrates were both obesogenic and diabetogenic while the very high-fat, carbohydrate-free diet was not obesogenic but led to insulin resistance and higher risk of cardiovascular disease. This finding may indicate that high-fat diets could easily be precipitated with carbohydrates to an unhealthy diet, pointing at the significance of macronutrient composition rather than caloric content in high-fat diets.

Comment

Dietary advice is vital for patients with Type 2 diabetes. Initial dietary advice was aimed at controlling the glucose concentration in the blood and the urine. A high-fat, low-carbohydrate diet was advocated for the treatment of Type 2 diabetes by Dr W. Osler and by other distinguished physicians at that time [START_REF] Osler | The Principles and Practice of Medicine[END_REF]. The diet recommended for Type 2 diabetics in 1923 contained approximately 75% of total energy intake from fat, 20% from protein and 5% from carbohydrates [START_REF] Osler | The Principles and Practice of Medicine[END_REF]. The current recommendations of the American Diabetes Association, (ADA) and the European Association for the Study of Diabetes, (EASD), on dietary composition to the treatment and prevention of diabetes mellitus are < 35% of total energy intake from fat (< 10% from saturated and transunsatured fat, < 10% from n-6 and n-3 polyunsaturated fat, 10-20% from cis monounsaturated fat), 10-20% from protein and 45-60% from carbohydrates [START_REF]American Diabetes Association Task Force for Writing Nutrition Principles and Recommendations for the Management of Diabetes and Related Complications: American Diabetes Association position statement: evidence-based nutrition principles and recommendations for the treatment and prevention of diabetes and related complications[END_REF][START_REF] Mann | The Diabetes and Nutrition Study Group (DNSG) of the European Association: Evidence-based nutritional approaches to the treatment and prevention of diabetes mellitus[END_REF]. Thus, current low-fat, high-carbohydrate diet recommendations are dramatically different from the high-fat, low-carbohydrate dietary recommendations from the 1920´s.

Excess weight is the principal risk factor for Type 2 diabetes. Visceral fat produces inflammatory activators, which, in turn, evoke insulin resistance. Obesity therefore promotes insulin resistance and glucose intolerance [START_REF] Weiss | Prediabetes in obese youth: a syndrome of impaired glucose tolerance, severe insulin resistance, and altered myocellular and abdominal fat partitioning[END_REF]. The cardiovascular and other metabolic complications resulting from abdominal visceral obesity and Type 2 diabetes are a significant cause of premature morbidity and mortality in industrialized countries [START_REF] Simone | Prognostic impact of metabolic syndrome by different definitions in a population with high prevalence of obesity and diabetes. The Strong Heart Study[END_REF]. Not surprisingly, the risk of contribution of high-fat food for weight gain and cardiovascular complications has been thoroughly investigated in population studies. Data from an 8-year follow-up in the Nurses´Health Study (NHS) showed that overall, percent of calories from fat only had a weak positive association with weight gain [START_REF] Field | Dietary fat and weight gain among women in the Nurses´Health Study[END_REF]. Increased intake of monosaturated and polysaturated fat were not associated with weight gain: however, percentage of fat from animal, saturated, and trans fat showed associations with greater weight gain. Furthermore, diets lower in carbohydrate and higher in vegetable sources of fat and protein have been shown to reduce glycaemic burden and the risk of coronary heart disease in women [START_REF] Halton | Low-carbohydrate-diet score and the risk of coronary heart disease in women[END_REF].

In this issue of Clinical Science, Sinitskaya et al. [START_REF] Sinitskaya | Increasing fat-to-carbohydrate ratio in a high-fat diet prevents the development of obesity but not prediabetic state in rats[END_REF] have demonstrated that, in rodents categorized into groups with high-fat, medium-carbohydrate (53/30 ratio of fat/CHO % of energy, 4.7 kcal/g), high-fat, low-carbohydrate (67/9 ratio of fat/CHO % of energy, 5.2

S t a g e 2 ( a ) P O S T -P R I N T

kcal/g) and high-fat carbohydrate-free (75/0 ratio of fat/CHO % of energy, 5.9 kcal/g) diets,

the high-fat diets that contained carbohydrates produced weight gain and abdominal obesity.

Interestingly, the high-fat, carbohydrate-free diet did not produce significantly higher body mass index or % adiposity than the control with a low-fat, high-carbohydrate, low-caloric (12/64 ratio of fat/CHO % of energy, 3.7 kcal/g) diet. Instead, the high-fat diets with low and medium-carbohydrate produced significantly higher BMI and % adiposity than the control diet. However, authors do not comment upon the fact that the high-fat, medium-carbohydrate diet (53/30 ratio of fat/CHO % of energy, 4.7 kcal/g) also contained 7-8% less metabolizable energy content in the form of protein compared to the other diets, possibly confounding the conclusions. In fact, population studies have shown that a lower content of protein in diet may not be favourable in terms of trying to maintain a thinner waist, instead a high energy intake from protein predicts an abdominal decrease [START_REF] Halkjaer | Intake of macronutrients as predictors of 5-y changes in waist circumference[END_REF]. Also, those eating a lot of refined grains and processed foods show a much larger increase in waist circumference than those eating carbohydrates rich in fibres and whole grains [START_REF] Halkjaer | Intake of macronutrients as predictors of 5-y changes in waist circumference[END_REF]. Furthermore, fat-rich diets can acutely induce insulin resistance, especially in subjects with already low adiponectin levels [START_REF] Thamer | Serum adiponectin levels predict the effect of short-term dietary interventions on insulin sensitivity in humans[END_REF].

Thus, it is possible that the restriction of dietary fat and a diet high in carbohydrates might be particularly effective in subjects with low adiponectin levels such as individuals with obesity and Type 2 diabetes. The high-fat diets in this study may, in part, be precipitated or accelerated in combination with carbohydrates and low protein content to an unhealthy diet, pointing at the significance of macronutrient composition rather than merely the caloric content in high-fat foods.

In current dietary recommendations for people with Type 2 diabetes, there is flexibility in the proportion of energy derived from monounsaturated fats and carbohydrate [START_REF]American Diabetes Association Task Force for Writing Nutrition Principles and Recommendations for the Management of Diabetes and Related Complications: American Diabetes Association position statement: evidence-based nutrition principles and recommendations for the treatment and prevention of diabetes and related complications[END_REF][START_REF] Mann | The Diabetes and Nutrition Study Group (DNSG) of the European Association: Evidence-based nutritional approaches to the treatment and prevention of diabetes mellitus[END_REF]. Provided that energy intake is controlled, the use of monounsaturated fat instead of carbohydrate as a replacement for saturated fat may cause an increase, as opposed to a decrease, in HDLcholesterol levels, which is of particular benefit in Type 2 diabetes and abdominal obesity.

Thus, monounsaturated fats are promoted as the main source of dietary fat because of their lower susceptibility to lipid peroxidation and consequent lower atherogenic potential [11].

The rationale for modifying fat distribution and reducing weight is based on an expected improvement in insulin sensitivity, β-cell function and insulin-sensitizing factors [START_REF] Guldstrand | Improved β-cell function after standardized weight reduction in severely obese subjects[END_REF].

Adiponectin, which is exclusively produced and secreted from the adipocyte, has putative anti-atherogenic properties and appears to ameliorate insulin resistance in both muscle and S t a g e 2 ( a ) P O S T -P R I N T liver [START_REF] Thamer | Serum adiponectin levels predict the effect of short-term dietary interventions on insulin sensitivity in humans[END_REF][START_REF] Guldstrand | Improved β-cell function after standardized weight reduction in severely obese subjects[END_REF]. This protein has now been extensively explored, and it is possible that a low plasma adiponectin concentration may be a convenient marker for identifying subjects with the metabolic syndrome who may progress to prediabetes on an unhealthy diet. Ways of preventing severe obesity, especially in childhood, by providing healthy dietary recommendations for individuals at risk, and also the role of calorically sweetened soft drinks and the increased total intake of caloric sweeteners contribution to the current obesity epidemic need to be subjected to intense scrutiny.

S t a g e 2 ( a ) P O S T -P R I N T

THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070263 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society


MD Manon V Van Hecke
email: m.vanhecke@vumc.nl

Jacqueline M Dekker

Giel Nijpels

Tom Teerlink

Cornelis Jacobs

Ronald P Stolk

Rob J Heine

Lex M Bouter

Coen Bettine Cp Polak

Da Stehouwer

C Jakobs

B C P Polak

C D A Stehouwer

the Hoorn Study

Keywords: homocysteine, S-adenosylmethionine, S-adenosylhomocysteine, methionine metabolism, retinal microvascular disease, retinopathy

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

Hyperhomocysteinemia has been associated with an increased risk for cardiovascular disease independently of conventional cardiovascular risk factors [START_REF]Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis[END_REF][START_REF] Welch | Homocysteine and atherothrombosis[END_REF]. It remains unclear, however, whether homocysteine itself is a direct cause of atherosclerosis. Other components of homocysteine metabolism, such as S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and folate, have also been suggested to play a role in, and possibly mediate, the relationship with cardiovascular disease [START_REF] Loria | Serum folate and cardiovascular disease mortality among US men and women[END_REF][START_REF] Kerins | Plasma Sadenosylhomocysteine is a more sensitive indicator of cardiovascular disease than plasma homocysteine[END_REF][START_REF] Becker | Plasma homocysteine and S-adenosylmethionine in erythrocytes as determinants of carotid intima-media thickness: different effects in diabetic and nondiabetic individuals. The Hoorn Study[END_REF][START_REF] Robinson | Low circulating folate and vitamin B6 concentrations: risk factors for stroke, peripheral vascular disease, and coronary artery disease[END_REF]. Methionine -homocysteine metabolism involves methionine transmethylation, which involves the formation of SAM and SAH, and leads to the production of homocysteine (Figure 1). Homocysteine can be remethylated to methionine, which requires vitamin B12 and folate, or can go through the transsulfuration pathway, which requires vitamin B6. However, the exact extent of contribution of these metabolites to cardiovascular disease risk has not yet been established [START_REF] Spijkerman | S-adenosylmethionine and 5methyltetrahydrofolate are associated with endothelial function after controlling for confounding by homocysteine: the Hoorn Study[END_REF].

Homocysteine is thought to increase cardiovascular risk by inducing endothelial dysfunction, increasing oxidative stress, and inducing thrombophilia and proliferation of smooth muscle cells, which can lead to narrowing of the intravascular lumen [START_REF] Welch | Homocysteine and atherothrombosis[END_REF][START_REF] Spijkerman | S-adenosylmethionine and 5methyltetrahydrofolate are associated with endothelial function after controlling for confounding by homocysteine: the Hoorn Study[END_REF][START_REF] Moat | Folate, homocysteine, endothelial function and cardiovascular disease[END_REF][START_REF] Van Guldener | Hyperhomocysteinemia, vascular pathology, and endothelial dysfunction[END_REF].

