Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque.

BACKGROUND: The heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition. METHODS: Weighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria. RESULTS: The CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice. CONCLUSIONS: The results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

Cathepsin B aggravates atherosclerosis in ApoE-deficient mice by modulating vascular smooth muscle cell pyroptosis through NF-κB / NLRP3 signaling pathway.

Atherosclerosis (AS) is a chronic inflammatory disease involving cell death and inflammatory responses. Pyroptosis, a newly discovered pro-inflammatory programmed cell death process, exacerbates inflammatory responses. However, the roles of cathepsin B (CTSB) in pyroptosis and AS remain unclear. To gain further insight, we fed ApoE-/- mice a high-fat diet to investigate the effects and mechanisms of CTSB overexpression and silencing on AS. We also explored the specific role of CTSB in vascular smooth muscle cells (VSMCs) in vitro. The study revealed that high-fat diet led to the formation of AS plaques, and CTSB was found to increase the AS plaque lesion area. Immunohistochemical and TUNEL/caspase-1 staining revealed the existence of pyroptosis in atherosclerotic plaques, particularly in VSMCs. In vitro studies, including Hoechst 33342/propidium iodide staining, a lactate dehydrogenase (LDH) release assay, detection of protein indicators of pyroptosis, and detection of interleukin-1ß (IL-1ß) in cell culture medium, demonstrated that oxidized low-density lipoprotein (ox-LDL) induced VSMC pyroptosis. Additionally, CTSB promoted VSMC pyroptosis. Ox-LDL increased the expression of CTSB, which in turn activated the NOD-like receptor protein 3 (NLRP3) inflammasome and promoted NLRP3 expression by facilitating nuclear factor kappa B (NF-κB) p65 nuclear translocation. This effect could be attenuated by the NF-κB inhibitor SN50. Our research found that CTSB not only promotes VSMC pyroptosis by activating the NLRP3 inflammasome, but also increases the expression of NLRP3.

FNDC5 Attenuates Atherosclerotic Plaque Formation and Regulates PPARα/HO-1 in ApoE-/- Mice.

INTRODUCTION: This study attempted to observe the role of fibronectin type III domain-containing protein 5 (FNDC5) in atherosclerosis development and the underlying mechanism. METHODS: After being fed a high-fat diet (HFD), ApoE-/- mice were injected with saline, control adenovirus (Ad-vector), or FNDC5 overexpressing adenovirus (Ad-FNDC5). ApoE-/- mice fed with a chow diet were considered the control. After 12 weeks of treatment, the levels of serum high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and irisin were detected by commercial kits. RESULTS: Compared with the control, the serum TG, TC, and LDL-C levels, aortic plaque area, and weight were significantly increased, while serum HDL-C and irisin levels were reduced in HFD mice. Treating with Ad-FNDC5 could alleviate these changes in HFD mice and cause the activation of PPARα/HO-1 signaling in aortic tissue. After co-treating with GW6471, a PPARα antagonist, the effects of Ad-FNDC5 on the weight, serum LDL-C, TC, TG, and HDL-C levels, and aortic plaque of HFD mice were partly blocked. CONCLUSION: Elevated FNDC5 has a delaying effect on atherosclerotic plaque formation, which may be related to the upregulation of PPARα/HO-1 signaling.

Dapagliflozin Attenuates NLRP3/Caspase-1 Signaling Pathway-Mediated Pyroptosis of Vascular Smooth Muscle Cells by Downregulating CTSB.

