BackgroundSpinal cord injury is a major cause of disability that has no clinically accepted treatment. Functional decline following spinal cord injury is caused by mechanical damage, secondary cell death, reactive gliosis and a poor regenerative capacity of damaged axons. Wnt proteins are a family of secreted glycoproteins that play key roles in different developmental processes although little is known of the expression patterns and functions of Wnts in the adult central nervous system in normal or diseased states.FindingsUsing qRT-PCR analysis, we demonstrate that mRNA encoding most Wnt ligands and soluble inhibitors are constitutively expressed in the healthy adult spinal cord. Strikingly, contusion spinal cord injury induced a time-dependent increase in Wnt mRNA expression from 6 hours until 28 days post-injury, and a narrow peak in the expression of soluble Wnt inhibitors between 1 and 3 days post-injury. These results are consistent with the increase in the migration shift, from day 1 to 7, of the intracellular Wnt signalling component, Dishevelled-3. Moreover, after an initial decrease by 1 day, we also found an increase in phosphorylation of the Wnt co-receptor, low-density lipoprotein receptor-related protein 6, and an increase in active β-catenin protein, both of which suffer a dramatic change, from a homogeneous expression pattern in the grey matter to a disorganized injury-induced pattern.ConclusionsOur results suggest a role for Wnts in spinal cord homeostasis and injury. We demonstrate that after injury Wnt signalling is activated via the Wnt/β-catenin and possibly other pathways. These findings provide an important foundation to further address the function of individual Wnt proteins in vivo and the pathophysiology of spinal cord injury.

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4 -5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4 -5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10ϩFNIII4 -5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.

1 repeats may also promote binding of the I 1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in combination, inhibited binding of 125 I-labeled 70-kDa fragments to cycloheximide-treated cells plated on the 160-kDa substrate, suggesting that additional I 1-5 binding sites, independent of the III 1 and III 12-14 repeats, may be involved in fibrillogenesis. Fibronectin is required for normal growth and development (1) and plays an important role in regulating cell attachment and movement, wound healing, and tumorigenesis (for review, see Refs. 2 and 3). It is a 500-kDa disulfide-bonded dimer consisting of similar subunits and is found as a soluble glyco-protein in blood and other body fluids and as an insoluble fibrous matrix component in tissues. Each subunit of fibronec-tin consists of three different types of repeating sequences, called types I, II, and III, which are arranged into discrete structural and functional modules. Assembly of dimeric fibronectin into the extracellular matrix involves multiple consecutive binding interactions with inte-grin receptors, with itself, and with matrix components such as type I collagen (for review, see Refs. 4-6). Although the 5 1 integrin appears to be the primary fibronectin receptor involved in matrix assembly (7-12), at least two other integrins, IIb 3 and v 3 , can also support fibronectin fibrillogenesis (13, 14). High affinity binding interactions between these integrins and the RGD site in the 10th type III repeat (III 10) of fibronec-tin are thought to promote fibrillogenesis by exposing appropriate self-assembly sites in fibronectin. Such sites may become exposed through local integrin-induced conformational changes in III 10 repeats (15) or through integrin-mediated stretching (reversible unfolding) of one or a whole array of type III repeats in fibronectin in response to cell movements (13, 16). Self-assembly of fibronectin dimers into fibrils is currently thought to involve primarily interactions between the first five type I repeats (I 1-5) and the first type III (III 1) repeat (17, 18). The I 1-5 repeats are critical for matrix assembly, i.e. peptides including these repeats block assembly of fibronectin into fibrils, and fibronectin dimers lacking these repeats will not be incorporated into fibrils (19-22). The III 1 repeats are also important for fibril formation, and either anti-III 1 monoclonal antibodies or peptides derived from III 1 repeats can block assembly of fibronectin into matrix (23, 24). The mechanism, however, by which I 1-5-III 1 interactions affect matrix assembly remains controversial. For example, Hocking et al. (25) have shown that III 1 repeats will interact not only with I 1-5 repeats but also with heat-denatured III 10 repeats. They have proposed that the latter interaction activates the III 1 repeat thereby allowing it to function as a receptor for the amino termini of a second fibronectin dimer. In contrast, Sechler et al. (26) have shown that fibronectin dimers lacking III 1-7 repeats are readily polymerized into fibrils. In fac...

Spinal cord injury (SCI) is a devastating event with huge personal and social costs, for which there is no effective treatment. Cell therapy constitutes a promising therapeutic approach for SCI; however, its clinical potential is seriously limited by their low survival in the hostile conditions encompassing the acute phase of SCI. Human HC016 (hHC016) cells, generated from expanded human adipose mesenchymal stem cells (hAMSCs) and pulsed with a patented protocol with hydrogen peroxide (H2O2), are expected to acquire improved resistance to oxidative environments which appears as a major limiting factor hampering the engrafting success. Our specific aim was to assess whether H2O2‐pulsed hHC016 cells had an improved survival and thus therapeutic efficacy in a rat contusion model of acute SCI when grafted 48 hr after injury. Functional recovery was evaluated up to 56 days post‐injury (dpi) by locomotor (open field test and CatWalk) and sensory (Von Frey and Hargreaves) tests. Besides, histological evaluation of transplanted cell survival and tissue protection/regeneration was also performed. Functional results showed a statistically significant improvement on locomotor recovery outcomes with hHC016 cells. Accordingly, superior cell survival in correlation with long‐term neuroprotection, higher axonal regeneration, and reduced astroglial and microglial reactivity was also observed with hHC016 cells. These results demonstrate an enhanced survival capacity of hHC016 cells resulting in improved functional and histological outcomes as compared with hAMSCs, indicating that hHC016 cell transplants may constitute a promising cell therapy for acute SCI.