Angiotensin-Converting Enzyme (ACE) in Gut Inflammation
Ferna do Magro
Gastroenterology Department and Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal
1. INFLAMMATORY BOWEL DISEASE AS A MODEL OF INFLAMMATION
Mucosal inflammation is almost always mediated by one of two pathways (Bouma et al 2003): T
H1- cell response or T
H2-cell response. The T
H1- cell response is associated with increased secretion of IL-12, IFN-γ and/or TNF-α and T
H2-cell response is associated with increased secretion of IL-4, IL-5 and/or IL-13 (Strober et al 2002). This dual type of response is shown in the TNBS and oxazolone colitis. In the former the TNBS induces an IL-12- mediated T
H1 – cell response characterized by transmural cellular infiltration associated with granulomas, in some cases, and can be abrogated with
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U. Lendeckel and Nigel M. Hooper (eds.), Proteases in Gastrointestinal Tissue, 301-314.
© 2006 Springer. Printed in the Netherlands
Inflammatory bowel disease (IBD) is a chronic relapsing disease and affects all parts of the gastrointestinal (GI) tract. The major clinical features of the disease include diarrhoea, abdominal pain, weight loss and bleeding.
The two main forms of idiopathic IBD, Crohn’s disease (DC) and ulcerative colitis (UC) can be distinguished by clinical, radiological, endoscopic and pathologic features. In CD the lesions extend throughout the GI and in UC are limited to colon. In UC the endoscopic lesions have a continuous involvement and in DC the wall is discontinuous affected, that is, in the same endoscopic area ulcers are surrounding by normal endoscopic mucosa. The inflammatory process in UC is confined to the inner lining of the gut (mucosa and submucosa) and is characterized by crypt abscesses and ulcerations.
Unlike UC, in DC the inflammatory process extends throughout the bowel
wall (transmural) and is characterized by the presence of granulomas and
fistulae.
antibodies specific for IL-12 (Neurath et al 1995). In the later the oxazolone induces inflammation characterized by superficial cellular infiltration associated with a greater infiltration of neutrophils (Boirivant et al 1998). This mucosal inflammation results from the induction of natural-killer T (NKT) cells producing IL-13 (Heller et al 2002). Also a large number of mice with different genetic defects develop spontaneous mucosal inflammation reinforcing the importance of inflammation in IBD. That is, colitis can be induced by different ways: due to defective induction of regulatory cells (CD4
+CD45RB
hi) (Powrie et al 1993), regulatory cell defects (IL-10-deficient mice, TGF- β- deficient mice) (Shull et al 1992; Kuhn et al 1993) or increased effector-cell responses (Stat4 transgenic mice, G-protein subunit α1,2-deficient mice) (Rudolph et al 1995; Wirtz et al 1999).
The data support the evidence that CD is a T
H1- mediated inflammation.
Various immunohistochemical studies indicate that in situ IL-12 is over- produced by macrophages (Monteleone et al 1997) and macrophages that are isolated from the inflammatory lesions of patients with CD produce increased amounts of IL-12 ex-vivo (Liu Z et al 1999). In addition, nuclear extracts of T cells from tissues of patients with CD contain increased amounts of activated STAT4, which is indicative of IL-12 signalling (Neurath et al 2002; Parrello et al 2000). Also T cells from CD patients produce markedly amounts of IFN- γ and markedly decreased of IL-4, T
H2 cytokine, compared with controls (Parronchi et al 1997; Fuss et al 1996). UC, T
H2- mediated inflammation, is associated with antibodies production (IgG1 and IgG4) and increased secretion of IL-5 (Fuss et al 1996; Kett et al 1987).
A rate-limiting step in the pathophysiology of IBD is the activation and recruitment of leukocytes. Tissue trafficking of leukocytes is orchestrated by a coordinated expression of chemokines and adhesion molecules. In fact, when the endothelial cells are activated they display surface markers such as P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and integrins. The adhesion of leukocytes to the endothelium is initiated by weak interactions that produce a characteristic rolling motion of the leukocytes on the endothelial surface (Ley et al 1995;
Lasky et al 1995). P-selectin, acting in cooperation with L-selectin, is implicated in the mediation of these initial interactions (True et al 1990).
