Le VEGF-111 comme nouvel outil thérapeutique des lésions tendineuses

in 3ème Congrès Commun SFMS - SFTS (2010, September 30)

Introduction : Les lésions tendineuses sont très fréquentes en traumatologie du sport et deviennent fréquemment chroniques. Pour ces raisons, de nouvelles thérapeutiques sont en cours de développement ... [more ▼]

Introduction : Les lésions tendineuses sont très fréquentes en traumatologie du sport et deviennent fréquemment chroniques. Pour ces raisons, de nouvelles thérapeutiques sont en cours de développement. Les injections de concentrés plaquettaires (platelet-rich plasma ou PRP) semblent constituer en ce sens une voie encourageante. Elles agissent par libération locale de divers facteurs de croissance parmi lesquels le VEGF-A (vascular endothelial growth factor-A), connu pour induire un effet positif sur la fonction vasculaire et l’angiogenèse, serait impliqué dans le processus cicatriciel des tendons. Récemment, une nouvelle isoforme du VEGF-A a été identifié : le VEGF-111. Celui-ci est une isoforme biologiquement active du VEGF-A, résistant à la protéolyse et aussi connu pour présenter un effet bénéfique sur les pathologies ischémiques. Pour ces raisons, nous avons pensé que le VEGF-111 pourrait avoir un intérêt thérapeutique pour les pathologies tendineuses. Matériel et méthode : 60 rats de souche Sprague-Dawley adultes ont été séparés en 2 groupes (A: groupe contrôle sans traitement et B: groupe traité par une injection de VEGF-111). Chez ces rats, un défaut de 5mm dans le tendon d’Achille a été réalisé après résection du tendon du plantaire grêle. Les 30 rats du groupe B ont alors bénéficié d’une injection in loco de 100ng de VEGF-111. Les rats ont été euthanasiés par groupe de 20 (10 du groupe A et 10 du groupe B) respectivement à J5, J15 et J30 et le tendon d’Achille en cours de régénération a été disséqué et prélevé. Une étude biomécanique de traction jusqu’à rupture a été réalisée à l’aide de mors type « cryo-jaw ». Résultats : L’analyse de nos résultats montre que la force nécessaire pour rompre le tendon lors du test de traction, était plus importante pour les tendons du groupe B. Ces résultats peuvent être observés dès le 5ème jour. Le rapport entre la force et la masse corporelle du rat augmente dans les 2 groupes avec le temps, mais cette augmentation est plus importante pour les tendons du groupe B. La surface de section du tendon de groupe B s’accroit plus rapidement entre les jours 5 et 15 et ensuite se stabilise. Après 30 jours, les sections tendineuses sont similaires dans les 2 groupes. Enfin, dans le groupe B, les contraintes nécessaires pour obtenir la rupture du tendon, en tenant compte de l’accroissement de sa section, sont similaires entre les jours 5 et 15 et augmentent après un mois. Conclusion : Cette expérience a démontré qu’une injection de 100ng de VEGF-111 stimulait le processus de cicatrisation tendineuse en augmentant la résistance du tendon et les contraintes nécessaires pour rompre celui-ci. D’autres expérimentations avec différentes concentrations de VEGF-111 sont actuellement en cours. [less ▲]

New use of VEGF in therapeutics: application in tendon lesions

in Clinical Chemistry (2010, July), 56(S6), 111

Introduction: As demonstrated in previous studies, mechanical overload, injury and inflammation, hypoxic condition or any combination of the above could lead to increased expression of VEGF in the tendon ... [more ▼]

Introduction: As demonstrated in previous studies, mechanical overload, injury and inflammation, hypoxic condition or any combination of the above could lead to increased expression of VEGF in the tendon. Thus, VEGF could participate in the healing of pathological tendons. Indeed, some authors are convinced that this neovascularization is the sign of a chronic tendinopathy while others plead in favour of it being a sign of healing processes. The VEGF111, which is a biologically active and proteolysis-resistant VEGF-A isoform, was recently identified. It is induced by ultraviolet B and genotoxic drugs. Experimentation shows that, in nude mice, tumors formed by HEK293 cells expressing VEGF111 develop a more widespread peritumoral neovascularisation than those expressing other VEGF isoforms. Good angiogenic activity and resistance to proteolysis makes VEGF111 a potential beneficial therapeutic option for ischemic diseases. The aim of our study was to determine whether if VEGF111 could have a therapeutic interest in the framework of tendinous pathology. Methods (*): A 5mm defect was surgically induced in Achilles tendon of 60 rats. Rats were divided into 2 groups of 30: A: a control group (no injection) and B: with a VEGF111 injection. The rats of group B received an injection of 100 ng of VEGF111 in situ 1 hour after surgery on the site of the tendon lesion. Afterwards, rats of both groups were placed in their cages without immobilization. After 5, 15 and 30 days, 10 rats of each group were euthanized. The traumatized Achilles tendon of each rat was dissected and removed. Immediately after sampling, tendons were submitted to a biomechanical tensile test up to rupture, using a tensile machine with “Cryo-jaw”. Statistical analyses were made with an ANOVA. Results: A significant increase over time of the force necessary to induce tendon rupture was observed for tendons which had been submitted to an injection of VEGF111 (p=0.016). The force required to break the tendon is always greater for the VEGF111 group (p<0.05). Discussion: We demonstrated that the force necessary to induce the rupture of a rat’s Achilles tendon during biomechanical tensile testing was greater for tendons which had been submitted to an injection of VEGF111. Thus, this experimentation showed that VEGF111 injections could accelerate the tendon healing process and increase the force needed to break tendons in their healing process. Conclusion: VEGF111 could be a new therapy for tendon lesions. However, other experimentation using a rat model with different concentrations of VEGF111 should be made to ascertain the best concentration for this healing process. Acknowledgement: This experimentation was partially financed by “Standard de Liège” and “Lejeune-Lechien” grants. (*) All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Liège. [less ▲]

Acide Hyaluronique et matrice extracellulaire : une molécule primitive ?

in Annales de Dermatologie et de Vénéréologie (2010), 137(supplément 1), 3-8

Hyaluronic acid, or hyaluronan, is a polymer made of the repetition of a unique disaccharidic unit, D-glucuronic acid and D-N-acetylglucosamine, that can reach a molecular mass of 10(7) daltons. This ... [more ▼]

Hyaluronic acid, or hyaluronan, is a polymer made of the repetition of a unique disaccharidic unit, D-glucuronic acid and D-N-acetylglucosamine, that can reach a molecular mass of 10(7) daltons. This primitive polymer has emerged as a remarkable extracellular matrix component by its viscoelastic properties, its hygroscopic capacities and the diversity of cell processes it controls. Identified in all vertebrate tissues, more than 50% of acid hyaluronic of the organism is present in skin. Having no protein core, its synthesis is performed through a unique process, depending on enzymatic activity of hyaluronan synthases acting at the internal face of the plasmatic membrane and extruding the nascent polymer to the extracellular medium. This polymer constitutes a scaffold on which a large number of sulfated proteoglycans, up to one hundred, can be linked. These supramolecular structures of considerable size are able to entrap large amounts of water and ions to provide tissues with hydration and turgescence. Hyaluronic acid is recognized by cell membrane receptors, notably CD44 which is the best known. Interaction of hyaluronic acid with its receptors triggers several intracellular signaling pathways regulating proliferation, migration and differentiation. Cell response is largely influenced by the size of the polymer and by that of the fragments generated upon degradation by hyaluronidases or free radicals. Hyaluronic acid is metabolically very active, as, for example, its half-life in skin is less than one day. Detected in epidermis where it could play a role in the control of proliferation and differentiation of basal cells, it is however prominent in dermis in association with versican. The remarkable physicochemical properties of hyaluronic acid as well as the diversity of biological processes it controls largely surpass the primitive character of this polymer. [less ▲]

Effect of β-cyclodextrin and its derivatives on caveolae disruption, relationships with their cholesterol extraction capacities

in Journal of Inclusion Phenomena and Macrocyclic Chemistry (2010), 67

Endothelial cells (HUVEC) were treated with b-cyclodextrin (b-CD) and hydroxypropylated or methylated derivatives solutions to confirm their lack of affinity with phospholipids and their specificity ... [more ▼]

Endothelial cells (HUVEC) were treated with b-cyclodextrin (b-CD) and hydroxypropylated or methylated derivatives solutions to confirm their lack of affinity with phospholipids and their specificity towards cholesterol. Further studies were performed on bovine aortic endothelial cells to assess the effect of b-CDs (mainly methylated derivatives) on membrane microdomains (lipid rafts or caveolae), by detecting the caveolae marker caveolin-1 in fractions of sucrose gradients. A displacement from the lighter to the heavier fractions, characteristic of caveolae disruption, was observed using CDs. The strongest effect was obtained with dimethyl-b-CD, for which an accumulation of caveolin-1 was observed in the bottom of the gradient. Crysmeb and trimethyl-b-CD seemed to have the weaker effects as a significative amount of caveolin-1 was still detected in the light fraction corresponding to caveolae. b-CD and CDs having a degree of methylation a bit lower than 2 showed intermediate effects. The results of the present study on microdomains seem in good correlation with the cell cholesterol extraction capacities of CDs previously determined. [less ▲]

Histological and transcriptional study of angiogenesis and lymphangiogenesis in uninvolved skin, acute pinpoint lesions and established psoriasis plaques: an approach of vascular development chronology in psoriasis

in Journal of Dermatological Science (2010), 57(3), 162-169

Background Dysregulation of angiogenesis and lymphangiogenesis could participate in psoriasis pathogenesis. Analysis of nascent psoriasis lesions should help at identifying early vascular anomalies ... [more ▼]

Background Dysregulation of angiogenesis and lymphangiogenesis could participate in psoriasis pathogenesis. Analysis of nascent psoriasis lesions should help at identifying early vascular anomalies. Objective To analyse vascular development, angiogenesis and lymphangiogenesis markers expression in uninvolved skin in psoriatic patients (N), early psoriasis lesions or pinpoints (PP) and psoriasis plaques (PSO). Methods Skin biopsies were taken in 17 patients in N and in PSO and/or PP. The mRNA steady-state level of angiogenesis and lymphangiogenesis markers was measured by RT-PCR. Immunohistochemistry was performed for von Willebrand factor, podoplanin, Ki-67 and VEGFR3. Blood (BV) and lymphatic (LV) vessels expansion was measured by computer-assisted morphometry. Results Clinical and epidermal aspects indicated that PP are intermediate between N and PSO. While total BV area was already increased in PP similarly to PSO as compared to N, LV area in PP was intermediate between N and PSO. Mean LV size was identical in N and PP and increased in PSO, mean BV size in PP being intermediate between N and PSO. VEGF-A 189 variant was increased in PP as compared to N and PSO. As compared to N, angiogenesis markers (VEGF-A isoforms, PlGF, VEGFR2, NRP-1), VEGF-C and NRP-2 were similarly increased in PP and PSO. Keratin 16 and the lymphangiogenesis markers (VEGFR3, prox-1) were intermediate in PP. Conclusion These data suggest that the expansion of lymphatic vessels occurs after blood vascular development in psoriasis. Expansion of BV in PP could be followed by vessel enlargement during progression to PSO, in parallel with a decreased VEGF-A 189/VEGF-A 121 balance in plaques [less ▲]

Study of the cholesterol extraction capacity of β-cyclodextrin and its derivatives, relationships with their effects on endothelial cell viability and on membrane models

in Journal of Inclusion Phenomena and Macrocyclic Chemistry (2009), 63(3-4), 225-231

Endothelial cells (HUVEC) were treated with β-cyclodextrin and hydroxypropylated or methylated derivatives solutions in order to quantify their cholesterol extraction capacity. Non-toxic concentrations of ... [more ▼]

Endothelial cells (HUVEC) were treated with β-cyclodextrin and hydroxypropylated or methylated derivatives solutions in order to quantify their cholesterol extraction capacity. Non-toxic concentrations of cyclodextrins (CDs) were determined following methyl thiazol tetrazolium (MTT) assays, total protein measurements, morphological observations and trypan blue assays. The residual cholesterol content of cells was measured and the extraction power of CDs compared to results obtained by phase solubility diagrams. Cholesterol was extracted with a dose-response relationship, the lowest residual cholesterol content being obtained with β-CD at 10 mM. Low substituted derivatives (Crysmeb® and hydroxypropyl-β-CD) maintained liposomes integrity (as shown before), were the less cytotoxic and presented the lowest affinity for cholesterol contrary to methylated derivatives with degrees of substitution around 2. [less ▲]

Cold shock (25°C) and re-warming (37°C) affect cell phenotype: impac on space biology experiments.

Conference (2009)

Altered expression of angiogenesis and lymphangiogenesis markers in the uninvolved skin of plaque-type psoriasis

in British Journal of Dermatology (2009), 160(3), 581-90

Background Vascular alterations are significant events in the pathomechanism of psoriasis. A disorder in the mechanisms regulating skin angiogenesis and lymphangiogenesis could participate in the ... [more ▼]

Background Vascular alterations are significant events in the pathomechanism of psoriasis. A disorder in the mechanisms regulating skin angiogenesis and lymphangiogenesis could participate in the pathogenesis of the disease. Objectives To quantify differences in the expression of angiogenesis and lymphangiogenesis growth factors, receptors, coreceptors as well as their antagonists in the uninvolved skin of patients with psoriasis compared with the skin of nonpsoriatic volunteers. Methods Skin biopsies were collected from the involved skin of 13 patients with untreated plaque-type psoriasis, from their nonlesional skin at distance from the lesions and from the skin of 16 healthy volunteers. The mRNA steady-state level of keratins 10, 14 and 16, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vimentin, collagen I and IV, proliferating cell nuclear antigen, the various splice variants of vascular endothelial growth factor, VEGF-A, VEGF-C and VEGF-D, their receptors VEGFR1, VEGFR2 and VEGFR3, neuropilin (NRP)-1 and its soluble forms, NRP-2, semaphorin 3A and prox-1, was measured by reverse transcription–polymerase chain reaction. Immunohistochemistry was performed for Ki-67, von Willebrand factor and D2-40. Blood and lymphatic vessel density, area and distance from epidermis were estimated by morphological analysis coupled to an original computer-assisted method of quantification. Results Skin from healthy volunteers and nonlesional skin from patients with psoriasis displayed similar histological, morphometric and proliferative features. However, a significant overexpression of VEGFR3, the VEGF-A isoform VEGF121, soluble 12 NRP-1 and GAPDH was observed in the nonlesional psoriatic skin as compared with that of normal volunteers. Conclusions These data point to significant differences in the blood and lymphatic vascular transcriptome between the clinically normal-appearing skin of patients with psoriasis and the skin of volunteers without psoriasis. [less ▲]

Determination of the ideal cyclodextrin concentrations to study their effect in the lipid rafts by the use of cholesterol extraction and cell viability studies in endothelial cells

Poster (2008, April)

VEGF111 : Dr Jekyll et Mr Hyde ?

in Medecine Sciences : M/S (2008), 24(6-7), 579-80