Heat shock attenuates VEGF expression in three-dimensional myoblast sheets deteriorating therapeutic efficacy in heart failure.

BACKGROUND: Myoblast sheet transplantation is a promising novel treatment for ischemic heart failure. The aim of this study was to test the hypothesis that heat shock (HS) pre-treatment affects the angiogenic properties of myoblast sheets in vivo and in vitro. MATERIAL/METHODS: We studied HS preconditioning of L6 myoblast sheets in relation to their apoptosis, proliferation, and vascular endothelial growth factor (VEGF)-associated responses under normoxia and under hypoxia in vitro. In vivo evaluation of their therapeutic effect was performed with 60 male Wistar rats divided into 3 groups (20 each): sole left anterior descending (LAD) ligation (control); LAD ligation and non-conditioned sheet transplantation (L6 No-Shock); and LAD ligation and L6-heat shock conditioned sheet transplantation (L6 Heat-Shock). Left ventricular function was evaluated by echocardiography after 3, 10, and 28 days. RESULTS: Expression of HSP70/72 was strongly induced 24 hours after HS, and thereafter it decreased notably during 72 hours in hypoxia. Under normal growth conditions, HSP70/72 expression remained stable. HS delayed apoptosis-associated caspase-3 expression during 24-hour hypoxia compared to non-treated controls. However, VEGF expression reduced significantly in the heat shock pretreated sheets. Ejection fraction of the L6-myoblast HS pre-treatment group (L6 Heat-Shock) decreased gradually during follow-up, in the same pattern as the controls. However, these functional parameters improved in the L6-myoblast normal sheet group (L6 No-Shock) at the tenth day and remained significantly better. CONCLUSIONS: HS protects myoblast sheets from hypoxia-associated apoptosis in vitro, but reduces VEGF expression of the sheet, leading to lower therapeutic effect in heart failure.

Heat shock enhances troponin expression and decreases differentiation-associated caspase-3 dependence in myoblasts under hypoxia.

BACKGROUND: Myoblast transplantation can functionally restore muscle tissues damaged by ischemic or other insults. Despite promising results in clinical trials, however, myoblast transplantation still presents several challenges, with effective differentiation under harsh conditions of the host tissue being one of the most demanding. In keeping with a straightforward clinical application, heat shock (HS) pretreatment as a nonviral method can be utilized with promising results in cell therapy. The aim of this study was to demonstrate whether HS-pretreated cells would receive a differentiation benefit under hypoxic conditions. MATERIALS AND METHODS: We studied HS preconditioning of C2C12 myoblasts in relation to their differentiation- and apoptosis-associated responses under normoxia or 1% hypoxia. RESULTS: HS induced long-lasting expression of Hsp70/72 and Hsp90. Although myoblast differentiation proceeded in HS-pretreated and control cells under both normoxia and hypoxia, expression of differentiation-associated troponin was enhanced in HS-preconditioned cells under hypoxia. This effect persisted when differentiation was inhibited by Z-DEVD-FMK, a caspase-3 inhibitor. CONCLUSIONS: HS preconditioning enhances expression of myoblast differentiation-associated troponin and may reduce dependence of differentiation on caspase-3.

Assessment of enzymatic cleaning agents and disinfectants against bacterial biofilms.

PURPOSE: Microbial biofilm has become difficult to control by antibiotic and biocide regimes that are effective against suspended bacteria. Their colonization of surfaces can be a problem and is generally controlled through cleaning and disinfection. This study was undertaken to examine the efficacy of the disinfectants including Bio-Ow, Econase CE, Gamanase GC 140, IndiAge 44L, Mannanase AMB, Multifect P-3000, Neutrase, Pandion, Paradigm, Pectinex Ultra SP-L, Promozyme, Resinase A2X, Spezyme AA300, Spezyme GA300 and Vinozym EC, and the proteinase against bacterial biofilms. METHODS: The effectiveness of 20 commercial disinfectants against Pseudomonas aeruginosa (P. aeruginosa) biofilms using a fluorometric technique was examined. Additionally the disinfectants were also tested against Lactobacillus bulgaricus (L. bulgaricus), Lactobacillus lactis (L. lactis) and Streptococcus thermophilus (S. thermophilus) isolates using microtitration tray based turbidimetric techniques. Escherichia coli (E. coli) was used as the test bacteria in the fluorometric control method. RESULTS: Among the first group of the enzymatic cleaning agents tested, four disinfectants (Pandion, Resinase A2X, Spezyme GA300 and Paradigm) were the most potent against bacterial biofilms after 30 min incubation time (residual bacterial count less than 10(3) CFU (colony forming units)/ml). However, only Resinase A2X and Paradigm showed a good effect on bacterial biofilms after 15 min incubation time. Proteinase disinfectants (alkalase, chymotrypsin, cryotin and krilltrypsin) from the second group of the disinfectants showed a good effect against P. aeruginosa biofilm when tested in the absence of milk. The performance of the disinfectants was reduced in the presence of milk. The minimum inhibitory concentration (MIC) of the cleaning agents was determined as the lowest concentration inhibiting bacterial growth. The MIC was tested on Lactobacillus bulgaricus (L. bulgaricus), Lactobacillus lactis (L. lactis) and Streptococcus thermophilus (S. thermophilus) isolates. The minimum inhibitory concentrations (MIC) for Paradigm against S. thermophilus and L. Lactis were lower than L. Bulgaricus. Whereas, the MIC of Pandion against L. bulgaricus was lower than MIC against L. lactis. Resinase A2X had no inhibitory effect on bacterial growth when the concentration was less than or equal to 2.4 mg/ml and Spezyme GA 300 concentration less than or equal to 7.3 mg/ml. Minimum inhibitory concentration of Pandion against L. bulgaricus was 2.7 microg/ml and against L. lactis 5.3 microg/ml. Growth of S. thermophilus was inhibited in all concentration of Pandion tested. CONCLUSIONS: The choice of disinfectant or cleaning agent along with the optimum concentration and the time of action is very important when destroying microbes. It is also important that the resistances of microbes to different disinfectants and cleaning agents be taken into account when planning the cleaning process