Experimental studies, mainly in rats, suggest that hyperhomocysteinemia may also play a role in the pathogenesis of renal microvascular disease [START_REF] Kumagai | Renal tubulointerstitial injury in weanling rats with hyperhomocysteinemia[END_REF][START_REF] De Vriese | Endothelium-derived hyperpolarizing factormediated renal vasodilatory response is impaired during acute and chronic hyperhomocysteinemia[END_REF][START_REF] Vermeulen | Homocysteine-lowering treatment with folic acid plus vitamin B6 lowers urinary albumin excretion but not plasma markers of endothelial function or C-reactive protein: further analysis of secondary end-points of a randomized clinical trial[END_REF]. In humans, however, little is known about the relationship of homocysteine and its metabolites with microangiopathy.

High levels of homocysteine were reported to be strongly associated with the development of microalbuminuria [START_REF] Hoogeveen | Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study[END_REF][START_REF] Jager | Serum homocysteine levels are associated with the development of (micro)albuminuria: the Hoorn Study[END_REF]. Hyperhomocysteinemia is also thought to be a strong risk factor for retinal vascular occlusive disease [START_REF] Cahill | Meta-analysis of plasma homocysteine, serum folate, serum vitamin B(12), and thermolabile MTHFR genotype as risk factors for retinal vascular occlusive disease[END_REF]. For retinopathy, this relationship is not very clear.

In the Hoorn study, we previously reported a relationship of hyperhomocysteinemia with retinopathy in type 2 diabetic individuals [START_REF] Hoogeveen | Hyperhomocysteinemia is associated with the presence of retinopathy in type 2 diabetes mellitus: the Hoorn Study[END_REF], whereas others did not find any relationship [START_REF] Hofmann | Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM[END_REF].

In light of these considerations, we hypothesized that homocysteine and (or) components of homocysteine metabolism are involved in the pathogenesis of microvascular disease. As the retina offers a unique opportunity to non-invasively analyze the microvasculature, we investigated the relationship of homocysteine and components of homocysteine metabolism with computer-assisted measurements of retinal vessel diameters, retinal sclerotic vessel abnormalities and retinopathy. Because hyperhomocysteinemia appears to be more strongly related to cardiovascular disease in the presence of type 2 diabetes [START_REF] Hoogeveen | Hyperhomocysteinemia is associated with an increased risk of cardiovascular disease, especially in non-insulin-dependent diabetes mellitus: a population-based study[END_REF], we studied the above-mentioned relationships in a population-based study of subjects with and without type 2 diabetes.

Methods

Study population

For the present study, we used data of the 2000-2001 Hoorn Study follow-up examination.

The Hoorn Study is a population-based cohort study of type 2 diabetes and its cardiovascular complications among 2484 Caucasian persons, aged 50-74 years, which started in 1989. Full details have been provided elsewhere [START_REF] Mooy | Prevalence and determinants of glucose intolerance in a Dutch caucasian population. The Hoorn Study[END_REF][START_REF] Henry | Type 2 diabetes is associated with impaired endothelium-dependent, flow-mediated dilation, but impaired glucose metabolism is not; The Hoorn Study[END_REF]. Fasting and 2-hour post-load plasma glucose levels after a 75-g oral glucose tolerance test (OGTT) were measured in plasma, and were used for classification into glucose tolerance categories [START_REF]World Health Organization: Definition, diagnosis and classification of diabetes mellitus and its complications[END_REF]. Subjects who were already known to have diabetes or use glucose-lowering treatment were excluded for the OGTT. At baseline, an age-, sex-, and glucose-tolerance-stratified subsample of 631 participants was extensively studied for reasons of efficiency. In the years 2000-2001, a follow-up examination was carried out among surviving participants who gave their permission to be re-contacted [START_REF] Henry | Type 2 diabetes is associated with impaired endothelium-dependent, flow-mediated dilation, but impaired glucose metabolism is not; The Hoorn Study[END_REF]. Of the 631 participants who had an ophthalmological examination at baseline, almost 60% dropped out for the follow-up examination, because of the following reasons: 119 (19%) persons had died, 49 (8%) had moved out of the region, and 207 (33%) did not participate because of mobility or health problems, or lack of motivation. Finally, 256 persons were included in the follow-up ophthalmological examination [START_REF] Van Leiden | Risk factors for incident retinopathy in a diabetic and nondiabetic population -The Hoorn Study[END_REF], 70 with normal glucose metabolism (NGM), 69 with impaired glucose metabolism (IGM), and 109 with type 2 diabetes [START_REF]World Health Organization: Definition, diagnosis and classification of diabetes mellitus and its complications[END_REF]. Written informed consent was obtained from all participants. The Ethical Review Committee of the VU University Medical Center (VUmc) in Amsterdam, the Netherlands, approved the Hoorn Study.

