Hydrogen Sulfide On Macrophage Derived Foam Cell Formation And Atherosclerotic Plaque Lipid Accumulation

Background：Endogenous hydrogen sulfide (H2S) is synthesized from L-cysteinemainly by cystathionine gamma lyase (CSE) and cystathionine-synthase(CBS). H2S, isproduced by various mammalian tissues and cells, exerts many effects. Therefore， H2S isreputed to be the third gastrotransmitter. A negative correlation was found between degree,number of coronary atherosclerotic lesions and plasma H2S level in patients of coronary heartdisease, but mechanism is not clear. Monocyte/macrophages are kinds of immune andinflammatory cells, play central roles in the initiation and progression of atherosclerosis. Ithas been evidenced that oxidized low density lipoprotein (oxLDL) is an independent riskfactor of atherosclerosis. It brings about atherogenic features such as the activation ofmacrophages. However, there are no reports to date on the production of H2S in humanmonocyte/macrophages and effect of oxLDL on above process.Objectives: To investigate the endogenous production of H2S in humanmonocyte-derived macrophages and whether oxLDL regulates H2S generation.Methods: The production of H2S in human monocyte-derived macrophages wasdetermined by spectrophotometry. The expression of cystathionine-γ-lyase (CSE) andcystathionine-synthase (CBS) was detect by RT-PCR and Western blot.Results: Human monocyte-derived macrophages had robust mRNA expression of CSEand slender mRNA expression of CBS, and generate endogenous H2S. L-cysteine (a substrate of CSE and CBS) increased endogenous H2S production and releasing in normalmacrophages. However, DL-propargylglycine (PPG, CSE inhibitor) and hydroxylamine(HA, CBS inhibitor) inhibited H2S production and releasing. OxLDL treatment caused asignificant reduction of H2S synthesizing activity in human monocyte-derivedmacrophages. OxLDL also decreased the H2S content in a dose and time dependentmanner in the cell supernatants. Moreover, oxLDL abolished the effect of L-cysteine onthe increased H2S production but had a synergistic inhibitory effect with PPG or HA.Furthermore, oxLDL treatment down-regulated the mRNA and protein expression of CSEand CBS.Conclusions: These results demonstrate that CSE is a main enzyme for H2S productionin huamn monocyte-derived macrophages. OxLDL can decrease the endogenousproduction of H2S in huamn monocyte-derived macrophages. Background：Studies have shown several antiatherogenic effects of H2S, includingsuppressing the proliferation of rat aortic smooth muscle cells(SMC), inhibiting adhesionmolecule expression in arterial endothelial cells. It is worthy of further investigating theunderlying anti-atherogenic effect of H2S due to the complexity of the atherogenic process.Formation of macrophage-derived foam cells is a crucial event in the development ofatherosclerosis. But no study has examined the role of H2S in foam cell formation.Objectives: To explore the effect of hydrogen sulfide (H2S) on the formation ofmacrophage-derived foam cells and its potential mechanisms.Methods: Monocytes(human primary monocytes or the monocytic THP-1cell line) weredifferentiated into monocyte-derived macrophages by treatment with phorbol12-myristate13-acetate. Intracellular cholesterol content, oxLDL binding and uptake, scavengerreceptor A (SR-A), CD36and acyl-coenzyme A:cholesterol acyltransferase-1(ACAT-1)expression, TNF-α production of the cell and signaling pathways of H2S were determined.Results: Incubation of monocyte-derived macrophages with oxLDL alone causedsignificant increases both in intracellular lipid revealed by Oil-red O staining and inintracellular total cholesterol (TC), cholesterol ester (CE) concentrations assessed by highperformance liquid chromatography (HPLC). Sodium hydrosulfide (NaHS, an H2S donor)remarkably abrogated oxLDL-induced intracellular lipid accumulation and attenuated TC,CE concentrations and CE/TC ratio, whereas, PPG (a H2S-generating enzymecystathionine gamma lyase inhibitor) exacerbated lipid accumulation and augmented TC,CE concentrations and CE/TC ratio. Incubation of DiI-oxLDL led to lipoprotein