Knockdown of HDAC6 alleviates ventricular remodeling in experimental dilated cardiomyopathy via inhibition of NLRP3 inflammasome activation and promotion of cardiomyocyte autophagy.

Histone deacetylases (HDACs) has been implicated in cardiac diseases, while the role of HDAC6 in dilated cardiomyopathy (DCM) remains obscure. The in silico analyses predicted potential association of HDAC6 with autophagy-related genes and DCM. Thus, we evaluated the functional relevance of HDAC6 in DCM in vivo and in vitro. We developed a rat model in vivo and a cell model in vitro by doxorubicin (DOX) induction to simulate DCM. HDAC6 expression was determined in myocardial tissues of DCM rats. DCM rats exhibited elevated HDAC6 mRNA and protein expression as compared to sham-operated rats. We knocked HDAC6 down and/or overexpressed NLRP3 in vivo and in vitro to characterize their roles in cardiomyocyte autophagy. It was established that shRNA-mediated HDAC6 silencing augmented cardiomyocyte autophagy and suppressed NLRP3 inflammasome activation, thus ameliorating cardiac injury in myocardial tissues of DCM rats. Besides, in DOX-injured cardiomyocytes, HDAC6 silencing also diminished NLRP3 inflammasome activation and cell apoptosis but enhanced cell autophagy, whereas ectopic NLRP3 expression negated the effects of HDAC6 silencing. Since HDAC6 knockdown correlates with enhanced cardiomyocyte autophagy and suppressed NLRP3 inflammasome activation through an interplay with NLRP3, it is expected to be a potential biomarker and therapeutic target for DCM. 1. HDAC6 was up-regulated in DCM rats. 2. HDAC6 knockdown promoted cardiomyocyte autophagy to relieve cardiac dysfunction. 3. HDAC6 knockdown inhibited NLRP3 inflammasome and promoted cardiomyocyte autophagy. 4. Silencing HDAC6 promoted autophagy and repressed apoptosis in cardiomyocytes. 5. This study provides novel therapeutic targets for DCM.

Atorvastatin suppresses vascular hypersensitivity and remodeling induced by transient adventitial administration of lipopolysaccharide in rats.

BACKGROUND: The phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative state markedly affects the pathophysiology of cardiovascular diseases. The adventitial inflammation can promote neointimal formation and vascular remodeling. We used direct administration of lipopolysaccharide (LPS) into the periphery of the carotid artery to investigate the influence of transient adventitial inflammation on vascular remodeling and its potential mechanism. METHODS: Male 15-week-old Wistar rats were randomly assigned to four groups with six rats in each group. The rats of groups I and II were administered distilled water, and group III and IV were treated with fasudil and atorvastatin respectively. All treatments were given daily for 11 days. On day 8, the adventitia in group I was injected with 5 µL sterile saline, and the group II-IV were injected with 5 µL sterilized LPS. The carotid blood flow and femoral blood pressure were measured in vivo, and the thickness of vascular intima and middle layer was measured in vitro. Serum interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were determined using enzyme-linked immunosorbent assay (ELISA) assay. And the Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), myosin phosphatase target subunit 1 (MYPT1), myosin light chain (MLC), myocardin, SM-α actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by western blot. The comparisons were made by one-way analysis of variance with Bonferroni's post hoc test. A value of P<0.05 was considered to represent a statistically significant difference. RESULTS: Transient adventitial inflammation induced by LPS caused no obvious change in basal blood flow, but did lead to vascular hypersensitivity to serotonin. Morphological examinations revealed that the medial layer was the only domain affected, and showed VSMC proliferation and rearrangement. LPS increased serum IL-6 and TNFα contents, ROCK2 expression and activity, and caused changes in the expression levels of some stereotypical VSMC genes. Similar to the Rho-kinase inhibitor fasudil, atorvastatin completely restored the morphological alterations, even increased blood flow. CONCLUSIONS: Our study confirms the beneficial effect of atorvastatin on the vascular system in terms of morphology and function.

Statin-ezetimibe versus statin lipid-lowering therapy in patients with acute coronary syndromes undergoing percutaneous coronary intervention.

BACKGROUND: Studies comparing the clinical efficacy and safety of intensive statin therapy with ezetimibe-statin combination therapy are still rare at present, especially in Asian population. METHODS: We enrolled 202 patients who suffered acute coronary syndrome (ACS) and underwent percutaneous coronary intervention (PCI) between May and July in 2016. Patients were allocated into three groups based on the lipid lowering strategy: moderate-intensity statin group (n=118), ezetimibe combined with moderate-intensity statin group (ezetimibe-statin combination, n=55) and intensive statin group (n=29). The lipid profiles and side effects were analyzed and compared among the patients in three groups at admission, 1 month and 3 months after PCI. The clinical outcomes of the patients were observed through 6-month follow-up. RESULTS: One month after PCI, the level of non-high density lipoprotein-cholesterol (non-HDL-C) was decreased by 41.9%, 21.6% and 29.8% by ezetimibe-statin combination therapy, moderate-intensity statin therapy and intensive statin therapy, respectively (P<0.05). The reduction percentages of TC and LDL-C were significantly higher in ezetimibe-statin combination group than in moderate-intensity statin group (P<0.001). The proportion of patients reaching LDL-C goal was higher in ezetimibe-statin combination group (69.1%, P=0.007) and intensive statin group (67.9%, P=0.047) compared with moderate-intensity statin group (46.9%) at 1 month after PCI. There was no significant difference among the three groups with respect to hepatic enzymes level, creatine kinase (CK) level and incidence of muscle symptoms. CONCLUSIONS: The reduction percentage of non-HDL-C was larger in ezetimibe-statin combination group than intensive statin group. This finding suggested that statin/ezetimibe combination therapy could be an alternative to intensive statin therapy in Chinese patients with atherosclerotic cardiovascular disease.

Efficacy and safety of ticagrelor versus clopidogrel with different dosage in high-risk patients with acute coronary syndrome.

BACKGROUND: Dual antiplatelet therapy is recommended as a standard antiplatelet strategy in acute coronary syndrome. For those with reduced pharmacologic response to clopidogrel, strengthening antiplatelet therapy (clopidogrel 150mg daily) may reduce adverse clinical events. Ticagrelor is a direct-acting inhibitor of the adenosine diphosphate receptor P2Y12 that has a more rapid onset and offset than clopidogrel. METHODS: In this retrospective study, we compared ticagrelor (180mg loading dose 90mg twice daily thereafter), clopidogrel (300mg loading dose, 75mg or 150mg daily thereafter) for the prevention of cardiovascular events in 273 high-risk patients admitted to coronary care unit with acute coronary syndrome. RESULTS: The rate of IST in hospital was significantly reduced in patients of ticagrelor group comparing with those receiving clopidogrel 75mg (0.69% vs 8.2%, p=0.009). Moreover, the TVR rate was less in the ticagrelor group than clopidogrel 75mg group (2.7% vs 13.1%, p=0.007) 6months follow-up. The incidence of MACCE has no difference between the two clopidogrel groups. Kaplan-Meier analysis of MACCE-free indicated that there was no difference between the three groups. Ticagrelor significantly increased the rate of minor bleeding compared with clopidogrel 75mg daily during hospital (45.5% vs 26.2%,p=0.012) and 6-month follow-up (66.9% vs 45.9%,p=0.004).Bleeding-free prognosis was significantly better in the clopidogrel 75mg daily group. CONCLUSIONS: In patients with acute coronary syndrome undergoing PCI, the rate of in-stent thrombosis and TVR were significantly reduced treated with ticagrelor compared with clopidogrel 75mg daily, without an increase of overall major bleeding, but with an increase of minor bleeding.

Gypenosides alleviate myocardial ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function in rat heart.

Gypenosides (GP) are the predominant components of Gynostemma pentaphyllum, a Chinese herb medicine that has been widely used for the treatment of chronic inflammation, hyperlipidemia, and cardiovascular disease. GP has been demonstrated to exert protective effects on the liver and brain against ischemia-reperfusion (I/R) injury, yet whether it is beneficial to the heart during myocardial I/R is unclear. In this study, we demonstrate that pre-treatment with GP dose-dependently limits infarct size, alleviates I/R-induced pathological changes in the myocardium, and preserves left ventricular function in a rat model of cardiac I/R injury. In addition, GP pre-treatment reduces oxidative stress and protects the intracellular antioxidant machinery in the myocardium. Further, we show that the cardioprotective effect of GP is associated with the preservation of mitochondrial function in the cardiomyocytes, as indicated by ATP level, enzymatic activities of complex I, II, and IV on the mitochondrial respiration chain, and the activity of citrate synthase in the citric acid cycle for energy generation. Moreover, GP maintains mitochondrial membrane integrity and inhibits the release of cytochrome c from the mitochondria to the cytosol. The cytoprotective effect of GP is further confirmed in vitro in H9c2 cardiomyoblast cell line with oxygen-glucose deprivation and reperfusion (OGD/R), and the results indicate that GP protects cell viability, reduces oxidative stress, and preserves mitochondrial function. In conclusion, our study suggests that GP may be of clinical value in cytoprotection during acute myocardial infarction and reperfusion.

Tongxinluo Induces nNOS Expression Through ERK Activation: Possible Contribution to the Effects of Tongxinluo to Attenuate Vasoconstriction.

BACKGROUND AND OBJECTIVE: Vasoconstriction and hypersensitivity to the vasoconstrictive action of serotonin occurs in the early stage of atherosclerosis. Vascular neural nitric oxide synthase (nNOS) plays an important role in the regulation of vascular tone and is vasoprotective against atherosclerosis. In this study, we intended to investigate the possible role of nNOS in mediating the effect of Chinese medicine Tongxinluo (TXL) to attenuate vasoconstriction induced by the chronic injury in the collared carotid artery. METHODS: Twenty-four male Wistar Kyoto rats were assigned to 2 treatments (n = 12): vehicle and TXL (400 mg·kg·d). After 2 weeks of treatment, adventitia injury was induced by placing a silicone collar around the left carotid artery for 2 weeks. Blood flow and vascular reactivity to serotonin were determined, and carotid arteries were harvested for morphometry, RT-PCR, and Western blotting analysis. Expression of nNOS and phosphorylated ERK1/2 was also analyzed in primary cultured vascular smooth muscle cells after TXL and/or ERK kinase inhibitor treatment. RESULTS: Adventitia injury induced by the placement of a silicone collar around the carotid artery for 2 weeks led to chronic vasoconstriction and vascular hypersensitivity to serotonin, which was attenuated by TXL treatment. TXL improved the carotid blood flow and normalized the vascular hypersensitivity to serotonin in collared carotid arteries. The expression of nNOS and phosphorylated ERK1/2 was increased by TXL treatment in both collared carotid artery and vascular smooth muscle cells indicating a possible contribution of ERK1/2 and nNOS signaling to the beneficial effects of TXL. Moreover, we showed that the effect of TXL to increase nNOS expression was mediated by the phosphorylated ERK1/2 since the effect could be abolished by the ERK kinase inhibitor PD98059. CONCLUSIONS: TXL increases nNOS expression in the collared carotid artery through activation of ERK1/2 signaling, which may have contributed to the attenuation of vasoconstriction induced by the collar-induced adventitia injury.

Novel nanoliposomal delivery system for polydatin: preparation, characterization, and in vivo evaluation.

BACKGROUND: The objective of this study was to develop a novel polydatin (PLD)-loaded liposome system using the thin film hydration technique. METHODS: The delivery system was characterized in terms of morphology, size, zeta potential, encapsulation efficiency, and in vitro release. In addition, a pharmacokinetic study was carried out in rats after oral administration of PLD-loaded liposomes in vivo. RESULTS: Transmission electron microscopy revealed that the PLD-loaded liposomes had a homogeneous size and spherical shape. Dynamic light scattering showed that the PLD-loaded liposomes had a smaller size with a mean value of 80.2±3.7 nm and a polydispersity index of 0.12±0.06. The encapsulation efficiency of the prepared liposomes was 88.4%±3.7%. During the release process, liposome showed two distinct phases. The first was characterized by rapid release during the first 2 hours, which could be related to the release of the drug adsorbed on the surface of liposomes. In the second phase, the release rate slowed down, demonstrating a typical sustained and prolonged drug-release behavior. The release kinetic model for the PLD-loaded liposomes fitted well with the Weibull distribution equation. In vivo, relative oral bioavailability of the encapsulated PLD was 282.9%, ie, significantly enhanced (P<0.05) compared with the free drug. No histological changes occurred in the organs after administration of PLD-loaded liposomes. CONCLUSION: PLD-loaded liposomes could significantly prolong the drug circulation time in vivo and increase the oral bioavailability of the drug.

High-dose insulin inhibits gap junction intercellular communication in vascular smooth muscle cells.

Gap junction intercellular communication (GJIC) is important in mediating intercellular substance and signal transmission. Connexin (Cx)43 is a major component involved in GJIC in vascular tissue and its abnormal expression is closely associated with various vascular diseases. Insulin resistance is the central component of metabolic syndrome, and high doses of insulin can affect vascular function through multiple pathways, resulting in cardiovascular disease. However, the effects of insulin on GJIC function and connexin (Cx)43 expression in vascular smooth muscle cells (VSMCs) remain unclear. Following treatment of VSMCs with different doses of insulin, a fluorescence recovery after photobleaching (FRAP) assay was performed to evaluate GJIC function in treated VSMCs. The results showed that high­dose insulin suppressed GJIC function. Western blot assays further demonstrated that high­dose insulin induced the phosphorylation of Cx43 at s368 and downregulated the expression of Cx43. H2O2 release assays demonstrated that high­dose insulin treatment significantly elevated the cellular H2O2 level. In addition, compared with cells treated with high­dose insulin, pretreatment with catalase significantly restored the cellular GJIC function, decreased the phosphorylation level of Cx43 at s368, and enhanced Cx43 expression. In conclusion, these data indicate that high­dose insulin inhibits cellular GJIC function through the oxidative stress­activated signaling pathway. This phenomenon may also constitute a potential mechanism underlying the pathogenesis of insulin resistance and its complications.

Development of lovastatin-loaded poly(lactic acid) microspheres for sustained oral delivery: in vitro and ex vivo evaluation.

BACKGROUND: A novel lovastatin (LVT)-loaded poly(lactic acid) microsphere suitable for oral administration was developed in this study, and in vitro and in vivo characteristics were evaluated. METHODS: The designed microspheres were obtained by an improved emulsion-solvent evaporation method. The morphological examination, particle size, encapsulation ratio, drug loading, and in vitro release were characterized. Pharmacokinetics studies were used to show that microspheres possess more advantages than the conventional formulations. RESULTS: By using the emulsion-solvent evaporation method, it was simple to prepare microspheres and easy to scale up production. The morphology of formed microspheres showed a spherical shape with a smooth surface, without any particle aggregation. Mean size of the microspheres was 2.65±0.69 µm; the encapsulation efficiency was 92.5%±3.6%, and drug loading was 16.7%±2.1%. In vitro release indicated that the LVT microspheres had a well-sustained release efficacy, and ex vivo studies showed that after LVT was loaded to microspheres, the area under the plasma concentration-time curve from zero to the last measurable plasma concentration point and the extrapolation to time infinity increased significantly, which represented 2.63-fold and 2.49-fold increases, respectively, compared to suspensions. The rate of ex vivo clearance was significantly reduced. CONCLUSION: This research proved that poly(lactic acid) microspheres can significantly prolong the drug circulation time in vivo and can also significantly increase the relative bioavailability of the drug.

[Effect of valsartan on vasoconstriction induced by the chronic injury of the adventitia in the rat collared carotid artery].

OBJECTIVE: Vasoconstriction and vascular hypersensitivity to serotonin were previously shown in animal models of adventitia injury. We investigated the contribution of angiotensin II (AngII)/AngII receptors and oxidative stress to vascular contractility and reactivity in this model. METHODS: Wistar Kyoto rats were divided into 3 groups: normal (n = 6, no any intervention, only for measuring the serum AngII concentration), vehicle (n = 12, collared), and valsartan (n = 12, collared + valsartan 30 mg×kg(-1)×d(-1)). After one week of treatment, adventitia injury was induced by positioning a silicone collar around the right carotid artery for one week. Blood flow and vascular reactivity to serotonin were determined one week after injury, the blood from left ventricle was taken to measure the serum AngII concentration by ELISA, and carotids were harvested for morphometry and Western blot analysis. RESULTS: Adventitia injury induced lumen cross-sectional area reduction (-44% vs. -5%), media diameter increase (62% vs. 10%), blood flow reduction [(2.79 ± 0.22) vs. (4.33 ± 0.84) ml/min] were significantly attenuated by valsartan. The increased vascular reactivity sensitivity to serotonin in vehicle group was also significantly reduced in valsartan group. Serum AngII concentration was significantly increased in vehicle group [(45.21 ± 4.52) pg/ml vs. (19.83 ± 0.5) pg/ml in normal rats, P = 0.0148] and the expression of AngII type 1 (AT(1)) receptor, AngII type 2 (AT(2)) receptor, as well as p22(phox) in collared arteries were significantly upregulated. Valsartan did not affect the AT(1) receptor expression but further increased serum AngII concentration [(89.73 ± 20.44) pg/ml vs. (45.21 ± 4.52) pg/ml, P = 0.001], and AT(2) receptor expression, while downregulated p22(phox) expressions. CONCLUSIONS: Collar-induced adventitia injury resulted in chronic vasoconstriction and vascular hypersensitivity to serotonin via increased serum AngII level, upregulated AngII receptors expression in the vascular well, and activated local oxidative stress. These changes could be blocked by valsartan suggesting a crucial role of AngII/AngII receptors on vascular contractility and reactivity changes in this model.

Involvement of Rho-kinase in collar-induced vasoconstriction and vascular hypersensitivity to serotonin in rat carotid.

BACKGROUND: Vasoconstriction and vascular hypersensitivity to serotonin has been described previously in animal models of adventitia injury. The present study was undertaken to investigate the contribution of the RhoA/Rho-kinase pathway to the collar-induced the change of vascular contractility and reactivity in rat carotid artery. METHODS: Wistar Kyoto rats were assigned to 4 treatments (n=12): vehicle, fasudil, valsartan, and fasudil plus valsartan. After 1 week of treatment, adventitia injury was induced by positioning a silicone collar around the right carotid artery for 1 week. Blood flow and vascular reactivity to serotonin was determined 1 week after injury, and carotids were harvested for morphometry and biochemical analysis. RESULTS: Adventitia injury leaded to histological changes of vasoconstriction with the percent lumen patency of 54.5 ± 4.3% (p<0.001) decreasing, accompanying by the reduction of the blood flow (2.79 ± 0.22 ml/min vs. 3.67 ± 0.26 mi/min/p<0.001) when compared to contralateral arteries. The increase of vascular reactivity sensitivity to serotonin was observed in the collared artery when compared with the contralateral artery. Treatment with valsartan and fasudil prevented the development of vasoconstriction, improved the carotid blood flow and normalized the hypersensitivity to serotonin. Injury increased Angiotensin II type 1(AT(1)receptor, Rho-kinase, and p-MYPT1(Thr696) expression. Valsartan lowered the Rho-kinase and p-MYPT1(Thr696) expression. Fasudil inhibited the p-MYPT1(Thr696) expression. CONCLUSIONS: Collar-induced adventitia injury resulted in the enhancement of vascular contractility and reactivity. The activation of RhoA/Rho-kinase signal pathway, stimulated by AT(1) receptor, plays an important role in the collar-induced the change of vascular contractility and reactivity.

Inflammation inhibitory effects of sirolimus and paclitaxel-eluting stents on interleukin-1ß-induced coronary artery in-stent restenosis in pigs.

BACKGROUND: Coronary artery in-stent restenosis (ISR) and late stent thrombosis remain as important complications of stenting. The inflammation reactions to sirolimus and paclitaxel-eluting stents were investigated in a swine stenosis model induced by interleukin (IL)-1ß. METHODS: Mini pigs (n = 12; 2-3 months old and weighing 25-30 kg) were subjected to thoracotomy. Segments (10 mm) of the mid left anterior descending coronary artery and left circumflex coronary artery were exposed and aseptically wrapped with a cotton mesh soaked with IL-1ß (5 µg). After 2 weeks, the animals were anesthetized and quantitative coronary arteriography (QCA) was performed. The stenosis sites were randomized into three groups for stent insertion: a sirolimus-eluting stent (SES) group (Firebird(TM), n = 7), a paclitaxel-eluting stent (PES) group (TAXUS(TM), n = 9), and a bare-metal stent (BMS) group (YINYITM, Dalian Yinyi Biomaterials Development Co., Ltd, China, n = 8). The three different stents were randomly implanted into stenosis segments. Expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), P-selectin and vascular cell adhesion molecule-1 (VCAM-1) was determined by reverse transcription-coupled polymerase chain reaction (RT-PCR). RESULTS: QCA showed severe stenosis in IL-1ß treated segments. The SES and PES groups showed lower 1-month angiographic late lumen loss (LLL) within the stent and the lesion compared with BMS (P < 0.05) by follow-up QCA. The SES showed lower LLL than that of PES in reducing 1-month inflammation lesions in pigs by follow-up QCA ((0.15 ± 0.06) mm vs. (0.33 ± 0.01) mm, P < 0.0001). The neointimal hyperplasia areas in SES and PES showed lower than those of BMS (SES (11.6 ± 1.7) mm(2), PES (27.2 ± 1.6) mm(2) vs. BMS (76.2 ± 1.3) mm(2), P < 0.0001). The mRNA expression of MCP-1 by RT-PCR in SES and PES showed lower than that of BMS at 30 days after stenting (SES 0.20 ± 0.03, PES 0.48 ± 0.49 vs. BMS 0.58 ± 0.07, P < 0.05). Levels of VCAM-1 in SES were significantly lower than those of PES and BMS (SES 0.35 ± 0.08 vs. PES 0.65 ± 0.13, BMS 0.70 ± 0.06, P < 0.05). Histochemical immunostaining of vessel walls showed lower inflammatory chemokine MCP-1 expression in the SES and PES groups compared with BMS. CONCLUSION: SESs were superior in reducing 1-month angiographic LLL in inflammation lesions in pigs, strongly suggesting that SESs can suppress inflammatory reactions in ISR at multiple points.

IL-1beta modulate the Ca(2+)-activated big-conductance K channels (BK) via reactive oxygen species in cultured rat aorta smooth muscle cells.

The large conductance Ca(2+)-activated K(+) (BK) channel, abundantly expressed in vascular smooth muscle cells, plays a critical role in controlling vascular tone. Activation of BK channels leads to membrane hyperpolarization and promotes vasorelaxation. BK channels are activated either by elevation of the intracellular Ca(2+) concentration or by membrane depolarization. It is also regulated by a diversity of vasodilators and vasoconstrictors. Interleukin-1beta (IL-1beta) is one of the cytokines that play important roles in the development and progression of a variety of cardiovascular diseases. The effects of IL-1beta on vascular reactivity are controversial, and little is known about the modulation of BK channel function by IL-1beta. In this study, we investigated how IL-1beta modulates BK channel function in cultured arterial smooth muscle cells (ASMCs), and examined the role of H(2)O(2) in the process. We demonstrated that IL-1beta had biphasic effects on BK channel function and membrane potential of ASMCs, that is both concentration and time dependent. IL-1beta increased BK channel-dependent K(+) current and hyperpolarized ASMCs when applied for 30 min. While long-term (24-48 h) treatment of IL-1beta resulted in decreased expression of alpha-subunit of BK channel, suppressed BK channel activity, decreased BK channel-dependent K(+) current and depolarization of the cells. H(2)O(2) scavenger catalase completely abolished the early effect of IL-1beta, while it only partly diminished the long-term effect of IL-1beta. These results may provide important molecular mechanisms for therapeutic strategies targeting BK channel in inflammation-related diseases.

[Inhibitory effect and acting mechanism of Tongxinluo on IL-1beta-mediated coronary intimal hyperplasia and 5-hydroxytryptamine-induced coronary vasospasm in small swine].

OBJECTIVE: To observe the inhibitory effect of Tongxinluo (TXL) on coronary vaso spasm in small swine in vivo, and to investigate its possible acting mechanism. METHODS: The model of coronary atherosclerosis in 16 male small swines was established by left thoracotomy after anesthesia, isolated the sections of left anterio-descending branch and proximal end of rotator branch with similar outer diameter, and encapsulated them with paper-towel holding 2.5 microg interleukin-1beta. Two weeks later, the condition of coronary vasospasm induced by catheter intra-coronary injection of 5-hydroxytryptamine (5-HT, 10 microg/kg) was observed through coronary artery contrast examination. The 12 swines with successfully formed coronary vaso spasm were randomly divided into 2 groups, the TXL group and the control group. They were fed with special diet, but TXL 1 g/(kg d) was administered additionally to the TXL group for 4 weeks. The observation on coronary vasospasm was repeated 1 week after discontinuation of TXL treatment, then the animals were sacrificed, their vascular sections enclosed with IL-1beta was taken to conduct the pathologic examination and to detect the expressions of Rho kinase mRNA and its substrate myosin- binding subunit phosphorylation (MBS-P) by RT-PCR and Western blot method. RESULTS: Coronary artery contrast showed that local coronary stenosis occurred in the 12 model swines to different extents (20% - 30%, and vascular spasm on them could be induced by 5-HT. At the time of repeating examination, 11 vascular sections in the control group still maintain their positive spasm reaction to 5-HT, but only 2 in the TXL group did so, the reaction turned to negative in 1 and 10 in the two groups respectively. Pathological examination showed that different degrees of macrophage aggregation could be found in both groups. The degree of lumen stricture and endometrial hyperplasia in the TXL group was obviously attenuated than those in the control group. The expressions of Rho kinase mRNA and MBS-P in the control group were up-regulated obviously. As compared with those in the control group, they were inhibited significantly in the TXL group, as (71.5 +/- 2.4) vs (98.2 +/- 7.7)% and 16,633 +/- 1,390 vs 25,818 +/- 4,745, respectively (all P < 0.05). CONCLUSION: TXL could obviously inhibit the coronary intimal hyperplasia mediated by IL-1beta and coronary vasospasm induced by 5-HT, one of its mechanisms is possibly the inhibition on the intracellular Rho kinase mRNA expression in the IL-1beta enclosed vascular section to decrease the level of MBS-P.

[Effects of rapamycin on Rho-kinase and p27 mRNA expressions in a porcine coronary intimal proliferation model induced by interleukin-1beta].

OBJECTIVE: To observe the effects of rapamycin on the expressions of Rho-kinase and p27 mRNA during vascular intimal proliferation in a porcine model of coronary stenosis induced by interleukin-1beta (IL-1beta). METHODS: The proximal segments of LAD and LCX were wrapped with cotton mesh that had absorbed sepharose bead solution with or without IL-1beta. Selective coronary angiography was performed two weeks later and the animals were killed for collecting the samples for histopathology and RT-PCR analyzing of Rho-kinase and p27 mRNA. RESULTS: The expressions of Rho-kinase and p27 mRNA could be visualized in normal coronary wall. The expression of Rho-kinase mRNA was significantly enhanced and the expression of p27 mRNA was significantly decreased during the process of intimal proliferation induced by IL-1beta. Rapamycin significantly inhibited the intimal proliferation, reduced the infiltration of inflammatory cells, reduced the expression of Rho-kinase mRNA and increased the expression of p27 mRNA. CONCLUSIONS: The expression of Rho-kinase mRNA is upregulated and p27 mRNA downregulated in coronary artery stenosis induced by IL-1beta and these effects could be abolished by cotreatment with rapamycin.

[Upregulated Rho-kinase and increased phosphorylation of myosin-binding subunit of myosin phosphates are key players in a porcine coronary artery spasm model with interleukin-1beta].

OBJECTIVE: Phosphorylation of myosin light chain (MLC) is one of the most important steps for vascular smooth muscle contraction and Rho-kinase is involved in this process. We investigated the role of Rho-kinase in a porcine coronary artery spasm model with interleukin-1beta. METHODS: Segments of left coronary artery adventitia were surrounded by normal saline (n = 8) or IL-1beta agarose microne (n = 8) for 2 weeks. Vasospastic responses to intracoronary serotonin or histamine then studied at the saline or IL-1beta-treated site. The Rho-kinase mRNA expression in the treated site was measured by reverse transcription-polymerase chain reaction analysis (RT-PCR). The extent of phosphorylation of myosin-binding subunit of myosin phosphates (MBS, one of the major substrates of Rho-kinase) were quantified by Western blot analysis. RESULTS: Intracoronary serotonin or histamine repeatedly induced coronary artery spasm and coronary arterial stenosis was evidenced at IL-1beta-treated site. Expression of Rho-kinase mRNA in IL-1beta-treated site was significantly increased compared to saline treated site (98.20% +/- 7.66% vs. 63.70% +/- 4.26%, P < 0.05). Western blot analysis showed that during the serotonin-induced contractions the extent of phosphorylation of MBS was also significantly increased in the spastic site (25,485 +/- 4745 vs. 6510 +/- 779, P < 0.05). CONCLUSION: Rho-kinase upregulation at the spastic site and increased phosphorylation of myosin-binding subunit of myosin phosphates are key players in inducing vascular smooth muscle hypercontraction in this porcine model.