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Keywords:

  • supraspinatus tendon;
  • proteoglycans;
  • glycosaminoglycans;
  • chondrogenesis;
  • HARP/pleiotrophin

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:61–71, 2012

Tendon is composed primarily of type 1 collagen molecules organized into fibrils that constitute the tension-bearing structure in association with biglycan and decorin. Decorin, a small dermatan sulfate (DS) rich proteoglycan (PG) associated with collagen fibrils, regulates their diameter and longitudinal organization into fibers.1, 2 The collagen fibrils are embedded in the colloid extra-cellular matrix, containing most of the 70–80% tendinous water bound to the compression-bearing sulfated glycosaminoglycans (GAGs) of PGs. Tendon cells are surrounded by versican, a large chondroitin sulfate (CS) rich PG, which buffers load transmitted from the collagen matrix.3 Overuse human chronic degenerative tendinopathy due to repetitive loading is characterized by structural and biochemical alterations including collagen fibrils disarray, separation, and disorganization, fibrocartilaginous cellular metaplasia, GAGs accumulation4, 5 and PGs variations at mRNA levels and protein content.6, 7 However tendinopathic biopsies are mostly available at late stages of pathology; knowledge of early pathological events is still incomplete.

A rat model of tendon overuse has been developed that generates changes in histology and mechanical properties reproducing key characteristics of human supraspinatus tendinopathy.8, 9 This early experimental tendinosis is associated with collagen fragmentation, GAG accumulation, proliferating tenocytes, and higher expression of cartilage matrix markers mRNA.10, 11 These events are not primarily mediated by the presence of inflammatory cells, although mRNAs of some inflammatory mediators were detected.12 Because of their long polysaccharidic anionic chains, GAGs retain water molecules that influence biomechanical tissue environment13 and also interact with proteins, such as cytokines, both mechanisms finally regulating tenocyte metabolism. It could thus be hypothesized that changes in GAG composition may be involved in early stages of tendon overuse pathology. These changes may temporarily interfere with the tendon's tensile load-bearing capacity, thus serving as a prelude to subsequent recurring collagen microtears and cell-mediated pathoetiological events.

The rat model appears suitable for studying the time course of early changes in tendon matrix composition and to establish the relationship between chemical and mechanical modulators of cellular phenotype. Our objective was to analyze when, in overused supraspinatus tendon, changes in GAGs composition occur and whether these changes are associated with previously suggested tenocyte phenotypic alterations.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Rat Running Protocol and Sample Collection

Male Sprague–Dawley rats (500 ± 20 g) (Janvier, Le Genest Saint-Isle, France) were used. Twelve control animals were allowed normal cage activity. Twelve other rats were subjected to daily treadmill downhill running.9 After 2 and 4 weeks, control (n = 6) and running (n = 6) rats were euthanized, and the supraspinatus tendons were dissected free from their muscular and bony attachments, weighed, frozen in liquid nitrogen, and stored at −80°C until use for biochemical studies. For immunohistochemistry on fixed rat supraspinatus tendon, paraffin blocks from a previous cohort of rats (n = 32) trained according to the same protocol were used.11 At each time (4, 8, 12, and 16 weeks), five running and three control rats were studied. We used these tissue blocks to reduce the required number of animals, following principles of the Canadian Council on Animal Care (192/2002).

GAG Extraction and Quantification

GAG extraction and quantification was performed according to.14 Aliquots of total GAG extracts were treated with nitrous acid or 0.5 U/ml chondroitinase B (Sigma, St-Quentin-Fallavier, France), and the remaining GAGs was quantified with the same DMMB method. Using these chemical and enzymatic treatments, the heparan sulfate (HS), DS, and CS contents were calculated:

  • equation image

Histological Analysis of Total Sulfated GAG by DMMB Complexation

At each time point, paraffin sections (7 µm) were deparaffinized, and DMMB solution was used labeling of total sulfated GAGs on tissue sections after incubation overnight in the dark at room temperature. The labeling was validated using chondroitinase ABC. A CoolSNAP camera (Princeton Instruments; Acton, MA) was used for images acquisition. Image analysis was performed using a color segmentation procedure programmed with ImageJ.15

Immunolabeling of GAGs and PGs

At each time point, deparaffinized sections were blocked with PBS–BSA 3% (w/v) and incubated overnight with phage display single chain anti-HS, anti-DS, and anti-CS (produced by T. Van Kuppevelt). Bound antibodies were detected with mouse anti-VSV tag IgG antibody P5D4. PGs were detected using anti-decorin, anti-versican, and anti-aggrecan antibodies, followed by Alexa 488-conjugated goat anti-mouse IgG (Fluoprobes Interchim, Montluçon, France). Fluorescence images were obtained using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD) fitted to a BH-2 epifluorescence microscope (Olympus; Rungis, France).

Protein Extraction and Western Blot Analysis

The frozen supraspinatus tendons were homogenized in RIPA buffer containing 60 mM Tris–HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate supplemented with 1/100 protease inhibitor cocktail (Sigma). Tissue was lysed overnight at 4°C then centrifuged at 12,000g for 10 min. Twenty micrograms proteins were analyzed by Laemmli SDS–PAGE (Biorad, Marne la Coquette, France) and then transferred to Immobilon-P PVDF membrane (Millipore, Guyancourt, France) for detection of: decorin (pAb LF-113, 1/5,000, kind gift from Larry Fisher, NIH, Bethesda, MD), versican (pAb, 1/200, Santa Cruz Biotechnology, Le Perray en Yvelines, France), aggrecan (pAb, 1/100, Santa Cruz Biotechnology), heparin affine regulatory peptide (HARP) (pAb, 1/1,000, R&D system, Inc, Oxon, UK), GAPDH (mAb, 1/10,000, Applied Biosystems, Courtaboeuf, France), and α-tubulin (pAb, 1/5,000, Sigma). Appropriate horseradish-peroxidase-conjugated secondary antibodies were visualized with BM chemiluminescence substrate (Roche Diagnostics, Meylan, France).

mRNA Isolation and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from supraspinatus tendons using Trizol reagent (Invitrogen, Cergy-Pontoise, France) according to manufacturer's instructions. Reverse transcription was performed on 1 µg mRNA extracts using the SuperScript II RNase H (Invitrogen). The resulting cDNA were used for PCR amplification. Primers were as shown in Table I. The method was validated by preliminary experiments that established the optimal conditions. RT-PCR products were subjected to electrophoresis on 2% (w/v) agarose gels containing 0.5 mg/ml ethidium bromide. Gels were photographed using a Chimigenius system (Syngene, Cambridge, UK) and band intensities were quantified using ImageJ.15

Table I. Primer Sequences and Products Size
GenesPrimersAnnealing temperature (°C)CyclesProduct size (bp)
Collagen 1α15′-CTGCTGGTCCTAAGGGAGAG-3′ (forward)6235372
5′-CAATACCAGGAGCACCATTG-3′ (reverse)
Collagen2α15′-GGAAGAGCGGAGACFACFGG-3′ (forward)6440390
5′-CCCTCATCTCCACATCATTG-3′ (reverse)
Collagen 3α15′-GATTCCCTGGATCTCCTGGTG-3′ (forward)6235309
5′-TCCTGGCTCTCCCTTTGCTC-3′ (reverse)
Collagen 6α15′-ATACCGGCGCAATTTCACGGC-3′ (forward)6440311
5′-AACCCTTCTCTCCACGGCTTCC-3′ (reverse)
Decorin5′-GGAAFGAAGGGTCTCGGAFA-3′ (forward)6235394
5′-GACFCACGGCAGFGTAGGAA-3′ (reverse)
Biglycan5′-TGFCCTTTTGGCTGCCACTGC-3′ (forward)6440350
5′-AACACGCCCFTGGGCACTTTG-3′ (reverse)
Versican5′-AGACATGA 1 GGGGAAGGAAG-3′ (forward)6440351
5′-AGAGGGAAGCATGTCTGGTT-3′ (reverse)
Aggrecan5′-ClGGCATTACGTTTGTGGAC-3′ (forward)6440374
5′-AGCAGTAGGAGCCAGGGTTA-3′ (reverse)
CHST145′-CGCAGTGACTTGGTGTTTCT-3′ (forward)3434363
5′-ATCAGCTTCCAGCCTCTCAT-3′ (reverse)
SOX95′-ATGACCGACGAGCAGGAGAAGG-3′ (forward)644037
5′-CGITGIGCAGATGCGGGIACTG-3′ (reverse)
HARP5′-TCGTCCCAGCAATACCAGCAGC-3′ (forward)6235390
5′-ACAGTCGGCATTGTGCAGAGC-3′ (reverse)
TFIID5′-CGTCTAGTGGCCCAGATCTGT-3′ (forward)6043347
5′-CGGTACIGCAFCFTGAFTGFCA-3′ (reverse)

