Gilles Carpentier

Heparan-like molecules induce the repair of skull defects

Heparin-binding growth factors (HBGFs) are known to stimulate bone repair when applied to bone lesions. Nevertheless, successful treatments are obtained with high protein doses since HBGFs are rapidly degraded in situ by multiple proteolytic activities associated with the inflammatory period of tissue healing. Like heparin or heparan sulfates, heparan-like molecules, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), are known to potentiate fibroblast growth factor activities by stabilizing them against pH, thermal or proteolytic denaturations, and by enhancing their binding with cell surface receptors. We have postulated that CMDBS stimulate in vivo bone healing by interacting with endogenous HBGFs, spontaneously released in the wounded site. The effect of CMDBS on bone repair was studied in a skull defect model in rats by computer-assisted radio-morphometry and histomorphometry. Single application of CMDBS in a collagen vehicle to skull defects induced a dose-dependent increase in bone defect closure and new bone formation after 35 days. Complete bony bridging occurred in defects treated with 3 micrograms CMDBS, whereas bone formation was not observed in vehicle-treated defects which contained only dense fibrous connective tissue between the defect margins. These results indicate that heparan-like molecules, such as CMDBS, are able to induce bone regeneration of skull defects. This action is possibly mediated by potentiation of endogenous growth factor activities and/or by neutralization of proteolytic activities.

A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins.

Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growth of tumor cell lines and that it also hampers angiogenesis. Additionally, we found that N6L is a proapoptotic molecule that increases Annexin V staining and caspase-3/7 activity in vitro and DNA fragmentation in vivo. Through affinity isolation experiments and mass-spectrometry analysis, we also identified nucleophosmin as a new N6L target. Notably, in mouse xenograft models, N6L administration inhibited human tumor growth. Biodistribution studies carried out in tumor-bearing mice indicated that following administration N6L rapidly localizes to tumor tissue, consistent with its observed antitumor effects. Our findings define N6L as a novel anticancer drug candidate warranting further investigation.

Alterations of overused supraspinatus tendon: A possible role of glycosaminoglycans and HARP/pleiotrophin in early tendon pathology

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 tenocytes 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.

Pleiotrophin Cellular Localization in Nerve Regeneration after Peripheral Nerve Injury

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.

Age-related Changes in Rat Myocardium Involve Altered Capacities of Glycosaminoglycans to Potentiate Growth Factor Functions and Heparan Sulfate-altered Sulfation

Background: Heparan sulfates (HS) are important cell behavior regulators. Results: With age, HS structural changes affect myocardial growth factor functionalities. Conclusion: This reveals the importance of HS on the control of essential tissue repair effectors during aging. Significance: Changes in cardiac HS may alter tissue homeostasis and impair heart function. This might also limit the success of protein therapies and implantation of therapeutic cells. Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF165 was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis.

Crucial events in myogenesis rely on the highly regulated spatiotemporal distribution of cell surface heparan sulfate proteoglycans to which are associated growth factors, thus creating a specific microenvironment around muscle cells. Most growth factors involved in control of myoblast growth and differentiation are stored in the extracellular matrix through interaction with specific sequences of glycosaminoglycan oligosaccharides, mainly heparan sulfate (HS). Different HS subspecies revealed by specific antibodies, have been shown to provide spatiotemporal regulation during muscle development. We have previously shown that glycosaminoglycan (GAG) mimetics called RGTA (ReGeneraTing Agent), stimulate muscle precursor cell growth and differentiation. These data suggest an important role of GAGs during myogenesis; however, little is yet known about the different species of GAGs synthesized during myogenesis and their metabolic regulation. We therefore quantified GAGs during myogenesis of C2.7 cells and show that the composition of GAG species was modified during myogenic differentiation. In particular, HS levels were increased during this process. In addition, the GAG mimetic RGTA, which stimulated both growth and differentiation of C2.7 cells, increased the total amount of GAG produced by these cells without significantly altering their rate of sulfation. RGTA treatment further enhanced HS levels and changed its sub-species composition. Although mRNA levels of the enzymes involved in HS biosynthesis were almost unchanged during myogenic differentiation, heparanase mRNA levels decreased. RGTA did not markedly alter these levels. Here we show that the effects of RGTA on myoblast growth and differentiation are in part mediated through an alteration of GAG species and provide an important insight into the role of these molecules in normal or pathologic myogenic processes.

Multivalent pseudopeptides targeting cell surface nucleoproteins inhibit cancer cell invasion through tissue inhibitor of metalloproteinases 3 (TIMP-3) release

