Protective and Regenerative Effects of Reconstituted HDL on Human Rotator Cuff Fibroblasts under Hypoxia: An In Vitro Study.

Hypoxia and hypo-high-density lipoproteinemia (hypo-HDLemia) are proposed risk factors for rotator cuff tear. HDL is recognized for its potential benefits in ischemia-driven angiogenesis and wound healing. Nevertheless, research on the potential benefits of reconstituted HDL (rHDL) on human rotator cuff fibroblasts (RCFs) under hypoxia is limited. This study investigates the cytoprotective and regenerative effects of rHDL, as well as N-acetylcysteine (NAC), vitamin C (Vit C), and HDL on human RCFs under hypoxic conditions. Sixth-passage human RCFs were divided into normoxia, hypoxia, and hypoxia groups pretreated with antioxidants (NAC, Vit C, rHDL, HDL). Hypoxia was induced by 1000 µM CoCl2. In the hypoxia group compared to the normoxia group, there were significant increases in hypoxia-inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1), and Bcl-2/E1B-19kDa interacting protein 3 (BNIP3) expressions, along with reduced cell viability, elevated reactive oxygen species (ROS) production, apoptosis rate, expressions of cleaved caspase-3, cleaved poly ADP-ribose polymerase-1 (PARP-1), vascular endothelial growth factors (VEGF), and matrix metalloproteinase-2 (MMP-2), as well as decreased collagen I and III production, and markedly lower cell proliferative activity (p ≤ 0.039). These responses were significantly mitigated by pretreatment with rHDL (p ≤ 0.046). This study suggests that rHDL can enhance cell proliferation and collagen I and III production while reducing apoptosis in human RCFs under hypoxic conditions.

In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon.

BACKGROUND: Bone mineral density of the humeral head is an independent determining factor for postoperative rotator cuff tendon healing. Bisphosphonates, which are commonly used to treat osteoporosis, have raised concerns regarding their relationships to osteonecrosis of the jaw and to atypical fracture of the femur. In view of the prevalence of rotator cuff tear in osteoporotic elderly people, it is important to determine whether bisphosphonates affect rotator cuff tendon healing. However, no studies have investigated bisphosphonates' cytotoxicity to human rotator cuff tendon fibroblasts (HRFs) or bisphosphonates' effects on rotator cuff tendon healing. The purpose of this study was to evaluate the cytotoxicity of alendronate (Ald), a bisphosphonate, and its effects on HRF wound healing. METHODS: HRFs were obtained from human supraspinatus tendons, using primary cell cultures. The experimental groups were control, 0.1 µM Ald, 1 µM Ald, 10 µM Ald, and 100 µM Ald. Alendronate exposure was for 48 h, except during a cell viability analysis with durations from 1 day to 6 days. The experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability. RESULTS: The following findings regarding the 100 µM Ald group contrasted with those for all the other experimental groups: a significantly lower rate of live cells (p < 0.01), a higher rate of subG1 population, a lower rate of Ki-67 positive cells, higher rates of apoptosis and necrosis, a higher number of cells with DNA fragmentation, higher caspase-3/7 activity (p < 0.001), and a higher number of caspase-3 positive staining cells. In scratch-wound healing analyses of all the experimental groups, all the wounds healed within 48 h, except in the 100 µM Ald group (p < 0.001). CONCLUSIONS: Low concentrations of alendronate appear to have little effect on HRF viability, proliferation, migration, and wound healing. However, high concentrations are significantly cytotoxic, impairing cellular proliferation, cellular migration, and wound healing in vitro.

N-acetylcysteine reduces glutamate-induced cytotoxicity to fibroblasts of rat supraspinatus tendons.

Purpose: Neuronal theory regarding rotator cuff degeneration has developed from the findings that glutamate, an amino acid and an excitatory neurotransmitter, is present in increased concentrations in tendon tissues with tendinopathy and that glutamate induces cell death in fibroblasts of origin in rat supraspinatus tendon. The purpose of the current study was to determine whether N-acetylcysteine (NAC) has cytoprotective effects against glutamate-induced fibroblast death. Materials and Methods: Primary cultured fibroblasts were obtained from rat supraspinatus tendons. Varying concentrations of glutamate (0.5, 1, 5, and 10 mM) and of NAC (0.5, 1, 2, and 5 mM) were used for evaluation of cytotoxicity. Cell viability, cell cycles, types of cell death, intracellular ROS production, expressions of caspase-3/7, and Ca2+ influx were evaluated. Results: Glutamate significantly induced cell death, apoptosis, and Ca2+ influx and significantly increased caspase-3/7 activity and intracellular ROS production (p < 0.001). NAC significantly reduced the glutamate-induced cell death, apoptosis, Ca2+ influx, caspase-3/7 activity, and intracellular ROS production (p < 0.001). Conclusions: The glutamate-induced cytotoxic effects can be reduced by NAC, an antioxidant, through the reduction of intracellular oxidative stress and/or Ca2+ influx.

