Jianting Yao

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.

Sonodynamic Therapy Inhibits Fibrogenesis in Rat Cardiac Fibroblasts Induced by TGF-β1

Background/Aims: Sonodynamic therapy (SDT) is a localized ultrasound-activated therapy for atherosclerosis when combined with a sonosensitizer, 5-aminolevulinic acid (ALA), but whether it can prevent cardiac fibrosis has not been studied. In the present study, we evaluated the effects SDT on fibrogenesis in rat cardiac fibroblasts. Methods: The primary cardiac fibroblasts were isolated from rats, and induced to fibrogenesis with TGF-β1. With this in vitro model, we tested the preventive effects of SDT on fibrogenesis and further the underlying mechanism. Results: TGF-β1 stimulation up-regulated α-SMA and COLI/III protein levels in cardiac fibroblasts, and enhanced the progression of cells from the G0/G1 phase to the S phase. SDT inhibited the TGF-β1 mediated cell proliferation and decreased the levels of α-SMA and COLI/III by activating AKT/GSK3β pathway and blocking TGF-β1/SMAD3 signaling. Conclusion: Our studies demonstrate an antifibrotic effect of SDT in rat cardiac fibroblasts, suggesting that SDT may intervene cardiac fibrogenesis by regulating myocardial fibrotic remodeling.

Non-Lethal Sonodynamic Therapy Inhibits Atherosclerotic Plaque Progression in ApoE-/- Mice and Attenuates ox-LDL-mediated Macrophage Impairment by Inducing Heme Oxygenase-1

Background: Previous studies from our group showed that low-intensity sonodynamic therapy (SDT) has protective effects on atherosclerosis (AS). However, because the intensity of ultrasound passing through tissue is attenuated, the consequences of very low-intensity SDT, referred to as non-lethal SDT (NL-SDT), on atherosclerotic plaques are unclear. The aim of this study was to determine whether NL-SDT affects atherosclerotic plaques and to elucidate the possible underlying mechanisms. Methods: An AS model was established using ApoE-/- mice fed a western diet. En face Oil Red O staining was used to measure atherosclerotic plaque size. Hematoxylin and eosin staining and immunohistochemical staining were used to observe plaque morphology and assess the location of macrophages and heme oxygenase 1 (HO-1). HO-1 mRNA and protein levels in AS plaques were evaluated by real-time PCR and western blotting. Human THP-1 cells and mouse peritoneal macrophages were used in this study. Western blotting was used to investigate the expression of cellular proteins after NL-SDT. Macrophage apoptosis was evaluated by TUNEL assays and flow cytometry with Annexin V/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with 2′-7′-dichlorofluorescein diacetate (DCFH-DA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) staining, respectively. Results: NL-SDT significantly inhibited AS progression and reduced the necrotic core area. NL-SDT induced HO-1 expression in lesional macrophages and in cultured macrophages. NL-SDT activated the protein kinase B (AKT) and extracellular signal-related protein kinase (ERK) pathways and the transcription factor NF-E2-related factor 2 (Nrf2).NL-SDT significantly reduced oxidized LDL (ox-LDL)-induced macrophage MMP collapse, ROS production and cell apoptosis. Zinc protoporphyrin (ZnPP), a HO-1-specific inhibitor, reversed the protective effects of NL-SDT. Conclusion: NL-SDT inhibits atherosclerotic plaque progression and increases plaque stability. In vitro, NL-SDT has a protective effect on ox-LDL-induced macrophage impairment via HO-1.

The decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals

The ultrasound activation of certain drugs, such as porphyrins, could cause synergistic cytotoxic effects on cells. Both sonomechanical and sonochemical effects occur and the latter play a critical role because antioxidant agents could exert significant protective effects against the cytotoxicity. To investigate the reactive oxygen species involved in the sonochemical effects, aqueous protoporphyrin IX (PpIX) solutions were characterized under ultrasound sonication in this study. Inertial cavitation was indirectly evaluated using terephthalic acid dosimetry. The fluorescence intensity of the PpIX was measured using a fluorescence spectrophotometer. The effects of PpIX concentration, ultrasound parameters and free radical scavengers on the PpIX activation by ultrasound were investigated. Our results showed that the increase in PpIX decomposition was significantly correlated with cavitation activities (R=0.9874, p<0.05), and the decomposing effect increases with ultrasound intensity (0.6-1.5 W/cm(2)), initial PpIX concentration (1-5 μM), duty cycle (10-100%) and the sonication duration (2-10 min). The fluorescence and absorption spectra of PpIX showed a decrease in the peak intensity without spectral shifts or new peak build-up after sonication. The PpIX decomposition was significantly inhibited by hydroxyl radical scavengers, histidine, mannitol, acetone, methanol and ethanol, but the decomposition was not inhibited by sodium azide, catalase or superoxide dismutase. These results suggest that the decomposition of protoporphyrin IX by ultrasound is dependent on the generation of hydroxyl radicals, which sheds some light on the sonochemical effects of the interaction between ultrasound and porphyrins.

