Jiali Cheng

Apoptosis of THP-1 macrophages induced by protoporphyrin IX-mediated sonodynamic therapy

Background Sonodynamic therapy (SDT) was developed as a localized ultrasound-activated cytotoxic therapy for cancer. The ability of SDT to destroy target tissues selectively is especially appealing for atherosclerotic plaque, in which selective accumulation of the sonosensitizer, protoporphyrin IX (PpIX), had been demonstrated. Here we investigate the effects of PpIX-mediated SDT on macrophages, which are the main culprit in progression of atherosclerosis. Methods and results Cultured THP-1 derived macrophages were incubated with PpIX. Fluorescence microscopy showed that the intracellular PpIX concentration increased with the concentration of PpIX in the incubation medium. MTT assay demonstrated that SDT with PpIX significantly decreased cell viability, and this effect increased with duration of ultrasound exposure and PpIX concentration. PpIX-mediated SDT induced both apoptosis and necrosis, and the maximum apoptosis to necrosis ratio was obtained after SDT with 20 μg/mL PpIX and five minutes of sonication. Production of intracellular singlet oxygen and secondary disruption of the cytoskeleton were also observed after SDT with PpIX. Conclusion PpIX-mediated SDT had apoptotic effects on THP-1 macrophages via generation of intracellular singlet oxygen and disruption of the cytoskeleton. PpIX-mediated SDT may be a potential treatment to attenuate progression of atherosclerotic plaque.

Interleukin-18 among atrial fibrillation patients in the absence of structural heart disease

AIMS Inflammation plays a role in the genesis and perpetuation of atrial fibrillation (AF). Interleukin (IL)-18 is a pleiotropic proinflammatory cytokine with a central role in the inflammatory cascade. We hypothesize that the circulating IL-18 concentration is elevated in AF patients. METHODS AND RESULTS In a case-control study design, 56 cases with AF and 26 controls were enrolled. All AF cases were categorized into paroxysmal and persistent AF or lone AF and AF with hypertension. Circulating levels of IL-18, tumour necrosis factor-α, high-sensitivity C-reactive protein, matrix metalloproteinase (MMP)-9, and tissue inhibitor of MMP-1 were measured. In adjusted analyses, only age, MMP-9, and IL-18 were independently associated with AF, in which IL-18 had the most significant association (P = 0.0011, standardized estimate &bgr = 1.76, OR = 1.02, 95% confidence interval: 1.01-1.03). Interleukin-18 levels in persistent AF patients were higher than those in paroxysmal ones (P = 0.0011). Patients who developed AF within 24 h prior to sampling displayed a higher level of IL-18 than those with sinus rhythm (P = 0.0027). Interleukin-18 was positively correlated with left atrial diameter (r = 0.33, P = 0.0117). CONCLUSION This study documents the elevated IL-18 in AF patients. Interleukin-18 may be superior to other inflammatory markers which are known to be elevated in AF.

Effects of 5-aminolevulinic acid-mediated sonodynamic therapy on macrophages

Background Inflammatory cells exhibit an elevated level of protoporphyrin IX (PpIX) after the administration of 5-aminolevulinic acid (ALA). Here, we investigate the sonodynamic effects of ALA-derived PpIX (ALA-PpIX) on macrophages, which are the pivotal inflammatory cells in atherosclerosis. Methods and results Cultured THP-1 macrophages were incubated with ALA. Fluorescence microscope and fluorescence spectrometer detection showed that intracellular PpIX increased with the concentration of ALA in the incubation solution in a time dependent manner; the highest level of intracellular PpIX was observed after 3-hour incubation. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays demonstrated that lower concentrations (less than 2 mM) of ALA have no influence on cell viability (more than 90% of cells survived), but sonodynamic therapy (SDT) with a low concentration of ALA significantly decreased the survival rate of cells, and the effect was increased with both ALA concentration and ultrasound exposure time. Cell apoptosis and necrosis induced by ALA-mediated SDT (ALA-SDT) were measured using Hoechst 33258 and propidium iodide assay. ALA-SDT induced both cell apoptosis and necrosis, and the maximum apoptosis/necrosis ratio was observed at 6 hours after SDT with 1 mM of ALA and 5 minutes of ultrasound exposure. Flow cytometry analysis showed that ALA-SDT significantly increased late stage apoptotic cells (about 10-fold control). Furthermore, ALA-SDT induced reactive oxygen species generation in THP-1 macrophages immediately after the treatment and a conspicuous loss of mitochondrial membrane potential (MMP) at 6 hours compared with that of the control, ALA alone, and ultrasound alone groups. Conclusion ALA-SDT exhibited synergistic apoptotic effects on THP-1 macrophages, involving excessive intracellular reactive oxygen species generation and MMP loss. Therefore, ALA-SDT is a potential treatment for atherosclerosis.

