Angiotensins II and IV modulate adrenocortical cell proliferation in ovariectomized rats.

Effects of angiotensins II (AngII), angiotensin IV (AngIV, 3-8 fragment of angiotensin II) and losartan (an antagonist of angiotensin receptor type 1) on the proliferation of adrenocortical cells in ovariectomized rats have been studied. The incorporation of bromodeoxyuridine (BrdU) into cell nuclei was used as an index of cell proliferation. AngIV decreased BrdU labeling index mainly in the reticularis zone and losartan (Los) was able to partially reverse this inhibitory effect of AngIV. AngII had no effect on the adrenocortical cell proliferation when given alone, however Los given simultaneously diminished BrdU incorporation into nuclei of glomerulosa and reticularis zones as compared with AngII. These findings suggest that AngII and AngIV modulate adrenocortical cell proliferation in ovariectomized rats.     

Comparative effects of angiotensin IV and two hemorphins on angiotensin-converting enzyme activity

The role of angiotensin IV (AngIV) in the regulation of angiotensin-converting enzyme (ACE) was studied in vitro. This study demonstrates that this active fragment appeared as a novel endogenous ACE inhibitor. Inhibitory kinetic studies revealed that AngIV acts as a purely competitive inhibitor with a K(i) value of 35 microM. AngIV was found to be quite resistant to ACE hydrolysis opposite to hemorphins which are both ACE inhibitors and substrates. In order to confirm a putative role of AngIV and hemorphins in the Renin-Angiotensin system (RAS) regulation, we studied their influence on AngI conversion. We noticed that 16.7 microM of both peptides decreased more than 50% of AngI conversion to AngII in vitro. The capacity of hemorphins, particularly LVVH-7, and AngIV to inhibit ACE activity here suggests a synergistic relation between these two peptides and the regulation of RAS.     

Angiotensin II protects against alpha-synuclein toxicity and reduces protein aggregation in vitro

In this study, we examined the effects of angiotensin II (AngII) in a genetic in vitro PD model produced by alpha-synuclein (alpha-syn) overexpression in the human neuroglioma H4 cell line. We observed a maximal decrease in alpha-syn-induced toxicity of 85% and reduction in inclusion formation by 19% when cultures were treated with AngII in the presence of the angiotensin type 1 (AT1) receptor antagonist losartan and AT2 receptor antagonist PD123319. When compared to AngII, the AT4 receptor agonist AngIV was moderately effective in protecting H4 cells against alpha-syn toxicity and did not significantly reduce inclusion formation. Here we show that AngII is protective against genetic, as well as neurotoxic models of PD. These data support the view that agents acting on the renin-angiotensin-system (RAS) may be useful in the prevention and/or treatment of Parkinson's disease.     

A role for the angiotensin IV/AT4 system in mediating natriuresis in the rat

Angiotensin II (AngII) or Angiotensin IV (AngIV) was infused into the renal artery of anesthetized rats while renal cortical blood flow was measured via laser Doppler flowmetry. The infusion of AngII produced a significant elevation in mean arterial pressure (MAP) with an accompanying decrease in cortical blood flow, glomerular filtration rate (GFR), urine volume, and urine sodium excretion. The infusion of AngIV induced significant increases in renal cortical blood flow and urine sodium excretion, without altering MAP, GFR, and urine volume. Pretreatment infusion with a specific AT1 receptor antagonist, DuP 753, blocked or attenuated the subsequent AngII effects, while pretreatment infusion with the specific AT4 receptor antagonist, Divalinal-AngIV, blocked the AngIV effects. These results support distinct and opposite roles for AngII and AngIV, i.e. AngII acts as an anti-natriuretic agent, while AngIV acts as a natriuretic agent.     

ACE inhibition lowers angiotensin II-induced chemokine expression by reduction of NF-kappaB activity and AT1 receptor expression

OBJECTIVE: Angiotensin converting enzyme (ACE) inhibitors significantly improve survival in patients with atherosclerosis. Although ACE inhibitors reduce local angiotensin II (AngII) formation, serine proteases form AngII to an enormous amount independently from ACE. Therefore, our study concentrates on the effect of the ACE-inhibitor ramiprilat on chemokine release, AngII receptor (ATR) expression, and NF-kappaB activity in monocytes stimulated with AngII. METHODS AND RESULTS: AngII-induced upregulation of IL-8 and MCP-1 protein and RNA in monocytes was inhibited by the AT1R-blocker losartan, but not by the AT2R-blocker PD 123.319. Ramiprilat dose-dependently suppressed AngII-induced upregulation of IL-8 and MCP-1. The suppressive effect of ramiprilat on AngII-induced chemokine production and release was in part caused by downregulation of NF-kappaB, but more by a selective and highly significant reduced expression of AT1 receptors as shown in monocytes and endothelial cells. CONCLUSION: In our study we demonstrated for the first time that ramiprilat reduced expression of AT1R in monocytes and endothelial cells. In addition, ramiprilat downregulated NF-kappaB activity and thereby reduced the AngII-induced release of IL-8 and MCP-1 in monocytes. This antiinflammatory effect, at least in part, may contribute to the clinical benefit of the ACE inhibitor in the treatment of coronary artery disease.     

A Specific Binding Site for Angiotensin II(3-8), Angiotensin IV,in Rabbit Cardiac Fibroblasts

Abstract

This study demonstrates the existence of a high affinity binding site on rabbit cardiac fibroblasts of the hexapeptide (3-8) fragment of angiotensin II (AngIV). [¹²⁵I]-AngIV binding is saturable, reversible and distinct from angiotensin II (AngII) receptors. At 37°C equilibrium of [¹²⁵I]-AngIV binding is reached within 2 h. AngIV displaces [¹²⁵I]-AngIV bound to cultured rabbit cardiac fibroblasts whereas AngII receptor-specific ligands ([Sar¹,IIe⁸]-AngII, Dup753, CGP42112A) do not. Scatchard plot analysis revealed that [¹²⁵I]-AngIV binds to a single class of sites with K_d = 4.87 ± 0.11 × 10^?9 mol/l, B_max = 371 ± 8.3 fmol/mg protein and a Hill coefficient of 0.92. In the presence of the non-hydrolyzable GTP analog GTP_γS [¹²⁵I]-AngIV binding in rabbit cardiac fibroblasts was not markedly affected, whereas binding of [¹²⁵I]-(Sar¹,IIe⁸)-AngII is reduced. The role of AngIV in the heart and in particular in cardiac fibroblasts is unknown, and the putative interaction of AngIV with AngII needs further characterization.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

Gender differences in steroid modulation of angiotensin II-induced protein kinase C activity in anterior pituitary of the rat

To investigate whether the various steroid hormones can modulate the basal and angiotensin II-induced protein kinase C (PKC) activity in the anterior pituitary of the rat, female and male intact and ovariectomized female Wistar rats were treated in vivo with estradiol (E2), progesterone (P), dehydroepiandrostendione sulfate (DHEA-S), and pregnenolone sulfate (PREG-S). Estradiol caused the increase of basal PKC activity in intact and ovariectomized females, but did not change the enzyme activity in males. In ovariectomized animals the increase of PKC activity was lower than in intact females. Progesterone decreased PKC activity only in intact animals. DHEA-S strongly enhanced activity of PKC in ovariectomized females. Pregnenolone sulfate did not significantly change PKC function of all studied groups. Incubation with AngII enhanced the PKC activity in intact (without steroid treatment) animals of both genders. In females, AngII and estradiol together rise the PKC-stimulated phosphorylation in greater degree than used separately. Treatment with other investigated steroids reduced the effect of AngII. In intact males every examined hormone turned back the stimulatory effect of AngII on PKC activity. These data suggest that gender differences in PKC activity are likely related to hormonal milieu of experimental animals and may depend in part on the basic plasma level of estrogens.     

Ethanol-induced suppression of LTP can be attenuated with an angiotensin IV analog

Hippocampal slices taken from animals chronically or acutely treated with ethanol exhibit significant inhibition of long-term potentiation (LTP). This inhibition appears to be associated with impaired activity of N-methyl-D-aspartate (NMDA) receptors, perhaps via ethanol-induced increases in GABAergic synaptic transmission. Recently, a role for the octapeptide angiotensin II (AngII) in ethanol's inhibition of LTP has been reported. Complementary to these findings our laboratory has shown that the application of the hexapeptide metabolite of AngII, angiotensin IV (AngIV), significantly facilitated normal tetanic-induced LTP in the hippocampal slice. This facilitation is presumably by activation of the angiotensin receptor subtype, AT(4). The present study tested whether an AT(4) receptor agonist could overcome ethanol-induced suppression of LTP. The results indicate that Nle(1)-AngIV could offset ethanol-induced suppression of LTP in the CA(1) region of the hippocampus. Pretreatment with the specific AT(4) receptor antagonist Nle(1), Leual(3)-AngIV blocked this facilitation implicating the involvement of the AT(4) receptor subtype. These results suggest that an AT(4) receptor agonist is effective in overcoming ethanol's suppressing influence on LTP, and encourage further investigation of the cognitive enhancing properties of such compounds.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

Degradation of alpha and beta neo-endorphin by rat brain membrane peptidases

Fractionation of Triton-solubilized rat brain membranes on diethylaminoethyl-cellulose resolves two peptidases which hydrolyze beta-neo-endorphin. One of these peptidases was identified as Angiotensin Converting Enzyme by (a) its sensitivity to inhibition by the specific inhibitors MK422 and captopril, (b) by the identification of reaction products, and (c) by comparison to authentic angiotensin converting enzyme. In contrast, alpha-neo-endorphin hydrolysis by angiotensin converting enzyme could not be detected. The second enzyme active on beta-neo-endorphin was identified as an aminopeptidase. This aminopeptidase is identical to the previously described enkephalin-degrading aminopeptidase. The possible involvement of these enzymes in the metabolism of opioid peptides is discussed.     

Engineering and characterization of a divalent single-chain Fv angiotensin II fusion construct of the monoclonal antibody CC49

For the therapy of solid tumors, co-administration of angiotensin II (AngII) results in an increased uptake of drugs into the tumor interstitium. We have engineered a dimeric sc(Fv)(2)-AngII fusion construct that combines the superior kinetics of covalent dimeric scFvs [sc(Fv)(2)], recognizing the pancarcinoma tumor-associated antigen 72 (TAG-72), with the advantageous intrinsic activity of AngII. The binding characteristics of the fusion construct were unaltered by the addition of the AngII sequence [affinity constant K(A) 1.18 x 10(7) and 8.42 x 10(6) M(-1) for sc(Fv)(2) and sc(Fv)(2)-AngII, respectively]. The binding of the fusion construct to the angiotensin receptor (AT(1)) was similar to AngII, and the arterial contraction was 16 +/- 1% of the response observed with norepinephrine. In animal studies, the radiolabeled sc(Fv)(2)-AngII construct exhibited similar uptake and a more homogeneous distribution within the tumor as compared to sc(Fv)(2).     

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.     

Lack of angiotensin II conversion to angiotensin III increases water but not alcohol consumption in aminopeptidase A-deficient mice

Elevated central concentrations of the vasopressor octapeptide angiotensin (Ang) II increase the water intake in mammals. Recently, we showed that central AngII is also crucial in alcohol-consuming behavior. Since the heptapeptide AngIII, an AngII metabolite, is discussed to mediate AngII-related effects, we investigated water and alcohol consumption in mice, genetically deficient in aminopeptidase A (APA), a peptidase responsible for AngII conversion to AngIII. Sixteen male APA-deficient mice and their age matched wild-type controls were monitored on their water intake under basal conditions and total fluid and alcohol intake before and after social stress in a two-bottle free-choice paradigm. Alterations were connected to the regulation in activity of Ang-related peptidases (APA, ACE; ACE2) in brain regions involved in alcohol intake and peripheral organs. In comparison to their wild-type controls, APA-deficient mice drank significantly more water but not more alcohol at all investigated time points. A reduction in water intake, as observed in wild-type animals after social stress, did not occur in knockout mice. However, the reduction in alcohol consumption after social stress was significantly reduced in both strains. Alcohol consumption upregulated all three peptidases in the kidney, but not in lung. Notable, renal ACE2 activity was significantly higher in APA-deficient mice under basal condition. While the inhibition of AngII metabolism to AngIII does not influence the alcohol intake, water consumption in mice deficient for APA was significantly elevated. These differences induced by an altered AngII/AngIII ratio oppose the hypothesis that central AngII and AngIII act in a congruent pattern.     

Demonstration of angiotensin II-induced Ras activation in the trans-Golgi network and endoplasmic reticulum using bioluminescence resonance energy transfer-based biosensors

Previous studies have demonstrated that molecules of the Ras signaling pathway are present in intracellular compartments, including early endosomes, the endoplasmic reticulum (ER), and the Golgi, and suggested that mitogens can regulate Ras activity in these endomembranes. In this study, we investigated the effect of angiotensin II (AngII) on intracellular Ras activity in living HEK293 cells expressing angiotensin type 1 receptors (AT(1)-Rs) using newly developed bioluminescence resonance energy transfer biosensors. To investigate the subcellular localization of AngII-induced Ras activation, we targeted our probes to various intracellular compartments, such as the trans-Golgi network (TGN), the ER, and early endosomes. Using these biosensors, we detected AngII-induced Ras activation in the TGN and ER, but not in early endosomes. In cells expressing a cytoplasmic tail deletion AT(1)-R mutant, the AngII-induced response was enhanced, suggesting that receptor internalization and β-arrestin binding are not required for AngII-induced Ras activation in endomembranes. Although we were able to demonstrate EGF-induced Ras activation in the plasma membrane and TGN, but not in other endomembranes, AG1478, an EGF receptor inhibitor, did not affect the AngII-induced response, suggesting that the latter is independent of EGF receptor transactivation. AngII was unable to stimulate Ras activity in the studied compartments in cells expressing a G protein coupling-deficient AT(1)-R mutant ((125)DRY(127) to (125)AAY(127)). These data suggest that AngII can stimulate Ras activity in the TGN and ER with a G protein-dependent mechanism, which does not require β-arrestin-mediated signaling, receptor internalization, and EGF receptor transactivation.     