bindingand uptake of macrophages, which was blunted by NaHS, but enhanced by PPG. Furthermore, OxLDL markedly induced CD36, SR-A and ACAT-1expression inmacrophages, which was abolished by NaHS (50to200μmol/L). Finally, thedown-regulations of TC, CE concentrations as well as CD36and ACAT-1expression byNaHS were abolished by glibenclamide, a KATPchannel blocker; but facilitated byPD98059, an ERK1/2inhibitor. On the other hand, NaHS not only significantly reducedTNF-α production and secretion, but also inhibited partially CD36expression induced byTNF-α in monocyte-derived macrophages.Conclusions: These results suggested that H2S inhibits foam cell formation bydown-regulating CD36, SR-A and ACAT-1expression in part via the KATP/ERK1/2pathway and in part via inhibiting TNF-α in human monocyte-derived macrophages. Background: Studies indicate that arterial tissues including aorta, coronary artery,arteriola can express CSE and product and release H2S, which suggests H2S involves inarterial function regulation by resembling paracrine/autocrine mechanis.Our previousfindings showed that H2S inhibits intracellular lipid accumulation in macrophagessuggesting furtherly its potential role in reducting lipid accumulation in atheroscleroticplaque.Objectives: To investigate the effect of H2S on lipid accumulation in atheroscleroticplaque.Methods:8-week-old C57BL/6J wild-type and apoE-/-mice received regular chow or ahigh-fat diet containing21%w/w fat and0.15%w/w cholesterol. ApoE-/-mice weredivided into3groups for treatment: intraperitoneal injections of56μmol/kg NaHS,37.5mg/kg PPG or vehicle(saline) alone once a day. After20weeks, mice were anesthetizedand various tissues such as heart, aorta, and blood were collected. Lipids in the aortic rootand en face aorta were viewed by Oil-red O staining and Sudan IV staining, respectively.Foam cell area within the aortic root lesion was examined by Movat’5and HE staining.The expression of CSE, SR-A, CD36, ACAT-1and macrophage content were detected byimmunohistochemistry or immunofluorescent staining. Messenger RNA levels ofSR-A,CD36,ACAT-1were assessed by real-time PCR. Serum TNF-α levels weredetected by ELISA. H2S level in plasma and H2S synthesizing activity in aortic tissue weredetermined by methylene blue method. Serum lipid levels were determined by enzymaticmethod.Results: There were no differences in serum lipid levels and bodyweight in any of thegroups of apoE-/-mice. CSE express in aortic root lesion in apoE-/-mice. Compared with C57BL/6J mice, H2S level in plasma was reduced in apoE-/-mice treated with vehicle.NaHS treatment caused a approximately21.4%increase and PPG treatment led to a22.4%decrease in plasma H2S level compared with vehicle treatment. Mice receiving NaHStreatment had a49.0%decrease in en face aortic lipid area and a23.2%decrease (P﹤0.05)in percentage of lipid-positive area in aortic root lesions as compared with vehicle-treatedmice.Whereas, mice treated with PPG exhibited a31.3%(P﹤0.01) increase in proportionof aortic root lesion area with lipid and a75.4%(P﹤0.01)increase in en face aortic areawith lipid as compared with vehicle treatment. In agreement with these results, there was a42.6%reduction in total atherosclerotic lesion area in NaHS treatment mice but a46.8%increase in PPG application compared with vehicle treatment mice. In addition, plaques ofNaHS-treated mice displayed a reduction of foam cell accumulation and notably smallerfoam cells, but advease results were observed in plaques of PPG-treated mice.Furthermore, reduction of macrophage accumulation in aortic lesions was observed inNaHS-treated mice, correlating with reduction of expression of SR-A, CD36and ACAT-1in plaque. In contrast, PPG increased macrophage accumulation and expression of SR-A,CD36and ACAT-1in aortic lesions. Finally, serum TNF-α levels were also reduced inNaHS-treated mice,but elevated in PPG-treated mice.Conclusions: Our results support a role for H2S in the regulation of accumulation oflipid in atherosclerotic lesion, possibly through reduced accumulation of macrophage andexpression of SR-A, CD36and ACAT-1and reduced levels of serum TNF-α.