Detection and photodynamic therapy of inflamed atherosclerotic plaques in the carotid artery of rabbits

Photodynamic therapy (PDT) has been applied in the treatment of artery restenosis following balloon injury. This study aimed to detect the accumulation of 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PpIX) in inflamed atherosclerotic plaque in rabbit model and evaluate the efficacy of PDT. The inflamed atherosclerotic plaque in the common carotid artery was produced by combination of balloon denudation injury and high cholesterol diet. After intravenous administration of ALA, the fluorescence of PpIX in plaque was detected. At the peak time, the correlation between the fluorescence intensity of PpIX and the macrophage infiltration extent in plaque was analyzed. Subsequently, PDT (635nm at 50J/cm(2)) on the atherosclerotic plaques (n=48) was performed and its effect was evaluated by histopathology and immunohistochemistry. The fluorescence intensity of PpIX in the plaque reached the peak 2h after injection and was 12 times stronger than that of adjacent normal vessel segment, and has a positive correlation with the macrophage content (r=0.794, P<0.001). Compared with the control group, the plaque area was reduced by 59% (P<0.001) at 4week after PDT, the plaque macrophage content decreased by 56% at 1week and 64% at 4week respectively, the smooth muscle cells (SMCs) was depleted by 24% at 1week (P<0.05) and collagen content increased by 44% at 4week (P<0.05). It should be pointed out that the SMC content increased by 18% after PDT at 4week compared with that at 1week (P<0.05). Our study demonstrated that the ALA-derived PpIX can be detected to reflect the macrophage content in the plaque. ALA mediated PDT could reduce macrophage content and inhibit plaque progression, indicating a promising approach to treat inflamed atherosclerotic plaques.

Anti-apoptotic action of hydrogen sulfide is associated with early JNK inhibition

The mechanism of action of Hydrogen sulfide (H2S) as a novel endogenous gaseous messenger and potential cardioprotectant is not fully understood. We therefore investigated the prevention of cardiomyocyte apoptosis by exogenous H2S and the signaling pathways leading to cardioprotection. Using a simulated ischemia–reperfusion (I/Re) model with primary cultured rat neonatal cardiomyocytes, I/Re induced a rapid, time‐dependent phosphorylation of c‐Jun N‐terminal kinase (JNK), with significant elevation at 0.25 h and a peak at 0.5 h during reperfusion. NaHS (H2S donor) significantly inhibited the early phosphorylation of JNK, especially at 0.5 h. Both NaHS and SP600125 (specific JNK inhibitor) decreased the number of apoptotic cells, lowered cytochrome C release and enhanced Bcl‐2 expression. When NaHS application was delayed 1 h after reperfusion, the inhibition of apoptosis by H2S was negated. In conclusion, this is novel evidence that early JNK inhibition during reperfusion is associated with H2S‐mediated protection against cardiomyocyte apoptosis.

The predominant pathway of apoptosis in THP-1 macrophage-derived foam cells induced by 5-aminolevulinic acid-mediated sonodynamic therapy is the mitochondria-caspase pathway despite the participation of endoplasmic reticulum stress.

Background: In advanced atherosclerosis, chronic endoplasmic reticulum (ER) stress induces foam cells apoptosis and generates inflammatory reactions. Methods: THP-1 macrophage-derived foam cells (FC) were incubated with 1 mM 5-aminolevulinic acid (ALA). After ALA mediated sonodynamic therapy (ALA-SDT), apoptosis of FC was assayed by Annexin V-PI staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were detected by staining with CellROX® Green Reagent and jc-1. Pretreatment of FC with N-acetylcysteine (NAC), Z-VAD-FMK or 4-phenylbutyrate (4-PBA), mitochondria apoptotic pathway associated proteins and C/EBP-homologous (CHOP) expressions were assayed by wertern blotting. Results: Burst of apoptosis of FC was observed at 5-hour after ALA-SDT with 6-hour incubation of ALA and 0.4 W/cm2 ultrasound. After ALA-SDT, intracellular ROS level increased and mitochondrial membrane potential collapsed. Translocations of cytochrome c from mitochondria into cytosol and Bax from cytosol into mitochondria, cleaved caspase 9, cleaved caspase 3, upregulation of CHOP, as well as downregulation of Bcl-2 after ALA-SDT were detected, which could be suppressed by NAC. Activation of mitochondria-caspase pathway could not be inhibited by 4-PBA. Cleaved caspase 9 and caspase 3 as well as apoptosis induced by ALA-SDT could be inhibited by Z-VAD-FMK. Conclusion: The mitochondria-caspase pathway is predominant in the apoptosis of FC induced by ALA-SDT though ER stress participates in.

Hematoporphyrin monomethyl ether-mediated photodynamic effects on THP-1 cell-derived macrophages

Photodynamic therapy (PDT) has been shown to attenuate atherosclerotic plaque progression and decrease macrophage-infiltration. The effectiveness of PDT depends strongly on the type of photosensitizers. Hematoporphyrin monomethyl ether (HMME) is a promising second-generation porphyrin-related photosensitizer for PDT. This study is designed to characterize effects of HMME-based PDT on THP-1 cell-derived macrophages and define the cell-death pathway. HMME was identified to accumulate in the macrophages by fluorescence microscopy and confocal scanning laser microscope. Our data demonstrated that the intensity of laser-induced HMME fluorescence in macrophages steadily increased with the increasing incubation concentration of HMME. The survival rate of macrophages determined by MTT assay decreased with the increasing HMME concentration and irradiation time. HMME-based PDT induced macrophage apoptosis via caspase-9 and caspase-3 activation pathway detected by caspase fluorescent assay kit and flow cytometer. The PDT increased the number of apoptotic macrophages by 14-fold at 12 h post irradiation by 9 J/cm(2) 635 nm diode laser. These results imply that photodynamic therapy with HMME may therefore be a useful clinical treatment for unstable atherosclerotic plaques.

Sonodynamic effect of an anti-inflammatory agent--emodin on macrophages.

Emodin has been used as an anti-inflammatory agent and inflammation is a crucial feature of atherosclerosis. Here, we investigated the sonodynamic effect of emodin on macrophages, the pivotal inflammatory cells in atherosclerotic plaque. THP-1 derived macrophages were cultured with emodin and exposed to ultrasound. Six hours later, unlike the cells treated for 5 and 10 min, the viability of cells treated for 15 min decreased significantly and the cells showed typical apoptotic chromatin fragmentation. The percentage of apoptotic and necrotic cells in the sonodynamic therapy (SDT) group was higher than that in the ultrasound group. Two hours after treatment for 15 min, the cytoskeleton lost its original features as the filaments dispersed and the cytoskeletal proteins aggregated. The percentage of cells with disturbed cytoskeletal filaments in the SDT group was higher than that in the ultrasound group. These results suggest emodin has a sonodynamic effect on macrophages and might be used as a novel sonosensitizer for SDT for atherosclerosis.

The Sonodynamic Effect of Curcumin on THP-1 Cell-Derived Macrophages

Curcumin is extracted from the rhizomes of the traditional Chinese herb Curcuma longa and has been proposed to function as a photosensitizer. The potential use of curcumin as a sonosensitizer for sonodynamic therapy (SDT) requires further exploration. This study investigated the sonodynamic effect of curcumin on macrophages, the pivotal inflammatory cells in atherosclerotic plaque. THP-1-derived macrophages were incubated with curcumin at a concentration of 40.7 μmol/L for 2 h and then exposed to pulse ultrasound irradiation (2 W/cm2 with 0.86 MHz) for 5–15 min. Six hours later, cell viability was decreased in cells that had been treated with ultrasound for 10 and 15 min. After ultrasound irradiation for 15 min, the ratio of apoptotic and necrotic cells in SDT group was higher than that in ultrasound group, and the ratio of apoptotic cells was higher than that of necrotic cells. Both loss of mitochondrial membrane potential and morphological changes of cytoskeleton were apparent 2 h after treatment with curcumin SDT. These findings support that curcumin had sonodynamic effect on THP-1-derived macrophages and that curcumin SDT could be a promising treatment for atherosclerosis.

5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Inhibits RIPK1/RIPK3-Dependent Necroptosis in THP-1-Derived Foam Cells.

Necroptosis, or programmed necrosis, contributes to the formation of necrotic cores in atherosclerotic plaque in animal models. However, whether inhibition of necroptosis ameliorates atherosclerosis is largely unknown. In this study, we demonstrated that necroptosis occurred in clinical atherosclerotic samples, suggesting that it may also play an important role in human atherosclerosis. We established an in vitro necroptotic model in which necroptosis was induced in THP-1-derived foam cells by serum deprivation. With this model, we demonstrated that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) inhibited necroptosis while promoting apoptosis. ALA-SDT activated the caspase-3 and caspase-8 pathways in foam cells, which is responsible for the switch from necroptosis to apoptosis. The inhibition of either caspase-8 or caspase-3 abolished the anti-necroptotic effect of ALA-SDT. In addition, we found that caspase-3 activation peaked 4 hours after ALA-SDT treatment, 2 hours earlier than maximal caspase-8activation. Taken together, our data indicate that ALA-SDT mediates the switch from necroptosis to apoptosis by activating the caspase-3 and caspase-8 pathways and may improve the prognosis of atherosclerosis.