Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-beta1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5 ؊/؊ mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced ␣-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGF␤ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5 ؊/؊ cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan hdf/؉ ) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5 ؊/؊ cells resulted from increased PCM versican content, we generated Adamts5 ؊/؊ ;Vcan hdf/؉ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5 ؊/؊ mice. In Adamts5 ؊/؊ fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.In addition to the role of cells in building specialized structural extracellular matrix, they maintain a pericellular matrix (PCM) 2 that is dynamic, provisional, and intimately associated with the cell surface (1, 2). Thus, it is appropriate to view a cell and its PCM as a functional unit. Processes that could potentially be regulated by PCM include macromolecular assembly (e.g. assembly of collagen, fibronectin fibrils, or fibrillin microfibrils), cell surface receptor-ligand interactions, binding of pathogens, and cell-matrix and cell-cell adhesion. PCM is sometimes referred to as the glycocalyx because of its abundant carbohydrate content (3). The PCM in many cell types such as chondrocytes, ova, neurons (in which PCM is also termed the perineuronal net), vascular smooth muscle cells, and fibroblasts contains aggregates of hyaluronan (HA) with chondroitin sulfate proteoglycans (CSPG) (4 -7). These aggregates are formed from non-covalent association of HA with large CSPGs such as aggrecan, versican, brevican, and neurocan. In contrast to the aforementioned cell types, heparan sulfa...

Neural stem/progenitor cells (NSCs) proliferate vigorously as neurospheres in medium containing basic fibroblast growth factor (FGF-2), but start differentiating into neurons, astrocytes or oligodendrocytes in FGF-2-free medium. An extract of royal jelly (RJ) significantly increased the percentage in the total cell population of not only neurons immunoreactive for class III β-tubulin (Tuj1) but also astrocytes immunoreactive for glial fibrillary acidic protein (GFAP), and oligodendrocytes immunoreactive for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) generated from NSCs, but decreased that of nestin-positive NSCs. These results highlight a novel and outstanding property of the RJ, i.e., that it facilitates the differentiation of all types of brain cells (neurons, astrocytes, and oligodendrocytes). On the other hand, 10-hydroxy-trans-2-decenoic acid (HDEA), an unsaturated fatty acid characteristic of RJ, increased the generation of neurons and decreased that of astrocytes from NSCs. These observations suggest that RJ contains plural components that differently influence neuronal and/or glial lineages and that HDEA is one of such components of RJ that facilitates neurogenesis by NSCs.

Objective. To identify the T cells responsive to ␤ 2 -glycoprotein I (␤ 2 GPI) that mediate antiphospholipid antibody production in patients with antiphospholipid syndrome (APS).Methods. In vitro proliferative responses and anti-␤ 2 GPI antibody production induced by ␤ 2 GPI were examined in peripheral blood mononuclear cell (PBMC) cultures from 12 APS patients, 13 systemic lupus erythematosus patients without APS, and 12 healthy donors.Results. Peripheral blood T cells from all subjects failed to respond to ␤ 2 GPI in its native form. In contrast, reduced ␤ 2 GPI was able to stimulate T cells not only from all 12 patients with anti-␤ 2 GPI antibodies, but also from 10 of 25 individuals without anti-␤ 2 GPI antibodies. The specificity of the responses to ␤ 2 GPI was confirmed by activation of the reduced ␤ 2 GPI-primed T cells by recombinant ␤ 2 GPI in secondary cultures. Characterization of the T cell response induced by ␤ 2 GPI revealed that the response was associated with the presence of the DR53-associated alleles, the responding T cells were CD4؉ and restricted by HLA class II, and antigenic peptides were located in domains IV and/or V. Anti-␤ 2 GPI antibody production was induced specifically in anti-␤ 2 GPI antibody-positive patients, in PBMC cultures with reduced ␤ 2 GPI. Anti-␤ 2 GPI antibodies produced in vitro recognized ␤ 2 GPI immobilized with cardiolipin or ␤ 2 GPI coated on "high-binding" polystyrene plates.Conclusion. These results strongly suggest that CD4؉ and HLA class II-restricted T cells responsive to ␤ 2 GPI are involved in the production of antiphospholipid antibodies in APS patients.

No abstract

Trimethyltin (TMT) is a toxic organotin compound that induces acute neuronal death selectively in the hippocampal dentate gyrus (DG) followed by cognition impairment; however the TMT-injured hippocampal DG itself is reported to regenerate the neuronal cell layer through rapid enhancement of neurogenesis. Neural stem/progenitor cells (NS/NPCs) are present in the adult hippocampal DG, and generate neurons that can function for the cognition ability. Therefore, we investigated whether royal jelly (RJ) stimulates the regenerating processes of the TMT-injured hippocampal DG, and found that orally administered RJ significantly increased the number of DG granule cells and simultaneously improved the cognitive impairment. Furthermore, we have already shown that RJ facilitates neurogenesis of cultured NS/NPCs. These present results, taken together with previous observations, suggest that the orally administered RJ may be a promising avenue for ameliorating neuronal function by regenerating hippocampal granule cells that function in the cognition process.

We showed earlier that neurite outgrowth of rat pheochromocytoma PC12 cells was stimulated by royal jelly extract (PERJ) or its unique component, AMP N 1 -oxide, via adenosine A2a receptors. In this study, we found that stimulated neurite outgrowth occurred in medium supplemented with serum, but not in serum-free medium. The pentapeptide GRGDS, which includes the RGD sequence commonly shared by extracellular matrix (ECM) components, could attenuate the effect of serum, suggesting that integrin receptor signaling was essential for the neurite outgrowth induced by PERJ or AMP N 1 -oxide. PERJ or AMP N 1 -oxide also activated extracellular signal-regulated kinases 1 or 2 (ERK1/2); however, this activation was not associated with the neurite outgrowth. As it is known that Mn 2+ induces neurite outgrowth from PC12 cells and activates ERK1/2 through integrin signals and that activation of ERK1/2 is essential for Mn 2+ -induced neurite outgrowth, a difference in the mechanism between Mn 2+ -induced and PERJ-or AMP N 1 -oxide-induced neurite outgrowth is suggested. Furthermore, we demonstrated that PERJ contained no ECM component-like substances. These results demonstrate that AMP N 1 -oxide and its analogues were the only entities in PERJ with neurite outgrowth-inducing activity and that they required integrin signaling in addition to activation of A2a receptors to induce neurite outgrowth.

Earlier we identified adenosine monophosphate (AMP) N1-oxide as a unique compound of royal jelly (RJ) that induces neurite outgrowth (neuritegenesis) from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK) but also that of cAMP/calcium-response element-binding protein (CREB) in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA) by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.