Synthesis and Evaluation of Neuroprotective α,β-Unsaturated Aldehyde Scavenger Histidyl-Containing Analogues of Carnosine

The synthesis, scavenging activity, and cytoprotective profiles of histidyl-containing carnosine analogues bearing hydrazide or 1,2-diol moieties is reported. Some compounds have demonstrated higher aldehyde-sequestering efficiency than carnosine and were also efficient in protecting SH-SY5Y neuroblastoma cells and rat hippocampal neurons from 4-hydroxy-trans-2,3-nonenal (HNE)-mediated death. The cytoprotective efficacy of these compounds suggests their potential use as therapeutic agents for disorders that involve excessive membrane lipids peroxidation and HNE-mediated neuronal toxicity

Identification of matrix metalloproteinase-2 (MMP-2)-specific cleavage of MMP-2-cleavable peptide complex, MFK902.

(A) Sequence of recombinant murine MFK902 consisting of MFK00 (murine 4th fas-1 peptide truncated for the H1 and H2 sequences) and an RGD motif linked by a MMP-2 substrate (GPLGVRG). (B) Schematic representation of the pET-29b(+) vector containing the MFK902 sequence. (C) Immunoblot of purified recombinant MFK902. (D) MMP-2–specific cleavage of MFK902 was performed as detailed in the Methods section.</p

Stem Cell-Derived Extracellular Vesicle-Bearing Dermal Filler Ameliorates the Dermis Microenvironment by Supporting CD301b-Expressing Macrophages

Hyaluronic acid-based hydrogels (Hyal-Gels) have the potential to reduce wrinkles by physically volumizing the skin. However, they have limited ability to stimulate collagen generation, thus warranting repeated treatments to maintain their volumizing effect. In this study, stem cell-derived extracellular vesicle (EV)-bearing Hyal-Gels (EVHyal-Gels) were prepared as a potential dermal filler, ameliorating the dermis microenvironment. No significant differences were observed in rheological properties and injection force between Hyal-Gels and EVHyal-Gels. When locally administered to mouse skin, Hyal-Gels significantly extended the biological half-life of EVs from 1.37 d to 3.75 d. In the dermis region, EVHyal-Gels induced the overexpression of CD301b on macrophages, resulting in enhanced proliferation of fibroblasts. It was found that miRNAs, such as let-7b-5p and miR-24-3p, were significantly involved in the change of macrophages toward the CD301bhi phenotype. The area of the collagen layer in EVHyal-Gel-treated dermis was 2.4-fold higher than that in Hyal-Gel-treated dermis 4 weeks after a single treatment, and the collagen generated by EVHyal-Gels was maintained for 24 weeks in the dermis. Overall, EVHyal-Gels have the potential as an antiaging dermal filler for reprogramming the dermis microenvironment

Down-regulation of Cpn10 promotes mitochondrial dysfunction in SK-N-MC cells.

<p>(A-C), SK-N-MC cells were transfected with a control scrambled (Sc) or Cpn10 siRNA (si #1, si #2). After 5 days, the alteration of mitochondrial membrane potential was monitored by the MitoProbe JC-1 assay (A). The cellular total ATP level was examined by an ATP bioluminescence detection assay (B). The Intracellular ROS level was measured by a DCFH-DA fluorescence ROS detection assay (C). Data are represented as the mean ± SEM (n>3), and were considered significant at a value of *<i>p</i><0.05.</p

Down-regulation of Cpn10 induces mitochondrial fragmentation in neuroblastoma cells.

<p>(A, B) SK-N-MC cells stably expressing mito-YFP (SK/mito-YFP) were transfected with either a control scrambled siRNA (Sc) or a specific siRNA against Cpn10 for 5 days. Then mitochondrial morphology (A) and mitochondrial length (B) were examined with a fluorescence microscope. Both Drp1 siRNA (siDrp1) and Opa1 siRNA (siOpa1) were used as positive controls. (C) The reduced expression of Cpn10 by siRNA was confirmed by Western blotting. (D) SK/mito-YFP cells were transfected with scrambled siRNA or Cpn10 siRNAs (si#1, si#2). And the mitochondrial fragmentation was observed by a fluorescence microscopy at the indicated time points. (E) SH-SY5Y cells were transfected with either a control scrambled siRNA (Sc) or specific siRNAs against Cpn10 (siCPN10 #1, #2). After 5 days, the cells were stained with Mito-tracker (100 nM), and the cells containing fragmented mitochondria were counted using a fluorescence microscope. Data are represented as the mean ± SEM. (n>3).</p

MFK902 inhibition of NIH3T3 cell adhesion and migration.