The selectins family is composed of three distinct carbohydrate receptors
expressed by endothelial cells (E-selectin), leukocytes (L-selectin), or
platelets and endothelium (P-selectin). E-selectin initiates the so-called
rolling of leukocytes on the endothelial surface, with subsequent expression
of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion
molecule-1 (VCAM-1) on the endothelium (Springer 1990). ICAM-1 and
VCAM-1 appear to be particularly important for the firm attachment and
transendothelial migration of leukocytes. ICAM-1 promotes the initial
interaction between macrophages and T-cells during immune activation
(Nielsen et al 1994) and the enhanced expression of VCAM-1 may facilitate the transmigration of monocytes and CD4
+memory/effector cells, since these cells express VLA-4, the ligand for VCAM-1 (Horgam et al 1992).
Soluble isoforms of E-selectin, P-selectin, ICAM-1 and VCAM-1 have been found in the plasma of normal individuals (Patel et al 1995), probably arising from proteolytic cleavage of the surface-expressed molecule (Gearing et al 1993). E-selectin is substantially lost from the surface of endothelial cells within 24 hours, and circulating P-selectin appears to be slightly smaller than native P-selectin. An alternatively spliced mRNA encoding a form of human P-selectin, lacking the transmembrane anchoring domain, has been reported for both megakaryocytes and endothelial cells (Gearing et al 1993), and evidence suggests that the majority of circulating soluble P- selectin originate in this manner.
Patel R et al (Patel et al 1995) in 83 IBD patients showed that the plasma levels of sICAM and sE-S were significantly higher in those with active UC and CD than those in controls and inactive disease. Nielsen et al (Nielsen in patients in remission and controls. However, in 93 IBD patients, Goke M et al (Goke et al 1997) did not find any relation between disease activity and the increased serum levels of sE-S, sP-S, s-ICAM and s-VCAM.
We stratified 218 patients ((145 with Crohn’s disease (CD) and 73 with ulcerative colitis (UC)) into three categories of activity - clinical remission, biochemical evidence of inflammation (biologically active patients) and
activity. This categorization help to show that in inactive IBD patients the serum levels of selectins and immunoglobulin superfamily molecules were lower than those found in controls (Magro et al 2004). The low serum levels of adhesion molecules in periods of remission in IBD patients suggest continuous leukocytic activation. This theory is in accordance with chemi- luminescent response of neutrophils from IBD patients (Faden et al 1985).
Moreover, low serum levels of L-selectin were found mainly in patients with inflammatory vascular discords or connective tissue diseases associated with vasculitis due to counter receptor-bearing cells.
2. ANGIOTENSIN, ACE AND BRADYKININ AS AGENTS OF INFLAMMATION
Angiotensinogen is the circulating protein substrate from which renin cleaves angiotensin I. In humans, the concentration of angiotensinogen in the circulation is less than Km of the renin-angiotensinogen reaction and is et al 1994) also found higher levels of s-ICAM in IBD patients (n = 58) than
mation were considered because they represent a group with sub-clinical
clinical evidence of activity. Patients with biochemical evidence of inflam-
therefore an important determinant of the rate of formation of angiotensin.
Angiotensin I has little or no biologic activity. Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that catalyzes the cleavage of dipeptides from the carboxyl terminal of certain peptides. The most important substrates are angiotensin I which is converts to angiotensin II, and bradykinin, which is inactivated (Nadel 1996). It also cleaves enkephalins and substance P (SP) (Nadel 1996) (Figure 1). The aim somatic ACE activity is associated with tissues such as the luminal side of the vascular endothelium or the renal epithelium. A soluble form of the enzyme that circulates in the plasma is also released after proteolytic cleavage of extracellular ACE (Beldent et al 1993).
A polymorphic marker, which correlates with circulant ACE, has been described (45). The marker consists of the presence (Insertion) or absence (Deletion) of a 287 bp repeat sequence (Tiret et al 1992). The Insertion/
The Ang II acts via two receptors: AT1 and AT2. AT1 receptors have a high affinity for losartan and a low affinity for PD 123177, while AT2 receptors have a high affinity to PD 123177. Most of the known actions of
(Lapteva et al 2001). It was found that Ang II-induced leukocyte rolling is mainly mediated by P-selectin and that firm adhesion is LFA-1-dependent in colonic venules. Moreover, this P-selectin-mediated rolling was found to be a precondition for the subsequent firm adhesion of leukocytes to the vascular endothelium in response to Ang II (Riaz et al 2004). In animal model, New Zealand White rabbits, Ang II was implicated in the recruitment of
levels of IL-10 and reduce metalloprotease-9 protein/activity in patients with angiographically documented coronary artery disease. Moreover, the AT1- receptor blockade specifically reduced thromboxane A2-dependent effects in platelets; an effect that has not been reported for ACE inhibitors.