Sample preparation and determination of homocysteine, SAM, SAH, folates and vitamins

All samples were processed within 30 minutes after collection, stored at -80 degrees Celsius and analyzed within two years. Ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood samples were put on ice for the determination of homocysteine, SAM and SAH. For SAM and SAH measurements, samples were immediately deproteinized by the addition of 0.625 ml of a 10% perchloric acid solution to 1 ml plasma and by adding 1 ml of 5% perchloric acid to 1 ml whole blood, followed by mixing [START_REF] Loehrer | Disturbed ratio of erythrocyte and plasma Sadenosylmethionine/S-adenosylhomocysteine in peripheral arterial occlusive disease[END_REF]. For the determination of total folate, 0.5 mg of ascorbic acid was added to 0.5 ml of serum [START_REF] Loehrer | Disturbed ratio of erythrocyte and plasma Sadenosylmethionine/S-adenosylhomocysteine in peripheral arterial occlusive disease[END_REF], and for the determination of total folate in erythrocytes, 1 ml of reagent with ascorbic acid, human serum albumin and sodium azide (ACS:180, Chiron Diagnostics) was added to 50 µl of whole blood. [CV = (standard deviation of the mean difference / ((square root of 2) x pooled mean)] was 4 %) [START_REF] Shipchandler | Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer[END_REF]. Tandem mass spectrometry was used to determine SAM and SAH in plasma and whole blood (intra-assay CV were 4% for both determinations, interassay CV were 8% and 6%, respectively) [START_REF] Struys | Determination of S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid by stable-isotope dilution tandem mass spectrometry[END_REF]. Erythrocyte concentration for SAM and SAH were calculated as:

[whole blood concentration -plasma concentration * (1 -hematocrit)] / hematocrit. Total folate in serum and erythrocytes and serum vitamin B12 were measured with automated chemiluminescence (Chiron Diagnostics ASC: 180 ® Automated Chemiluminescence Systems). Intra-and interassay CV were 4% and 5% for total folate, respectively. 5-Methyltetrahydrofolate (5-MTHF), the active form of folate, was measured by highperformance liquid chromatography [START_REF] Leeming | Measurement of 5methyltetrahydrofolic acid in man by high-performance liquid chromatography[END_REF], which was also used to measure plasma vitamin B6 [START_REF] Ubbink | Stability of pyridoxal-5-phosphate semicarbazone: applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status[END_REF]. The interassay CV was 7%.

Ophthalmological examination and retinal vessel diameter measurements

After mydriasis with 0.5% tropicamide and 2.5% phenylephrine eye drops, the retina was examined by funduscopy and fundus photography, as previously described [START_REF] Van Leiden | Risk factors for incident retinopathy in a diabetic and nondiabetic population -The Hoorn Study[END_REF]. In short, fundus photography was performed with a 45º CR5 non-mydriatic retinal camera (Canon Inc., Tokyo, Japan), interfaced to a 3CCD Color Video Camera (Sony Corp., Japan). The quality of each photo was checked immediately on a connected color video monitor (Trinitron, Sony, Tokyo, Japan) and a new photograph was taken if the quality was insufficient. The photographs were digitized, compressed (10:1 JPEG), and stored on a magneto-optical disc using the TEAC MV-300P Viewfile system (TEAC Corp.,Tokyo, Japan). Two photographs were made of each eye, one centered on the macula and one centered on the optic disc.

Methods used to measure retinal vessel diameters from digitized photographs followed a standardized protocol, which has been described elsewhere [START_REF] Hubbard | Methods for evaluation of retinal microvascular abnormalities associated with hypertension/sclerosis in the Atherosclerosis Risk in Communities Study[END_REF]. Briefly, one investigator (M.V.H.) independently measured all arterioles and venules 0.5 to 1 disc diameter from the optic disc margin using a computer-imaging program (Retinal Analysis, Optimate, Madison, Wisconsin), masked to participant identity. The branches of arterioles were also measured if the trunk measures were >= 85 µm. Computer-assisted measurements of the diameters of arterioles and venules were obtained and combined according to the revised formulas of Parr and Hubbard [START_REF] Hubbard | Methods for evaluation of retinal microvascular abnormalities associated with hypertension/sclerosis in the Atherosclerosis Risk in Communities Study[END_REF][START_REF] Parr | Mathematic relationships between the width of a retinal artery and the widths of its branches[END_REF][START_REF] Parr | General caliber of the retinal arteries expressed as the equivalent width of the central retinal artery[END_REF][START_REF] Knudtson | Revised formulas for summarizing retinal vessel diameters[END_REF], which account for magnification differences and the number of vessels combined into an arteriole-to-venule ratio (AVR). An AVR of 1.0 indicates that the diameters of the arterioles are approximately equal to the diameters of the venules, whereas a smaller AVR indicates narrower arterioles or wider venules. For each subject, one photograph centered on the optic disc was used, alternately selected of the left and right eye (ratio: 50%-50%). In the case of insufficient quality, the photograph of the other eye was examined. The intra-observer intersession CV were 5% for CRVE, 8% for CRAE and 9% for AVR.

Both fundus photographs were independently analyzed by two individuals (H.A.L. en A.C.M.) to grade retinal sclerotic vessel abnormalities and retinopathy. In the case of disagreement, the judgment of a third investigator (B.C.P.P.) was taken to be decisive. Retinal sclerotic vessel abnormalities were defined as the presence of venous beading, focal narrowing, arteriovenous crossing changes, "copper" or "silver" wiring, dilated or tortuous retinal veins, or central or branch venular occlusion. Retinopathy was defined as the presence of one or more microaneurysms, hemorrhages, or hard exudates, possibly in combination with areas of neovascularization, fibrous proliferation, pre-retinal or vitreous hemorrhages and/or laser coagulation scars in at least one eye according to the Eurodiab classification [START_REF] Van Leiden | Risk factors for incident retinopathy in a diabetic and nondiabetic population -The Hoorn Study[END_REF][START_REF] Aldington | Methodology for Retinal Photography and Assessment of Diabetic-Retinopathy -the Eurodiab IDDM Complications Study[END_REF].

