David E. Dostal

The Cardiac Renin-Angiotensin System: Conceptual, or a Regulator of Cardiac Function?

Angiotensin II, the effector peptide of the renin-angiotensin system, regulates cellular growth in response to developmental, physiological, and pathological processes. The identification of renin-angiotensin system components and angiotensin II receptors in cardiac tissue suggests the existence of an autocrine/paracrine system that has effects independent of angiotensin II derived from the circulatory system. To be functional, a local renin-angiotensin system should produce sufficient amounts of the autocrine and/or paracrine factor to elicit biological responses, contain the final effector (angiotensin II receptor), and respond to humoral, neural, and/or mechanical stimuli. In this review, we discuss evidence for a functional cardiac renin-angiotensin system.

Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts.

Angiotensin II has been reported to be a hormonal stimulus of cardiac growth, a response that may involve myocyte hypertrophy as well as growth of nonmyocytes. This study was designed to determine whether neonatal rat cardiac fibroblasts have an angiotensin II receptor that is coupled with hypertrophic and/or proliferative growth. Competitive radioligand binding studies showed that cardiac fibroblasts have a single class of high-affinity (IC50, 1.0 nM) angiotensin II binding sites (Bmax, 778 fmol/mg protein) that are sensitive to the competitive nonpeptide AT1 receptor antagonist losartan (IC50, 13 nM). Other angiotensin peptides competed for [125I]angiotensin II binding in the following rank order: angiotensin II > angiotensin III > angiotensin I > > [des-Asp1-des-Arg2]angiotensin II. A nonhydrolyzable analogue of guanosine triphosphate increased the dissociation rate of bound [125I]angiotensin II and decreased hormone binding to the receptor at equilibrium. The angiotensin II receptor was coupled with increases in intracellular calcium. Incorporation of precursors into protein, DNA, and RNA in response to angiotensin II was determined. In serum-deprived cultures, a 24-hour exposure to 1 microM [Sar1]angiotensin II increased rates of phenylalanine, thymidine, and uridine incorporation by 58%, 103%, and 118%, respectively. These increases were blocked by the noncompetitive AT1 receptor antagonist EXP3174. After 48 hours, [Sar1]angiotensin II increased total protein and DNA of cardiac fibroblasts by 23% and 15%, respectively, with no change in the protein/DNA ratio. [Sar1]Angiotensin II increased cell number by 138% after a 24-hour exposure, without affecting cell area. In summary, cardiac fibroblasts have G protein-linked AT1 receptors that are coupled with proliferative growth. These results suggest that angiotensin II-induced cardiac hypertrophy is, in part, secondary to stimulated increases in nonmyocyte cellular growth.

Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy

Angiotensin II (Ang II) has a significant role in regulating cardiac homeostasis through humoral, autocrine and paracrine pathways, via binding to the plasma membrane AT1 receptor. Recent literature has provided evidence for intracrine growth effects of Ang II in some cell lines, which does not involve interaction with the plasma membrane receptor. We hypothesized that such intracrine mechanisms are operative in the heart and likely participate in the cardiac hypertrophy induced by Ang II. Adenoviral and plasmid vectors were constructed to express Ang II peptide intracellularly. Neonatal rat ventricular myocytes (NRVMs) infected with the adenoviral vector showed significant hypertrophic growth as determined by cell size, protein synthesis and enhanced cytoskeletal arrangement. Adult mice injected with the plasmid vector developed significant cardiac hypertrophy after 48 h, without an increase in blood pressure or plasma Ang II levels. This was accompanied by increased transcription of transforming growth factor-beta (TGF-beta) and insulin-like growth factor-1 (IGF-1) genes. Losartan did not block the growth effects, excluding the involvement of extracellular Ang II and the plasma membrane AT1 receptor. These data demonstrate a previously unknown growth mechanism of Ang II in the heart, which should be considered when designing therapeutic strategies to block Ang II actions.

Canine Ventricular Myocytes Possess a Renin-Angiotensin System That Is Upregulated With Heart Failure

Abstract— Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT1 receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II–mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT1 and AT2 receptors was evaluated by reverse transcriptase–polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT1 receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT1. Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT1 were markedly increased in paced myocytes. Conversely, chymase and AT2 proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.

Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

The cardiac renin–angiotensin system: novel signaling mechanisms related to cardiac growth and function

Angiotensin II, the effector peptide of the renin-angiotensin system, has been demonstrated to be involved in the regulation of cellular growth of several tissues in response to developmental, physiological, and pathological processes. The recent identification of renin-angiotensin system components and localization of angiotensin II receptors in cardiac tissue suggests that locally synthesized Ang II can modulate functional and growth responses in cardiac tissue. In this review, regulation of the cardiac RAS is discussed, with an emphasis on growth-related Ang II signal transduction systems.

Cardiotrophin-1 Increases Angiotensinogen mRNA in Rat Cardiac Myocytes Through STAT3: An Autocrine Loop for Hypertrophy

-Cardiotrophin-1, an interleukin-6-related cytokine, stimulates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and induces cardiac myocyte hypertrophy. In this study, we demonstrate that cardiotrophin-1 induces cardiac myocyte hypertrophy in part by upregulation of a local renin-angiotensin system through the JAK/STAT pathway. We found that cardiotrophin-1 increased angiotensinogen mRNA expression in cardiac myocytes via STAT3 activation. Tyrosine phosphorylation of STAT3 by cardiotrophin-1 treatment resulted in STAT3 homodimer binding to the St-domain in the angiotensinogen gene promoter, which lead to promoter activation in a transient transfection assay. Cardiotrophin-1-induced STAT3 tyrosine phosphorylation and binding to the St-domain were suppressed by AG490, a specific JAK2 inhibitor, which also attenuated cardiotrophin-1-stimulated angiotensinogen promoter activity. Cardiotrophin-1 did not activate the angiotensinogen gene promoter that contained a substitution mutation within the St-domain. Finally, losartan, an angiotensin II type 1 receptor antagonist, significantly attenuated cardiotrophin-1-induced hypertrophy of neonatal rat cardiac myocytes. Angiotensin II is known to induce cardiac myocyte hypertrophy by activating the G-protein-coupled angiotensin II type 1 receptor. Our results suggest that upregulation of angiotensinogen and angiotensin II production contribute to cardiotrophin-1-induced cardiac myocyte hypertrophy and emphasize an important interaction between G-protein-coupled and cytokine receptors.

Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2–4 μm2, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl−/HCO3− AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl−/HCO3− exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Angiotensin II is a potent stimulator of MAP-kinase activity in neonatal rat cardiac fibroblasts

We have previously shown that angiotensin II (AII) is a mitogen for neonatal rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to AII remain to be elucidated. Mitogen-activated protein (MAP) kinases are cytosolic serine/threonine kinases which have been shown to be activated in quiescent cells by diverse growth stimuli, thereby being linked to growth regulatory pathways. This study was designed to determine whether MAP-kinase activation occurred in response to AII/receptor coupling in neonatal rat cardiac fibroblasts and the role of MAP-kinase activation in the AII-induced proliferation of these cells. Immunoblot analysis of MAP-kinase isoforms revealed predominantly p44 with less p42 MAP-kinase in rat cardiac fibroblasts. Both isoforms were activated upon stimulation of the cells with AII for 5 min or platelet derived growth factor-BB for 10 min. Angiotensin II stimulated MAP-kinase in a dose-dependent fashion with an EC50 of 2.5 nM. Two minutes following stimulation with 1 microM AII MAP-kinase activity increased from 90 +/- 17.9 to 477.5 +/- 75.9 pmol/min/mg protein, P < 0.05, n = 4. A smaller, sustained, secondary increase in MAP-kinase activity from 37.7 +/- 5.3 to 110.9 +/- 15.3 pmol/min/mg protein, P < 0.05, n = 4, was observed in response to AII between 120-150 minutes following receptor occupancy. The responses to AII were markedly attenuated by the AT1 receptor antagonist EXP3174. Stimulation of the cells with carbachol induced the first but not the second phase of MAP-kinase activity and this compound had no effect on cellular growth. The second phase of MAP-kinase activity 2-2.5 h after AII stimulation, paralleled data demonstrating that a 2-3 h receptor occupancy with AII was necessary to induce DNA synthesis and fibroblast proliferation. These results indicate that AII stimulates a biphasic activation of MAP-kinase by the AT1 receptor and that this pathway may participate in the AII induced mitogenic response in cardiac fibroblasts.

Regulation of Cardiac Collagen Angiotensin and Cross-Talk With Local Growth Factors

In the myocardium, collagen fibers provide a supporting framework for myocytes and blood vessels and act as lateral connections between muscle bundles. These functional properties of collagen serve to maintain tissue architecture and to coordinate the delivery of force generated by myocytes on the ventricular chamber. The accumulation of excess collagen is believed to be an important pathophysiological process that contributes to diastolic heart failure. Diastolic heart failure accounts for 30% to 50% of heart failure in clinical practice, and hypertensive disease is the major cause of this type of heart failure.1 The precise mechanisms responsible for excess fibrillar collagen accumulation in the pathological heart are poorly understood. Fibrosis of both the injured and noninjured myocardium2 indicates that humoral mechanisms are responsible for this process. In the failing heart, several humoral, autocrine, and paracrine systems are activated,3 suggesting that cross-talk between synergistic and opposing signaling pathways constitutes the predominant form of regulation under these conditions. Several factors have been identified as potentially important mediators of cardiac collagen production. In vitro studies of neonatal and adult rat cardiac fibroblasts have shown that angiotensin II (Ang II) directly stimulates cardiac fibroblast proliferation and collagen synthesis via Ang II type 1 (AT1) receptors.4 5 6 In this issue of Hypertension , Pathak et al7 provided evidence that a myocyte cofactor was an important mediator of Ang II–induced collagen type I and type III mRNA synthesis in a rat cell coculture model. This work, together with other studies, provides strong evidence that Ang II indirectly regulates cardiac fibroblast function via specific growth factors.8 9 10 11 12 13 14 15 16 17 18 19 20 21 Although the primary autocrine and paracrine mediators of Ang II effects on fibrillar collagen synthesis remain to be elucidated, principal candidates …