Furthermore, stimulation of monocytes with Ang II causes the activation of NF-kB and subsequent increases the TNF- α production (Ruiz-Ortega et al 2000). Ang II increases the family member of PYRIN-containing Apaf1-like proteins in DC cells (Lapteva et al 2002). The family of PYRIN-containing Apaf1-like proteins participates in inflammatory signalling by regulating the activation of NF-kB and cytokine processing (Wnag et al 2002).
Ang II are mediated by AT1 receptor, a G protein-coupled receptor (Dinh renin-binding protein, ACE, and angiotensin II/vasopressin receptor et al 2001). Dendritic cells (DC) have mRNA expression of angiotensinogen,
monocytes into the vessel wall (Hernandez-Presa et al 1997) and Schieffer
et al (Schieffer et al 2004) showed that AT1-receptor blockade enhances serum
Insertion genotype has the lowest circulant ACE levels while the DD
genotype has the highest. The serum ACE level tend to be higher according
to ACE genotype in order II, <ID<DD (Furuya et al 1996). In the
physiological point of view the activity of ACE is very interesting because
the levels of three important substances are directly modulated for it, namely
angiotensin II, bradykinin and SP (Figure 1).
By activating in neurokinin 1 receptors (NK-1R), SP is capable of inducing a number of inflammatory responses including plasma extravasation, leukocyte activation, endothelial cell adhesion molecules expression, cytokine production, and mast cell activation (Quartara et al 2002). In addition, there is increasing evidence that SP is capable, directly or Moreover, human and murine lymphocytes express NK-1R an SP augments the mitogen-and-Ag-induced proliferation and IL-2 secretion of T Lymphocytes. Also SP is capable of directly inducing T cell IFN-γ that is known to suppress IL-4 and the development of a Th2 cell phenotype. A recent in vitro study demonstrated that the addition of a specific NK1-R antagonist partly reduced T cell proliferation during the interaction with syngenic or allogenic DC cells and in the absence of DC cells, proliferation of T cells induced by direct CD3/CD28 ligation, was partly dependent on signalling thought NK1-R, revealing an autocrine effect of SP.
Bradykinin (BK) is a powerful mediator capable of promoting symptoms of inflammation, including vasodilatation, plasma extravasion, and pain by the activating bradykinin receptors (B-R), in particular B2-R (Hall 1992).
Activation of B2-R on sensory nerves triggers the release of neuropeptides
An interesting fact is the relationship between ACE and thrombotic mediators. ACE is associated with stimulation of the production of plasminogen activator inhibitor-1 (PAI-1) (Nishimura et al 1999). Elevated levels of PAI- 1 have been implicated in the pathogenesis of thromboembolic diseases (Vaughn 1997). Enhancing the activity of PAI-1 can result in a reduction in the breakdown of fibrin strands and thus an acceleration of thrombus formation (Moriyama et al 1997).
ACE inhibitors (captopril, enalapril and cilazapril) suppress the synthesis of TNF and IL-1 in mononuclear cells (Fukuzawa et al 1997). Moreover, captopril reduces the level of tissue factor expression in endotoxin- stimulated mononuclear leukocytes (Napolene et al 2000) and significantly inhibited LPS-induced production of TNF- α, IL-12 and IL-18 (Lapteva et al 2002).The immunomodulatory actions of captopril have been explained by several mechanisms, including anti-proliferation, anti-oxidant activity, inhibition of metalloproteases, and elevation of prostaglandin synthesis. The inhibitory properties of captopril may be related to the presence of thiol groups in the captopril structure as well as the indirect suppression of expression of the gene encoding ACE.
such as SP and calcitonin gene-related peptide, which results in amplifi- cation of neurogenic inflammation (Geppetti 1993). Moreover, bradykinin is a potent vasodilator and exerts its action by stimulation of endothelial receptors to release prostacyclin and NO (Linz et al 1995).
indirectly, of acting on T lymphocytes (Payan et al 1984; Lai et al 1982).