Other measurements

Brachial systolic and diastolic blood pressures, glycated hemoglobin, fasting insulin, serum total, high-density and low-density lipoprotein cholesterol, serum triglycerides, serum creatinine, serum albumin, body mass index, waist and hip circumferences, smoking and prior cardiovascular disease (CVD) were determined according to methods described elsewhere [START_REF] Beks | Peripheral arterial disease in relation to glycaemic level in an elderly Caucasian population: the Hoorn Study[END_REF][START_REF] Henry | Arterial stiffness increases with deteriorating glucose tolerance status: the Hoorn Study[END_REF][START_REF] Jager | Microalbuminuria and peripheral arterial disease are independent predictors of cardiovascular and all-cause mortality, especially among hypertensive subjects: five-year follow-up of the Hoorn Study[END_REF]. Hypertension was defined as diastolic blood pressure >= 90 mmHg, systolic blood pressure >= 140 mmHg and/or use of anti-hypertensive medication [START_REF] Chobanian | The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report[END_REF].

Statistical analyses

Clinical and ophthalmological characteristics, expressed as mean ± SD, percentage, or median (interquartile range) in the case of a skewed distribution, were computed according to tertiles of levels of total plasma homocysteine. Overall group differences in continuous variables were tested by means of analyses of variance, and differences in categorical measures were tested with Pearson's chi-square test.

Multivariable linear regression analyses were used to calculate the associations of homocysteine and its metabolites with AVR, CRAE, and CRVE. To make the results of linear regression models comparable among different determinants, we report standardized beta retinopathy (odds ratios were reported per 1 SD). Because all determinants showed linearity with AVR, CRAE, CRVE, retinal sclerotic vessel abnormalities and retinopathy, we used homocysteine and its metabolism components as continuous variables in the models. First, we adjusted the associations for the stratification variables, age, sex and glucose tolerance status.

Then, we additionally adjusted the associations for the other components of the homocysteine metabolism, and for other potential confounders, i.e. glycated hemoglobin, systolic and diastolic blood pressure, body mass index, total cholesterol, microalbuminuria, and prior CVD.

Effect-modification by type 2 diabetes was investigated by entering product terms of diabetes yes/no times the predictor variable in the regression models. P-values less than 0.05 were considered statistically significant. All analyses were performed in SPSS 12.0 for Windows 98.

Results

Of the 256 participants, 6 had photographs that could not be graded for AVR and CRAE, and 2 had photographs that could not be graded for CRVE. Furthermore, 4 subjects had missing data of total plasma homocysteine and 68 subjects had missing data of one or more components of the homocysteine metabolism. Subjects with one or more missing variables did not differ substantially from subjects with available data (data not shown), and therefore, were not excluded from further analyses. Mean (± SD) CRAE (in µm) was 170.6 ± 18.9 (range: 119.9 -225.1), mean (± SD) CRVE (in µm) was 231.2 ± 27.1 (range: 148.7 -333.5), and mean (± SD) AVR was 0.75 ± 0.10 (range: 0.49 -1.31). Of the 256 subjects, 31 had retinal sclerotic vessel abnormalities, and 32 had retinopathy.

Clinical characteristics

Table 1 presents the clinical and ophthalmological characteristics according to tertiles of total plasma homocysteine levels. AVR and CRAE were inversely correlated with levels of homocysteine.

Associations of homocysteine, SAM and SAH with retinal vessel diameter

Figure 2a shows the distribution of homocysteine levels against levels of CRAE for persons with normal glucose metabolism, impaired glucose metabolism and diabetes. Figure 2b shows that, after adjustment for age, sex, and glucose tolerance status, subjects in the highest tertile of homocysteine had an 8 µm lower CRAE than subjects with homocysteine levels in the lowest tertile (p < 0.05). Indeed, when we analyzed homocysteine as a continuous variable, total plasma homocysteine was inversely associated with CRAE and AVR after adjustment for age, sex, and glucose tolerance status (st.ß (95%CI) -0.20 (-0.33;-0.07) and -0.15 (-0.28 ; -0.01), respectively). This implies that CRAE decreases with 3.78 µm per 1 SD increase of homocysteine level (= 4.6 µmol/l) (Table 2). After additional adjustments for SAM-plasma, 5-MTHF, and cardiovascular risk factors, the association of total plasma homocysteine with small retinal arteriolar diameter did not change (Table 3). Furthermore, individual adjustment for other components of the homocysteine metabolism and other cardiovascular risk factors, such as lipid levels or current smoking, also did not affect the results (data not shown).

High SAM-plasma and SAH-plasma were both associated with lower AVR after adjustment for age, sex and glucose tolerance status (Table 2). After adjustment for 5-MTHF, plasma homocysteine, SAM-plasma or SAH-plasma, body mass index, waist-to-hip ratio, waist circumference or microalbuminuria the associations lost statistical significance. After adjustment for total folate in serum and erythrocytes, or vitamins B6 and B12, the association of SAM and SAH in plasma with lower AVR remained statistical significant (data not shown).

Associations of SAM and SAH with retinal sclerotic vessel abnormalities and retinopathy

The SAM/SAH ratio in plasma was lower in subjects with retinal sclerotic vessel abnormalities than in subjects without retinal sclerotic vessel abnormalities (median (interquartile range): 5.6 (4.5-6.5) vs. 6.0 (5.1-7.0), p=0.05). In addition, SAH in plasma was significantly higher, and the SAM/SAH ratio in plasma lower, in subjects with retinopathy as compared with subjects without retinopathy (SAH-plasma: 17.6 (15.6-25.6) vs. 15.3 (12.5-20.2) nmol/l, p=0.003; SAM/SAH ratio in plasma: 5.1 (4.2-5.8) vs. 6.0 (5.1-7.0), p<0.001).

After adjustment for age, sex and glucose tolerance status, the SAM/SAH ratio in plasma was inversely and strongly associated with retinal sclerotic vessel abnormalities and retinopathy (ORs (95% CI): 0.61 (0.39-0.96) and 0.50 (0.30-0.83), respectively) (Table 2).

Adjustments for other homocysteine metabolism components showed little impact on both associations. Further adjustments for cardiovascular risk factors did not change the results In addition, SAM/SAH ratio in plasma showed a borderline association with microalbuminuria after adjustments for age, sex and glucose tolerance status ( OR (95% CI) microalbuminuria (yes/no): 0.81 (0.62-1.06)). The standardized beta for microalbuminuria as a linear variable was -0.153 (p=0.029, SD=28.1) (data not shown).

S

Additional analyses

The presence of type 2 diabetes did not statistically significantly modify the associations of homocysteine metabolism components with AVR, CRAE, CRVE, retinal sclerotic vessel abnormalities or retinopathy (p>0.05). SAM and SAH in erythrocytes, the SAM/SAH ratio in erythrocytes, 5-MTHF, total folate in serum and in erythrocytes, and vitamins B12 and B6

were not associated with AVR, CRAE, CRVE, retinal sclerotic vessel abnormalities and retinopathy (data not shown).

Discussion

The present study, which included subjects with and without type 2 diabetes, had two main findings. First, a high total plasma homocysteine was significantly associated with retinal arteriolar narrowing, independently of other components of the homocysteine metabolism or cardiovascular risk factors. Second, a lower SAM/SAH ratio in plasma was independently associated with retinal sclerotic vessel abnormalities and retinopathy. These results did not differ between subjects with and without type 2 diabetes.

The present study is the first to show an independent association between total plasma homocysteine and retinal arteriolar narrowing. This finding is in line with the general concept that homocysteine has vasculotoxic properties. Homocysteine is considered an independent risk factor for atherosclerotic vascular disease and mortality [START_REF]Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis[END_REF][START_REF] Welch | Homocysteine and atherothrombosis[END_REF]. In addition, hyperhomocysteinemia has been associated with retinal vascular occlusive disease, which was recently shown in a meta-analysis [START_REF] Cahill | Meta-analysis of plasma homocysteine, serum folate, serum vitamin B(12), and thermolabile MTHFR genotype as risk factors for retinal vascular occlusive disease[END_REF]. In contrast with our results, one study including 84 recently diagnosed type 2 diabetic individuals and 115 non-diabetic individuals did not find a relationship of plasma homocysteine levels with AVR [START_REF] Wirta | Methylenetetrahydrofolate reductase gene polymorphism, hyperhomocysteinemia and occlusive retinal vascular disease in type 2 diabetic and non-diabetic subjects[END_REF]. This study, however, did not use a computer-assisted method to accurately measure arteriolar and venular diameters, as we did.

Further support for an association between hyperhomocysteinemia and microvascular disease derives from studies of renal microvascular disease [START_REF] De Vriese | Endothelium-derived hyperpolarizing factormediated renal vasodilatory response is impaired during acute and chronic hyperhomocysteinemia[END_REF][START_REF] Hoogeveen | Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study[END_REF][START_REF] Jager | Serum homocysteine levels are associated with the development of (micro)albuminuria: the Hoorn Study[END_REF], which show an independent association of homocysteine with the presence and development of microalbuminuria [START_REF] Hoogeveen | Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study[END_REF][START_REF] Jager | Serum homocysteine levels are associated with the development of (micro)albuminuria: the Hoorn Study[END_REF]. subjects with cerebral microangiopathy, compared with subjects without cerebrovascular disease [START_REF] Fassbender | Homocysteine in cerebral macroangiography and microangiopathy[END_REF]. Therefore, our and previous studies suggest that hyperhomocysteinemia may not only be a risk factor for macrovascular disease, but may also be associated with microvascular disease in the retina and elsewhere.

Our results showed that subjects with homocysteine levels in the highest tertile had 8 µm smaller retinal arteriolar diameter compared with subjects with homocysteine levels in the lowest tertile. To put this into perspective, higher blood pressure (a known determinant of retinal arteriolar narrowing) was, in our study, associated with decreases of 0.6 and 1.1 µm in retinal arteriolar diameter per 10 mmHg higher systolic and diastolic blood pressure, respectively, which is comparable to previous findings [START_REF] Ikram | Are retinal arteriolar or venular diameters associated with markers for cardiovascular disorders? The Rotterdam Study[END_REF][START_REF] Leung | Relationships between age, blood pressure, and retinal vessel diameters in an older population[END_REF]. Therefore, these data suggest that homocysteine could be a biologically relevant risk factor for retinal arteriolar narrowing.

The exact mechanism by which homocysteine affects the vascular system is not completely understood. However, homocysteine is thought to exert its toxic effects on the vascular wall by impairing endothelial function, increasing oxidative stress, decreasing availability of nitric oxide, inducing a prothrombotic state, and by inducing proliferation of smooth muscle cells [START_REF] Welch | Homocysteine and atherothrombosis[END_REF][START_REF] Van Guldener | Hyperhomocysteinemia, vascular pathology, and endothelial dysfunction[END_REF]. This may cause vasoconstriction, intimal thickening, medial hyperplasia, hyalinization and sclerosis, consequently leading to a smaller arteriolar lumen.

We did not find any association between homocysteine and prevalent retinopathy.

