BACKGROUND: Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. METHODS: We generated an microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t -test were used to analyze the data. RESULTS: Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. CONCLUSIONS Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.
Animals ; MicroRNAs/metabolism* ; Angiotensin II/toxicity* ; Mice ; Renal Insufficiency, Chronic/chemically induced* ; Mice, Knockout ; Disease Models, Animal ; Male ; Signal Transduction/genetics* ; LIM Domain Proteins/genetics* ; Mice, Inbred C57BL ; Cell Line ; Humans

The study aims to examine the effects and potential mechanisms of disulfiram (DSF) on cardiac hypertrophic injury, focusing on the role of transforming growth factor-β-activated kinase 1 (TAK1)-mediated pan-apoptosis (PANoptosis). H9C2 cardiomyocytes were treated with angiotensin II (Ang II, 1 µmol/L) to establish an in vitro model of myocardial hypertrophy. DSF (40 µmol/L) was used to treat cardiomyocyte hypertrophic injury models, either along or in combination with the TAK1 inhibitor, 5z-7-oxozeaenol (5z-7, 0.1 µmol/L). We assessed cell damage using propidium iodide (PI) staining, measured cell viability with CCK8 assay, quantified inflammatory factor levels in cell culture media via ELISA, detected TAK1 and RIPK1 binding rates using immunoprecipitation, and analyzed the protein expression levels of key proteins in the TAK1-mediated PANoptosis pathway using Western blot. In addition, the surface area of cardiomyocytes was measured with Phalloidin staining. The results showed that Ang II significantly reduced the cellular viability of H9C2 cardiomyocytes and the binding rate of TAK1 and RIPK1, significantly increased the surface area of H9C2 cardiomyocytes, PI staining positive rate, levels of inflammatory factors [interleukin-1β (IL-1β), IL-18, and tumor necrosis factor α (TNF-α)] in cell culture media and p-TAK1/TAK1 ratio, and significantly up-regulated key proteins in the PANoptosis pathway [pyroptosis-related proteins NLRP3, Caspase-1 (p20), and GSDMD-N (p30), apoptosis-related proteins Caspase-3 (p17), Caspase-7 (p20), and Caspase-8 (p18), as well as necroptosis-related proteins p-MLKL, RIPK1, and RIPK3]. DSF significantly reversed the above changes induced by Ang II. Both 5z-7 and exogenous IL-1β weakened these cardioprotective effects of DSF. These results suggest that DSF may alleviate cardiac hypertrophic injury by inhibiting TAK1-mediated PANoptosis.
Animals ; MAP Kinase Kinase Kinases/physiology* ; Rats ; Myocytes, Cardiac/pathology* ; Disulfiram/pharmacology* ; Cardiomegaly ; Apoptosis/drug effects* ; Cell Line ; Angiotensin II ; Necroptosis/drug effects* ; Interleukin-1beta/metabolism* ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism* ; Lactones ; Resorcinols ; Zearalenone/administration & dosage*

Objective To investigate the effects of ROCK-siRNA transfection on endothelial cell senescence and endothelial microparticles (EMPs) induced by angiotensin II (Ang II). Methods Human umbilical vein endothelial cells (HUVECs) were treated with Ang II (1.0 μmo/L) to induce cellular senescence models, followed by transfection with ROCK-siRNA. The cells were divided into four groups: control group, model group, negative transfection control group (Ang II combined with NC-siRNA), and ROCK-siRNA transfection group (Ang II combined with ROCK-siRNA). Cellular senescence was assessed by SA-β-Gal staining. EMP levels in cell supernatants and intracellular reactive oxygen species (ROS) levels were assessed using flow cytometry. The expression levels of silenced information regulator 1(SIRT1) and p53 protein in each group were analyzed by Western blotting. Results Following ROCK-siRNA transfection, the number of senescent cells induced by Ang II was significantly reduced, accompanied by decreased CD31⁺ EMP levels and suppressed intracellular ROS levels. Meanwhile, the expression levels of SIRT1 were up-regulated, while the expression levels of p53 were down-regulated. Conclusion Silencing ROCK expression suppresses EMP release, reduces ROS generation, regulates the expression of SIRT1 and p53, and ultimately attenuates Ang II-induced endothelial cell senescence.
Humans ; Angiotensin II/pharmacology* ; Cellular Senescence/genetics* ; Human Umbilical Vein Endothelial Cells/cytology* ; RNA, Small Interfering/metabolism* ; Reactive Oxygen Species/metabolism* ; Sirtuin 1/genetics* ; Transfection ; Tumor Suppressor Protein p53/genetics* ; Cell-Derived Microparticles/drug effects* ; rho-Associated Kinases/metabolism* ; Endothelial Cells/metabolism* ; Cells, Cultured

OBJECTIVE: To explore the main components and potential mechanisms of Sangqi Qingxuan Liquid in the treatment of arterial vascular endothelial cells (AVECs) injury in hypertension through network pharmacology. METHODS: Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID) were used to screen the active components of Sangqi Qingxuan Liquid (SQQX), which met the oral utilization rate and drug similarity criteria. An active component-target network was constructed using Cytoscape 3.6 software. A protein-protein interaction (PPI) network of targets associated with SQQX treatment for hypertension was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The Metascape database was used to perform enrichment analysis of gene ontology biological functions and MSigDB pathway enrichment analysis of proteins in the PPI network. Further analysis of the main components of SQQX was performed using UPLC-MS. Based on the results of network pharmacology, the mechanism of SQQX to improve the injury of AVECs in hypertension was verified through lentiviral transfection by Wnt/ β -catenin signaling pathway. AVECs induced by angiotensin II (Ang II ) was used to establish a model of endothelial function injury in hypertension. Cell viability, intracellular nitric oxide content, malonaldehyde content, and superoxide dismutase activity were measured to determine the optimal induction conditions. The optimal intervention conditions for SQQX were determined based on cell viability, cellular DNA activity, and the gradient method. The cells were further divided into blank, model, overexpression lentivirus negative control, overexpression lentivirus, overexpression lentivirus + SQQX intervention (2.47 mg/mL, 12 h), inhibition lentivirus negative control, inhibition lentivirus, and inhibition lentivirus + SQQX intervention (2.47 mg/mL, 12 h) groups. Finally, quantitative real-time PCR and Western blotting were performed to analyze the molecular mechanisms of SQQX in the Wnt/ β -catenin signaling pathway. RESULTS: The main SQQX components were betaine, buddleoside, and chlorogenic acid, in descending order. Network pharmacology analysis screened 12 pathways associated with the hypertensive vascular endothelium. The results showed that 1 µ mol/L for 12 h was the optimal condition for Ang II to induce AVECs injury, and 2.47 mg/mL SQQX intervention for 12 h was the optimal condition for treating AVECs injury. In the experimental validation based on the interaction network of the Wnt/ β -catenin signaling pathway, SQQX significantly decreased the expressions of β -catenin, Smad2, peroxisome proliferator-activated receptors (PPARs), endothelial nitric oxide synthase (eNOS), and endothelin-1 (ET-1) caused by the β -catenin overexpression lentivirus (P<0.05 or P<0.01). The function of vascular endothelial cells can be improved by the β -catenin inhibition lentivirus, and no obvious changes were observed after further intervention with SQQX. CONCLUSION SQQX may protect against AVECs injury by regulating the Wnt/β -catenin signaling pathway.
Drugs, Chinese Herbal/therapeutic use* ; beta Catenin/metabolism* ; Hypertension/metabolism* ; Endothelial Cells/metabolism* ; Protein Interaction Maps/drug effects* ; Humans ; Wnt Signaling Pathway/drug effects* ; Network Pharmacology ; Endothelium, Vascular/injuries* ; Cell Survival/drug effects* ; Angiotensin II/pharmacology* ; Nitric Oxide/metabolism*

OBJECTIVE: To explore the functional roles and underlying mechanisms of neferine in the context of angiotensin II (Ang II)-induced hypertension and vascular dysfunction. METHODS: Male mice were infused with Ang II to induce hypertension and randomly divided into treatment groups receiving neferine or a control vehicle based on baseline blood pressure using a random number table method. The hypertensive mouse model was constructed by infusing Ang II via a micro-osmotic pump (500 ng/kg per minute), and neferine (0.1, 1, or 10 mg/kg), valsartan (10 mg/kg), or double distilled water was administered intragastrically once daily for 6 weeks. A non-invasive blood pressure system, ultrasound, and hematoxylin and eosin staining were performed to assess blood pressure and vascular changes. RNA sequencing and network pharmacology were employed to identify differentially expressed transcripts (DETs) and pathways. Vascular ring tension assay was used to test vascular function. A7R5 cells were incubated with neferine for 24 h and then treated with Ang II to record the real-time Ca²⁺ concentration by confocal microscope. Immunohistochemistry (IHC) and Western blot were used to evaluate vasorelaxation, calcium, and the extracellular signal-regulated kinase (ERK)1/2 pathway. RESULTS: Neferine treatment effectively mitigated the elevation in blood pressure, pulse wave velocity, aortic thickening in the abdominal aorta of Ang II-infused mice (P<0.05). RNA sequencing and network pharmacology analysis identified 355 DETs that were significantly reversed by neferine treatment, along with 25 potential target genes, which were further enriched in multiple pathways and biological processes, such as ERK1 and ERK2 cascade regulation, calcium pathway, and vascular smooth muscle contraction. Further investigation revealed that neferine treatment enhanced vasorelaxation and reduced Ca²⁺-dependent contraction of abdominal aortic rings, independent of endothelium function (P<0.05). The underlying mechanisms were mediated, at least in part, via suppression of receptor-operated channels, store-operated channels, or voltage-operated calcium channels. Neferine pre-treatment demonstrated a reduction in intracellular Ca²⁺ release in Ang II stimulated A7R5 cells. IHC staining and Western blot confirmed that neferine treatment effectively attenuated the upregulation of p-ERK1/2 both in vivo and in vitro, which was similar with treatment of ERK1/2 inhibitor PD98059 (P<0.05). CONCLUSIONS Neferine remarkably alleviates Ang II-induced elevation of blood pressure, vascular dysfunction, and pathological changes in the abdominal aorta. This beneficial effect is mediated by the modulation of multiple pathways, including calcium and ERK1/2 pathways.
Animals ; Angiotensin II ; Male ; Benzylisoquinolines/therapeutic use* ; Network Pharmacology ; Blood Pressure/drug effects* ; Sequence Analysis, RNA ; Mice ; Hypertension/chemically induced* ; Mice, Inbred C57BL ; Calcium/metabolism*

OBJECTIVES: To explore the mechanism of Circ-MYOCD back-splicing and its regulatory role in myocardial hypertrophy. METHODS: Sanger sequencing and RNase R assays were performed to verify the circularity and stability of Circ-MYOCD, whose subcellular distribution was determined by nuclear-cytoplasmic fractionation. Bioinformatics analysis and mass spectrometry from pull-down assays were conducted to predict the RNA-binding proteins (RBPs) interacting with Circ-MYOCD. In rat cardiomyocytes H9C2 cells, the effects of HNRNPH1 and HNRNPL knockdown and overexpression on Circ-MYOCD back-splicing were evaluated. In a H9C2 cell model of angiotensin II (Ang II)-induced myocardial hypertrophy, the expression of HNRNPH1 was detected, the effects of HNRNPH1 knockdown and overexpression on progression of myocardial hypertrophy were assessed, and the regulatory effect of HNRNPH1 on Circ-MYOCD back-splicing was analyzed. RESULTS: Sanger sequencing confirmed that the junction primers could amplify the correct Circ-MYOCD sequence. RNase R and nuclear-cytoplasmic fractionation assays showed that Circ-MYOCD was stable and predominantly localized in the cytoplasm. Bioinformatics analysis and mass spectrometry from the Circ-MYOCD pull-down assay identified HNRNPH1 and HNRNPL as the RBPs interacting with Circ-MYOCD. In H9C2 cells, HNRNPH1 knockdown significantly enhanced while its overexpression inhibited Circ-MYOCD back-splicing; HNRNPH1 overexpression obviously increased the expressions of myocardial hypertrophy markers ANP and BNP, while its knockdown produced the opposite effect. In Ang II-induced H9C2 cells, which exhibited a significant increase of HNRNPH1 expression and increased expressions of ANP and BNP, HNRNPH1 knockdown obviously increased Circ-MYOCD expression, decreased MYOCD expression and lowered both ANP and BNP expressions. CONCLUSIONS HNRNPH1 regulates Circ-MYOCD back-splicing to influence the progression of myocardial hypertrophy.
Animals ; Rats ; RNA, Circular/genetics* ; Cardiomegaly/metabolism* ; Myocytes, Cardiac/metabolism* ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism* ; Cell Line ; RNA Splicing ; Angiotensin II ; RNA-Binding Proteins

Astragali Radix (AR) and Notoginseng Radix et Rhizoma (NR) are frequently employed in cardiovascular disease treatment. However, the efficacy of the AR-NR medicine pair (AN) in improving cardiac remodeling and its underlying mechanism remains unclear. This study aimed to evaluate AN's cardioprotective effect and potential mechanism on cardiac remodeling using transverse aortic constriction (TAC) in mice and angiotensin II (Ang II)-induced neonatal rat cardiomyocytes (NRCMs) and fibroblasts in vitro. High-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) characterized 23 main components of AN. AN significantly improved cardiac function in the TAC-induced mice. Furthermore, AN considerably reduced the serum levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), cardiac troponin T (CTn-T), and interleukin-6 (IL-6) and mitigated inflammatory cell infiltration. Post-AN treatment, TAC-induced heart size approached normal. AN decreased cardiomyocyte cross-sectional area and attenuated the upregulation of cardiac hypertrophy marker genes (ANP, BNP, and MYH7) in vivo and in vitro. Concurrently, AN alleviated collagen deposition in TAC-induced mice. AN also reduced the expression of fibrosis-related indicators (COL1A1 and COL3A1) and inhibited the activation of the transforming growth factor-β1 (TGF-β1)/mothers against decapentaplegic homolog 3 (Smad3) pathway. Thus, AN improved TAC-induced cardiac remodeling. Moreover, AN downregulated p-dynamin-related protein (Drp1) (Ser616) expression and upregulated mitogen 2 (MFN-2) and optic atrophy 1 (OPA1) expression in vivo and in vitro, thereby restoring mitochondrial fusion and fission balance. In conclusion, AN improves cardiac remodeling by regulating mitochondrial dynamic balance, providing experimental data for the rational application of Chinese medicine prescriptions with AN as the main component in clinical practice.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Myocytes, Cardiac/metabolism* ; Mice ; Rats ; Male ; Mitochondrial Dynamics/drug effects* ; Ventricular Remodeling/drug effects* ; Astragalus Plant/chemistry* ; Mice, Inbred C57BL ; Rhizome/chemistry* ; Panax notoginseng/chemistry* ; Rats, Sprague-Dawley ; Natriuretic Peptide, Brain/genetics* ; Humans ; Angiotensin II ; Astragalus propinquus

The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 μmol/L) with or without Ang II (0.4 μmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.
Rats ; Mice ; Animals ; Rats, Sprague-Dawley ; Angiotensin II/pharmacology* ; Fibroblasts ; Mice, Inbred C57BL ; Fibrosis ; Collagen/pharmacology* ; Collagen Type I/metabolism* ; RNA, Messenger/metabolism* ; Myocardium/pathology*