Background: Atherosclerosis is a chronic inflammatory disease. Pyroptosis triggers and amplifies the inflammatory response and plays an important role in atherosclerosis. Cathepsin B (CTSB) can promote atherosclerosis and activate NOD-like receptor protein 3 (NLRP3) to mediate pyroptosis. Dapagliflozin (DAPA) can inhibit cell pyroptosis to improve atherosclerosis. This study aimed to explore the effect of DAPA on oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis of vascular smooth muscle cells (VSMCs) and its underlying mechanism. Objective: We aimed to investigate the effect of DAPA on ox-LDL-induced pyroptosis of VSMCs in mice and its underlying mechanism. Methods: VSMCs were transfected with CTSB-overexpressing and -silencing lentiviral vectors. VSMCs were treated with different concentrations of ox-LDL (0, 50, 100 and 150 µg/ml ). Then, Hoechst 33342/PI double staining, interleukin (IL)-1ß and lactate dehydrogenase (LDH) release assay were used to detect cell pyroptosis. Western blotting was used to detect pyroptosis indicators protein, based on which the appropriate concentration of ox-LDL was selected. After VSMCs were treated with different concentrations of DAPA (0.1 µM, 1.0 µM, 5.0 µM, 10 µM, 25 µM and 50 µM), the proliferative activity of VSMCs was detected using Cell Counting Kit-8 (CCK8) assay. After VSMCs were pretreated with different DAPA concentrations (0.1 µM, 1.0 µM, 5.0 µM and 10 µM) for 24 hours and then treated with 150 µg/mL ox-LDL for 24 hours, the effects of different concentrations of DAPA on pyroptosis of VSMCs were detected, based on which the appropriate DAPA concentration was selected. After lentivirus transfected VSMCs were treated with 150 µg/mL ox-LDL for 24 hours, the effects of overexpression and silencing of CTSB in pyroptosis were observed. On the basis of DAPA (0.1 µM)- and ox-LDL(150 µg/mL)-treated VSMCs, overexpression and silencing of CTSB were used to observe the effects of DAPA and CTSB on ox-LDL-mediated VSMCs pyroptosis. Results: (1) VSMCs stably transfected with CTSB-overexpressing and -silencing lentiviruses were obtained; 150 µg/mL was the optimal concentration of ox-LDL for inducing pyroptosis of VSMCs, and 0.1 µM was the optimal concentration of DAPA for ameliorating pyroptosis of VSMCs. (2) Ox-LDL-induced pyroptosis of VSMCs was worsened by CTSB overexpression but suppressed by CTSB silencing. (3) DAPA attenuated ox-LDL-induced pyroptosis of VSMCs through downregulating CTSB and NLRP3. (4) Overexpression of CTSB based on DAPA intervention aggravated ox-LDL-induced pyroptosis of VSMCs. Conclusion: DAPA attenuates NLRP3/caspase-1 pathway-mediated pyroptosis of VSMCs through downregulating CTSB.

FNDC5/PPARa Pathway Alleviates THP-1-derived Macrophage Pyroptosis and Its Mechanism.

Context: Atherosclerosis (AS) is a chronic inflammatory disease. Pyroptosis is a newly discovered, pro-inflammatory cell death that can trigger and amplify the occurrence and progression of AS. Researchers are still uncertain about the anti-atherosclerotic mechanism of "fibronectin type III domain-containing protein 5" (FNDC5). Objective: The study aimed to investigate the ability of FNDC5-mediated, "peroxisome proliferator activated receptor alpha" (PPARa) to inhibit oxidized low-density lipoprotein (ox-LDL)-induced, THP-1-derived macrophage pyroptosis and to determine a potential molecular mechanism at the cellular level. Design: The research team performed a laboratory study. Setting: The study took place in the Department of Cardiovascular Medicine at the Affiliated Hospital of Guzhou Medical University at the Medical Research Institute at Guizhou Medical University in Guiyang, Guizhou, China. Outcome Measures: The research team: (1) constructed and stably transfected FNDC5 gene-overexpressing and FNDC5 gene-silencing lentiviral vectors into THP-1 cells; (2) observed the cell morphology under an inverted fluorescence microscope and screened the stably transfected THP-1 cells with puromycin; (3) verified the transfection efficiency using quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot; (4) used phorbol to induce THP-1 cells into macrophages; (5) cultured the THP-1-derived macrophages with different concentrations of ox-LDL-25, 50, 75, and 100 µg/ml-for 24 h; (6) performed Hoechst 33342/ propidium iodide (PI) double staining and examined lactate dehydrogenase (LDH) and interleukin-1 beta (IL-1ß) activity to determine the effects of ox-LDL on THP-1-derived macrophage pyroptosis; (7) selected the optimal ox-LDL concentration; (8) divided the THP-1-derived macrophages into seven groups: NC group (no ox-LDL intervention), ox-LDL group, PBS group, Mock1 group, Ad-FNDC5 group, Mock2 group, and Sh-FNDC5 group; (9) examined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and "nuclear factor kappa-light chain enhancer of activated B cells P65" (NF-κB P65), and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining, respectively; (10) treated the THP-1-derived macrophages with FNDC5 expression with GW6471, a specific PPARα antagonist; (11) determined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and NF-κB P65, and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining and detection of the LDH and IL-1ß activity, respectively. Results: With the stably transfected THP-1 cells with FNDC5 overexpression or silencing the ox-LDL-induced, THP-1-derived, macrophage pyroptosis occurred in a concentration-dependent manner. Compared with the ox-LDL, phosphate buffered saline (PBS), Mock1, and Mock2 groups, the Ad-FNDC5 group had a significant increase in expression of FNDC5 and of peroxisome proliferator activated receptor alpha (PPARa) proteins (P < .05). The "nuclear factor kappa-light chain enhancer of activated B cells P65: (NF-κB P65), NOD-like receptor thermal protein domain associated protein 3, (NLRP3), Caspase-1, gasdermin D (GSDMD, IL-1ß and IL-18 protein expressions, percentage of PI-positive cells, LDH activity, and IL-1ß activity decreased significantly (P < .05); the results in the Sh-FNDC5 group were opposite to those in the Ad-FNDC5 group. 3. Intervention with GW6471 (PPARa antagonist) in the stably transfected THP-1-derived macrophages with FNDC5 overexpression abolished the protective effect of FNDC5 against ox-LDL-induced THP-1-derived macrophage pyroptosis. Conclusions: Irisin/PPARa inhibited THP-1-derived macrophage pyroptosis and inflammation and delayed AS by inhibiting the NF-κB/NLRP3 pathway.

Association between AT1 receptor gene polymorphism and left ventricular hypertrophy and arterial stiffness in essential hypertension patients: a prospective cohort study.

BACKGROUND: AT1 receptor gene (AGTR1) is related to essential hypertension (EH), and left ventricular hypertrophy (LVH) and arterial stiffness are common complications of EH. This study aimed to explore the association between AGTR1 genotype and LVH and arterial stiffness in EH patients. METHODS: A total of 179 EH patients were recruited in this study. Oral exfoliated cells were collected from each patient, and the genetic polymorphism of AGTR1(rs4524238) was assessed using a gene sequencing platform. The outcomes were LVH and arterial stiffness. RESULTS: Among 179 patients, 114 were with AGTR1 genotype of GG (57 males, aged 59.54 ± 13.49 years) and 65 were with AGTR1 genotype of GA or AA (36 males, aged 61.28 ± 12.79 years). Patients with AGTR1 genotype of GG were more likely to have LVH (47 [41.23%] vs. 14 [21.54%], P = 0.006) and arterial stiffness (30 [26.32%] vs. 8 [12.31%], P = 0.036). The AGTR1 polymorphism frequency was in accordance with Hardy-Weinberg equilibrium (P = 0.291). The multivariate logistic regression showed that AGTR1 genotype of GA or AA was independently associated with lower risk of LVH (OR = 0.344, 95%CI 160~0.696, P = 0.003) and arterial stiffness (OR = 0.371, 95%CI 0.155~0.885, P = 0.025) after adjusting for gender, age, and diabetes. CONCLUSION: EH patients with the AGTR1 genotype of GA or AA were at lower risk for LVH and arterial stiffness than those with the GG genotype.

Protective activity of Malus doumeri leaf extract on H2O2-induced oxidative injury in H9C2 rat cardiomyocytes.

In this study, Malus doumeri leaf extract (MDLE) was used to test its anti-oxidation capacity in vitro, it has been preliminarily analyzed for H2O2-induced oxidative damage in H9C2 cells and its main active components. The antioxidant capacity through DPPH (1, 1-Diphenyl-2-Picrylhydrazyl), ABTS+• [2,2,2'-azino-BIS-(3-ethylbenzo-thiazoline-6-sulfonic acid)] radical ion, •OH (hydroxyl radical), and • O 2 - (superoxide anion) were determined in vitro. The proliferation of H9C2 cells was examined by MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-Tetrazolium bromide]. MDA (malondialdehyde), SOD (superoxide dismutase), CAT (catalase), GSH (glutathione), and GSH-Px (glutathione peroxidase) were determined by colorimetry. Apoptosis induced by oxidative damage was detected by flow cytometry. The mRNA expression of antioxidant related genes of SOD, CAT, GSH, and GSH-Px were checked by qRT-PCR (quantitative real-time polymerase chain reaction). The MDLE main active components were analyzed by HPLC (high-performance liquid chromatography). MDLE had significant scavenging effects on DPPH, ABTS+•, •OH, and superoxide anion radicals in a concentration-dependent manner. H2O2 treatment could significantly lead to oxidative stress injury of H9C2 cells, and MDLE treatment significantly improved the degree of H9C2 cell damage, and showed a positive correlation with concentration. MDLE can also reduce apoptosis caused by oxidative damage. MDLE treatment could significantly reduce MDA content and increase CAT, SOD, GSH, and GSH-Px contents and expression. In addition, by HPLC analysis, the following six bioactive components were detected from MDLE: chlorogenic acid, isoquercitrin, quercetin, baicalin, and phloretin. Therefore, MDLE has a good protective effect on myocardial cells.

Case Report: Passive Handstand Promotes Cerebrovascular Elasticity Training and Helps Delay the Signs of Aging: A 40-Year Follow-Up Investigation.

Background: There are no long-term (>10 years) follow-up evaluations of the effects of handstand exercise or studies on the use of equipment for passive handstand exercise. Objective: To report a 40-year follow-up investigation of a Chinese man who has been practicing passive handstand for 40 years. Design: This observational investigation was conducted in Guizhou Province, China. Participant: A (currently) 66-year-old Chinese man who had been practicing passive handstand exercise for 40 years was followed up. Interventions: Physical and auxiliary examinations were carried out to determine the effects of long-term passive handstand exercise on the human body. Main Measures: The participant's cerebrovascular, spinal health, mental health, and visual acuity as well as the presence of facial aging were examined. Key Results: His cerebral vessels were healthy, he appeared younger than his peers, his cervical spondylosis improved, and his mental state and cognitive function were good. Conclusion: Long-term passive handstand exercise can promote cerebrovascular elasticity training and delay signs of aging. We recommend promoting this passive handstand exercise to the public.

MicroRNA-23b prevents aortic aneurysm formation by inhibiting smooth muscle cell phenotypic switching via FoxO4 suppression.

AIMS: Phenotypic switching of vascular smooth muscle cells (VSMCs) is essential for the formation of abdominal aortic aneurysms (AAAs). MicroRNA-23b (miR-23b) has recently been shown to play a vital role in maintaining the VSMC contractile phenotype; however, little is known about the role of miR-23b in the formation of AAAs. Here, we investigated whether miR-23b prevents AAA formation by inhibiting VSMC phenotypic switching. MATERIALS AND METHODS: We administered angiotensin II (Ang II, 1000 ng/kg/min) or vehicle to 10-12-week-old male apolipoprotein E knockout (ApoE-/-) or C57BL/6J mice via subcutaneous osmotic minipumps for 4 weeks. KEY FINDINGS: The expression of miR-23b was significantly reduced in the aorta during the early onset of AAA in angiotensin II-treated ApoE-/- mice and in human AAA samples. In vitro experiments showed that the suppression of SMC contractile marker gene expression induced by Ang II was accelerated by miR-23b inhibitors but inhibited by mimics. In vivo studies revealed that miR-23b deficiency in Ang II-treated C57BL/6J mice aggravated the formation of AAAs in these mice compared with control mice; the opposite results were observed in miR-23b-overexpressing mice. Mechanistically, miR-23b knockdown significantly increased the expression of the transcription factor forkhead box O4 (FoxO4) during VSMC phenotypic switching induced by Ang II. In addition, a luciferase reporter assay showed that FoxO4 is a target of miR-23b in VSMCs. SIGNIFICANCE: Our study revealed a pivotal role for miR-23b in protecting against aortic aneurysm formation by maintaining the VSMC contractile phenotype.

Heme oxygenase-1 (HO-1) alleviates vascular restenosis after balloon injury in a rabbit carotid artery model.

Percutaneous coronary intervention (PCI) is used commonly for coronary artery disease (CAD); however, restenosis is a proliferative response and frequent sequela to this treatment. Although the introduction of drug-eluting stents has convincingly reduced the incidence of vascular restenosis, restenosis remains a problem. The present study was designed to investigate the effects of the heme oxygenase-1 (HO-1) on restenosis formation after balloon injury in a rabbit carotid artery model. We found that involvement of the HO-1 in defensive restenosis formation was independent of the levels of blood lipid. Activation of HO-1 induced by chlorhematin treatment alleviated vascular restenosis after balloon injury in a rabbit carotid artery model, whereas inhibition of HO-1 by zinc protoporphyrin treatment exacerbated restenosis formation. Furthermore, overexpression of HO-1 inhibited nuclear factor kappa B subunit 1 (NF-кB) activity and decreased tumor necrosis factor-alpha (TNF-α) and endothelin 1 (ET-1) expression. In conclusion, our study provides preliminary data suggesting that HO-1 alleviates vascular restenosis after balloon injury in a rabbit carotid artery model by inhibiting NF-кB, TNF-α and ET-1 expression, indicating induction of HO-1 activation may be a feasible therapeutic target for treating vessels resistant to restenosis.

Intractable angina pectoris after coronary artery bypass surgery in Takayasu arteritis involving the aorta ventralis and main coronary artery in a young girl.

We report a case of sudden death in Takayasu arteritis after coronary artery bypass. A 22-year-old girl visited our hospital in June 2009 because of paroxysmal chest tightness and shortness of breath for 2 years. She was diagnosed as Takayasu arteritis, the limited stenosis of upper aorta ventralis, low perfusion pressure changes of double renal artery and double lower limbs artery, left ventricular mural thrombus and patent foramen ovale. The coronary artery bypass grafting was conducted. However, the symptoms were recurrent 2 months later.

Effects of induction/inhibition of endogenous heme oxygenase-1 on lipid metabolism, endothelial function, and atherosclerosis in rabbits on a high fat diet.

The heme oxygenase-1 (HO-1) / carbon monoxide (CO) system has been presumed as a therapeutic target for preventing atherosclerosis. However, the exact mechanism(s) underlying this system remains largely undefined. This study aims to examine the influence of induction/inhibition of HO-1 on atherosclerotic plaque using pharmacological approaches and to elucidate potential mechanisms. Rabbits were randomly assigned to receive a standard diet (control group), high fat diet (HFD), HFD plus HO inducer hemin (HFD + H group), and HFD plus an HO inhibitor, zinc protoporphyrin-9 (ZnPP9, HFD + Z group). Atherosclerotic plaque was evaluated using oil red O staining and histological analyses. Immunohistochemistry, western blotting, and RT-PCR were employed to study the expression of HO-1 and endothelin-1 (ET-1). Levels of CO, nitric oxide (NO), eNOS/iNOS activities, NF-κB activity, and TNF-α level were determined. No significant differences of serum lipid levels were observed among the HFD, HFD + Z, and HFD + H groups. In rabbits, HFD induced typical atherosclerotic plaque and increased intima/media thickness ratio, which was markedly reduced in the HFD + H group and further aggravated in the HFD + Z group. Furthermore, hemin increased HO-1 expression, CO levels, and eNOS activity, while decreasing iNOS levels, ET-1 expression, NF-κB activity, and TNF-α level. ZnPP9 caused opposite effects. Induction of the endogenous HO-1/CO system by hemin can prevent atherosclerosis though increasing CO levels, regulating eNOS activity, NF-κB activity, TNF-α levels, and ET-1 levels in rabbits. Our results add new evidence for the importance of HO-1 in the genesis and development of atherosclerosis and provide several possible mechanisms underlying the anti-atherosclerosis effects of HO-1.

Effects of Induction/Inhibition of Endogenous Heme Oxygenase-1 on Lipid Metabolism, Endothelial Function, and Atherosclerosis in Rabbits on a High Fat Diet.

The heme oxygenase-1 (HO-1) / carbon monoxide (CO) system has been presumed as a therapeutic target for preventing atherosclerosis. However, the exact mechanism(s) underlying this system remains largely undefined. This study aims to examine the influence of induction/inhibition of HO-1 on atherosclerotic plaque using pharmacological approaches and to elucidate potential mechanisms. Rabbits were randomly assigned to receive a standard diet (control group), high fat diet (HFD), HFD plus HO inducer hemin (HFD + H group), and HFD plus an HO inhibitor, zinc protoporphyrin-9 (ZnPP9, HFD + Z group). Atherosclerotic plaque was evaluated using oil red O staining and histological analyses. Immunohistochemistry, western blotting, and RT-PCR were employed to study the expression of HO-1 and endothelin-1 (ET-1). Levels of CO, nitric oxide (NO), eNOS/iNOS activities, NF-κB activity, and TNF-α level were determined. No significant differences of serum lipid levels were observed among the HFD, HFD + Z, and HFD + H groups. In rabbits, HFD induced typical atherosclerotic plaque and increased intima/media thickness ratio, which was markedly reduced in the HFD + H group and further aggravated in the HFD + Z group. Furthermore, hemin increased HO-1 expression, CO levels, and eNOS activity, while decreasing iNOS levels, ET-1 expression, NF-κB activity, and TNF-α level. ZnPP9 caused opposite effects. Induction of the endogenous HO-1/CO system by hemin can prevent atherosclerosis though increasing CO levels, regulating eNOS activity, NF-κB activity, TNF-α levels, and ET-1 levels in rabbits. Our results add new evidence for the importance of HO-1 in the genesis and development of atherosclerosis and provide several possible mechanisms underlying the antiatherosclerosis effects of HO-1.

[Prevention of atherosclerotic plaque development by modulating heme oxygenase-1-endogenous carbon monoxide system in rabbit model].

OBJECTIVE: To investigate the effect of heme oxygenase/carbon monoxide (HO-1/CO) system on lipid deposition at aortic intima and the mechanism involved in hyperlipidemic rabbits. METHODS: Totally 32 rabbits, were divided into four groups. One group as control. Three groups for the following treatments: 1.5% cholesterol ration (Ch group, n = 8); 1.5% cholesterol ration plus HO-1 inducer hemin (Hm group, n = 8); and instead of hemin, the HO-1 inhibitor, zinc protoporphyrin IX (Zn group, n = 8) was given by injection into the abdominal cavity. Experiments were lasted for 12 weeks. Rabbit aortas were then isolated as the samples for histopathologic and ultrastructural examination. The protein expressions of HO-1 and endothelin-1 (ET-1) were investigated by immunohistochemical staining and Western blot analysis. RESULTS: Comparing with the Ch group, rabbits of the Hm group showed a remarkably less extent of lipid deposition at the aortic intima [(17.9 ± 3.0)% vs (54.0 ± 4.2)%], and rabbits of the Zn group had a marked extent of lesion development [(61.1 ± 3.5)%]. Lipid deposition, endothelial damage and neo-intimal formation were less severe in rabbits of the Hm group than those in the Zn or Ch group, respectively. Comparing with the control group, rabbits of the Ch group showed a significant decrease of aortic NO production and cNOS activity. However, there were an enhancement of CO production and HO-1 activity (P < 0.01). Compared with Ch group, rabbits of the Hm group showed a remarkable elevation of aortic HO activity and CO production, whereas rabbits of the Zn group showed a marked decrease of both parameters. Compared with the Ch group, rabbits of the Hm group demonstrated a marked reduction of aorta ET-1 expression, whereas Zn group had a significantly higher ET-1 expression. CONCLUSIONS: Modulation of HO-1/CO system may improve vascular endothelial function and inhibit smooth muscle cell proliferation in hypercholesterolemic rabbits, likely through a compensatory mechanism and a reduction of ET-1 expression, eventually leading to an inhibition of atherosclerotic plaque development.

[Influences of heme oxygenase-1, carbon monoxide and nitric oxide synthase, nitrogen monoxide systems on vascular remodeling of injured balloon carotid artery in rabbits and the intercorrelations among the two systems].

The aim of this study was to investigate the influences of heme oxygenase-1, carbon monoxide and nitricoxide synthase, nitrogen monoxide systems on vascular remodeling of injured balloon carotid artery in rabbits and the intercorrelations among the two systems after balloon angioplasty. Seventy rabbits were randomly divided into seven groups, i. e., control group, SH group, Chol group, Arg group, L-NAME group, Hem group, and Znpp group. The control group received normal chow, while all the rabbits the rest six groups received 1.5% cholesterol diet. Among the six test groups, to those in Chol group and SH group nothing else was added except the 1.5% cholesterol. L-arginine or L-nitro-arginine methylester was added to those in the Arg group and in the L-NAME group with drinking water. Hemin or zincprotoporphyrin IX was added to those in Hem group and in Znpp group by injecting the medicine into the abdominal cavity. After two weeks, the experimental groups underwent balloon injury at one side common carotid artery. Compared to Chol group, the HO-1 activity and CO production increased significantly. The intima area was reduced distinctly in Hem group, while there were opposite results in Znpp group. Compared with that in Chol group, the NF-kappaB activity of Arg group and Hem group were lower significantly. That of L-NAME group and Znpp group were higher significantly. Compared with that in the Chol group, the cNOS activity and NO production were eleveated markedly in Arg group while they were decreased markedly in L-NAME group. The intima area was reduced significantly in Arg group, while in L-NAME group they were not different from those in Chol group. These results suggested that the reciprocal relationship between HO-1/CO and NOS/NO system in restenosis may play the inhibitory role against neointimal proliferation and vascular wall remodeling after balloon angioplasty.

Effect of the haeme oxygenase-1/endogenous carbon monoxide system on atherosclerotic plaque formation in rabbits.

BACKGROUND: To investigate the effect of the haeme oxygenase-1/carbon monoxide (HO-1/CO) system on atherosclerotic plaque formation and its possible mechanism. METHODS: For 12 weeks, rabbits were given a 1.5% cholesterol diet (Ch group, n = 8) or a 1.5% cholesterol diet plus an HO-1 inducer, haemin (Hm group, n = 8), or an HO-1 inhibitor, zinc protoporphyrin IX (Znpp-IX, Zn group, n = 8) by intraperitoneal injection. RESULTS: Compared with the normal control group (C group, n = 8), serum levels of lipids and oxidised low-density lipoproteins (ox-LDL) increased significantly in all experimental groups (p < 0.01). However, no significant differences were observed among the three experimental groups (p > 0.01). Compared with the control group, aortic nitric oxide (NO) production and nitric oxide synthase (cNOS) activity decreased markedly, whereas carbon monoxide (CO) production and HO-1 activity increased markedly in the Ch group (p < 0.01). This was associated with an increase in the area of aortic plaque of 54.00 ± 4.16%. Compared with the Ch group, CO production and HO-1 activity increased markedly, while aortic HO activity and CO production decreased significantly in the Hm group. The area of aortic plaque was significantly reduced in the Hm group (17.88 ± 3.01%), whereas the area of aortic plaque was significantly increased in the Zn group (61.13 ± 3.50%). Compared with the Ch group, aortic endothlin-1 expression in the Hm group reduced significantly, while in the Zn group it was significantly higher than in the Ch group (p < 0.01). CONCLUSION: The HO-1/CO system plays an inhibitory role in atherosclerotic plaque formation. This role was not mediated by regulating serum lipids and ox-LDL, but was related to the reciprocal relationship between the HO-1/CO and NOS/NO systems in atherosclerosis and the down-regulated expression of endothlin-1 (ET-1), which inhibits the proliferation of vascular smooth muscle cells.

[Heme oxygenase-1 and carbon monoxide are key mediators for vascular smooth muscle cells proliferation induced by insulin-like growth factor-I].

OBJECTIVE: To determine the role and related mechanisms of heme oxygenase-1/carbon monoxide (HO-1/CO) on VSMCs proliferation induced by insulin-like growth factor-I (IGF-I). METHODS: VSMCs isolated from rabbit aorta were cultured in vitro and proliferation was induced by IGF-I. Hemin (a substrate and inducer of HO-1) or zinc protoporphyrin-IX (Znpp-IX, an inhibitor of HO-1) was added to stimulate or inhibit the expression of HO-1. The mRNA and protein expressions of HO-1 were detected by RT-PCR and Western blot analysis. CO released into the culture media was quantitated by measuring carbon monoxide hemoglobin (COHb), VSMCs proliferation and cell cycle were determined by (3)H-TdR incorporation assay and flow cytometry, respectively. RESULTS: The HO-1 mRNA and protein expressions in VSMCs and the amount of COHb in the culture media were significantly increased and the IGF-I-induced (3)H-TdR incorporations of VSMCs significantly reduced by hemin in a dose-dependent manner (P < 0.01). Furthermore, VSMCs in the G(0)/G(1) phase were increased and in the S and G(2)/M phase decreased by hemin (P < 0.01). Opposite results were observed in VSMCs treated with Znpp-IX. CONCLUSIONS: Endogenous HO-1 and CO are important mediators for inhibiting IGF-I induced VSMCs proliferation by reducing VSMCs DNA synthesis and decelerating cell cycle progression.