Protective effects of icariin on human umbilical vein endothelial cell injured by angiotensin Ⅱ

To investigate the effects of icariin （ICA） on angiotensin Ⅱ（Ang Ⅱ）-induced injury in human umbilical vein endothelial cells line （ECV-304）. The ECV-304 cells were cultured in vitro. After 24 h incubating with icariin, the model of AngⅡ-induced injury in ECV-304 was established. The cell viability （MTT method）, Lactate dehydrogenase （LDH） release and Nitric oxide （NO） production in the medium, the capacity of scavenging superoxide anion radicals （O2^-） and hydroxyl radicals （.OH） were measured. The activities of superoxide dismutase （SOD）, total nitric oxide synthase （T-NOS）, inducible nitric oxide synthase （iNOS） and constitutive nitric oxide synthase （cNOS） in the cells were determined. Compared with the Ang Ⅱ-treated group, ICA can significantly raise the viability of EC, increase the activities of SOD, T-NOS and cNOS, increase the production of NO, enhance the capacity of scavenging superoxide anion radicals （ O2^- ） and hydroxyl radicals（.OH）, and lower LDH leakage and iNOS activity. The results suggest that ICA can protect endothelial cells （ECV-304） from Ang II-induced injury.

Relationship between angiotensin-(1-7) and angiotensin Ⅱ correlates with hemodynamic changes in human liver cirrhosis

AIM： To measure circulating angiotensins at different stages of human cirrhosis and to further evaluate a possible relationship between renin angiotensin system （RAS） components and hemodynamic changes. METHODS： Patients were allocated into 4 groups： mild-to-moderate liver disease （MLD）, advanced liver disease （ALD）, patients undergoing liver transplantation, and healthy controls. Blood was collected to determine plasma renin activity （PRA）, angiotensin （Ang） Ⅰ, Ang Ⅱ, and Ang-（1-7） levels using radioimmunoassays. During liver transplantation, hemodynamic parameters were determined and blood was simultaneously obtained from the portal vein and radial artery in order to measure RAS components. RESULTS： PRA and angiotensins were elevated in ALD when compared to MLD and controls （P 〈 0.05）. In contrast, Ang Ⅱ was significantly reduced in MLD. Ang-（1-7）/Ang Ⅱ ratios were increased in MLD when compared to controls and ALD. During transplantation, Ang Ⅱ levels were lower and Ang-（1-7）/Ang Ⅱ ratios were higher in the splanchnic circulation than in the peripheral circulation （0.52 ± 0.08 vs 0.38 ±0.04, P 〈 0.02）, whereas the peripheral circulating Ang Ⅱ/Ang Ⅰ ratio was elevated in comparison to splanchnic levels （0.18 ±0.02 vs 0.13 ±0.02, P 〈 0.04）. Ang-（1-7）/ Ang Ⅱ ratios positively correlated with cardiac output （r = 0.66） and negatively correlated with systemic vascular resistance （r = -0.70）. CONCLUSION： Our findings suggest that the relationship between Ang-（1-7） and Ang Ⅱ may play a role in the hemodynamic changes of human cirrhosis.

Changes of c-fos and c-jun mRNA Expression in Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy and Effects of Sodium Tanshinone ⅡA Sulfonate

The changes of proto-oncogene c-fos and c-jun mRNA expression in angiotensin Ⅱ （AngⅡ）-induced hypertrophy and effects of sodium tanshinone ⅡA sulfonate （STS） in the primary culture of neonatal rat cardiomyocytes were investigated. Twelve neonatal clean grade Wistar rats were selected. The cardiomyocytes were isolated, cultured and divided according to different treatments in the medium. The cardiomyocyte size was determined by phase contrast microscope, and the rate of protein synthesis was measured by [3H]-Leucine incorporation. The c-fos and c-jun mRNA expression in cardiomyocytes was detected by reverse transcription polymerase chain reaction （RT-PCR）. It was found after cardiomyocytes were treated with AngⅡ for 30 min, the c-fos and c-jun mRNA expression in cardiomyocytes was increased significantly （P〈0.01）. After treatment with AngⅡ for 24 h, the rate of protein synthesis in AngⅡ group was significantly increased as compared with control group （P〈0.01）. After treatment with AngⅡ for 7 days, the size of cardiomyocytes in AngⅡ group was increased obviously as compared with control group （P〈0.05）. After pretreatment with STS or Valsartan before AngⅡ treatment, both of them could inhibit the above effects of AngⅡ （P〈0.05 or P〈0.01）. It was suggested that STS could ameliorate AngⅡ-induced cardiomyocyte hy- pertrophy by inhibiting c-fos and c-jun mRNA expression and reducing protein synthesis rate of cardiomyocytes.

Association of Polymorphisms in Angiotensin-converting Enzyme and Type 1 Angiotensin Ⅱ Receptor Genes with Coronary Heart Disease and the Severity of Coronary Artery Stenosis

To explore the relation of angiotensin-converting enzyme （ACE） and angiotensin Ⅱ type 1 receptor （AT1R） gene polymorphism with coronary heart disease （CHD） and the severity of coronary artery stenosis, 130 CHD patients who underwent coronary angiography were examined for the number of affected coronary vessels （≥75% stenosis） and coronary Jeopardy score. The insertion/deletion of ACE gene polymorphism and AT1R gene polymorphism （an A→C transversion at nucleotide position 1166） were detected by using polymerase chain reaction （PCR） and restriction fragment length polymorphism （RFLP） in CHD patients and 90 healthy serving as controls. The resuits showed that DD genotype and of ACE were more frequent in CHD patients than that in control group （38.5% vs 14.4%, P〈0.001）. The frequency of the ATIR A/C genotypes did not differ between the patients and the controls （10% vs 13.1%, P〉0.05）. The relative risk associated with the ACE-DD was increased by AT1R-AC genotype. Neither the number of affected coronary vessels nor the coronary score differed among the ACE I/D genotypes （P〉0.05）. But the number of affected coronary vessels and the coronary score were significantly greater in the patients with the AT1R-AC genotype than in those with the AA genotype （P〈0.05）. In conclusion, DD genotype may be risk factor for CHD and MI in Chinese people, and is not responsible for the development of the coronary artery stenosis. The AT1R-C allele may increase the relative risk associated with the ACE-DD genotype, and may be involved in the development of the stenosis of coronary artery.

Valsartan Inhibits Angiotensin Ⅱ-induced Proliferation of Vascular Smooth Muscle Cells via Regulating the Expression of Mitofusin 2

Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.

Construction of shRNA Targeted to the Rat Angiotensin Ⅱ Type 1 Receptors and Its RNAi in Cytoplasma

The expression vector of shRNA targeted to the rat angiotensin Ⅱ receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in the in vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat （SHR） at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin Ⅱ receptor mRNA were synthesized individually according to the sequence of the rat angiotensin Ⅱ receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases. RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35.5%±3.0 %, and the levels reached their lowest point after 72 h （20.7% ±4 % of control）. At 24 and 48 h, AT1 protein was reduced to 46.9%±4.2% and 36.98%±3.7% respectively compared to control and a maximum reduction was observed after 72 h of incubation （28.1%± 4% compared to controls）. Plasmid-based shRNA expression systems targeted against the rat angiotensin Ⅱ receptor gene were generated successfully. The shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA （siRNA） by the Dicer. The in vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.

Expression of Angiotensin Ⅱ Receptors in Aldosterone-producing Adenoma of the Adrenal Gland and Their Clinical Significance

The expression of angiotensin Ⅱ type 1 receptor (AT1R) and angiotensin Ⅱ type 2 receptor (AT2R) in aldosterone-producing adenoma (APA) of the adrenal gland was detected, and their relationship with clinical indexes of APA was analyzed. The mRNA expression of AT1R and AT2R in 50 cases of APA and tissues adjacent to tumors and 12 cases of normal adrenal tissues was detected by using reverse transcriptase polymerase chain reaction (RT-PCR). The expression of AT1R and AT2R proteins in paraffin-embedded slices of tissue was detected by immunohistochemistry. The expression of AT1R in adenoma, tissues adjacent to tumor, and normal tissues of the adrenal gland showed no significant differences. The expression of AT2R in APA tissue was lower than that in normal adrenal gland tissues (P<0.05). Correlation analysis of the mRNA expression level of AT2R and clinical data from patients demonstrated that AT2R expression was negatively related to plasma aldosterone concentration (PAC) (r=-0.467, P<0.05), but positively related with plasma renin activity (PRA) (r=0.604, P<0.05). It is concluded that down-regulation of the AT2R expression is possibly related with the tumorigenesis of APA.

Effects of angiotensin Ⅱ receptor antagonist, Losartan on the apoptosis, proliferation and migration of the human pancreatic stellate cells

AIM： To investigate the effects of AT, （Type 1 angiotensin Ⅱ receptor） antagonist （Losartan） on the apoptosis, proliferation and migration of the human pancreatic stellate cells （hPSCs）. METHODS： hPSCs were isolated from pancreatic sample of patients with pancreatic carcinoma using radioimmunoassay （RIA） technique to detect the concentration of AngⅡ in culture media and cell homogenate. Immunocytochemistry （ICC） and in situ hybridization （ISH） methods were utilized to test AT1 expression in hPSCs. Effects of Losartan on hPSCs proliferation, apoptosis and migration were investigated using BrdU incorporation, TUNEL, flow cytometry （FCM）, and phase-contrast microscope separately when cells treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type Ⅰ collagen in hPSCs. RESULTS： There exists AT1 expression in hPSCs, while no AngⅡ was detected in culture media and cell homogenate. Losartan induces cell apoptosis in a doseand time-dependent manner （apparently at 10^-5 mol/L）, no pro-proliferative effect was observed in the same condition. Corresponding dosage of Losartan can also alleviate the motion capability and type Ⅰ collagen content of hPSCs compared with AngⅡ treatment and non-treatment control groups. CONCLUSION： These findings suggest that paracrine not autocrine functions of AngⅡ may have effects on hPSCs, which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion and partly by inducing apoptosis.

Rapamycin Treatment Attenuates Angiotensin Ⅱ-induced Abdominal Aortic Aneurysm Formation via VSMC Phenotypic Modulation and Down-regulation of ERK1/2 Activity

The aim of the present study is to address the effect of rapamycin on abdominal aortic aneurysm（AAA） and the potential mechanisms. A clinically relevant AAA model was induced in apolipoprotein E-deficient（ApoE-/-） mice, in which miniosmotic pump was implanted subcutaneously to deliver angiotensin Ⅱ（Ang Ⅱ） for 14 days. Male ApoE-/-mice were randomly divided into 3 groups： saline infusion, Ang Ⅱ infusion, and Ang Ⅱ infusion plus intraperitoneal injection of rapamycin. The diameter of the supra-renal abdominal aorta was measured by ultrasonography at the end of the infusion. Then aortic tissue was excised and examined by Western blotting and histoimmunochemistry. Ang Ⅱ with or without rapamycin treatment was applied to the cultured vascular smooth muscle cells（VSMCs） in vitro. The results revealed that rapamycin treatment significantly attenuated the incidence of Ang Ⅱinduced-AAA in ApoE-/-mice. Histologic analysis showed that rapamycin treatment decreased disarray of elastin fibers and VSMCs hyperplasia in the medial layer. Immunochemistry staining and Western blotting documented the increased phospho-ERKl/2 and ERK1/2 expression in aortic walls in Ang Ⅱ induced-AAA,as well as in human lesions. Whereas in the rapamycintreated group, decreased phospho-ERK1/2 expression level was detected. Moreover, rapamycin reversed Ang Ⅱ-induced VSMCs phenotypic change both in vivo and in vitro. Based on those results, we confirmed that rapamycin therapy suppressed Ang Ⅱ-induced AAA formation in mice, partially via VSMCs phenotypic modulation and down-regulation of ERK1/2 activity.

