Non-clinical assessment of lubrication and free radical scavenging of an innovative non-animal carboxymethyl chitosan biomaterial for viscosupplementation: An in-vitro and ex-vivo study.

OBJECTIVE: Lubrication and free radical scavenging are key features of biomaterials used for viscosupplementation (VS) of joints affected by osteoarthritis (OA). The objective of this study was to describe the non-clinical performance characterization of KiOmedine® CM-Chitosan, a non-animal carboxymethyl chitosan, in order to assess its intended action in VS and to compare it to existing viscosupplements based on crosslinked hyaluronan (HA) formulations. METHOD: The lubrication capacity of the tested viscosupplements (VS) was evaluated in-vitro and ex-vivo. In-vitro, the coefficient of friction (COF) was measured using a novel tribological system. Meanwhile, an ex-vivo biomechanical model in ovine hindlimbs was developed to assess the recovery of join mobility after an intra-articular (IA) injection. Free radical scavenging capacity of HA and KiOmedine® CM-Chitosan formulations was evaluated using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. RESULTS: In the in-vitro tribological model, KiOmedine® CM-Chitosan showed high lubrication capacity with a significant COF reduction than crosslinked HA formulations. In the ex-vivo model, the lubrication effect of KiOmedine® CM-Chitosan following an IA injection in the injured knee was proven again by a COF reduction. The recovery of joint motion was optimal with an IA injection of 3 ml of KiOmedine® CM-Chitosan, which was significantly better than the crosslinked HA formulation at the same volume. In the in-vitro TEAC assay, KiOmedine® CM-Chitosan showed a significantly higher free radical scavenging capacity than HA formulations. CONCLUSION: Overall, the results provide a first insight into the mechanism of action in terms of lubrication and free radical scavenging for the use of KiOmedine® CM-Chitosan as a VS treatment of OA. KiOmedine® CM-Chitosan demonstrated a higher capacity to scavenge free radicals, and it showed a higher recovery of mobility after a knee lesion than crosslinked HA formulations. This difference could be explained by the difference in chemical structure between KiOmedine® CM-Chitosan and HA and their formulations.

Safety and efficacy of a carboxymethyl chitosan dermal injection device for the treatment of skin defects: a first-in-man, pilot, comparative, split-body study.

Injectable soft-tissue devices are increasingly used for improving skin defects and deficiencies related to ageing. To assess the safety and efficacy of KIO015, a new injectable soft-tissue device formulated with carboxymethyl chitosan for the intradermal treatment of skin defects associated with ageing. Twenty-two subjects (40-65 years) were randomized to receive injections in the neckline of KIO015 and a non-cross-linked HA-based device, and were followed for up to 10 months. Injection site reactions (ISRs) and adverse events (AEs) were documented. Skin improvement was assessed instrumentally and clinically. Skin biopsies at injection zones in the lower back were taken at Day 28 for histopathology and immunohistochemistry analyses, to further assess product performance. Histomorphometric analyses on rabbits and in vitro assessment of KIO015 antioxidant capacity were also conducted. KIO015 was very well tolerated. Only expected and transient ISRs were observed; mainly erythema and hematoma. No adverse local effects or foreign body granuloma were observed histologically. Both clinical and instrumental evaluations confirmed the performance of KIO015. The skin was firmer and more elastic. Skin hydration showed significant improvement three days after injection. KIO015 exhibited superior overall maintenance of skin hydration after 10 months as compared to HA. These clinical results were supported by in vitro trials and implantation tests in the rabbit. The results from this pilot study support the use of KIO015 as an innovative alternative to HA-based devices for intradermal treatment of skin disorders.

Soluble biomarkers development in osteoarthritis: from discovery to personalized medicine.

CONTEXT: Specific soluble biomarkers could be a precious tool for diagnosis, prognosis and personalized management of osteoarthritic (OA) patients. OBJECTIVE: To describe the path of soluble biomarker development from discovery to clinical qualification and regulatory adoption toward OA-related biomarker qualification. METHODS AND RESULTS: This review summarizes current guidance on the use of biomarkers in OA in clinical trials and their utility at five stages, including preclinical development and phase 1 to phase 4 trials. It also presents all the available regulatory requirements. CONCLUSIONS: The path through the adoption of a specific soluble biomarker for OA is steep but is worth the challenge due to the benefit that it can provide.

