The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.

Objective. To clarify the consequences of the valine/leucine polymorphism at position 247 of the ␤ 2 -glycoprotein I (␤ 2 GPI) gene in patients with antiphospholipid syndrome (APS), by investigating the correlation between genotypes and the presence of anti-␤ 2 GPI antibody.

IgG anti-laminin-1 auto-Abs are associated with recurrent miscarriages and the subsequent pregnancy outcome of recurrent aborters.

␤ 2 -Glycoprotein I ( ␤ 2 -GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that ␤ 2 -GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for ␤ 2 -GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by ␤ 2 -GPI and subsequently by anti-␤ 2 -GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1liposomes was enhanced more than 10-fold in the presence of both ␤ 2 -GPI and an anti-␤ 2 -GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-␤ 2 -GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with ␤ 2 -GPI. We suggest that autoimmune atherogenesis linked to ␤ 2 -GPI interaction with oxLDL and Abs may be present in APS.

Beta2-glycoprotein I (beta2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of beta2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193-K246, D222-K317 and E228-K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-beta2-GPI auto-antibodies to solid-phase beta2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-beta2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure.

Binding of beta(2)-glycoprotein I (beta(2)-GPI)-dependent anticardiolipin antibodies (aCL) derived from antiphospholipid syndrome (APS) is significantly reduced in aCL ELISA due to loss of the phospholipid (PL) binding property of beta(2)-GPI by plasmin treatment. In the present study, the treatment generated a nicked form of beta(2)-GPI and resulted in loss of antigenicity for the autoantibodies detected in ELISA, using an beta(2)-GPI directly adsorbed polyoxygenated carboxylated plate, the assay system of which was not related to PL binding. The nicked form bound to neither Cu(2+)-oxidized low-density lipoprotein (oxLDL) nor to beta(2)-GPI-specific lipid ligands isolated from oxLDL, the result being a complete loss of subsequent binding of anti-beta(2)-GPI autoantibodies. The conformational change in the nicked domain V was predicted from its intact structure determined by an X-ray analysis (implemented in Protein Data Bank: 1C1Z), molecular modeling and epitope mapping of a monoclonal anti-beta(2)-GPI antibody, i.e. Cof-18, which recognizes the related structure. The analysis revealed that novel hydrophobic and electrostatic interactions appeared in domain V after the cleavage, thereby affecting the PL binding of beta(2)-GPI. Such a conformational change may have important implications for exposure of cryptic epitopes located in the domains such as domain IV.

No abstract

In ventricular myocytes, spontaneous release of calcium (Ca 2+) from the sarcoplasmic reticulum via ryanodine receptors ("Ca 2+ sparks") is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca 2+ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca 2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca 2+ sparks and ROS that occurs with stretch. Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca 2+ sparks (4.6 µM Fluo-4, 10 min) or ROS (1 µM DCF, 20 min), exposed to normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia [15 mM potassium], acidosis [6.5 pH], and metabolic inhibition [1 mM sodium cyanide, 20 mM 2-deoxyglucose]), and subjected to sustained stretch by the carbon fiber technique (∼10% increase in sarcomere length, 15 s). Ca 2+ spark rate and rate of ROS production were measured by confocal microscopy. Results: Baseline Ca 2+ spark rate was greater in SI (2.54 ± 0.11 sparks•s −1 •100 µm −2 ; n = 103 cells, N = 10 mice) than NT (0.29 ± 0.05 sparks•s −1 •100 µm −2 ; n = 33 cells, N = 9 mice; p < 0.0001). Stretch resulted in an acute increase in Ca 2+ spark rate in both SI (3.03 ± 0.13 sparks•s −1 •100 µm −2 ; p < 0.0001) and NT (0.49 ± 0.07 sparks•s −1 •100 µm −2 ; p < 0.0001), with the increase in SI being greater than NT (+0.49 ± 0.04 vs. +0.20 ± 0.04 sparks•s −1 •100 µm −2 ; p < 0.0001). Baseline rate of ROS production was also greater in SI (1.01 ± 0.01 normalized slope; n = 11, N = 8 mice) than NT (0.98 ± 0.01 normalized slope; n = 12, N = 4 mice; p < 0.05), but there was an acute increase with stretch only in SI (+12.5 ± 2.6%; p < 0.001). Conclusion: Ischemia enhances the stretch-induced increase of Ca 2+ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia.