Estradiol increases apoptosis in human coronary artery endothelial cells by up-regulating Fas and Fas ligand expression

CONTEXT: In animal models, estrogen inhibits atherogenesis by inhibiting many of the early steps of atherosclerotic plaque formation. However, the lack of cardioprotective effect by postmenopausal hormone replacement therapy and possible increase in cardiovascular events observed during the first year after the initiation of hormone replacement therapy may suggest that once the plaque is formed, estrogen may have additional effects that may counteract its beneficial outcomes. Indeed, the effect of estrogen on plaque stability has not been identified. OBJECTIVE: We hypothesized that 17beta-estradiol (E2) may cause increased apoptosis in human coronary artery endothelial cells (HCAECs). This effect would explain an adverse effect on plaque stability in vivo. INTERVENTION(S) AND MAIN OUTCOME MEASURE(S): The effect of E2 on apoptosis, cell proliferation, and expression of proapoptotic molecules Fas and Fas ligand (FasL) in cultured HCAECs was evaluated. RESULTS: HCAECs in culture treated with E2 showed an increase in DNA strand breaks and nuclear fragmentation indicative of apoptosis. E2 treatment also induced a significant concentration-dependent increase in Fas mRNA and protein expressions in HCAECs. Moreover, the expression of FasL mRNA and secretion of FasL protein by HCAECs were enhanced in response to E2 treatments. CONCLUSIONS: E2 increases the apoptosis in cultured HCAECs. Enhanced Fas and FasL expressions in response to E2 suggest that activation of the Fas/FasL pathway may be a mediator of the proapoptotic effects of E2 in these cells.     

Statins inhibit oxidized-LDL-mediated LOX-1 expression, uptake of oxidized-LDL and reduction in PKB phosphorylation

OBJECTIVES: LOX-1, a lectin-like receptor on endothelial cells, facilitates the uptake of oxidized-LDL. Expression of LOX-1 is involved in the pathobiological effects of oxidized-LDL in endothelial cells, including apoptosis, suppression of cNOS activity and cell adhesion. Recent studies show that intracellular signal protein kinase B (PKB) is involved in the regulation of cNOS. Further, HMG CoA reductase inhibitors (statins) may affect LOX-1 expression. In this study, we examined the modulation of LOX-1 expression and PKB activity in response to oxidized-LDL by two different statins (simvastatin and atorvastatin). METHODS AND RESULTS: Cultured human coronary artery endothelial cells (HCAECs) were used in this study. Oxidized-LDL (40 microg/ml) was found to upregulate the expression of LOX-1 (mRNA and protein), enhance [125I]-ox-LDL uptake and to reduce the phosphorylation of PKB (p-PKB). Two different statins, simvastatin and atorvastatin (each 1 and 10 microM), upregulated the activity of PKB and decreased LOX-1 expression and [125I]-ox-LDL uptake. A high concentration of statins (10 microM) gave a more potent effect than the low concentration (1 microM). The effects of the two different statins were similar. CONCLUSIONS: These observations show that statins decrease LOX-1 expression, a novel oxidized-LDL endothelial receptor, and uptake of oxidized-LDL in HCAECs. The effect of statins on LOX-1 expression is associated with an increase in PKB activity in HCAECs.     

腺病毒介导血管平滑肌细胞转染表达血管紧张素Ⅱ2型受体的研究

目的体外培养大鼠血管平滑肌细胞（VSMC）并以腺病毒介导转染表达血管紧张素Ⅱ（AngⅡ）2型受体（AT     

Novel role of macrophage migration inhibitory factor in angiotensin II regulation of neuromodulation in rat brain

Previously we determined that angiotensin II (Ang II) activates neuronal AT(1) receptors, located in the hypothalamus and the brainstem, to stimulate noradrenergic pathways. To link Ang II to the regulation of norepinephrine metabolism in neurons cultured from newborn rat hypothalamus and brainstem we have used cDNA arrays for high throughput gene expression profiling. Of several genes that were regulated, we focused on macrophage migration inhibitory factor (MIF), which has been associated with the modulation of norepinephrine metabolism. In the presence of the selective AT(2) receptor antagonist PD123,319 (10 microM), incubation of cultures with Ang II (100 nM; 1-24 h) elicited an increase in MIF gene expression. Western immunoblots further revealed that Ang II (100 nM; 1-24 h) increased neuronal MIF protein expression. This effect was inhibited by the AT(1) receptor antagonist losartan (10 microM), the PLC inhibitor U-73122 (10 or 25 microM), the PKC inhibitor chelerythrine (10 microM), and the Ca(2+) chelator 1,2-bis-[2-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (10 microM). Taken together with our observation that MIF is expressed in the terminal fields of noradrenergic neurons (hypothalamus) and that Ang II increases the expression of MIF in this region in vivo, our data may suggest a novel role of Ang II in norepinephrine metabolism.     

Angiotensin II upregulates LDL receptor-related protein (LRP1) expression in the vascular wall: a new pro-atherogenic mechanism of hypertension

AIMS: Hypertension is a risk factor for atherothrombotic vascular events. Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, has been associated with the development and progression of atherosclerosis. However, it is not fully known how Ang II contributes to lipid-enriched atherosclerotic lesion formation. In human vascular smooth muscle cells (VSMC), low density lipoprotein (LDL) receptor-related protein (LRP1) internalizes cholesteryl esters (CE) from extracellular matrix-bound aggregated LDL (agLDL). The aim of this study was to investigate the effect of Ang II on LRP1 expression and function in VSMC. METHODS AND RESULTS: Here, we report for the first time that Ang II induces the upregulation of LRP1 expression in VSMC. Ang II (1 microM) induced maximal LRP1 mRNA expression at 12 h and maximal protein overexpression (by 4.10-fold) at 24 h in cultured human VSMC. Ang II effects were functionally translated into an increased CE accumulation from agLDL uptake (by two-fold at 50 microg/mL) that was prevented by the LRP1 ligand lactoferrin and by siRNA-LRP1 treatment. Ang II-LRP1 upregulation and excess CE accumulation from agLDL were prevented by losartan (an AT1 blocker) but not by PD123319 (a specific AT2 blocker). Additionally, in a normolipidaemic rat model, Ang II infusion produced a significant increase in aortic LRP1 expression and lipid infiltration in the arterial intima. CONCLUSION: The in vitro and in vivo data reported here indicate that Ang II upregulates LRP1 receptor expression and LRP1-mediated aggregated LDL uptake in vascular cells.     

血管紧张素Ⅱ及其受体拮抗剂对心肌细胞肥大的影响

目的观察血管紧张素Ⅱ（AngⅡ）、AT1受体拮抗剂氯沙坦和AT2受体拮抗剂PD123177对心肌细胞蛋白质合成速率和AT1受体mRNA表达的影响。方法采用3H-亮氨酸掺入法测定培养的心肌细胞蛋白质合成速率,RT-PCR方法检测心肌细胞AT1受体mRNA表达。结果在培养的心肌细胞中加入AngⅡ可明显增加心肌细胞3H-亮氨酸的掺入量,并呈剂量依赖性,氯沙坦可显著抑制AngⅡ引起的蛋白质合成增加,而PD123177对其无影响;AngⅡ上调AT1受体基因表达,氯沙坦抑制其上调,PD123177无影响。结论AngⅡ可通过上调AT1受体引起心肌细胞肥大,氯沙坦下调AT1受体,抑制心肌细胞肥大。     

Oxidized-LDL through LOX-1 increases the expression of angiotensin converting enzyme in human coronary artery endothelial cells