Heparin Versus GAGs Competition Assay Towards Growth Factors

A competition ELISA using HARP, also called pleiotrophin or fibroblast growth factor-2 (FGF-2) was performed. This assay allows evaluation of the ability of GAGs from tendons, taken as competitor, to prevent the binding of HARP or FGF-2 to heparin.16 This is a qualitative assay for GAGs, which competitive power depends on their composition. A heparin–BSA complex was coated overnight at 4°C on 96-well plates. Wells were blocked with PBS–BSA 3% (w/v). HARP, or FGF-2 (50 ng/ml) in PBS–BSA 1% (w/v) was added to wells simultaneously with GAGs (4 µg/ml) from each time point. Plates were incubated overnight at 4°C for competition then washed and further incubated for 2 h at room temperature with pAb against HARP or FGF-2 (R&D system, Inc), then incubated for 2 h with the appropriate peroxidase-labeled secondary antibody. Peroxidase activity was determined according to manufacturer's protocol (Thermo Scientific, Brebières, France). Binding of HARP or FGF-2 to heparin in the absence of GAG competitor was defined as 100% binding.

Statistical Analysis

Data are expressed as mean ± SD. Means were compared with Student's t-test using GraphPad software (San Diego, CA). Significant differences were determined at p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Changes in Extra-Cellular Matrix Components in Overused Supraspinatus Tendon

Compared to controls, Col 1α1 mRNA expression slightly decreased at 2 weeks, then more substantially at 4 weeks (by ∼4 fold). Col 2α1 transcripts increased progressively from 2 to 4 weeks, whereas Col 3α1 and Col 6α1 mRNA expressions significantly increased at 2 weeks but returned to the control level at 4 weeks (Fig. 1). Levels of decorin and biglycan transcripts increased at both 2 and 4 weeks of overuse compared to controls (Fig. 2a). The levels of mRNA of the large PGs versican and aggrecan also increased at both times (Fig. 2a). The corresponding protein levels increased concomitantly (biglycan could not be tested) (Fig. 3a). These increases persisted for at least 8 weeks (Fig. 3b). The mRNA of transcription factor Sox9 almost absent in controls was highly expressed at 2 weeks and remained high at 4 weeks (Fig. 2b). Table II shows results for all the molecules examined.

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Figure 1. Expression of different collagens in overused supraspinatus tendons (R) compared to controls (C). Col 1α1, 2α1, 3α1, and 6α1 mRNA levels were determined by semi-quantitative RT-PCR. TFIID was used as an internal control. Results are representative of six tendons from three rats.

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Figure 2. Expression of PGs in overused tendons compared to control. mRNA levels of decorin, biglycan, versican, aggrecan (a), and Sox9 (b) were determined by semi-quantitative RT-PCR. TFIID was used as an internal control. Results are representative of six tendons from three rats.

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Figure 3. Western blots analysis in control and overused tendons at 2 and 4 weeks (a) and immunolabeling at 4 and 8 weeks (b) of decorin, versican, and aggrecan. GAPDH or α-tubulin were used as loading controls. Results are representative of six tendons from three rats. Scale bar 50 µm.

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Table II. Overview Changes of all Analyzed Molecules
MoleculesRunners 2 weeks versus controlsRunners 4 weeks versus controls
  1. Levels of expression; - or - -: less, =: equivalent, + or + +: more than controls.

Collagens
 1α1 mRNA-- -
 2α1 mRNA++
 3α1 mRNA+=
 6α1 mRNA+ +=
Decorin
 mRNA++ +
 Protein=+ +
Biglycan
 mRNA+ ++ +
Versican
 mRNA+ ++ +
 Protein=+ +
Aggrecan
 mRNA+ ++ +
 Protein+ ++ +
SOX9
 mRNA+ ++
Glycosaminoglycans
 Total=+ +
 DS=+ +
 CS=+
 HS==
 CHST14+ +=
HARP
 mRNA==
 Protein++ +

Changes in Sulfated GAGs in Overused Tendon

Since overuse enhanced the expression of several PGs, we examined the amount and composition of GAGs in overused tendons and control animals (Fig. 4 and Table II). Whereas after 2 weeks of running the amount of total sulfated GAGs measured biochemically was not significantly altered compared to controls, it increased by almost three times in 4 weeks-overused tendons (Fig. 4a). This increase was also observed by histology. The high level of sulfated GAGs observed at 4 weeks then decreased progressively but still remained significantly higher than in controls at 16 weeks (Fig. 5). In controls, the main GAG species was DS (∼80%), whereas HS or CS each represented ∼10% of total GAG (Fig. 4a). In overused tendons, the proportion of CS slightly increased whereas that of DS decreased at 2 weeks, and more at 4 weeks (Fig. 4a). However, these GAG species increased by ∼2.5-fold at 4 weeks and thus accounted for the observed GAG accumulation. This was corroborated by the increase in the 4S sulfotransferase (CH4ST) mRNA as early as 2 weeks (Fig. 4b); CHST14 is specifically involved in DS synthesis.

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Figure 4. Sulfated GAGs quantification in controls and overused tendons. Total GAGs, DS, CS, and HS were quantified as detailed in the text. GAGs species shown as amount and percent of total GAGs (a). mRNA levels of dermatan 4-O-sulfotransferase 1 (CHST14) (b). TFIID was used as an internal control for RT-PCR. Results are representative of six tendons from three rats.

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Figure 5. Total sulfated GAGs in supraspinatus tendons after long term training (4, 8, 12, and 16 weeks). Results are representative of 10 runners and 6 controls tendons for each time point from different rats. Scale bar 50 µm.

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The use of phage display antibodies against HS, CS, and DS showed a labeling of parallel long stripes located between collagen fibers in the environment of tenocytes (Fig. 6). The enhanced CS and DS labeling compared to controls at 4 weeks was still visible at 8 weeks. Altogether, GAGs accumulation appeared to correspond to the observed increase in transcripts and core protein of PGs.

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Figure 6. Immunolabeling of HS, CS, and DS in long term of training (4 and 8 weeks) using phage display antibodies (Ab). Ab specificity was checked by enzymatic or chemical treatments. Nuclei were stained with DAPI. Results are representative of 10 runners and 6 controls tendons for each time point from rats. Scale different bar 50 µm.

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HARP Protein is Enhanced and Sequestered in Overused Supraspinatus Tendon

HARP is well known as a chondrogenesis promoting factor during development. Although the level of HARP transcript was not altered during the first 4 weeks of training, the protein level was increased up to threefold in tendon extracts at 2 and 4 weeks (Fig. 7a). Since HARP has affinity to HS/DS GAG species,17 we investigated the possibility that GAG composition and affinity to HARP was altered in overused tendons.

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Figure 7. Expression of HARP at 2 and 4 weeks of overuse at transcript and protein levels. TFIID and GAPDH were used as loading controls (a). Data represent the amounts of FGF-2 or HARP that bind to heparin in the presence of GAGs taken as a competitor (b). For each time point, data acquired in triplicate are from six tendons from three rats.

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One possibility to detect such qualitative GAG changes towards HARP is to perform an ELISA competition assay based on the ability of GAGs from tendons, taken as competitor, to prevent the binding of HARP to heparin. As comparison, similar test was conducted in the presence of FGF-2. GAGs from either controls or overused tendons prevented FGF-2 binding to heparin by <10%, whereas they reduced by 20% the binding of HARP to heparin at 2 weeks of overuse. Thus, these GAGs displayed a low affinity for FGF-2. In contrast, GAGs from tendons at 4 weeks reduced the binding of HARP to heparin by 50% (Fig. 7b). These results demonstrate that GAG affinity for HARP from overused tendons increased from 2 to 4 weeks in comparison to controls.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Understanding early biological events, which may evolve towards extensive injury-repair processes, is necessary to prevent the development of disabling tendinopathy pathology. We used a rat overuse rotator cuff model to analyze early changes in GAGs, PGs, and collagen composition related to phenotypic cellular alteration. Total sulfated GAG significantly increased after 4 weeks in overused tendons but not at an earlier time point. This increase lasted up to 16 weeks as shown by histology. The increase followed up-regulations at 2 weeks of the PGs transcripts of decorin, biglycan, versican, and aggrecan, which characterizes cartilage tissue. At this time point, collagen 2, collagen 3, and especially collagen 6 mRNAs were also up-regulated, whereas collagen 1 transcript decreased from 2 to 4 weeks. Interestingly, the transcription factor Sox9, known to regulate chondrogenesis, was also highly up-regulated as early as 2 weeks. These molecular alterations were characteristic of a phenotypic shift of tendon cells toward chondrogenesis as observed previously study.10

Biochemical data show that in tendons, the main GAG species is DS. At 4 weeks, overused tendons, DS, and CS GAGs species both accounted for GAGs accumulation, and their respective proportions slightly changed. These results differ from those in human post mortem supraspinatus tendon in which CS was found as the most abundant GAG species.18 This discrepancy might arise from different biomechanical environments of rat and human tendons or from recent improvements in the technology used to discriminate GAG species. The increase in total sulfated GAGs was preceded in time by up-regulations of both small (decorin and biglycan) and large PGs (versican and aggrecan), these biochemical and molecular data being obtained from the whole tendon. Changes in PGs and GAGs expressions in overused tendon may be part of an adaptive response to local modifications of compression-elongation exerted on tenocytes. One might argue that up-regulation of decorin and versican would occur in the tensile region, whereas the up-regulation of biglycan and aggrecan would occur in the osteotendinous fibrocartilaginous region. Alternatively, a shift of tendon to fibrocartilage may occur with overuse. Histology at 8 weeks of overuse showed that GAGs increased at the middle part of the tendon along with pericellular labeling of CS, versican, and aggrecan. These observations are in favor of chondrocyte metaplasia in the middle part of the tendon, known to be the most altered region with overuse.11

Alterations of collagens with overuse, particularly of collagen 2, confirm the fibrocartilaginous alteration in the pericellular matrix. Collagen 6 accumulation is also a feature of chondrogenesis,19 forming a hexagonal network organized by biglycan20 and connecting with collagen 2 fibers, aggrecan, decorin, and biglycan serving as adaptors.21 The chondrogenic transcription factor Sox9, which increased 2 weeks of overuse, is up-regulated in tendon cells in association with mechanical stress22 or cyclic hydrostatic pressure.23 It can be also induced in tendon cells by TGF-β.24

We further examined which cytokines might promote chondrogenesis in overused tendon. Several cytokines, including TGF-β or FGF family members, involved in developmental chondrogenesis,25 are altered in overused supraspinatus tendon.26 Insulin-like growth factor 1 (IGF-1) is another cytokine that more specifically increases in longer term mechanically loaded tendons (>4 weeks) and may favor tenocytes proliferation and survival.11 Our results suggest that another growth factor, HARP, may also be involved in the observed change in tendon phenotype. The amount of HARP protein was increased in overused tendons especially at 4 weeks. Changes in HARP protein was probably a local response of cells, presumably tenocytes, to overuse, since the amount of HARP did not increase in blood plasma (data not shown). Whereas HARP transcript was not increased, the increased amount of HARP protein, in keeping with Western blot data, suggested an enhanced sequestration of this growth factor in extra cellular matrix with overuse. The overall proportions of GAG species changed slightly with overuse, but subtle changes in the structure of GAG molecules may have also occurred. Using competitive ELISA technique, we showed that indeed GAGs from overused tendons at 4 weeks present an increased affinity for HARP protein compared to controls. Interestingly, GAG affinity for FGF-2 was not altered with overuse. An increased expression of the N-galactosamine 4-O-sulfotransferase transcript (CHST14) was also observed at 2 weeks. This increase, which probably leads to higher enzymatic activity, might contribute to an over-sulfation of CS/DS GAGs with a resulting higher affinity to HARP.27 This might contribute to an enhanced sequestration of HARP in the matrix.

Since it could not be excluded that the up-regulation of HARP could be secondary to overuse, it can be hypothesized that HARP is, at least partially, the cause of increased GAGs, collagen type 2, and PGs such as biglycan as observed in bovine cartilage28 or in human fibroblasts.29 Indeed this cytokine is known to be involved in chondrogenesis during development.30 Our results are the first to indicate the role of HARP in adult tendon. Further studies are ongoing to clarify its role in a tenocyte phenotypic shift to chondrocyte phenotypes.

In conclusion, we present new information on the role of GAGs and HARP in early molecular events associated with supraspinatus tendon overuse. In the context of high frequency repeated mechanical loading, the increase in amount and sulfation of GAGs may enhance the water retention and thus the viscoelasticity of the tendon.1 These responses may cause an imbalance in the synthesis and degradation of matrix components, eventually leading to structural alterations and degeneration of tendon. Anticipated detection of these events, some of which may be reversible, could help to develop injury prevention strategies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Mohamed Attia has PhD financial support from COGITOBIO and ANRT (Association Nationale de la Recherche et de la Technologie). Alexander Scott received support from the Michael Smith Foundation for Health Research. We thank Eric Huet, Suzanne Menashi, Dulce Papy-Garcia, and Jean Delbé for their valuable discussions and help.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES