The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (KCa) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K+ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance KCa channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single KCa channels in SMCs. Furthermore, the effect of CO on KCa channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, Gi/Go or Gs) in excised membrane patches. Our results suggest that the direct modulation of high-conductance KCa channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO.

1 Carbon monoxide (CO) induced a concentration-dependent relaxation of isolated rat tail artery tissues which were precontracted with phenylephrine or U-46619. This vasorelaxing e ect of CO was independent of the presence of the intact endothelium. 2 The CO-induced vasorelaxation was partially inhibited by the blockade of either the cyclicGMP pathway or big-conductance calcium-activated K (K Ca ) channels. When both the cyclicGMP pathway and K Ca channels were blocked, the CO-induced vasorelaxation was completely abolished. 3 Incubation of vascular tissues with hemin, in order to enhance the endogenous production of CO, suppressed the phenylephrine-induced vasocontraction in a time-and concentration-dependent manner. The hemin-induced suppression of the vascular contractile response to phenylephrine was abolished after the vascular tissues were co-incubated with either oxyhaemoglobin or zinc protoporphyrin-IX, suggesting an induced endogenous generation of CO from vascular tissues.4 The e ect of hemin incubation on vascular contractility did not involve the endogenous generation of nitric oxide. 5 Our results suggest that CO may activate both a cyclicGMP signalling pathway and K Ca channels in the same vascular tissues, and that the endogenously generated CO may signi®cantly a ect the vascular contractile responses.

Rationale: The NLRP3 (NLR [NOD-like receptor] family, pyrin domain containing 3) inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophages. However, whether the NLRP3 inflammasome is regulated by CMA, and the role of CMA in atherosclerosis remains unclear. Objective: To determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis. Methods and Results: The expression of CMA marker, LAMP-2A (lysosome-associated membrane protein type 2A), was first analyzed in ApoE −/− mouse aortas and human coronary atherosclerotic plaques, and a significant downregulation of LAMP-2A in advanced atherosclerosis in both mice and humans was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A knockout mouse strains in C57BL/6 mice and ApoE −/− mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE −/− mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL (interleukin)-1β in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A–deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway. Conclusions: CMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1β, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1β–driven diseases.

Rationale: Abnormal autophagic death of endothelial cells is detrimental to plaque structure as endothelial loss promotes lesional thrombosis. As emerging functional biomarkers, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular. This study is aimed to determine the role of hsa_circ_0030042 in abnormal endothelial cell autophagy and plaque stability. Methods: circRNA sequencing and quantitative polymerase chain reaction were performed to detect hsa_circ_0030042 expression in coronary heart disease (CHD) and human umbilical vein endothelial cells (HUVECs). Transfection of stubRFP-sensGFP-LC3 adenovirus, flow cytometry, and electron microscopy were used to identify the role of hsa_circ_0030042 in ox-LDL‒induced abnormal autophagy in vitro. Bioinformatic analysis, RNA immunoprecipitation, immunofluorescence assay and other in vitro experiments were performed to elucidate the mechanism underlying hsa_circ_0030042-mediated regulation of autophagy. To evaluate the role of hsa_circ_0030042 in atherosclerotic plaques and endothelial function, we measured the carotid artery tension and performed histopathology and immunohistochemistry analysis. Results: hsa_circ_0030042 was significantly downregulated in CHD, while upon overexpression, it acted as an endogenous eukaryotic initiation factor 4A-III (eIF4A3) sponge to inhibit ox-LDL-induced abnormal autophagy of HUVECs and maintain plaque stability in vivo. Furthermore, hsa_circ_0030042 influenced autophagy by sponging eIF4A3 and blocking its recruitment to beclin1 and forkhead box O1 (FOXO1) mRNA, while hsa_circ_0030042-induced inhibition of beclin1 and FOXO1 was counteracted by eIF4A3 overexpression or decreased hsa_circ_0030042 binding. In high-fat-diet fed ApoE-/- mice, hsa_circ_0030042 also ameliorated plaque stability and counteracted eIF4A3-induced plaque instability. Conclusions: These results demonstrate a novel pathway involving hsa_circ_0030042, eIF4A3, FOXO1, and beclin1; hence, modulating their levels may be a potential therapeutic strategy against CHD.

Carbon monoxide (CO) is an endogenous gaseous factor that relaxes vascular tissues by acting on both the cGMP pathway and calcium-activated K + (K Ca ) channels. Whether the vascular effect of CO is altered in diabetes had been unknown. It was found that the CO-induced relaxation of tail artery tissues from streptozotocininduced diabetic rats was significantly decreased as compared with that of nondiabetic control rats. The blockade of the cGMP pathway with ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one) completely abolished the CO-induced relaxation of diabetic tissues but only partially inhibited the CO effect in normal tissues. Single-channel conductance of K Ca channels in diabetic smooth muscle cells (SMCs) was not different from that of normal SMCs. However, the sensitivity of K Ca channels to CO in diabetic SMCs was significantly reduced. CO (10 µmol/l) induced an 81 ± 24% increase in the mean open probability of single K Ca channels in normal SMCs but had no effect in diabetic SMCs. Longterm culture of normal vascular SMCs with 25 mmol/l glucose or 25 mmol/l 3-OMG (3-O-methylglucose) but not 25 mmol/l mannitol significantly reduced the sensitivity of K Ca channels to CO. On the other hand, the sensitivity of K Ca channels to CO was regained in diabetic SMCs that were cultured with 5 mmol/l glucose for a prolonged period. The decreased vasorelaxant effect of CO in diabetes represents a novel mechanism for the vascular complications of diabetes, which could be closely related to the glycation of K Ca channels in diabetic vascular SMCs. Diabetes 50: [166][167][168][169][170][171][172][173][174] 2001 T he vasorelaxant effects of carbon monoxide (CO) have been demonstrated (1-4), and heme oxygenase (HO) that cleaves the heme ring to form biliverdin and CO has been located in many different types of vascular smooth muscles (5,6). The production of CO from vascular tissues has also been directly measured (7). These studies emphasize the importance of CO as an endogenous vasorelaxant factor under physiological (8) or pathophysiological (9,10) conditions. The elevation of cellular cGMP levels and the opening of plasma membrane K + channels are the main mechanisms that have been proposed to explain the vascular effects of CO. CO may increase cGMP content via its stimulatory interaction with the heme in the regulatory subunit of guanylyl cyclase. Increased cGMP would consequently decrease the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in smooth muscle cells (SMCs) through the inhibition of inositol triphosphate formation, the activation of Ca 2+ -ATPase, and the inhibition of Ca 2+ channels. The opening of K + channels leads to membrane hyperpolarization, which in turn inhibits the agonist-induced increase in inositol triphosphate, reduces Ca 2+ sensitivity and resting Ca 2+ level, and relaxes SMCs (11). Our studies have demonstrated that CO directly enhanced the activity of the big-conductance calcium-activated K + (K Ca ) channels in rat tail artery SMCs via a cGMP-independent mechanism (2,3). Whethe...

Both HIV and antiretroviral therapy could induce vascular aging with unclear mechanisms. In this study, via microarray analysis, we identified, for the first time, that miR-34a expression was significantly increased in both HIV-infected, and antiretroviral agents-treated vessels and vascular endothelial cells (ECs) from these vessels. In cultured ECs, miR-34a expression was significantly increased by HIV-Tat protein and by the antiretroviral agents, lopinavir/ritonavir. Both HIV-Tat protein and antiretroviral agents could induce EC senescence, which was inhibited by miR-34a inhibition. In contrast, EC senescence was exacerbated by miR-34a overexpression. In addition, the vascular ECs isolated from miR-34a knockout mice were resistant to HIV and antiretroviral agents-mediated senescence. In vivo, miR-34a expression in mouse vascular walls and their ECs was increased by antiretroviral therapy and by HIV-1 Tat transgenic approach. miR-34a inhibition could effectively inhibit both HIV-Tat protein and antiretroviral therapy-induced vascular aging in mice. The increased miR-34a was induced via p53, whereas Sirt1 was a downstream target gene of miR-34a in both HIV-Tat protein and antiretroviral agents-treated ECs and vessels. The study has demonstrated that miR-34a is a common link in both HIV and antiretroviral therapy-mediated vascular aging.

Spontaneously rhythmic contraction of peripheral blood vessels actively modulates the peripheral circulation and blood pressure. However, the underlying mechanisms for the complex rhythmic contraction patterns of various vascular tissues are not yet fully understood. In the present study, the tetraethylammonium (TEA)-induced spontaneously oscillatory contractions of isolated rat tail artery tissues were examined. It was found that TEA evoked arterial oscillatory contractions in a concentration-dependent, but endothelium-independent manner. The voltage-dependent K+ (Kv) channel specific blocker, 4-aminopyridine (4-AP), induced a sustained, but not oscillated, vascular contraction. The presence of 4-AP had no effect on the TEA-induced oscillatory contractions. The blockade of KCa channels with charybdotoxin or apamin did not affect the basal force of vascular tissues. Neither the TEA-induced oscillatory contraction was affected by these blockers. The opening of KATP channels by levcromakalim or their blockade by glybenclamide ceased or increased, respectively, the oscillation of TEA-induced contractions. The absence of Ca2+ or the presence of nifedipine in the bath solution completely abolished the effects of TEA. The inhibition of Ca2+-ATPase in the sarcoplasmic reticulum with micromolar concentrations of thapsigargin or cyclopiazonic acid either abolished or enhanced, respectively, the TEA-induced oscillatory contractions. Ryanodine did not affect the TEA-induced oscillatory contraction. In conclusion, the TEA-induced oscillatory contraction may be initiated by the blockade of the TEA-sensitive delayed rectifier K+ channels and maintained by the TEA-insensitive but ATP-sensitive K+ channels. This K-K model presents a novel mechanism for the depolarization-induced rhythmic contractions of small arteries.

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