Ciprofloxacin Accelerates Angiotensin-II-Induced Vascular Smooth Muscle Cells Senescence Through Modulating AMPK/ROS pathway in Aortic Aneurysm and Dissection.

Aortic aneurysm and dissection (AAD) is a cardiovascular disease that poses a severe threat to life and has high morbidity and mortality rates. Clinical and animal-based studies have irrefutably shown that fluoroquinolones, a commonly prescribed antibiotic for treating infections, significantly increase the risk of AAD. Despite this, the precise mechanism by which fluoroquinolones cause AAD remains unclear. Therefore, this study aims to investigate the molecular mechanism and role of Ciprofloxacin definitively-a type of fluoroquinolone antibiotic-in the progression of AAD. Aortic transcriptome data were collected from GEO datasets to detect the genes and pathways expressed differently between healthy donors and AAD patients. Human primary Vascular Smooth Muscle Cells (VSMCs) were isolated from the aorta. After 72 h of exposure to 110ug/ml Ciprofloxacin or 100 nmol/L AngII, either or combined, the senescent cells were identified through SA-ß-gal staining. MitoTracker staining was used to examine the morphology of mitochondria in each group. Cellular Reactive Oxygen Species (ROS) levels were measured using MitoSox and DCFH-DA staining. Western blot assay was performed to detect the protein expression level. We conducted an analysis of transcriptome data from both healthy donors and patients with AAD and found that there were significant changes in cellular senescence-related signaling pathways in the latter group. We then isolated and identified human primary VSMCs from healthy donors (control-VSMCs) and patients' (AAD-VSMCs) aortic tissue, respectively. We found that VSMCs from patients exhibited senescent phenotype as compared to control-VSMCs. The higher levels of p21 and p16 and elevated SA-ß-gal activity demonstrated this. We also found that pretreatment with Ciprofloxacin promoted angiotensin-II-induced cellular senescence in control-VSMCs. This was evidenced by increased SA-ß-gal activity, decreased cell proliferation, and elevation of p21 and p16 protein levels. Additionally, we found that Angiotensin-II (AngII) induced VSMC senescence by promoting ROS generation. We used DCFH-DA and mitoSOX staining to identify that Ciprofloxacin and AngII pretreatment further elevated ROS levels than the vehicle or alone group. Furthermore, JC-1 staining showed that mitochondrial membrane potential significantly declined in the Ciprofloxacin and AngII combination group compared to others. Compared to the other three groups, pretreatment of Ciprofloxacin plus AngII could further induce mitochondrial fission, demonstrated by mitoTracker staining and western blotting assay. Mechanistically, we found that Ciprofloxacin impaired the balance of mitochondrial fission and fusion dynamics in VSMCs by suppressing the phosphorylation of AMPK signaling. This caused mitochondrial dysfunction and ROS generation, thereby elevating AngII-induced cellular senescence. However, treatment with the AMPK activator partially alleviated those effects. Our data indicate that Ciprofloxacin may accelerate AngII-induced VSMC senescence through modulating AMPK/ROS signaling and, subsequently, hasten the progression of AAD.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

USP18 Curbs the Progression of Metabolic Hypertension by Suppressing JAK/STAT Pathway.

Hypertension is a pathological state of the metabolic syndrome that increases the risk of cardiovascular disease. Managing hypertension is challenging, and we aimed to identify the pathogenic factors and discern therapeutic targets for metabolic hypertension (MHR). An MHR rat model was established with the combined treatment of a high-sugar, high-fat diet and ethanol. Histopathological observations were performed using hematoxylin-eosin and Sirius Red staining. Transcriptome sequencing was performed to screen differentially expressed genes. The role of ubiquitin-specific protease 18 (USP18) in the proliferation, apoptosis, and oxidative stress of HUVECs was explored using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. Moreover, USP18 downstream signaling pathways in MHR were screened, and the effects of USP18 on these signaling pathways were investigated by western blotting. In the MHR model, total cholesterol and low-density lipoprotein levels increased, while high-density lipoprotein levels decreased. Moreover, high vessel thickness and percentage of collagen were noted along with increased malondialdehyde, decreased superoxide dismutase and catalase levels. The staining results showed that the MHR model exhibited an irregular aortic intima and disordered smooth muscle cells. There were 78 differentially expressed genes in the MHR model, and seven hub genes, including USP18, were identified. USP18 overexpression facilitated proliferation and reduced apoptosis and oxidative stress in HUVECs treated with Ang in vitro. In addition, the JAK/STAT pathway was identified as a USP18 downstream signaling pathway, and USP18 overexpression inhibited the expression of JAK/STAT pathway-related proteins. Conclusively, USP18 restrained MHR progression by promoting cell proliferation, reversing apoptosis and oxidative stress, and suppressing the JAK/STAT pathway.

New nitric oxide donors based on ruthenium complexes

Nitric oxide (NO) donors produce NO-related activity when applied to biological systems. Among its diverse functions, NO has been implicated in vascular smooth muscle relaxation. Despite the great importance of NO in biological systems, its pharmacological and physiological studies have been limited due to its high reactivity and short half-life. In this review we will focus on our recent investigations of nitrosyl ruthenium complexes as NO-delivery agents and their effects on vascular smooth muscle cell relaxation. The high affinity of ruthenium for NO is a marked feature of its chemistry. The main signaling pathway responsible for the vascular relaxation induced by NO involves the activation of soluble guanylyl-cyclase, with subsequent accumulation of cGMP and activation of cGMP-dependent protein kinase. This in turn can activate several proteins such as K+ channels as well as induce vasodilatation by a decrease in cytosolic Ca2+. Oxidative stress and associated oxidative damage are mediators of vascular damage in several cardiovascular diseases, including hypertension. The increased production of the superoxide anion (O2-) by the vascular wall has been observed in different animal models of hypertension. Vascular relaxation to the endogenous NO-related response or to NO released from NO deliverers is impaired in vessels from renal hypertensive (2K-1C) rats. A growing amount of evidence supports the possibility that increased NO inactivation by excess O2- may account for the decreased NO bioavailability and vascular dysfunction in hypertension.

Influencia del sistema nitridérgico en la respuesta contráctil a fenilefrina de anillos de vasos usados en revascularización coronaria / Modification of phenylephrine induced contraction of human vessel rings by L-arginine and L-arginine methyl ester

Background: Endothelial dysfunction is associated to a lower production of nitric oxide and a reduction of endothelium mediated vasodilation. Aim: To study the effects of pharmacological agents that modify nitric oxide synthetase (NOS) activity on tension changes induced by phenylephrine in rings of internal mammary and radial arteries and saphenous vein. Material and methods: Vessel rings of 7 to 10 mm length were obtained from 32 patients subjected to coronary vascular surgery Fourteen samples of radial artery, 12 samples of internal mammary artery and 15 samples of saphenous vein were obtained. A maximal contraction was induced with KC1 and dose response curves for phenylephrine (FE) in the absence or presence of L-arginine and L-arginine methyl ester (L-NAME), were constructed. Results: The tension induced by FE in internal mammary artery and saphenous vein reached a maximum, near 90 percent of 80 mM KCl-induced contraction, but in the radial artery, it reached a maximum of 63 percent (p <0.05). In all vessels, the dose response curves were significantly shifted to the right by L-arginine and to the íeft by L-NAME. Conclusions: Pre-incubation of human rings with L-ARG or L-NAME, changed the response to FE induced contraction, which may be related to different degrees of endothelial nitric oxide production or NO sensitivity. The basal NO production in radial artery seems to be larger than the other vessels.