<p>βig-h3-derivatives, including RGD (GGRGDSP), MFK00, and MFK902 peptides, were prepared as described in the Methods section. Inhibition of adhesion by (A) the RGD and RGE, (B) MFK00, and (C) MFK902 peptides. Inhibition of migration by (D) the RGD and RGE, (E) MFK00, and (F) MFK902 peptides. *<i>p</i> < 0.05. Values are presented as the mean ± SEM.</p

Amelioration of histopathologic severity in mice with CIA after MFK902 treatment.

(A) Representative histopathologic findings of ankle joints from control and MFK902-treated mice on day 50. Upper panel, H&E-stained joints. Lower panels, Immunohistochemical staining for CD3, CD31, and ICAM-1 (brown). (B) Histologic scoring of arthritis severity in control and MFK902-treated mice (mg/kg). Histologic scores for synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion (range: 0–3 for each parameter) were quantified as described in the Methods section. *p < 0.05 vs. controls. Data are expressed as the mean ± SEM. ICAM-1, intercellular adhesion molecule-1. Scale bar: 50 μm</p

Modulation of clinical arthritis in mice with collagen-induced arthritis (CIA) after MFK902 treatment.

<p>(A) Distribution of Cy5.5-labeled MFK902 in the mice with active CIA. MFK902-Cy5.5 or free Cy5.5 were intravenously injected into mice and then near-infrared fluorescence (NIRF) images were captured using an eXplore Optix system. Representative color-coded images and total photon counts (mean at ± SD) indicated times are shown. (B-C) Mice with CIA were treated intraperitoneally with MFK902 daily at indicated doses, methotrexate (MTX, 1 mg/kg twice weekly), or phosphate-buffered saline (Control) beginning on day 23 after the first immunization. Arthritis scores are shown for 7–8 mice per group. (B) Clinical arthritis index. (C) Incidence of paw involvement. *<i>p</i> < 0.05. Data are expressed as the mean ± SEM.</p

Suppression of Cpn10 exacerbates 3-NP-mediated mitochondrial dysfunction in SK-N-MC cells.

<p>(A) SK-N-MC cells were transfected with either scrambled siRNA or Cpn10 siRNA (si#1, si#2) with or without Drp1 siRNA (siDrp1) for 5 days. The cells were further exposed to 3-NP (10 mM) for 8 hr, then cells with fragmented mitochondria were counted with a fluorescence microscope. (B, C) SK-N-MC cells were transfected with either scrambled siRNA or Cpn10 siRNA (si#1, si#2) with or without Drp1 siRNA (siDrp1) for 5 days, then cells were further exposed to 3-NP (10 mM) for 8 hr. The total cellular ATP level was measured by an ATP bioluminescence detection assay (B). The intracellular ROS level was determined by a DCFH-DA fluorescence ROS detection assay (C). Data are represented as the mean ± SEM (n>3), and were considered significant at a value of *<i>p</i><0.02, **<i>p</i><0.05.</p

Loss of Cpn10 potentiates 3-NP-mediated cell death in SK-N-MC cells.

<p>(A) SK/mito-YFP cells were transfected with scrambled siRNA (Sc) or Cpn10 siRNA (si#1, si#2). After 5 days, the cells were further incubated with 3-NP (10 mM) in the presence or absence of a ROS inhibitor, NAC (1 mM) for 24 hr. Then cells were stained with Annexin V and propidium iodide to measure cell viability by a flow cytometric analysis. Data are represented as the mean ± SEM (n>3), and were considered significant at a value of *<i>p</i><0.02.</p