Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture

Angiotensin II has been identified immunohistochemically in the ovaries of both rats and humans. Here we present evidence that angiotensin II (an extremely vasoactive agent in a wide range of tissues) may be involved in the regulation of the major steroidogenic enzyme in the ovary, cholesterol side chain cleavage cytochrome P-450 (P-450scc), as well as of basic fibroblast growth factor (bFGF), which has been implicated as an angiogenic factor in the bovine corpus luteum. We have used primary cultures of bovine luteal cells to examine the effect of angiotensin II and its receptor antagonist, saralasin, on expression of mRNA encoding bFGF as well as on progesterone production and the expression of mRNA encoding cholesterol side chain cleavage cytochrome P-450 (P-450scc). Neither angiotensin II nor saralasin when added alone to the culture medium had any effect on basal progesterone production. Luteinizing hormone (LH) caused a 15-fold increase in progesterone accumulation after 24 h of exposure which was reduced to 5-fold in the presence of angiotensin II. This appeared to be receptor-mediated in that although saralasin alone had no effect on LH-stimulated progesterone accumulation, it significantly reversed the inhibition by angiotensin II. This pattern was mirrored by the levels of mRNA encoding P-450scc, i.c., LH induced the highest levels of expression of this message, these levels were reduced by angiotensin II, and saralasin partially overcame this reduction. Levels of mRNA encoding bFGF were elevated by both LH and angiotensin II. Treatment with saralasin, however, resulted in complete inhibition of bFGF mRNA expression in the presence of both LH and angiotensin II. These results suggest a role for angiotensin II to mediate the action of LH as a regulator of bFGF expression and hence, potentially, angiogenesis. Local production of angiotensin II might also contribute to the refractoriness of luteal progesterone secretion to LH at the time of luteal regression.     

Exercise-induced apoptosis in rat kidney is mediated by both angiotensin II AT1 and AT2 receptors

Excessive physical exercise may lead to disturbance of the entire homeostasis in the body, including damage not only in skeletal muscles but also in many distant organs. The mechanisms responsible for the exercise-induced changes could include oxidative stress or angiotensin II. We previously showed that acute exercise led to apoptosis in kidney but not as a result of oxidative stress. In this study, we examined the role of angiotensin II and its AT1 and AT2 receptors in mediation of exercise-induced apoptosis in kidney. We clearly demonstrated that acute physical exercise induced apoptosis in renal cells of distal convoluted tubuli and cortical and medullary collecting ducts. Moreover, the cells displayed an increased expression of both AT1 and AT2 angiotensin II receptors and of p53 protein. The results suggest that angiotensin II could upregulate p53 expression in renal distal convoluted tubular cells and in the cells collecting ducts via both AT1 and AT2 receptors, which might be the crucial apoptosis-mediating mechanism in kidneys after excessive exercise.     

Regulation of angiotensinogen by angiotensin II in mouse primary astrocyte cultures

Astrocytes are the major source of angiotensinogen in the brain and play an important role in the brain renin-angiotensin system. Regulating brain angiotensinogen production alters blood pressure and fluid and electrolyte homeostasis. In turn, several physiological and pathological manipulations alter expression of angiotensinogen in brain. Surprisingly, little is known about the factors that regulate astrocytic expression of angiotensinogen. There is evidence that angiotensinogen production in both hepatocytes and cardiac myocytes can be positively regulated via the angiotensin type 1 receptor, but this effect has not yet been studied in astrocytes. Therefore, the aim of this project was to establish whether angiotensin II modulates angiotensinogen production in brain astrocytes. Primary astrocyte cultures, prepared from neonatal C57Bl6 mice, expressed angiotensinogen measured by immunocytochemistry and real-time PCR. Using a variety of approaches we were unable to identify angiotensin receptors on cultured astrocytes. Exposure of cultured astrocytes to angiotensin II also did not affect angiotensinogen expression. When astrocyte cultures were transduced with the angiotensin type 1A receptor, using adenoviral vectors, angiotensin II induced a robust down-regulation (91.4% ± 1.8%, p < 0.01, n = 4) of angiotensinogen gene expression. We conclude that receptors for angiotensin II are present in extremely low levels in astrocytes, and that this concurs with available data in vivo. The signaling pathways activated by the angiotensin type 1A receptor are negatively coupled to angiotensinogen expression and represent a powerful pathway for decreasing expression of this protein, potentially via signaling pathways coupled to Gα(q/11) .     