Previously, Hoogeveen et al. found a significant relationship of homocysteine with retinopathy in diabetic subjects, but not in non-diabetic subjects in the Hoorn Study population of 1989 [START_REF] Hoogeveen | Hyperhomocysteinemia is associated with the presence of retinopathy in type 2 diabetes mellitus: the Hoorn Study[END_REF]. These discordant findings might probably be due to limited power of the present study (N retinopathy = 32), which might also explain the absence of interaction of diabetic status. Moreover, selective mortality of diabetic individuals with hyperhomocysteinemia [START_REF] Hoogeveen | Hyperhomocysteinemia increases risk of death, especially in type 2 diabetes : 5-year follow-up of the Hoorn Study[END_REF] may have resulted in a 'healthy survivor effect', and may have affected our results. On the other hand, data on the association between homocysteine and diabetic retinopathy are not consistent [START_REF] Vaccaro | Plasma homocysteine and microvascular complications in type 1 diabetes[END_REF][START_REF] Smulders | Fasting and post-methionine homocysteine levels in NIDDM. Determinants and correlations with retinopathy, albuminuria, and cardiovascular disease[END_REF]. Unlike total plasma homocysteine, a lower SAM/SAH ratio in plasma was strongly associated with retinal sclerotic vessel abnormalities and retinopathy. Previous studies have shown that a low SAM/SAH ratio was associated with end-stage renal failure [START_REF] Loehrer | Evidence for disturbed Sadenosylmethionine : S-adenosylhomocysteine ratio in patients with end-stage renal failure: a cause for disturbed methylation reactions?[END_REF], peripheral arterial occlusive disease [START_REF] Loehrer | Disturbed ratio of erythrocyte and plasma Sadenosylmethionine/S-adenosylhomocysteine in peripheral arterial occlusive disease[END_REF], and vascular disease (stroke and myocardial infarction) [START_REF] Castro | Increased Homocysteine and S-Adenosylhomocysteine Concentrations and DNA Hypomethylation in Vascular Disease[END_REF].

Our additional results that SAM/SAH ratio in plasma was borderline associated with microalbuminuria support these findings. Indeed, the ratio of SAM and SAH is crucial in the regulation of multiple enzymatic transmethylation reactions [START_REF] Finkelstein | Pathways and regulation of homocysteine metabolism in mammals[END_REF]. A decrease of this ratio may result in inhibition of transmethylation reactions, which affects the biosynthesis of proteins, hormones, phospholipids, neurotransmitters, RNA and DNA [START_REF] James | Elevation in Sadenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteinerelated pathology[END_REF][START_REF] Finkelstein | Methionine metabolism in mammals[END_REF], and consequently might lead to inhibition of vascular endothelial cell growth [START_REF] Wang | Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine[END_REF] and impairment of endothelial function. In addition, increased SAH, which could be caused by elevated homocysteine concentrations, also potentially acts as an inhibitor of transmethylation reactions [START_REF] Finkelstein | Pathways and regulation of homocysteine metabolism in mammals[END_REF]. Taken together, we hypothesize that a reduction of transmethylation reactions may be an important factor in the pathogenesis of retinal microvascular disease.

In the present study, we used a detailed measurement of retinal microvessel diameters by a computer-assisted method on fundus photographs made in mydriasis. Moreover, we used the revised formulas of Parr and Hubbard to quantify vessel caliber, by which the vessels were measured independently of image scale and the number of measured vessels [START_REF] Knudtson | Revised formulas for summarizing retinal vessel diameters[END_REF]. A second advantage of this study is its population-based design, including subjects with and without type 2 diabetes. This study has also limitations. The results that homocysteine is associated with retinal arteriolar narrowing and SAM/SAH ratio in plasma is only associated with sclerotic vessel abnormalities and retinopathy might be confusing, suggesting different pathophysiological mechanisms. As the complete pathophysiological pathway of the association of homocysteine/methionine metabolism with microangiopathy is still unclear, our results may represent different pathophysiological pathways, however we cannot exclude the possibility that these differences are cause by chance. Due to the cross-sectional design of this study, we cannot interpret the present results as cause-and-effect relationships. In addition, the study population consisted of individuals aged 60 to 85 with a considerable prevalence of cardiovascular risk factors, and we do not know whether the results of this study can be generalized to a younger or healthier population or to other ethnicities. However, an older population has also advantages in studying homocysteine and its components, because homocysteine levels increase with age, and low to low-normal concentrations or deficiencies of folate and vitamin B12 are relatively common in this age group, which provide wider ranges of concentrations to study in the elderly.

In conclusion, this is the first population-based study that shows an independent relationship of total plasma homocysteine with retinal arteriolar narrowing. In addition, the SAM/SAH ratio in plasma was strongly associated with retinal sclerotic vessel abnormalities and retinopathy. This suggests that homocysteine, and, possibly, reduced transmethylation reactions may be involved in the pathogenesis of retinal microvascular disease.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Total plasma homocysteine was determined with an automated fluorescence polarisation immunoassay on an Abbott Imx analyzer (interassay coefficient of variation (CV)

  in photographs. Average diameters of arterioles (central retinal arteriolar equivalents (CRAE)) and venules (central retinal venular equivalents (CRVE)) in one eye were assessed, and S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  values. A standardized beta of 0.1 indicates that if the determinant at issue increases by 1 SD, S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society the outcome increases by 0.1 SD. Furthermore, we report change of AVR and change in µm CRAE and CRVE. Logistic regression analyses were used to calculate the associations of components of homocysteine metabolism with retinal sclerotic vessel abnormalities and

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society substantially, except for prior CVD, which slightly attenuated the association for retinal sclerotic vessel abnormalities (data not shown).

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society In addition, Fassbender et al. demonstrated that homocysteine levels were increased among 82

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Further studies are needed to clarify the microvascular effects of homocysteine-lowering treatment S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society with folic acid, which may also increase transmethylation reactions [50], in order to create new treatment modalities for (retinal) microvascular disease.

Figure 1 :

 1 Figure 1: Methionine-homocysteine metabolism

Figure 2a :Figure 2b :

 2a2b Figure 2a: scatterplot of central retinal arteriolar equivalent against homocysteine levels with different symbols assigned to persons with normal glucose metabolism (NGM), impaired glucose metabolism (IGM), and diabetes (DM).

45 ) 16 4

 4516 Data are calculated with linear regression analyses (AVR, CRAE, and CRVE), and with logistic regression analyses (RSVC and RP), and are shown as standardized betas (St.ß )(95% confidence interval (CI)) / change in AVR or µm CRAE/CRVE and odds ratios (OR) per 1 SD (95% CI). * SAM/SAH ratio in plasma; SAM: Sadenosylmethionine; SAH: S-adenosylhomocysteine; 5-MTHF: 5-Methyltetrahydrofolate; AVR: arteriole-to-venule diameter; CRAE: central retinal arteriolar equivalent; CRVE: central retinal venular equivalent; RSVC: retinal sclerotic vessel abnormalities; RP: retinopathy S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical SocietyTable 3 Multivariate associations of homocysteine with the central retinal arteriolar equivalent (CRAE) Model Added variables CRAE St.ß (95% CI) / change in µm 1 Homocysteine, age, sex, glucose tolerance status -0.20 (-0.33 ; -0.07) / -3.78 2 As model 1 + SAM-plasma and 5-MTHF -0.25 (-0.39 ; -0.09) / -4.73 3 As model 1 + glycated hemoglobin, systolic and diastolic blood pressure, body mass index, total cholesterol, microalbuminuria and prior CVD -0.22 (-0.35 ; -0.09) / -4.As model 2 + glycated hemoglobin, systolic and diastolic blood pressure, body mass index, total cholesterol, microalbuminuria and prior CVD -0.25 (-0.41 ; -0.09) / -4.73 Data are calculated with linear regression analyses, and are showed as standardized betas (95% confidence interval) and change in µm CRAE. SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; 5-MTHF: 5-Methyltetrahydrofolate S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 : Baseline characteristics according to tertiles of homocysteine levels

 1

	Homocysteine	Homocysteine	Homocysteine	P trend
	<10.0 µmol/l	10.0-12.7 µmol/l	>= 12.8 µmol/l

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 : Age-, sex-, and glucose-tolerance-status-adjusted associations between homocysteine, components of homocysteine metabolism and retinal vascular abnormalities

 2

		Standard	AVR	CRAE	CRVE	RSVA	RP
		deviation
	Standard		0.1	18.9 µm	27.1 µm
	deviation
			St.ß (95% CI)/ change in ratio	St.ß (95% CI) / change in µm St.ß (95% CI) / change in µm OR (95% CI)	OR (95% CI)
	Homocysteine	4.6 µmol/l	-0.15 (-0.28 ; -0.01) / -0.02	-0.20 (-0.33 ; -0.07) / -3.78	-0.04 (-0.17 ; 0.09) / -1.03	0.97 (0.65-1.45)	0.98 (0.65-1.47)
	SAM-plasma	28.5 nmol/l	-0.13 (-0.26 ; 0.00) / -0.01	-0.05 (-0.18 ; 0.07) / -0.95	0.10 (-0.02 ; 0.23) / 2.71	0.92 (0.62-1.36)	0.94 (0.64-1.40)
	SAH-plasma	9.2 nmol/l	-0.15 (-0.29 ; -0.01) / -0.02	-0.12 (-0.25 ; 0.01) / -2.27	0.06 (-0.07 ; 0.19) / 1.63	1.17 (0.85-1.62)	1.16 (0.84-1.60)
	SAM/SAH ratio* 1.5	0.05 (-0.10 ; 0.20) / 0.01	0.09 (-0.05 ; 0.23) / 1.70	0.02 (-0.12 ; 0.16) / 0.54	0.61 (0.39-0.96)	0.50 (0.30-0.83)
	5-MTHF	8.9 nmol/l	0.02 (-0.13 ; 0.16) / 0.00	-0.00 (-0.14 ; 0.13) / -0.07	0.00 (-0.14 ; 0.14) / -0.11	0.98 (0.67-1.44)	0.92 (0.59-1.45)
	Vitamin B12	101.1 pmol/l	-0.01 (-0.14 ; 0.13) / -0.00	-0.03 (-0.16 ; 0.09) / -0.57	-0.02 (-0.15 ; 0.12) / -0.54	1.11 (0.74-1.64)	1.01 (0.68-1.49)
	Vitamin B6	37.2 nmol/l	0.03 (-0.10 ; 0.16) / 0.00	-0.06 (-0.18 ; 0.06) / -1.13	-0.10 (-0.22 ; 0.03) / -2.71	1.30 (0.97-1.74)	1.01 (0.70-1.

Acknowledgements

We thank H.A. van Leiden and A.C. Moll for their important contribution to the retinal examination of the participants of the present study. Also, we would like to thank M.K. Ikram and J.R. Vingerling for their help with judging the retinal vessel diameter measurements. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 24 Oct 2007 as manuscript CS20070275 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070275 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society