Chenghai Peng

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.

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.

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.

Effects of pravastatin on the function of dendritic cells in patients with coronary heart disease.

The aim of the study was to investigate the functional profile of dendritic cells in patients with coronary heart disease and the effects of pravastatin on this. Forty-eight patients with coronary heart disease were divided into three groups: 16 treated with pravastatin 10 mg/day, 16 treated with pravastatin 20 mg/day and 16 not treated with pravastatin. Dendritic cells from 48 patients with coronary heart disease (before and 4 weeks after the treatment) and 16 healthy individuals were derived from peripheral blood. CD86 of dendritic cells was assessed by flow cytometry. Immunostimulatory capacity of dendritic cells was measured by mixed lymphocyte reaction. The levels of cytokines in the medium of mixed lymphocyte reaction were analysed. Blood lipids and high-sensitivity C-reactive protein were measured. Compared to normal group, more CD86+ dendritic cells were expressed in coronary heart disease and greater immunostimulatory capacity of dendritic cells in coronary heart disease was demonstrated. T lymphocytes in coronary heart disease in mixed lymphocyte reaction secreted higher levels of pro-inflammation cytokines and lower levels of anti-inflammation cytokines. CD86 expression significantly correlated with C-reactive protein, but did not correlate with low-density lipoprotein cholesterol. Both dosages of pravastatin markedly inhibited the function of dendritic cells and lowered C-reactive protein, which is independent of plasma cholesterol lowering. The anti-inflammatory effect of pravastatin showed no obvious difference between the two dosage groups. In conclusion, dendritic cells were activated in coronary heart disease and dendritic cell-mediated immune mechanisms may be involved in the pathogenesis of coronary heart disease. Pravastatin can inhibit dendritic cell activation, which is independent of plasma cholesterol lowering. Pravastatin in different doses showed no apparent differences in the inhibition of dendritic cell functions.

An animal model of atherosclerotic plaque disruption and thrombosis in rabbit using pharmacological triggering to plaques induced by perivascular collar placement

INTRODUCTION Limited availability of suitable animal model of plaque disruption and thrombosis has hampered the study of mechanism and preclinical evaluation of plaque-stabilizing therapies. This study aims to develop an animal model of atherosclerotic plaque disruption and thrombosis in rabbit femoral artery. METHODS Silastic collars were placed around the bilateral femoral arteries of rabbits, which had been fed with atherogenic diet for 7 days. After 28 days on the same diet, the rabbits received pharmacological triggering by intraperitoneal injection of Russells viper venom (RVV, 0.15 mg/kg) followed by intravenous injection of histamine (0.02 mg/kg), and the animals were then processed for imageological and histological examinations. RESULTS Perivascular collar placement of the femoral artery in high-cholesterol-fed rabbits for 28 days induced marked intimal hyperplasia, which was a lipid- and collagen-rich lesion that contained substantial amount of macrophages and smooth muscle cells. Subsequent histological analysis showed that the pharmacological triggering evoked plaque disruption and platelet- and fibrin-rich thrombi in the collared femoral arteries. CONCLUSION We demonstrated, for the first time, a rabbit model of plaque disruption and thrombosis induced by the combination of perivascular collar placement, RVV, and histamine injections. This model can be rapidly formed, easily operated, and site controlled.

Apoptosis of vascular smooth muscle cells induced by photodynamic therapy with protoporphyrin IX

Photodynamic therapy (PDT) had been shown effective in the treatment of intimal hyperplasia, which contributes to restenosis, by eradicating cells in the vessel wall. This study is designed to evaluate the effects of PDT with protoporphyrin IX (PpIX) on the viability of vascular smooth muscle cells (SMCs) and to define the cell-death pathway. Fluorescence microscopy and laser-induced fluorescence spectroscopic detection showed that SMCs selectively uptake PpIX, and the intracellular PpIX concentration increases with the amount of PpIX in the incubation solution. PDT with PpIX impaired cellular viability from 93+/-3.4% to 36+/-3.9% when the light intensity increases from 2 to 9J/cm(2) and intracellular PpIX concentration increases from 0.5 to 20 microg/ml. Although PDT induced both apoptosis and necrosis, the ratio of apoptotic cells increased with light dosage or intracellular PpIX concentration. The loss of mitochondrial membrane potential coincided with the apoptotic ratio. Our results indicated that the induction of apoptosis of SMCs may be one of the mechanisms by which PDT inhibits restenosis in vivo.

A novel model of intimal hyperplasia with graded hypoosmotic damage

BACKGROUND The purpose was to develop a rabbit model of intimal hyperplasia with controllable lesion. METHODS Following 1 week of a 2% cholesterol diet, 32 New Zealand White male rabbits underwent right femoral arteries surgical perfusion with distilled water for 1, 3, 5, or 7 min (n=8/group). After a further 4 weeks of the same diet, serum total cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein were measured in all rabbits. Intimal hyperplasia in histological sections of arteries were assessed by intimal proliferation ratio. Macrophage numbers and levels of proteins matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions were analyzed by immunohistochemistry. RESULTS Serum lipids levels showed no statistical difference between experimental groups. Intimal proliferation ratio increased gradually with perfusion time, and a positive linear correlation was calculated between intimal proliferation ratio and duration of distilled water perfusion. Similarly, number of macrophages and levels of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions increased with perfusion time. CONCLUSIONS A novel model of intimal hyperplasia was established by intravascular distilled water perfusion in high-cholesterol-fed rabbits. Importantly, this model exhibits time-dependent neointimal proliferation lesions that can be readily controlled in terms of extent, thus providing an avenue for further studies.

HEMATOPORPHYRIN MONOMETHYL ETHER-MEDIATED PHOTODYNAMIC EFFECTS ON MACROPHAGES

Objectives 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 investigate HMME mediated Photodynamic therapy on macrophage and define the cell-death pathway. Methods Fluorescence of HMME with different incubated time was tested. Use confocal scanning laser microscope to investigate HMME subcellular distribution. THP-1 derived macrophages were cultured with HMME for 2 h and then exposed to 635 nm diode lasers for 1.5 min and 3 min. Six-hours later, cell viability analysis was performed with 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and annexin V/PI staining by flow cytometer. The activities of caspase-9 and caspase-3 were measured using the fluorescent assay kits. Results 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 1.5 min group increased the number of apoptotic macrophages 5.36±3.24 at 12 h post irradiation (mean±SD, n=3, p<0.05), whereas the 3 min group increased the number of apoptotic macrophages 67.18±5.17 (mean±SD, n=3, p<0.001). Conclusions Our data demonstrated that HMME could accumulate in macrophages and HMME-mediated PDT induced macrophage apoptosis. These results imply that photodynamic therapy with HMME may therefore be a useful clinical treatment for unstable atherosclerotic plaques.

Rapid inhibition of atherosclerotic plaque progression by sonodynamic therapy

Aims Currently, efficient regimens to reverse atherosclerotic plaques are not available in the clinic. Herein, we present sonodynamic therapy (SDT) as a novel methodology to rapidly inhibit progression of atherosclerotic plaques. Methods and results In atherosclerotic rabbit and apoE-deficient mouse models, SDT efficiently decreased the atherosclerotic burden within 1 week, revealing a decrease in the size of the atherosclerotic plaque and enlarged lumen. The shrunken atherosclerotic plaques displayed compositional alterations, with a reduction in lesional macrophages and lipids. The rapid efficacy of SDT may be due to its induction of macrophage apoptosis, enhancement of efferocytosis, and amelioration of inflammation in the atherosclerotic plaque. Compared with atorvastatin, the standard of care for atherosclerosis, SDT showed more significant plaque shrinkage and lumen enlargement during 1 week treatment. Furthermore, SDT displayed good safety without obvious side effects. In a pilot clinical trial recruiting the patients suffering atherosclerotic peripheral artery disease, combination therapy of SDT with atorvastatin efficiently reduced progression of atherosclerotic plaque within 4 weeks, and its efficacy was able to last for at least 40 weeks. Conclusion SDT is a non-invasive and efficacious regimen to inhibit atherosclerotic plaque progression.