Comparison study on the influence of the central metal ions in palladium(II)- and gadolinium(III)-porphyrins for phosphorescence-based oxygen sensing

The luminescence and oxygen sensing properties of a series of palladium(II)- and gadolinium(III)-porphyrins were evaluated and compared. Spectral analysis indicates that absorption and luminescence of Gd-porphyrins are red-shifted compared to those of Pd-porphyrins. This demonstrates that the energy levels of excited states in Gd-porphyrins are lower than those in the corresponding Pd-porphyrins. Phosphorescence quantum yield of Pd-hematoporphyrin monomethyl ether (HMME) was about 3-fold higher than that of Gd-HMME due to the decreased non-radiative decay rate of triplet states in Pd-HMME, which was confirmed by the longer phosphorescence lifetime of Pd-HMME. This is attributed to the higher first excited triplet state (T1) in Pd-HMME as compared to that of Gd-HMME. However, the phosphorescence intensity and lifetime responses of Gd-HMME to oxygen were larger than those of Pd-HMME. To understand this difference, oxygen quenching constants (kq) of Gd- and Pd-HMME were evaluated and were found to be 4972.9 and 26.4 s−1, respectively. The huge difference in kq between the two species is responsible for their disparate oxygen responses. We suggest that the greater kq of Gd-HMME results from the better energy matching between its T1 (12 658–14 006 cm−1) and the second excited state (13 123 cm−1) of oxygen, which was demonstrated by the higher singlet oxygen quantum yield of Gd-HMME. The same phenomena (spectral red-shifts, lower phosphorescence quantum yields, shorter lifetimes, and larger oxygen responses) were also observed upon substituting Pd(II) by Gd(III) in other porphyrins studied in this work.

Photophysical properties of sinoporphyrin sodium and explanation of its high photo-activity

Sinoporphyrin sodium (DVDMS) is a novel photosensitizer with high photodynamic therapy (PDT) effect. Reasons for its high photo-activity were investigated according to the study of photophysical characteristics of DVDMS. Extinction coefficients (e) of DVDMS at 405 nm and 630 nm are 4.36 × 105 and 1.84 × 104 M−1.cm−1; fluorescence quantum yield (ΦF) is 0.026; quantum yield of lowest triplet state formation is 0.94 and singlet oxygen quantum yield (ΦΔ) is 0.92. Although ΦΔ of DVDMS is only 10% higher than that of Photofrin®; (0.83), the extinction coefficient of DVDMS at 630 nm is 10-fold greater than that of Photofrin®;. This leads to its higher singlet oxygen generation efficiency (eΦΔ). The higher eΦΔ of DVDMS can result in an effective reduction of dosage (1/10 of Photofrin®;) reaching the same cytotoxic effect as Photofrin®;. Even though ΦF is approximately equal to that of Photofrin®;, brightness (eΦF) of DVDMS is 10-fold greater than that of Photofrin®; because of the 10-fold greater extinction coeff...

5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Alleviates Atherosclerosis via Enhancing Efferocytosis and Facilitating a Shift in the Th1/Th2 Balance Toward Th2 Polarization

Background/Aims: We and other groups have demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (ALA-SDT) induces macrophage and foam cell apoptosis and stabilizes atherosclerosis (AS) plaques in animal models. Lymphocytes also play vital roles in the development of AS. The primary purpose of the present study was to investigate the effects of ALA-SDT on T helper (Th) cell fate and function, Th subset differentiation, and atherosclerotic lesion stability. Methods: We utilized ALA-SDT on Western diet-fed apoE-/-mice in vivo and human Jurkat cells in vitro. Hematoxylin and eosin staining and TUNEL assays were used to evaluate the atherosclerotic plaque size and apoptosis within the atheroma. ALA induced cytotoxicity on cultured Jurkat cells was determined with CCK-8 assay. To address the mechanisms, levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (MPTP) opening were evaluated by staining with fluorescent probes. Western blot analysis and confocal microscopy were used to analyze the protein levels of caspases, Bax and cytochrome c and the release of cytochrome c. Cell apoptosis and necrosis and phagocytosis were examined by flow cytometry. ELISAs and immunofluorescent staining were used to assess the corresponding cytokine levels and Th subset cell numbers within the atheroma. Results: Our studies revealed that ALA-SDT significantly enhanced CD4+ cell apoptosis and macrophage-mediated phagocytosis and hence reduced the necrotic core size. ALA-SDT activated the mitochondrial apoptotic signaling pathway with minimal necrosis in Jurkat cells. ALA-SDT inhibited the Th1 response and enhanced the Th2 response. These effects of ALA-SDT were mediated primarily through the generation of ROS. Conclusion: ALA-SDT alleviates AS by enhancing cytotoxic effects on Th cells, subsequently stimulating efferocytosis and facilitating a shift in the Th1/Th2 balance toward Th2 cells, a discovery that might help elucidate the mechanism underlying SDT as a potential treatment to prevent atherothrombotic events.

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.

5-AMINOLEVULINIC ACID-DERIVED PROTOPORPHYRIN IX MEDIATED FLUORESCENCE DIAGNOSIS AND PHOTODYNAMIC THERAPY OF MACROPHAGES WITHIN THE ATHEROSCLEROTIC PLAQUE

Objectives To detect the accumulation of ALA-PpIX in the plaque macrophages and evaluate the effects of PDT on macrophages. Methods The fluorescence of PpIX was detected by fluorescence microscope in THP-1 macrophages and plaque fresh-frozen sections of rabbit carotid artery. Immunofluorescent staining of plaque macrophages and SMCs was performed to locate ALA-PpIX. Plaque burden was evaluated by Haematoxylin and eosin (H&E) staining. Plaque macrophages and SMCs content was determined by immunohistochemical staining. Results PpIX was found accumulated in the cytoplasm of THP-1 macrophages. In the plaque fresh-frozen sections, the fluorescence of ALA-PpIX was detected where macrophages immunofluorescence staining was positive. Compared with the control group, the plaque area was reduced by 59% p<0.01) at 4 week after PDT, the plaque macrophages content decreased by 56% p<0.001) at 1 week and 64% p<0.001) at 4 week respectively. Conclusions ALA-PpIX is preferentially accumulated in the macrophages of plaque and ALA-PpIX mediated PDT significantly decreases macrophages content, indicating a promising strategy of atherosclerotic plaque macrophages diagnosis and therapy.