Cytoskeletal remodeling in vascular smooth muscle cells in response to angiotensin II-induced activation of the SHP-2 tyrosine phosphatase

Angiotensin II is an octapeptide that regulates diverse cellular responses including the actin cytoskeletal organization. In this study, stable cell lines overexpressing wild-type or catalytically inactive SHP-2 were employed to elucidate the signaling pathway utilized by the SHP-2 tyrosine phosphatase that mediates an angiotensin II-induced reorganization of the actin cytoskeleton in vascular smooth muscle cells (VSMC). The expression of wild-type SHP-2 prevented an angiotensin II dependent increase in stress fiber formation. In contrast, the catalytically inactive mutant SHP-2 increased stress fiber formation. Additional observations further established that SHP-2 regulates the reorganization of the actin cytoskeleton through RhoA- and Vav2-dependent signaling pathways. The expression of wild-type SHP-2 caused a dephosphorylation of several focal adhesion associated proteins including paxillin, p130Cas, and tensin in VSMC. This dephosphorylation of focal adhesion associated proteins was accompanied by significantly decreased numbers of focal adhesions within cells. These results demonstrate a unique role for SHP-2 in the regulation of the cellular architecture of VSMC, suggesting the possibility that this phosphatase might be instrumental in vascular remodeling.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Expression and function of osteopontin in vascular adventitial fibroblasts and pathological vascular remodeling

Osteopontin is known to play important roles in various diseases including vascular disorders. However, little is known about its expression and function in vascular adventitial fibroblasts. Adventitial fibroblasts have been shown to play a key role in pathological vascular remodeling associating with various vascular disorders. In this study, we measured activation of Osteopontin and its biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results showed that angiotensin II and aldosterone increased Osteopontin expression in adventitial fibroblasts in a time- and concentration-dependent manner. MAPKs and AP-1 pathways were involved in Osteopontin upregulation. In addition, Adventitial fibroblast migration stimulated by Angiotensin II and aldosterone required OPN expression. Perivascular delivery of antisense oligonucleotide for Osteopontin suppressed neointimal formation post-injury. We concluded that upregulation of Osteopontin expression in adventitial fibroblasts might be important in the pathogenesis of vascular remodeling after arterial injury.     

肾素 - 血管紧张素 - 醛固酮系统阻滞的研究进展

肾素-血管紧张素-醛固酮系统起初被认为是较简单的神经体液调节机制之一。但是,这一想法随着RAAS阻滞剂:肾素阻滞剂、血管紧张素转换酶抑制剂(ACEI)、AT1受体拮抗剂及盐皮质激素受体拮抗剂的深入研究而受到挑战。因此,RAAS的组成、以上药物发挥作用的具体通路及副作用均得到重新定义。在RAAS阻滞剂的应用过程中,机体肾素水平升高,并刺激肾素原受体(即无活性的肾素前体,PRR),进而对机体造成不良影响。同理,在AT1受体拮抗剂的应用过程中,血浆血管紧张素II的水平升高,并与2型血管紧张素II(AT2)受体结合,进而对机体产生有利作用。此外,随着ACEI及ARB的应用,血管紧张素1-7水平升高,其与Mas受体结合,发挥心脏及肾脏保护的作用,还可通过刺激干细胞发挥组织修复作用。     

Wnt/β-catenin regulates blood pressure and kidney injury in rats

Activation of the renin-angiotensin system (RAS) plays a pivotal role in mediating hypertension, chronic kidney and cardiovascular diseases. As Wnt/β-catenin regulates multiple RAS genes, we speculated that this developmental signaling pathway might also participate in blood pressure (BP) regulation. To test this, we utilized two rat models of experimental hypertension: chronic angiotensin II infusion and remnant kidney after 5/6 nephrectomy. Inhibition of Wnt/β-catenin by ICG-001 blunted angiotensin II-induced hypertension. Interestingly, angiotensin II was able to induce the expression of multiple Wnt genes in vivo and in vitro, thereby creating a vicious cycle between Wnt/β-catenin and RAS activation. In the remnant kidney model, renal β-catenin was upregulated, and delayed administration of ICG-001 also blunted BP elevation and abolished the induction of angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II type 1 receptor. ICG-001 also reduced albuminuria, serum creatinine and blood urea nitrogen, and inhibited renal expression of fibronectin, collagen I and plasminogen activator inhibitor-1, and suppressed the infiltration of CD3⁺ T cells and CD68⁺ monocytes/macrophages. In vitro, incubation with losartan prevented Wnt/β-catenin-mediated fibronectin, α-smooth muscle actin and Snail1 expression, suggesting that the fibrogenic action of Wnt/β-catenin is dependent on RAS activation. Taken together, these results suggest an intrinsic linkage of Wnt/β-catenin signaling with BP regulation. Our studies also demonstrate that hyperactive Wnt/β-catenin can drive hypertension and kidney damage via RAS activation.     

Regulation of the expression of the rat angiotensin II receptor mRNA.

Regulation of the expression levels of the rat angiotensin II receptor mRNA in the adrenal, aorta, kidney, and brain was assessed by the competitive polymerase chain reaction method. The bilateral nephrectomy or the administration of Dup753 markedly reduced the expression levels of this receptor mRNA in the adrenal and brain stem, but not in the kidney nor aorta. A continuous infusion of angiotensin II increased the expression level of this receptor mRNA in the adrenal but not in the other tissues. It is suggested that the expression level of this receptor mRNA in the adrenal is dependent on the renin angiotensin aldosterone system.     