Effect of Microinfusion of Angiotensin Ⅱ into the RVLM in Rats on the Baroreceptor Reflex Sensitivity

Objective: The present study was designed to investigate the effect of microinfusion angiotensin Ⅱ(Ang Ⅱ),Ang Ⅱ type 1(AT_1)receptor antagonist losartan into the rostral ventrolateral medulla(RVLM)on the baroreceptor reflex sensitivity(BRS)in urethane-anesthetized rats. Methods: Reflex changes in heart rate(HR)were elicited by bolus intravenous injection of phenylephrine before and during RVLM microinfusion of saline(0.5 μl/h),Ang Ⅱ (1.5 nmol/h),losartan(250 nmol/h),and Ang Ⅱ(1.5 nmol/h)pretreated with microinjection of losartan (50 nmol/0.51 μl)into the RVLM.The average ratio between changes in HR in beats per minute(beats·min -1)and changes in mean arterial pressure [MAP,mmHg(1 mmHg=0.133 kPa)] was used as an index of BRS. Results: Ang Ⅱ resulted in a significant decrease in the BRS for reflex bradycardia compared with control(-2.1±0.1 vs-3.9±0.4 beats·min -1·mmHg -1).Microinfusion of losartan had no significant effect on BRS for reflex bradycardia.The effect of Ang Ⅱ was almost completely abolished by pretreatment with microinjection of losartan. Conclusion:These results showed that the exogenous Ang Ⅱ in the RVLM produces inhibitory modulation of BRS,which is mediated by AT_1 receptor.However,AT_1 receptor in the RVLM is not involved in the tonic control of BRS.

Astilbin Inhibits Proliferation of Rat Aortic Smooth Muscle Cells Induced by Angiotensin Ⅱ and Down-regulates Expression of Protooncogene

This study examined the effect of astilbin on the proliferation of rat aortic smooth muscle cells (RASMCs) induced by angiotensin Ⅱ (AngⅡ) and explored the possible mechanisms. Cell proliferation model of RASMCs was induced by treatmente with AngⅡ. Cells were randomly divided to 8 groups. Normally cultured VSMCs serves as blank control group; in AngⅡ model group, cells were treated with AngⅡ at 10–7 mol/L; in three astilbin groups, cells were treated with 10, 15, 30 mg/L of astilbin; in three AngⅡ+astilbin groups, cells were treated with AngⅡ (at 10–7 mol/L) and astilbin at 10, 15, 30 mg/L. Cell proliferation ability was detected by MTT method and the cell cycles and proliferation index were flow cytometrically determined. The expression of c-myc mRNA was assessed by using reverse transcription polymerase chain reaction (RT-PCR), and the expression of NF-κB in RASMCs was immunocytochemically observed. Our results showed that MTT metabo-lism in RASMCs in the basic and AngII stimulated situation was inhibited by astilbin, and the cells numbers of G0/G1 phase were increased and that of G2/S phase were decreased markedly. Not only highly expression of c-myc gene stimulated by AngⅡ could be inhibited by Astilbin significantly, but also the expression of NF-κB protein can be down regulated by Astilbin. We are led to conclude that astilbin astilbin can inhibit the AngⅡ-mediated proliferation of RASMCs by blocking the transition of RASMCs from G0/G1 phase to S phase and by down-regulating the expression of NF-κB, c-myc gene.

FOLLICULO-STELLATE CELLS OF THE RAT ANTERIOR PITUITARY RESPONDED TO ANGIOTENSIN Ⅱ BY INCREASING INTRACELLULAR Ca^(2+) CONCENTRATION

Objective The main purpose of this study was to investigate whether the folliculo stellate cells (FSC) respond to angiotensin(Ang)Ⅱ by increasing intracellular free Ca 2+ concentration ([Ca 2+ ]i),and where the origin of Ca 2+ mobilization is if that has occurred.Methods Pituitary cells in primary culture were prepared from male Wister rats(250g) by a conventional method and cultured in MEM supplemented with 4% normal rat serum.After 2 days in culutre,cells were loaded with 1 μmol/L fura PE3/AM for 1 h and subjected to a Ca 2+ imaging experiment with Quanti Cell 700 system.Excitation wavelengths of 340 and 380 nm were selected by means of a computer controlled filterwheel.Results The [Ca 2+ ]i of FSC in the rat anterior pituitary was elevated by Ang Ⅱ.The elevation of [Ca 2+ ]i of FSC induced by 0.1,1.0,10 and 100 nmol/L Ang Ⅱ was (56.33±6.18)( ±s ),(117.07±36.07),(175.59±40.01) and (216.02±11.52) nmol/L,respectively.The increase of [Ca 2+ ]i of FSC induced by 100 nmol/L Ang Ⅱ was not influenced by the medium without Ca 2+ (0Ca),but significantly suppressed by thapsigargin(TG),an inhibitor of ATPase.The rate of responsive FSC to Ang Ⅱ (100 nmol/L) was 61.84% which was obviously higher than that of pituitary endocrine cells(43.49%).Conclusion The present experiment demonstrates that the FSC in the rat anterior pituitary responds to Ang Ⅱ by increasing [Ca 2+ ]i,which raises the possibility that Ang Ⅱ released from either lactotrophs or gonadotrophs affects FSC through paracrine mechanism.The elevation of [Ca 2+ ]i induced by Ang Ⅱ presents a dosage dependent relation, and is possibly because of the release of Ca 2+ from an intracellular Ca 2+ pool(s).Fashions of Ca 2+ release are relative to the concentration of Ang Ⅱ.The results indicate that Ang Ⅱ functions as a paracrine factor among pituitary cells including FSC.

Regulatory roles of nitric oxide and angiotensin Ⅱ on renal tubular transport

Renal tubules regulate blood pressure and humoral homeostasis. Mediators that play a significant role in regulating the transport of solutes and water include angiotensin Ⅱ （AngⅡ） and nitric oxide （NO）. AngⅡcan signifcantly raise blood pressure via effects on the heart, vasculature, and renal tubules. AngⅡ generally stimulates sodium reabsorption by triggering sodium and fuid retention in almost all segments of renal tu-bules. Stimulation of renal proximal tubule （PT） trans-port is thought to be essential for AngⅡ-mediated hy-pertension. However, AngⅡ has a biphasic effect on in vitro PT transport in mice, rats, and rabbits： stimulation at low concentrations and inhibition at high concentra-tions. On the other hand, NO is generally thought to inhibit renal tubular transport. In PTs, NO seems to be involved in the inhibitory effect of AngⅡ. A recent study reports a surprising fnding： AngⅡ has a mono-phasic stimulatory effect on human PT transport. De-tailed analysis of signalling mechanisms indicates that in contrast to other species, the human NO/guanosine 3’,5’-cyclic monophosphate/extracellular signal-regulat-ed kinase pathway seems to mediate this effect of Ang Ⅱ on PT transport. In this review we will discuss recent progress in understanding the effects of AngⅡ and NO on renal tubular transport.

The changes of serum nitric oxide, angiotensin Ⅱ and superoxide anion in renal artery hypertension rat

Objectives To study the changes of nitric oxide, angiotensin Ⅱ and superoxide anion in renal artery hypertension pathogenesis. Methods Male Wistar rats weighing 256 -285g were divided into 5 groups randomly, 10 rats of each group. Control group: false operation was made and routine diet was given; Ligature group: left renal artery was ligatured uncompletely and routine diet was given; Ligature + Losartan group: left renal artery was ligatured uneompletely and Losartan 20mg · kg^(-1) · d^(-1) was added in the drinking water; Ligature + L -Arg group: left renal artery was ligatured uncompletely and L -Arg 2g · kg^(-1) · d^(-1) was added in the drinking water; Ligature + L - Arg + Losartan group: left renal artery was ligatured uncompletely and L - Arg 2g· kg^(-1)· d^(-1) and Losartan 20mg · kg^(-1)· d^(-1) was added in the drinking water. Blood pressure and heart rate were measured before and at the end of the experiment. One week after ligature, blood was drawn to determine angiotensin Ⅱ, cGMP, nitric oxide, nitric oxide synthase (NOS), O_2^-, superoxide dismutase (SOD). Results Systolic blood pressure was higher in ligature group than that in control group (p <0.05), systolic blood pressure was much lower in ligature + Losartan group than that in ligature group. Heart rate did not change significantly after experiment (p > 0. 05). AngⅡ was higher in ligature group than that in control group, even much higher in ligature + Losartan group (p < 0. 01 ). There was no difference of cGMP in each group (p >. 05). The concentration of NO was lower in ligature group (p <0.05), NO was higher in ligature + L - Arg + Losartan group than that in ligature group (p <0.05). O_2^- was higher in ligature group and ligature + L - Arg group than that in control group (p < 0. 05), O_2^- was lower in ligature + Losartan group than that in ligature group (p <0. 05). The level of SOD was lower in ligature group than that in control group (p <0.05), higher in ligature + L- Arg group and ligature + L - Arg + Losartan group than that in ligature group (p <0. 05). Conclusions AngⅡ,O_2^- and NO imbalance play an important role in hypertension pathogenesis, LArg and losartan may have protective effect.

Angiotensin Ⅱ/AT1及Apelin/APJ系统与血管钙化关系的研究进展

心血管疾病（cardiac vascular disease,CVD）是慢性肾脏病（chronic kidney disease,CKD）患者死亡的首要原因,血管钙化和CVD发生率、死亡率密切相关[1]。在未接受透析的CKD 3~5期患者中,血管钙化发生率为30%~65%,而在维持性血液透析人群中高达50%~80%[2]。CKD时RAS系统激活,Ang II是RAS系统的核心成员。

Effects of Cyclosporine A on Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy in Neonatal Rats

Summary： The effects of cyclosporine A （CsA） on Angiontensin Ⅱ （Ang Ⅱ ）-induced protein contents, c los protein levels and cytosolic Ca^2＋ level （[Ca^2＋]i） in cultured eardiomyocytes of neonatal rats were observed. Total protein contents were determined by Bradford method. The expression of c-fos protein was detected by Western blot. （[Ca^2＋]i） labeled with fluorescent probe Fluo-3/AM was measured under a laser scanning confoeal microscope. The results revealed that as compared with control, the total protein contents were increased in cardiomyocytes treated with Ang Ⅱ （10-1 mol/ L）, which could be inhibited by CsA in a dose-dependent manner. It was found that Ang Ⅱ could increase the c-los protein expression, which could be inhibited by CsA in a dose-dependent manner. Ang Ⅱ induced the [Ca^2＋]i elevation in cardiomyocytes. CsA did not influence the resting intracellular Ca^2＋ , but inhibited significantly the Ang Ⅱ-induced [Ca^2＋]i elevation. It was concluded that CsA can suppress the Ang Ⅱ-induced c-fos protein expression and [Ca^2＋]i elevation in single cardiomyocyte, which might play a role in the prevention of Ang Ⅱ-induced cardiomyocyte hypertrophy by CsA.