Cerebroprotective effect of angiotensin IV in experimental ischemic stroke in the rat mediated by AT(4) receptors.

Recent studies have reported potential roles of angiotensins in an adaptative physiological mechanism of protection against cerebral ischemia-induced neurological damages. In the present study, we examined the protective role of angiotensin IV (AngIV) in a rat model of embolic stroke induced by intracarotid injection of calibrated microspheres (50 microm). Internal carotid infusions of increasing doses of AngIV (0.01, 0.1 and 1 nmol/0.1 mL saline) dose dependently decreased mortality, neurological deficit and cerebral infarct size at 24 hours. With the highest dose of AngIV, mortality was reduced from 55 % in saline infused controls to 10 % (p=0.003), neurological deficit was reduced from 3.8 +/- 0.3 to 1.4 +/- 0.3 , (p<0.0001) and cerebral infarct size at 24 hours was decreased from 432 +/- 26 mm(3) to 185 +/- 19, (p=0.0001). The AT(4) antagonist divalinal-AngIV (10(-9) mol/0.1 mL), or pretreatment with L-NAME (10(-7) mol/0.1 mL), both completely abolished the protective effect of AngIV (1 nmol). The AT(2) antagonist PD123319 (10(-7) mol/0.1 mL) partially prevented the protective effect of AngIV on the neurological score. Sequential cerebral arteriographies revealed that AngIV induced a redistribution of blood flow to the ischemic areas within minutes. These results suggest that pharmacological doses of AngIV are protective against acute cerebral ischemia by triggering an AT(4)-mediated, NO-dependent intracerebral hemodynamic mechanism.     

Role of protein kinase C-δ in angiotensin II induced cardiac fibrosis

Previous studies have demonstrated a role for angiotensin II (AngII) and myofibroblasts (myoFb) in cardiac fibrosis. However, the role of PKC-δ in AngII mediated cardiac fibrosis is unclear. Therefore, the present study was designed to investigate the role of PKC-δ in AngII induced cardiac collagen expression and fibrosis. AngII treatment significantly (p < 0.05) increased myoFb collagen expression, whereas PKC-δ siRNA treatment or rottlerin, a PKC-δ inhibitor abrogated (p < 0.05) AngII induced collagen expression. MyoFb transfected with PKC-δ over expression vector showed significant increase (p < 0.05) in the collagen expression as compared to control. Two weeks of chronic AngII infused rats showed significant (p < 0.05) increase in collagen expression compared to sham operated rats. This increase in cardiac collagen expression was abrogated by rottlerin treatment. In conclusion, both in vitro and in vivo data strongly suggest a role for PKC-δ in AngII induced cardiac fibrosis.     

Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1–7). Co-expression of AngII receptors (AT₁ and AT₂) and Ang(1–7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1–7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1–7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1–7)-Mas antagonizes the antiadipogenic effect of AngII-AT₁ by inhibiting the AngII-AT₁-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT₁-ACE2-Ang(1–7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.     

Angiotensin II regulates tyrosine hydroxylase activity and mRNA expression in rat mediobasal hypothalamic cultures: the role of specific protein kinases

Dopamine secreted by hypothalamic neurons is crucial in regulating prolactin secretion from the pituitary. We have examined the ability of angiotensin II (AngII) to regulate the activity of these dopaminergic neurons and thus act as a potential physiological regulator of prolactin secretion. Using a hypothalamic cell culture preparation we determined the effect of AngII on tyrosine hydroxylase activity and expression (TOH). This is important because TOH is the rate-limiting enzyme in dopamine biosynthesis. AngII stimulated a time- and concentration-dependent increase in TOH activity which was suppressed by inhibitors able to act on protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMPKII). An inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD 98059, reduced basal TOH activity but the AngII response was still detectable. AngII stimulation enhanced the phosphorylation of TOH at Ser19, Ser31 and Ser40. AngII also induced a time-dependent increase in TOH mRNA expression which was unaffected by inhibitors able to act on PKA and CaMPKII, but was abolished by inhibitors able to act on ERK and PKC. AngII responses were very much larger in cultures prepared from female when compared to male rat pups. Data from adult hypothalamic slices confirmed this sexual dimorphism and supported the role of the protein kinases noted above. Therefore AngII can regulate both the activity and expression of TOH in hypothalamic neurons employing multiple, but only partially overlapping, signaling pathways.