OBJECTIVE: To explore the protective effect and mechanism of Kuntai (KT) Capsule on angiotensin II (Ang II)-induced hypertension in ovariectomized (OVX) rats. METHODS: Fifty-four rats were randomly divided into 6 groups according to a random number table, 9 in each group: control, OVX sham+Ang II, OVX, OVX+Ang II, OVX+Ang II +E₂, and OVX+Ang II +KT. OVX rats model was constructed by retroperitoneal bilateral ovariectomy. After 4 weeks of pretreatment with KT Capsule [0.8 g/(kg·d) and 17- β -estradiol (E₂, 1.2 mg/(kg·d)] respectively, Ang II was injected into a micro-osmotic pump with a syringe to establish a hypertensive rat model. Blood pressure of rat tail artery was measured in a wake state of rats using a non-invasive sphygmomanometer. Blood pressure changes were compared between the intervention groups (OVX+Ang II +KT, OVX+Ang II +E₂) and the negative control group (OVX+Ang II). Serum malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected respectively. The expressions of oxidative stress-related protein superoxide dismutase2 (SOD2) and anti-thioredoxin (TRX), autophagy marker protein [beclin1, light chain (LC) 3 II/I ratio and autophagy canonical pathway protein phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR)] were evaluated by Western blotting. RESULTS: Compared with the OVX+Ang II group, the systolic blood pressure of OVX+Ang II +KT group was significantly lowered (P<0.05) but not the diastolic blood pressure. Besides, SOD2 and TRX protein levels in mycardial tissues were significantly reduced in the OVX+Ang II +KT group compared with the OVX+Ang II group (P<0.05). Oxidative stress serum markers MDA and SOD were down- and up-regulated in the OVX+Ang II +KT group, respectively (P<0.05). Compared with OVX+Ang II group, the levels of cardiac proteins beclin-1 and LC3II/LC3 I in OVX+Ang II +KT group were also up-regulated (P<0.05), and the expression levels of p-PI3K, p-AKT and mTOR protein were down-regulated (P<0.05). CONCLUSION KT could protect blood pressure of Ang II-induced OVX rats by inhibiting oxidative stress and up-regulating protective autophagy.
Female ; Rats ; Animals ; Humans ; Angiotensin II ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Hypertension/drug therapy* ; Estradiol/pharmacology* ; Superoxide Dismutase ; Ovariectomy ; Mammals/metabolism*

Objective: To investigate the role and related mechanism of ubiquitin-like protein FAT10 in the angiotensin Ⅱ (AngⅡ)-induced endothelial cell inflammatory responses. Methods: The Western blot was used to detect the protein expression of FAT10 in 16-weeks old WKY rat carotid artery, thoracic aorta artery, renal artery and vascular smooth muscle cells (VSMC), human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MDA-MB-231). The optimal concentration and stimulation time of AngⅡ on inducing the highest FAT10 in HUVEC were determined. The following plasmids were constructed: control plasmid, overexpression FAT10 plasmid (Flag-FAT10), invalid interference plasmid, and interference FAT10 plasmid (sh-FAT10). These plasmids were then transfected into HUVEC cells and divided into following groups: control group, Flag-FAT10 group, invalid interference group, and sh-FAT10 group. After culturing with 100 nmol/L AngⅡ for 36 h, the control group and the Flag-FAT10 group were treated with reactive oxygen species scavenger N-acetyl-L-cysteine ​​(NAC), the protein expression levels of the inflammatory factor monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were measured. Laser confocal microscopy was used to detect the generation levels of reactive oxygen species in the cells of vrious groups. Results: FAT10 was expressed in carotid artery, thoracic aorta, and renal artery of normal blood pressure rats and expressed in HUVEC, VSMC, MDA-MB-231. The expression level of FAT10 gradually increased in proportion to the increase of the time and concentration of AngⅡ stimulation in HUVEC, and the expression level of FAT10 was the highest when the HUVEC was treated with 100 nmol/L AngⅡ for 36 h (P<0.01). The protein expression level of MCP-1 (P<0.001) and TNF-α (P<0.01) was higher in AngⅡ treated HUVEC with FAT10 overexpression, while the expression level of MCP-1 and TNF-α protein was lower in AngⅡ treated HUVEC with FAT10 knockdown (all P<0.01). The level of intracellular reactive oxygen species (ROS) production was significantly increased with FAT10 overexpression (P<0.001), and the level of ROS was decreased when the expression of FAT10 was interfered (P<0.05). The increased level of MCP-1 and TNF-α proteins in FAT10 overexpressed HUVEC was reversed by NAC (all P<0.05). Conclusion: FAT10 promotes the release of inflammatory factors induced by AngⅡ in endothelial cells by increasing the level of intracellular ROS production.
Humans ; Rats ; Animals ; Reactive Oxygen Species/pharmacology* ; Cells, Cultured ; Angiotensin II/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Rats, Inbred WKY ; Human Umbilical Vein Endothelial Cells ; Inflammation ; Ubiquitins/pharmacology*