Background: NucAnt 6L (N6L) binds to nucleoproteins and inhibits tumor growth. Results: N6L bound to sulfated glycosaminoglycans, induced TIMP-3 release, and inhibited cell invasion. Silencing of TIMP-3 abolished N6L effect on cell invasion. Conclusion: N6L inhibits cell invasion through the release of TIMP-3. Significance: TIMP-3 released by N6L inhibits cell invasion. Sulfated glycosaminoglycans are presented as new receptors for N6L. Blockage of the metastasis process remains a significant clinical challenge, requiring innovative therapeutic approaches. For this purpose, molecules that inhibit matrix metalloproteinases activity or induce the expression of their natural inhibitor, the tissue inhibitor of metalloproteinases (TIMPs), are potentially interesting. In a previous study, we have shown that synthetic ligands binding to cell surface nucleolin/nucleophosmin and known as HB 19 for the lead compound and NucAnt 6L (N6L) for the most potent analog, inhibit both tumor growth and angiogenesis. Furthermore, they prevent metastasis in a RET transgenic mice model which develops melanoma. Here, we investigated the effect of N6L on the invasion capacity of MDA-MB-435 melanoma cells. Our results show that the multivalent pseudopeptide N6L inhibited Matrigel invasion of MDA-MB-435 cells in a modified Boyden chamber model. This was associated with an increase in TIMP-3 in the cell culture medium without a change in TIMP-3 mRNA expression suggesting its release from cell surface and/or extracellular matrix. This may be explained by our demonstrated N6L interaction with sulfated glycosaminoglycans and consequently the controlled bioavailability of glycosaminoglycan-bound TIMP-3. The implication of TIMP-3 in N6L-induced inhibition of cell invasion was evidenced by siRNA silencing experiments showing that the loss of TIMP-3 expression abrogated the effect of N6L. The inhibition of tumor cell invasion by N6L demonstrated in this study, in addition to its previously established inhibitory effect on tumor growth and angiogenesis, suggests that N6L represents a promising anticancer drug candidate warranting further investigation.

Antitumor and Angiostatic Activities of the Antimicrobial Peptide Dermaseptin B2

Recently, we have found that the skin secretions of the Amazonian tree frog Phyllomedusa bicolor contains molecules with antitumor and angiostatic activities and identified one of them as the antimicrobial peptide dermaseptin (Drs) B2. In the present study we further explored the in vitro and in vivo antitumor activity of this molecule and investigated its mechanism of action. We showed that Drs B2 inhibits the proliferation and colony formation of various human tumor cell types, and the proliferation and capillary formation of endothelial cells in vitro. Furthermore, Drs B2 inhibited tumor growth of the human prostate adenocarcinoma cell line PC3 in a xenograft model in vivo. Research on the mechanism of action of Drs B2 on tumor PC3 cells demonstrated a rapid increasing amount of cytosolic lactate dehydrogenase, no activation of caspase-3, and no changes in mitochondrial membrane potential. Confocal microscopy analysis revealed that Drs B2 can interact with the tumor cell surface, aggregate and penetrate the cells. These data together indicate that Drs B2 does not act by apoptosis but possibly by necrosis. In conclusion, Drs B2 could be considered as an interesting and promising pharmacological and therapeutic leader molecule for the treatment of cancer.

Beneficial use of fibroblast growth factor 2 and RGTA, a new family of heparan mimics, for endothelialization of PET prostheses.

We have studied the endothelialization of polyethylene terephtalate (PET) prostheses coated with collagen by adult human saphenous endothelial cells (EC) under various in vitro conditions. Collagenous PET was impregnated either by Fibroblast Growth Factor 2 (FGF2), heparin, a synthetic heparan sulfate mimic named RGTA 11 (for ReGeneraTing Agent), or combinations of these products. RGTA 11 belongs to a new family of drugs, which have been previously described as stabilizer and protector of heparin binding growth factors (HBGF), and to act in vivo as to stimulate wounded tissue repair. As endothelialization of prosthesis can be obtained in vivo after EC seeding and/or by transanastomotic, as well as by transprosthetic EC migrations, we have designed in vitro models to study the growth of EC seeded on PET, the EC colonization of an acellular area on PET, and the migration of EC from a collagen gel through the prosthesis. The combinations of either RGTA11 or heparin with FGF2 enhanced after a week by 5-fold the growth of seeded EC compared to RGTA or heparin alone and by 3-fold compared to FGF2 alone (p < 0.05). More than 80% of the colonization of an acellular area was achieved within 6 days when FGF2 was combined with RGTA11 or heparin. In contrast, colonization was only of 20% promoted in presence of FGF2 alone and not promoted in the presence of RGTA or heparin alone (p < 0.05). In addition, transprosthetic migration of EC and endothelialization of the luminal side were observed only when gel contained RGTA11 or heparin in combination with FGF2. The present work did strongly indicate that RGTA11 could be used in vivo as to improve endothelialization and should be the focus of continued investigation.

Motoneuron morphological alterations before and after the onset of the disease in the wobbler mouse

The wobbler mutant mouse displays a recessively inherited neurological disease with degeneration of motoneurons and is considered to be an animal model for human motoneuron diseases. Mutant mice can be clinically recognised at about 3-4 weeks of age but a polymorphic marker close to the wobbler gene offers the opportunity of a preclinical diagnosis. Using this polymorphic marker we performed morphometric (cell size) analysis of spinal cord motoneurons from 10 to 40 days post natal (PN). We observed at day 16 PN a transient appearance of swollen motoneurons, probably those that present vacuolar degeneration a little later and possibly die. One week later, from 21 days onwards, we found that the subpopulation of large motoneurons was depleted in the mutant mice. The absence of large motoneurons may have important physiological consequences and the loss or absence of differentiation of this particular subpopulation of motoneurons may be a key event in the course of the disease.