BACKGROUND AND OBJECTIVES: Our previous studies have shown that oxidized low-density lipoprotein (ox-LDL) and angiotensin II (Ang II) influence each other's action in endothelial cells. This study was designed to examine the regulation by ox-LDL of the expression of angiotensin converting enzyme (ACE) gene in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of the HMG CoA reductase inhibitor simvastatin on this interaction. METHODS AND RESULTS: Cultured HCAECs were incubated with ox-LDL (10-80 microg/ml) for 1-24 h. Ox-LDL increased the expression of ACE in a concentration- and time-dependent fashion. The upregulation of ACE expression in response to ox-LDL was mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 prevented the expression of ACE (P<0.01). Native-LDL had no significant effect on ACE expression. In this process, ox-LDL-induced activation of mitogen-activated protein kinase (MAPK p42/44) played an important role, since pretreatment of HCAECs with the MAPK p42/44 inhibitor (PD98059, 10 microM) inhibited MAPK activation and subsequently attenuated the expression of ACE (P<0.01 vs. ox-LDL alone). In other experiments, we pretreated HCAECs with simvastatin (10 microM) and then exposed the cells to ox-LDL. Simvastatin markedly attenuated ox-LDL-induced MAPK activation, and concurrently reduced ACE expression (P<0.01 vs. ox-LDL alone). CONCLUSIONS: Our observations provide direct evidence that ox-LDL via LOX-1 activation induces ACE gene expression in HCAECs, and MAPK activation plays a signal transduction role in this process. Simvastatin, which inhibits MAPK activation, also blocks ox-LDL-mediated upregulation of ACE.     

The third cytoplasmic loop of the angiotensin II type 1 receptor exerts differential effects on extracellular signal-regulated kinase (ERK1/ERK2) and apoptosis via Ras- and Rap1-dependent pathways

The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT(1)) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT(1) sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT(1) into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH(2)-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca(2+), whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca(2+) dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca(2+) and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT(1).     

Influence of the 4G/5G PAI-1 genotype on angiotensin II-stimulated human endothelial cells and in patients with hypertension

BACKGROUND: We examined the influence of the 4G/5G PAI-1 (plasminogen activator inhibitor) genotype on Angiotensin II (Ang II)-induced PAI-1 expression by human endothelial cells (HUVEC) in the presence and absence of AT1-receptor blocker losartan, and screened for this polymorphism in relation to plasma PAI-1 and arterial pressure in apparently healthy subjects. METHODS AND RESULTS: Genotyped cultured HUVEC were incubated with Ang II (10(-8) M) with or without losartan up to 24 h. PAI-1 mRNA was determined in cell extracts and protein and activity assessed in supernatants and extracellular matrix (ECM). Ang II increased PAI-1 mRNA and activity in a genotype-dependent manner, higher values observed for 4G/4G HUVEC compared with 4G/5G and 5G/5G genotypes (p<0.05). Laser confocal microscopy and Western blot analysis showed increased PAI-1 protein within ECM in Ang II-stimulated cultures. PAI-1 expression and protein secretion induced by Ang II in 4G/4G HUVEC was completely inhibited by preincubation with 0.05 microM losartan (p<0.01), indicating an AT1-mediated effect. In a group of hypertensives homozygous for the 4G allele, PAI-1 antigen was significantly increased (51.0+/-10.1 ng/ml) compared with normotensives (28.3+/-4.0 ng/ml) and hypertensives carrying the 5G allele (p<0.05). CONCLUSIONS: The 4G/5G PAI-1 polymorphism determines the endothelial PAI-1 upregulation by Ang II and the inhibitory response to losartan. Analysis of PAI-1 genotypes may help identifying subgroups of hypertensives at higher cardiovascular risk.     

Renin-angiotensin system expression in rat bone marrow haematopoietic and stromal cells

The existence of a bone marrow renin-angiotensin system (RAS) is evidenced by the association of renin, angiotensin converting enzyme (ACE), and angiotensin (Ang) II and its AT(1) and AT(2) receptors with both normal and disturbed haematopoiesis. The expression of RAS components by rat unfractionated bone marrow cells (BMC), haematopoietic-lineage BMC and cultured marrow stromal cells (MSC) was investigated to determine which specific cell types may contribute to a local bone marrow RAS. The mRNAs for angiotensinogen, renin, ACE, and AT(1a) and AT(2) receptors were present in BMC and in cultured MSC; ACE2 mRNA was detected only in BMC. Two-colour flow fluorocytometry analysis showed immunodetectable angiotensinogen, ACE, AT(1) and AT(2) receptors, and Ang II, as well as binding of Ang II to AT(1) and AT(2) receptors, in CD4(+), CD11b/c(+), CD45R(+) and CD90(+) BMC and cultured MSC; renin was found in all cell types with the exception of CD4(+) BMC. Furthermore, Ang II was detected by radioimmunoassay in MSC homogenates as well as conditioned culture medium. The presence of Ang II receptors in both haematopoietic-lineage BMC and MSC, and the de novo synthesis of Ang II by MSC suggest a potential autocrine-paracrine mechanism for local RAS-mediated regulation of haematopoiesis.     

Enhancement of aminopeptidase A expression during angiotensin II-induced choriocarcinoma cell proliferation through AT1 receptor involving protein kinase C- and mitogen-activated protein kinase-dependent signaling pathway

Angiotensin II (Ang II) is a bioactive peptide of the renin-angiotensin system, exerting its actions not only as a vasoconstrictor, but also as a growth promoter. In human placenta, type 1 Ang II receptors (AT(1)R) are predominantly expressed in trophoblasts, and we previously reported that aminopeptidase A (APA), a cell surface peptidase that converts Ang II to Ang III, is also expressed in both normal and neoplastic trophoblasts. However, the roles of Ang II and APA in trophoblast function remain to be clarified. In the present study we examined the effects of Ang II on proliferation and APA expression in trophoblast-like BeWo choriocarcinoma cells. Treatment of BeWo cells with Ang II significantly increased DNA synthesis in a dose-dependent manner. Ang II also enhanced APA mRNA and cell surface expression in BeWo cells analyzed by Northern blotting, flow cytometry, and enzyme activity assay. The Ang II-induced proliferation and APA up-regulation were blocked by the AT(1)R antagonist candesartan, but not by the AT(2)R antagonist PD123319. Furthermore, these Ang II effects were abolished by the protein kinase C inhibitor bisindolylmaleimide I and the MAPK inhibitor PD98059. Immunohistochemistry using choriocarcinoma tissues demonstrated that APA was expressed on the cell surface of AT(1)R-positive cytotrophoblastic cells in vivo. With these findings we demonstrate that Ang II stimulates the proliferation of trophoblastic cells via AT(1)R that are linked to protein kinase C /MAPK-dependent signaling pathways, and that the Ang II-degrading enzyme APA is up-regulated during Ang II-induced cell proliferation. These observations suggest the possible regulatory mechanism by the local renin-angiotensin system, especially the Ang II-AT(1)R-APA system, for the growth of human choriocarcinoma cells.     

Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells

Angiostensin II (Ang II) regulates the migration and proliferation of vascular smooth muscle cells. Recent studies indicate that intermediate-conductance Ca2+ -activated K+ (IKca) channels have an important role in cell migration and proliferation. It is not known, however, whether the action of Ang II is linked to IKca channel regulation. Here, we investigated the modulation of IKca channels by Ang II in artery smooth muscle cells. Functional IKca channel expression in cultured embryonic rat aorta smooth muscle (A10) cells was studied using the patch-clamp technique. These cells predominantly express IKca channels. In contrast, large-conductance Ca2+ -activated K+ (BKca) currents were rarely observed in excised patches. Ang II increased the IKca current in a contration-dependent manner. Losartan (1.0 microM), an AT1 selective antagonist, abolished the activation of IKca channels by Ang II. Pretreatment with 100 microM myristoylated protein kinase C inhibitor peptide 20-28 or 10 microM GF109203X completely abolished the AngII-induced activation of IKca currents, whereas the action of Ang II was not prevented in the presence of 100 microM Rp-cyclic 3', 5'-hydrogen phosphotiate adenosine triethylammonium, a protein kinase A inhibitor, or 1.0 microM KT-5823, a protein kinase G inhibitor. A membrane permeant analogue of diacylglycerol 1, 2-dioctanoyl-sn-glycerol (10 microM) induced the activation of IKca currents. These data suggest that Ang II activates IKca channels through the activation of protein kinase C, and the AT1 receptor is involved in the regulation of these channels.