Inhibition of angiotensin Ⅱ and blockade of endothelin receptors reduce arterial calcification in rats

Objective To examine whether the two vascular paracrine/autocrine factors,angiotensin Ⅱ(Ang Ⅱ)and endothelin,participate in the pathogenesis of arterial calcification.Methods Nicotine and vitamin D_3 treated rats were studied.Vascular calcification was confirmed by using Von Kossa staining,measurement of calcium content, ^(45)Ca^(2+)uptake assay and alkaline phosphatase(ALP)activity.The plasma and vascular Ang Ⅱ and endothelin levels were measured by using radioimmunoassay.Angiotensinngen and endothehn mRNA levels were determined by RT- PCR.Results The arterial calcium content,^(45)Ca^(2+)uptake and ALP activity were increased in calcification groups compared with control(P<0.01).Administration of the angiotensin receptor antagonist losartan,the endothelin receptor antagonist bosentan,and the angiotensin-converting enzyme inhibitor captopril reduced significantly the arterial calcium content,^(45)Ca^(2+)uptake and ALP activity.In addition,the plasma and aortic Ang Ⅱ and endothelin contents,and vascular angiotensinngen and endothelin mRNA expression were significantly up-regulated(P<0.05). Conclusions These findings suggest that functional renin-angiotensin system and endothelin pathway are involved in vascular calcification,and that activation of these systems could potentiate pathogenesis of arterial calcification.(J Geriatr Cardiol 2004;1(2):108-113.)

Effect of Angiotensin Ⅱ on Cord Blood CD^(34+) Cells Expansion in vitro

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34 + cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1±1.8 times to 9 2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

Suppression of angiotensin Ⅱ stimulated responses in aortic vascular smooth muscle cells of experimental cirrhotic rats

Functional responses to angiotensin Ⅱ (AT-Ⅱ) were determined in aortic vascular smooth muscle cells (VSMCs) from experimental cirrhotic rats. Our data showed that AT-Ⅱ-stimulated extracellular acidification rate (ECAR), which was measured by Cytosensor microphysiometry, was significantly reduced in the aortic VSMCs from the cirrhotic rats as compared to those from the control animals. The ability of AT-Ⅱ to promote formation of inositol phosphates, the second messenger produced by the activation of Gq-coupled receptors, was also considerably suppressed in the cirrhotic VSMCs. Furthermore, the maximal p42/44 MAPK phosphorylation stimulated by AT-Ⅱ was significantly reduced in the cirrhotic VSMCs in contrast to that in the normal VSMCs. Taken together, our data clearly demonstrated that the functional responses to AT-Ⅱ was severely suppressed in aortic VSMCs in cirrhosis, indicating the impairment of general Gq-coupled receptor signaling and subsequent biological function in the cirrhotic VSMCs.

MULTIPLE ENHANCER ELEMENTS MEDIATE THE INDUCTION OF C-FOS BY ANGIOTENSIN Ⅱ IN VASCULAR SMOOTH MUSCLE CELLS

Previous work from this laboratory has demonstrated that the addition of angiotensin(Aug)Ⅱresults in the rapid transcriptional activation of early growth response gene c-fos.Blockage of this increase completely inhibits the Aug Ⅱinduced increase in vascular smooth muscle cell(VSMC)growth.To explore the molecular mechanism responsible for the induction of c-fos in VSMC,a series of constructs containing portions of c-fos promoter linked to the reporter gene chloramphenicol acetyltransferase(CAT)were used in transient transfection assays.When a construct containing both the well described serum response element(SRE)and the cyclic AMP response element(CRE)was used,no endogenous CAT activity was observed in serum starved cells.The addition of either Ang Ⅱor serum resulted in a marked increase in CAT activity.Mutations in either the SRE or CRE alone which have been demonstrated to inactivate these elements in number of cell types had no effect on c-fos inducibility by either Angll or serum. In contrast,if both elements were mutated in the same construct,inducibility was reduced by 75 %￣80%.Using a construct in which the SRE has been deleted,a mutation in the CRE completely abolished induction of c-fos by either Aug Ⅱor serum. Mobility shift assays demonstrated that tow proteins bind specifically to the SRE and three proteins to CRE. These data demonstrate that the induction of c-fos in VSMC's can be mediated by two distinct enhancer elements each of which can act independently. Future research will be aimed at identifying the proteins that interact with these elemetns delineating the mechanisms by which Ang Ⅱstimulates their activity.