Mitoproteome plasticity of rat brown adipocytes in response to cold acclimation.

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.

Proton leak induced by reactive oxygen species produced during in vitro anoxia/reoxygenation in rat skeletal muscle mitochondria.

Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenation in vitro. Production of superoxide anion was detected after several cycles of anoxia/reoxygenation. Concomitantly, a decrease of state 3 respiration and phosphorylation yield (ADP/O) were observed. The latter resulted from a proton leak. The presence of palmitic acid during anoxia/reoxygenation cycles led to a dose-dependent inhibition of superoxide anion production together with a partial protection of the ADP/O ratio measured after anoxia/reoxygenation. The ADP/O decrease was shown to be due to a permeability transition pore-sustained proton leak, as it was suppressed by cyclosporine A. The permeability transition pore activation was induced during anoxia/reoxygenation by superoxide anion, as it was cancelled by the spin trap (POBN), which scavenges superoxide anion and by palmitic acid, which induces mitochondrial uncoupling. It can be proposed that the palmitic acid-induced proton leak cancels the production of superoxide anion by mitochondria during anoxia/reoxygenation and therefore prevents the occurrence of the superoxide anion-induced permeability transition pore-mediated proton leak after anoxia/reoxygenation.

Mitochondrial UCPs: new insights into regulation and impact.

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.

Mitochondrial uncoupling proteins: new insights from functional and proteomic studies.

Mitochondria are the major sites of ATP synthesis through oxidative phosphorylation, a process that is weakened by proton leak. Uncoupling proteins are mitochondrial membrane proteins specialized in inducible proton conductance. They dissipate the proton electrochemical gradient established by the respiratory chain at the expense of reducing substrates. Several physiological roles have been suggested for uncoupling proteins, including roles in the control of the cellular energy balance and in preventive action against oxidative stress. This review focuses on new leads emerging from comparative proteomics about the involvement of uncoupling protein in the mitochondrial physiology. A brief overview on uncoupling proteins and on proteomics applied to mitochondria is also presented herein.

Saccharomyces cerevisiae mitoproteome plasticity in response to recombinant alternative ubiquinol oxidase.

The energy-dissipating alternative oxidase (AOX) from Hansenula anomala was expressed in Saccharomyces cerevisiae. The recombinant AOX was functional. A comparative analysis by two-dimensional differential in-gel electrophoresis (2D-DIGE) of mitochondrial protein patterns found in wild-type and recombinant AOX strains was performed. 60 proteins exhibiting a significant difference in their abundance were identified. Interestingly, proteins implicated in major metabolic pathways such as Krebs cycle and amino acid biosynthesis were up-regulated. Surprisingly, an up-regulation of the respiratory-chain complex III was associated with a down-regulation of the ATP synthase complex.

Uncoupling protein 1 affects the yeast mitoproteome and oxygen free radical production.

Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.

Steatosis-induced proteomic changes in liver mitochondria evidenced by two-dimensional differential in-gel electrophoresis.

Steatosis encompasses the accumulation of droplets of fats into hepatocytes. In this work, we performed a comparative analysis of mitochondrial protein patterns found in wild-type and steatosis-affected liver using the novel technique two-dimensional differential in-gel electrophoresis (2D-DIGE). A total of 56 proteins exhibiting significant difference in their abundances were unambiguously identified. Interestingly, major proteins that regulate generation and consumption of the acetyl-CoA pool were dramatically changed during steatosis. Many proteins involved in the response to oxidative stress were also affected.

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria.

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL.

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein.

Mitochondrial respiratory chain complex patterns from Acanthamoeba castellanii and Lycopersicon esculentum: comparative analysis by BN-PAGE and evidence of protein-protein interaction between alternative oxidase and complex III.

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria.

Redox state of endogenous coenzyme q modulates the inhibition of linoleic acid-induced uncoupling by guanosine triphosphate in isolated skeletal muscle mitochondria.

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria.

Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast.

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.