3. ANGIOTENSIN, ACE, BRADYKININ AND SP IN MODELS OF INFLAMMATION
The icatibant, a bradykinin B2 receptor antagonist, significantly suppressed shortening of the large intestine, the onset of diarrhoea, and worsening of the general health in dextran sulphate sodium-induced colitis in mice (a model of UC). Moreover, in kininogen deficient rats the severity of colitis was decreased as well as the severity of colitis. Also the FR173657, an active B2 antagonist, decreased the inflammation, putting in evidence the role of endogenous kinins generated from the kallikrein-kinin system (Kamata et al 2002). The mutation Ser511Asn increases the susceptibility to inflammation by increasing the cleavage of high molecular weight kininogen in rats genetically susceptible (Isordia-Salas et al 2003). Moreover, a specific plasma kallikrein inhibitor modulated chronic granulomatous enterocolitis and systemic inflammation in genetically susceptible Lewis rats (Stadnicki et al 1998).
Substantial evidence supports a role of SP in the pathophysiology of colitis (Mantyh et al 1988). Stucchi et al (Stucci et al 2000) showed that the that gene expression of ACE was up-regulated, the plasma levels of Ang II were increased and captopril (an ACE inhibitor) reduced leukocyte responses (64% reduction). Moreover, it was found that inhibition of AT1 receptor attenuated I/R inflammation (Riaz et al 2004). In granulomatous response to Schistosoma mansoni the SQ 14225, an inhibitor of ACE, partially inhibited the granulomatous response to Schistosoma eggs and the pathological manifestations associated (Weinstock et al 1981). Also chronic captopril treatment improved portal pressure, body and liver weight, and produced sustained granulomatous response in Schistosoma – infected mice (Weinstock et al 1981). In experimental colitis the prophylactic administration of captopril was effective in preventing colonic fibrosis in TNBS-induced colitis. The antifibrotic action of captopril could be due to the blockage of TGB- β overexpression, and/or to a direct down-regulation of TGB- β transcript (Wengrower et al 2004). In pancreatic inflammation model lisinopril alleviated chronic pancreatitis and fibrosis in male WBN/kob rats and suppressed the expression of TGB- β1 mRNA, preventing pancreatic stellate cell activation (Kuno et al 2003). Furthermore, the degree of TNBS colitis in angiotensinogen gene knockout mice was impaired as well as the expression of proinflammatory cytokines - IL-1beta and Interferon-gamma. As opposite, the expression of IL-4 and IL-10 in the colon of Ag-/Ag- mice was increased. Also subcutaneous infusion of losartan suppressed colitis and the production of proinflammatory cytokines (IL-1 β and IFN-γ) (Inokochi et al 2005).
In a mouse model of ischemia/reperfusion (I/R) in the colon it was found
early administration of neurokinin 1 receptor (NK-1R) antagonist (NK-1RA) is efficacious in reducing experimentally induced colitis. Also in the treatment of ileal pouch inflammation the intraperitoneal administration of NK-1RA antagonist (CJ-12,255) diminished the physical signs of clinical pouchitis and the rise in MPO levels were prevented (Stucci et al 2003). Weistoch et al showed that SP regulates Th1-type of colitis in IL-10 Knockout mice and the NK-1R antagonist reverses ongoing intestinal inflammation (Weinstock et al 2003).
4. ANGIOTENSIN, ACE, BRADYKININ AND SP IN IBD
Stadnicki et al (Stadnicki et al 2003) showed that in human intestinal tissue kallikrein is in the goblet cells of normal and inflamed colon and in macrophages of IBD patients. The plasma cells on the border of the granuloma contain large amounts of kallikrein. This observation emphasizes the close relationship between the immune response and the kallikrein-kinin system.
Kallikrein and the intestinal kallistatin levels were significantly decreased in
inflamed intestinal tissue. Stadnicki et al (Stadnicki et al 2005) using
specific immunostaining and image quantification, observed in the normal
tissue B2R and the inducible B1R. In general, in the normal condition B1R
is present in most tissue only in minimal amounts but is induced during
inflammatory process. The same group found in inflamed active IBD tissue
significantly higher B1R protein levels than in the controls and also observed
An increase in SP receptor binding sites was reported by Mantyh et al
(Mantyh et al 1995) in small blood vessels, lymphoid aggregates, and enteric
neurones of the small and large bowel of patients with CD and UC. In a
subsequent study, the same group showed that whereas the ectopic expression
of NK-1R in UC is confined to active disease. In DC up-regulation of NK-1R
was evident in positive and negative samples of the small and large bowel
(Mantyh et al 1995). Renzi et al showed that in normal human intestine NK-1R
and NK-2R are expressed in multiple cell types and both receptors are up-
regulated in patients with CD and UC (Renzi et al 2000). Moreover, they
found direct evidence of NK-1R up-regulation on inflammatory cells of the
lamina propria, as well as on epithelial cells lining the mucosal surface and
crypts. Up-regulated expression of NK-1R may have important implications
in the pathophysiology of IBD. NK-1R expression on lamina propria
inflammatory cells and in the endothelium of submucosal venulas may
participate in the margination and extravascular migration of granulocytes,
lymphocytes, and monocytes into inflamed tissues.
co-localization of kallistatin as well as kinin receptor proteins in the endothelium of intestinal vessel and in the macrophages forming granulomas (Stadnicki et al 2005).
Jaszewski et al (Jaszewski et al 1990) found that in colonic mucosa the levels of angiotensin I and II were greater in patients with CD than in controls and these levels were also higher in Crohn’s colitis than in UC. The mucosal levels of angiotensin I and II correlated well with the degree of macroscopic inflammation.
The significance of serum ACE levels in patients with CD and UC has been controversial. Physiologically sex and age affect the serum ACE level.
The serum ACE levels in men are higher than that in women and the levels decrease with age. Nunes-Gornes et al (Nunes-Gornes et al 1981) reported that only serum ACE levels of patients receiving steroids were decreased and they concluded that the decreased of serum ACE levels was due to the administration of steroids. Silverstein et al (Silverstein et al 1981) found that enzyme activity tended to be depressed in Crohn’s ileitis and colitis, but not in Crohn’s ileocolitis and UC. They concluded that granulomatous inflammation in Crohn’s disease differs from that in sarcoidosis, in which striking elevation of angiotensin-converting enzyme is present in granulomatous tissue and frequently in serum. D’Onofrio et al. determined the serum ACE activity and found that it was depressed in active Crohn’s disease compared to either controls subjects or patients with inactive disease (D`onofrio et al 1984). Sommer et al. (Sommer et al 1986) showed that serum ACE was significantly lowered in active Crohn’s disease and in UC as long as the ileum or cecum was affected. Also Takeuchi et al (Takeuchi et al 1992), found decreased ACE levels in Crohn’s disease compared with healthy controls and significant negative regression between ACE and CDAI was observed. Matsuda et al (Matsuda et al 2001) showed significantly decreased ACE levels in patients with CD with steroid therapy whose genotype was II, and in those with UC whose genotype was DD, but even among patients without steroid treatment. The serum ACE levels were significantly lower in patients with IBD than in controls for each genotype.
A polymorphism marker of ACE, which correlates with circulant levels, consists of the presence (insertion) or absence (deletion) of a 287 bp repeat sequence. Those with insertion/insertion genotypes have the lowest circulant levels. The PAI-1 is the fast-acting inhibitor of tissue plasminogen activator (t-PA). Assays in vivo of promoter activity demonstrated that under conditions of cytokine stimulations, as those found in IBD, the 4G allele has significant more activity than the 5G allele. In our patients the allelic frequencies of ACE Del and PAI-1 4G were higher in IBD patients than in reference population (Magro et al 2003) (Table 1).
In conclusion, the ACE is an essential step in kinin-kallikrein system,
Angiotensin and SP. There is evidence of the involvement of this enzyme in
the inflammatory process, experimental colitis and IBD, and a new target is open for new therapies.
Table 1: Allelic frequency in Inflammatory Bowel disease (n = 116) and reference population (n = 141) of ACE Del and PAI-1 4G
Crohn s disease
Ulcerative colitis
Reference population
P*
ACE Del 0.58 0.50 0.19 0.001
PAI-1 4G 0.46 0.49 0.15 0.001
*