Genistein Supplementation Inhibits Atherosclerosis with Stabilization of the Lesions in Hypercholesterolemic Rabbits

The effect of genistein on aortic atherosclerosis was studied by immunohistochemistry with RAM-11 and HHF-35 antibodies and western blotting for matrix metalloproteinase-3 (MMP-3) in New Zealand White rabbits. After provocation of atherosclerosis with hyperlipidemic diet, the rabbits were divided as hyperlipidemic diet group (HD), normal diet group (ND) and hyperlipidemic plus genistein diet group (HD+genistein) for 4 and half months. The average cross sectional area of atherosclerotic lesion was 0.269 mm2 after provocation. The lesion was progressed by continuous hyperlipidemic diet (10.06 mm2) but was increased mildly by genistein (0.997 mm2), and decreased by normal diet (0.228 mm2). The ratio of macrophages to smooth muscle cells in the lesion was not changed by genistein supplementation. The western blotting showed reduction of MMP-3 expression in HD+genistein and ND groups than HD group. The inhibition of atherogenesis by genistein was might be due to improve the endothelial dysfunction rather than direct action on macrophages and/or smooth muscle cells in the lesion, since endothelial dysfunction by lipid peroxidation was the main atherogenic factor in the hypercholesterolemicrabbits. The genistein supplementation also suggests that it helps the stabilization of the atherosclerotic lesion by inhibition of MMP-3 expression.

Expresión de COX-2 en arterias y células vasculares de pacientes diabéticos y no diabéticos / COX-2 expression in arteries and vascular cells from diabetic and nondiabetic patients

Introducción. El objetivo de este trabajo fue estudiar la expresión de ciclooxigenasa 2 (COX-2) en arterias y células de músculo liso vascular provenientes de pacientes diabéticos y no diabéticos, así como su inducción tras incubar células de pacientes no diabéticos con un preparado de triglicéridos. Material y métodos. Se utilizaron segmentos de arterias mamarias internas usadas en cirugía de revascularización coronaria, y se procedió a homogeneizarlos, fijarlos o cultivarlos para obtener células de músculo liso vascular. La expresión de COX-2 se analizó por Western blot y microscopia confocal. Para inducir in vitro la expresión de COX-2, se incubaron las células de pacientes no diabéticos con Intralipid®. Resultados. Todas las medidas indicaron una mayor expresión de COX-2 tanto en arterias como en células de pacientes diabéticos. La expresión de COX-2 se correlacionó con el índice EuroSCORE, como índice pronóstico de mortalidad precoz y de complicaciones mayores en la cirugía coronaria, menos favorable. Asimismo, la expresión de COX-2 se incrementó de forma significativa con concentraciones crecientes de Intralipid®. Conclusiones. La expresión de COX-2 en arterias mamarias internas se relaciona con la diabetes mellitus y un índice EuroSCORE más desfavorable. También es posible aumentar la expresión de COX-2 incubando las células de pacientes no diabéticos con triglicéridos, lo que refuerza la asociación entre hipertrigliceridemia, diabetes tipo 2, inflamación vascular y un peor pronóstico clínico (AU)

Introduction. The aim of this study was to investigate cyclooxygenase (COX)-2 expression in arteries and vascular smooth muscle cells from diabetic and nondiabetic patients. A further aim was to analyze COX-2 induction after cells from nondiabetic patients were incubated with a triglyceride-rich solution. Matherial and methods. Segments from internal mammary arteries discarded after coronary artery bypass grafting were used. Arterial segments were homogenized, fixed, or prepared for vascular smooth muscle cell cultures. COX-2 expression was assessed by both Western blot and confocal microscopy. For in vitro induction of COX-2 expression, cells from nondiabetic patients were incubated with Intralipid®. Results. All measurements showed increased COX-2 expression in the arteries and cells from diabetic patients. COX-2 expression was correlated with a poorer prognosis measured by the EuroSCORE scale. In addition, COX-2 expression was significantly enhanced when cells were incubated with increasing Intralipid® concentrations. Conclusions. COX-2 expression in internal mammary arteries is related to type 2 diabetes and a poorer EuroSCORE. In addition, COX-2 expression was experimentally increased when cells from nondiabetic patients were incubated with triglycerides, which enhances the associations among hypertriglyceridemia, type 2 diabetes, vascular inflammation, and a poorer clinical prognosis (AU)

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24 h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24 h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.(AU)