血管紧张素Ⅱ对培养心肌细胞c-fos表达和蛋白质合成的作用

本实验在培养新生大鼠心肌细胞上,探讨血管紧张素Ⅱ对心肌细胞ｃ－ｆｏｓ ｍＲＮＡ表达和蛋白质合成的影响。结果显示,血管紧张素Ⅱ能诱导ｃ－ｆｏｓ ｍＲＮＡ的表达,增加蛋白质含量,并呈量－效关系,还能加速＾３Ｈ－亮氨酸的掺入速度。上述这些作用可血管紧张素Ⅱ受体拮抗ｓａｒａｌａｓｉｎ所阻断,提示这些作用是受体介导的。     

Growth stimulatory angiotensin II type-1 receptor is upregulated in breast hyperplasia and in situ carcinoma but not in invasive carcinoma

Two different receptors which bind angiotensin II specifically have been identified in humans and were designated angiotensin II type-1 receptor (AT1) and angiotensin II type-2 receptor (AT2). They only have 34% sequence homology and act through different signalling pathways. AT1 stimulation has been implicated in hypertrophy and hyperplasia in various tissues. In order to study the involvement of AT1 in tissues from controls (n=10) and patients with hyperplasia (n=33), ductal carcinoma in situ (DCIS) (n=23) and invasive carcinoma of the breast (n=25), we tested biopsies and breast-derived cell lines using immunocytochemistry, in situ hybridisation and cell proliferation techniques. The results show specific overexpression of AT1 receptor on the cytoplasmic membrane of cells of hyperplastic lesions with and without atypia and on DCIS of the breast. Evidence for growth stimulation is provided by in vitro experiments showing growth induction by angiotensin II of T47D cells which express the AT1 but not the AT2 receptor. The expression of AT1 on the cell membrane disappears in invasive breast cancer cells suggesting a regulatory pathway which is no longer needed in invasive carcinoma. The specific AT1 expression upregulation might well be an important step in the pathogenesis of hyperplasia of the breast, which is regarded as a precursor lesion for breast cancer.     

Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells

The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.     

Monocyte chemoattractant protein-1 mediates angiotensin II-induced vascular smooth muscle cell proliferation via SAPK/JNK and ERK1/2

Abnormal vascular smooth muscle cells proliferation is the pathophysiological basis of cardiovascular diseases, such as hypertension, atherosclerosis, and restenosis after angioplasty. Angiotensin II can induce abnormal proliferation of vascular smooth muscle cells, but the molecular mechanisms of this process remain unclear. Here, we explored the role and molecular mechanism of monocyte chemotactic protein-1, which mediated angiotensin II-induced proliferation of rat aortic smooth muscle cells. 1,000 nM angiotensin II could stimulate rat aortic smooth muscle cells' proliferation by angiotensin II type 1 receptor (AT(1)R). Simultaneously, angiotensin II increased monocyte chemotactic protein-1 expression and secretion in a dose-and time-dependent manner through activation of its receptor AT(1)R. Then, monocyte chemotactic protein-1 contributed to angiotensin II-induced cells proliferation by CCR2. Furthermore, we found that intracellular ERK and JNK signaling molecules were implicated in angiotensin II-stimulated monocyte chemotactic protein-1 expression and proliferation mediated by monocyte chemotactic protein-1. These results contribute to a better understanding effect on angiotensin II-induced proliferation of rat smooth muscle cells.     

Effect in vivo of beta-adrenergic stimulation, angiotensin II, dibutyryl cyclic AMP, and theophylline on tonin concentration in rat saliva and submaxillary gland.

Tonin (an enzyme present in rat submaxillary gland and saliva) has previously been shown to be able, unlike renin and reninlike substances, to release angiotensin II either directly by acting on an appropriate substrate or from angiotensin I. The administration of a beta-adrenergic drug, isoproterenol, produces a rise of tonin concentration in saliva without affecting its concentration in the submaxillary gland. Prior administration of a beta blocker, propranolol, partially prevents this effect. The administration of theophylline increases the tonin concentration in both saliva and the submaxillary gland, whereas dibutyryl cyclic AMP increases tonin concentration in the former. These results suggest that beta-adrenergic stimulation enhances both tonin release into the saliva and tonin synthesis in the submaxillary gland, and that these effects might be mediated by cyclic AMP. Infusion of angiotensin II blocked the stimulatory effect of isoproterenol on salivary tonin. 1Sar-8Ile-angiotensin II is both a weak antagonist of angiotensin II in this respect and a strong agonist in terms of blocking the effect of isoproterenol another role mirrored in other physiological mechanisms of derivatives of angiotensin II.     

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 microM and was inhibited by the specific angiotensin II receptor antagonist, [Sar1, Ile8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c-fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c-fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vasoconstrictor.     

Angiotensin II stimulates nitric oxide production in pulmonary artery endothelium via the type 2 receptor

We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone.