Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody.

BACKGROUND: Mounting experimental evidence has underscored the remarkable role played by the Wnt family of proteins in the spinal cord functioning and therapeutic potential in spinal cord injury (SCI). We aim to provide a therapeutic prospect associated with the modulation of canonical Wnt signaling, examining the spatio-temporal expression pattern of Dickkopf-1 (Dkk1) and its neutralization after SCI. We employ an intraparenchymal injection of the clinically validated Dkk1-blocking antibody, BHQ880, to elucidate its effects in SCI. METHODS: A rat model of contusion SCI was used. Histological analyses were performed, wherein Dkk1 protein was sought, and ELISA analyses were employed for Dkk1 detection in cerebrospinal fluid and serum. To ascertain the BHQ880 therapeutic effect, rats were subjected to SCI and then injected with the antibody in the lesion epicenter 24 hours post-injury (hpi). Subsequent evaluation of motor functional recovery extended up to 56 days post-injury (dpi). qRT-PCR and histological analyses were conducted. RESULTS: We demonstrate the presence of Dkk1 in the healthy rat spinal cord, with pronounced alterations observed following injury, primarily concentrated in the epicenter regions. Notably, a significative upregulation of Dkk1 was detected at 24 hpi, peaking at 3 dpi and remaining elevated until 42 dpi. Moreover, we revealed that early administration of BHQ880 considerably improved motor functional recovery, promoted preservation of myelinated tissue, and reduced astroglial and microglia/macrophage reactivity. Furthermore, there was a decrease in the acute expression of different inflammatory genes. CONCLUSIONS: Collectively, our findings highlight the therapeutic potential of BHQ880 treatment in the context of SCI.

Silk-Elastin-like Polymers for Acute Intraparenchymal Treatment of the Traumatically Injured Spinal Cord: A First Systematic Experimental Approach.

Despite the promising potential of hydrogel-based therapeutic approaches for spinal cord injury (SCI), the need for new biomaterials to design effective strategies for SCI treatment and the outstanding properties of silk-elastin-like polymers (SELP), the potential use of SELPs in SCI is currently unknown. In this context, we assessed the effects elicited by the in vivo acute intraparenchymal injection of an SELP named (EIS)2-RGD6 in a clinically relevant model of SCI. After optimization of the injection system, the distribution, structure, biodegradability, and cell infiltration capacity of (EIS)2-RGD6 were assessed. Finally, the effects exerted by the (EIS)2-RGD6 injection-in terms of motor function, myelin preservation, astroglial and microglia/macrophage reactivity, and fibrosis-were evaluated. We found that (EIS)2-RGD6 can be acutely injected in the lesioned spinal cord without inducing further damage, showing a widespread distribution covering all lesioned areas with a single injection and facilitating the formation of a slow-degrading porous scaffold at the lesion site that allows for the infiltration and/or proliferation of endogenous cells with no signs of collapse and without inducing further microglial and astroglial reactivity, as well as even reducing SCI-associated fibrosis. Altogether, these observations suggest that (EIS)2-RGD6-and, by extension, SELPs-could be promising polymers for the design of therapeutic strategies for SCI treatment.

Efficacy of human HC016 cell transplants on neuroprotection and functional recovery in a rat model of acute spinal cord injury.

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 (H2 O2 ), 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 H2 O2 -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.

Influence of the Cation Adducts in the Analysis of Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry Data from Injury Models of Rat Spinal Cord.

Imaging mass spectrometry (IMS) is quickly becoming a technique of reference to visualize the lipid distribution in tissue sections. Still, many questions remain open, and data analysis has to be optimized to avoid interpretation pitfalls. Here we analyze how the variation on the [Na+]/[K+] relative abundance affects the detection of lipids between sections of spinal cord of (uninjured) control rats and of models of spinal cord demyelination and traumatic contusion injury. The [M + Na]+/[M + K]+ adducts ratio remained approximately constant along transversal and longitudinal sections of spinal cord from control animals, but it strongly changed depending on the type of lesion. A substantial increase in the abundance of [M + Na]+ adducts was observed in samples from spinal cord with demyelination, while the intensity of the [M + K]+ adducts was stronger in those sections from mechanically injured spinal cords. Such changes masked the modifications in the lipid profile due to the injury and only after summing the signal intensity of all adducts and corresponding monoprotonated molecular ions of each detected lipid in a single variable, it was possible to unveil the real changes in the lipid profile due to the lesion. Such lipids included glycerophospholipids (both diacyl and aryl-acyl), sphingolipids, and nonpolar lipids (diacyl and triacylglycerols), which are the main lipid classes detected in positive-ion mode. Furthermore, the results demonstrate the sensitivity of the technique toward modification in tissue homeostasis and that the [M + Na]+/[M + K]+ ratio may be used to detect alterations in such homeostasis.

Differential expression of Wnts after spinal cord contusion injury in adult rats.

BACKGROUND: Spinal 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. FINDINGS: Using 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 ß-catenin protein, both of which suffer a dramatic change, from a homogeneous expression pattern in the grey matter to a disorganized injury-induced pattern. CONCLUSIONS: Our results suggest a role for Wnts in spinal cord homeostasis and injury. We demonstrate that after injury Wnt signalling is activated via the Wnt/ß-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.

Inadequate activation of the GTPase RhoA contributes to the lack of fibronectin matrix assembly in von Hippel-Lindau protein-defective renal cancer cells.

The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL+ cells. We have previously reported that VHL(-) cells fail to form beta1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.

The heparin III-binding domain of fibronectin (III4-5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly.

Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4-5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4-5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4-5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4-5. These interactions were specific since FNIII4-5 did not bind to the FNIII7-10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4-5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4-5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated alpha4beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135-142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4-5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimer.

Activation pathways of alpha4beta1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation.

Alpha4beta1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. Alpha4beta1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti-beta1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of 41 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, alpha-CD3 mAb or the chemokine SDF-1alpha followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (alpha4beta1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the alpha4beta1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of alpha4beta1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements.

Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration.

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 FNIII7-10+FNIII4-5. 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.

A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells.

Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with alpha5beta1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with alpha5beta1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7-FNIII10 (FNIII7-10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7-10 or adding soluble HBP/III5 to cells prespread on FNIII7-10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to alpha5beta1 for the progression of the cytoskeletal response and that these include activation of RhoA.