N-acetyl cysteine protects cells from chondrocyte death induced by local anesthetics.

Local anesthetics (LA) are among the drugs most frequently used for musculoskeletal problems, in procedures ranging from diagnosis to postoperative pain control. Chondrocyte toxicity induced by LA is an emerging area of concern. The purpose of this study was to determine whether N-acetyl cysteine (NAC), an antioxidant, will exert cytoprotective effects against chondrocyte death induced by LA. Primary cultured human chondrocytes were used for this study. This study used control, NAC, LA, and NAC-LA groups. Cytotoxicity was induced in the LA subgroups and their paired NAC-LA subgroups through exposure to ropivacaine (0.075%), bupivacaine (0.05%), or lidocaine (0.2%) for 24 h. The NAC-LA subgroups were exposed to 10 mM NAC for 1 h, before LA exposure. These study groups were evaluated for rates of cell viability, apoptosis, necrosis, intracellular ROS production, and caspase-3/7 activity. Cell viability in all LA subgroups was significantly lower than in the control group (p < 0.001). Cell viability in the NAC-LA subgroups was significantly higher than in their paired LA subgroups (p < 0.001). In the LA subgroups, rates of apoptosis and necrosis, intracellular ROS production, and caspase-3/7 activity were significantly higher than in the control group (p ≤ 0.029). In the NAC-LA subgroups, rates of apoptosis and necrosis, intracellular ROS production, and caspase-3/7 activity were significantly lower than in their paired LA subgroups (p ≤ 0.023). These results indicate that N-acetyl cysteine, an antioxidant, has cytoprotective effects against LA-induced toxicity to chondrocytes in vitro. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:297-303, 2017.

Cytoprotective Mechanism of Cyanidin and Delphinidin against Oxidative Stress-Induced Tenofibroblast Death.

Age-related rotator cuff tendon degeneration is related to tenofibroblast apoptosis. Anthocyanins reduce oxidative stress-induced apoptotic cell death in tenofibroblasts. The current study investigated the presence of cell protective effects in cyanidin and delphinidin, the most common aglycon forms of anthocyanins. We determined whether these anthocyanidins have antiapoptotic and antinecrotic effects in tenofibroblasts exposed to H2O2, and evaluated their biomolecular mechanisms. Both cyanidin and delphinidin inhibited H2O2-induced apoptosis in a dose-dependent manner. However, at concentrations of 100 µg/ml or greater, delphinidin showed cytotoxicity against tenofibroblasts and a decreased antinecrotic effect. Cyanidin and delphinidin both showed inhibitory effects on the H2O2-induced increase in intracellular ROS formation and the activation of ERK1/2 and JNK. In conclusion, both cyanidin and delphinidin have cytoprotective effects on cultured tenofibroblasts exposed to H2O2. These results suggest that cyanidin and delphinidin are both beneficial for the treatment of oxidative stress-mediated tenofibroblast cell death, but their working concentrations are different.

Antioxidant's cytoprotective effects on rotator cuff tenofibroblasts exposed to aminoamide local anesthetics.

Local anesthetics (LA) are among the drugs most frequently used for musculoskeletal problems, in procedures ranging from diagnosis to postoperative pain control. The cytotoxicity of LA is an emerging area of concern. The purpose of this study was to determine whether cyanidin, an antioxidant, exerts cytoprotective effects against tenofibroblast death induced by LA. Primary cultured human rotator cuff tenofibroblasts were used to evaluate the cytotoxicity of these LA: Ropivacaine (0.075%), Bupivacaine (0.05%), and Lidocaine (0.2%). The effects of cyanidin (100 µg/ml) on the cytotoxicity induced by these LA were investigated. Cell viability, ROS production, caspase-3/7 activity, and expressions of phospho-extracellular signal-regulated kinases (ERK), phospho-p38, phospho-c-Jun N-terminal kinase (JNK), and cleaved PARP-1 were evaluated. Exposure to LA significantly induced cell death (p < 0.001), ROS production (p ≤ 0.04), the activation of caspase-3/7 (p < 0.001), and the increased expressions of phospho-ERK, phospho-p38, phospho-JNK, and cleaved PARP-1. These LA-induced cytotoxic effects were reduced by cyanidin. These data indicate that cyanidin, an antioxidant, has cytoprotective effects against LA-induced cytotoxicity to rotator cuff tenofibroblasts.

Cytotoxic effects of ropivacaine, bupivacaine, and lidocaine on rotator cuff tenofibroblasts.

BACKGROUND: Concern has recently arisen over the safety of local anesthetics used on human tissues. HYPOTHESIS: Aminoamide local anesthetics have cytotoxic effects on human rotator cuff tenofibroblasts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultured human rotator cuff tenofibroblasts were divided into control, phosphate buffered saline (PBS), and local anesthetic study groups; the PBS study group was further subdivided by pH level (pH 7.4, 6.0, and 4.4). The 6 local anesthetic subgroups (0.2% and 0.75% ropivacaine, 0.25% and 0.5% bupivacaine, and 1% and 2% lidocaine) were also studied at 10% dilutions of their original concentrations. Exposure times were 5, 10, 20, 40, or 60 minutes for the higher concentrations and 2, 6, 12, 24, 48, or 72 hours for the lower concentrations. Cell viability was evaluated through live, apoptotic, and necrotic cell rates using the annexin V-propidium iodide double-staining method. Intracellular reactive oxygen species (ROS) and the activity of mitogen-activated protein kinases (MAPKs) and caspase-3/7 were investigated. RESULTS: The control and PBS groups showed no significant differences in cell viability (P > .999). In the local anesthetic study groups, cell viability decreased significantly with increases in anesthetic concentrations (P < .001) and exposure times (P < .001), with the exception of the lidocaine subgroups, where this effect was masked by the very high cytotoxicity of even low concentrations. Among the studied local anesthetic subgroups, 0.2% ropivacaine was the least toxic. The levels of intracellular ROS of each local anesthetic subgroup also increased significantly (P < .05). The studied local anesthetics showed increases in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 as well as in levels of caspase-3/7 activity (P < .001). CONCLUSION: The cytotoxicity of the anesthetics studied to tenofibroblasts is dependent on exposure time and concentration. Of the evaluated anesthetics, ropivacaine is the least toxic in the clinically used concentration. The studied anesthetics induce tenofibroblast cell death, mediated by the increased production of ROS, by the increased activation of ERK1/2, JNK, and p38 and by the activation of caspase-3/7. CLINICAL RELEVANCE: This study identified the cytotoxic mechanisms of aminoamide local anesthetics acting on rotator cuff tenofibroblasts. The greatest margin of safety was found in lower anesthetic concentrations in general and more specifically in the use of ropivacaine.

Do antioxidants inhibit oxidative-stress-induced autophagy of tenofibroblasts?

Recent research on tendinopathy has focused on its relationship to programmed cell death. Increased autophagy has been observed in ruptured rotator cuff tendon tissues, suggesting a causal relationship. We investigated whether autophagy occurs in human rotator cuff tenofibroblast death induced by oxidative stress and whether antioxidants protect against autophagic cell death. We used H2 O2 (0.75 mM) as oxidative stressor, cyanidin (100 µg/ml) as antioxidant, zVAD (20 µM) as apoptosis inhibitor, and 3-MA (10 mM) as autophagy inhibitor. We evaluated cell viability and known autophagic markers: LC3-II expression, GFP-LC3 puncta formation, autolysosomes, and Atg5-12 and Beclin 1 expression. H2 O2 exposure increased the rates of cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. After we induced apoptosis arrest using zVAD, H2 O2 exposure still induced cell death, LC3-II expression, and GFP-LC3 puncta formation. H2 O2 exposure also increased Atg5-12 and Beclin 1 expressions, indicating autophagic cell death. However, cyanidin treatment reduced H2 O2 -induced cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. Cyanidin and 3-MA similarly reduced the cell-death rate, and Atg5-12 and Beclin 1 expression. This study demonstrated that H2 O2 , an oxidative stressor, induces autophagic cell death in rotator cuff tenofibroblasts, and that cyanidin, a natural antioxidant, inhibits autophagic cell death.