Research background and significance - 血管收縮素對心臟細胞的第二型血管收縮素轉化酶表現調節與第二型基質金屬蛋白酶表現之影響

1. Research background and significance

1-1. Heart remodeling and cardiac fibrosis

Organ fibrosis is a complex process that is defined as excess ECM deposited and

accumulated in the tissues including skin, heart, lung, kidney and vessels (Jinnin, 2010). Heart remodeling occurs in response to injury and an increase in wall stress plays a key role in the progressive deterioration of cardiac function that leads to heart failure (Pfeffer and Braunwald, 1990; Sharpe, 2000). Remodeling is characterized by cardiac hypertrophy and dilatation as well as conformational changes in the shape of the heart (Fig. 1-1). The remodeling process consists of a series of timed molecular events that include the inflammatory response to injury, proliferation of cardiac fibroblasts and differentiation to myofibroblasts, and formation of the fibrotic scar tissue in defected myocardium. Myocardium is comprised of a number of cell types such as cardiomyocytes, cardiofibroblasts, endothelial cells and smooth muscle cells, cardiac fibroblasts are also the highest cell population in the myocardium, accounting for about two-thirds of the cells (Camelliti et al., 2005). Cardiac fibroblasts are a critical element of myocardial repair that produce collagens, providing the tensile strength for cardiac tissue (Camelliti et al., 2005). The morphology of cardiac fibroblasts is the flat and spindle shaped cell in myocardium, it is the only cell population lack a basement membrane in the

myocardium. Furthermore, cardiac fibroblasts has more function such as homeostasis and remodeling of the cardiac ECM, electrical activity, production of growth factors and cytokines, and intercellular signaling with cardiomyocytes, endothelial or smooth muscle cells to impact cellular angiogenesis, cell proliferation, cardiomyocyte hypertrophy or apoptosis, appear that play a key role during pathological remodeling of the heart by maintaining normal cardiac structure, function, biochemical and electrical features of the heart (Fan et al., 2012).

Fig. 1-1. Pathologic cardiac intercellular communication. In response to injury, mechanical stretch, and ischemia, the cardiac fibroblast undergoes a phenotypic transition to a

myofibroblast, releasing a variety of growth factors, chemokines and cytokines that act both in an autocrine and paracrine fashion. Stimulation of cardiac fibroblast results in a positive feedback loop to further enhance their activation, collagen deposition, and cytokine release, resulting in fibrosis and chronic inflammation. Cytokine effects on cardiomyocytes leads to pathologic effects including hypertrophy, apoptosis, and impaired contractile responses.

[Martin and Blaxall, 2012]

In response to cardiac injury or stress, cardiac fibroblasts have been triggered and differentiated into myofibroblasts, which have greater synthetic ability to produce ECM

proteins, chemokines and cytokines such as IL-1α, IL-1β, IL-6, IL-10, TGF-β1 and TNF-α (Petrov et al., 2002; Baum and Duffy, 2011). These ECM, chemokines and cytokines mediate migration and contractile of cardiac fibroblasts and myofibroblasts at the site of injury, and maintain the inflammatory response to injury (Eghbali, 1992; Baum and Duffy, 2011). In addition, the TGF-β1 that cardiac fibroblasts and myofibroblasts released accelerate differentiation of cardiac fibroblasts into myofibroblasts and increase collagen expression (Butt et al., 1995; Walker et al., 2004), the accumulation of fibrotic depositions that can interrupts the connection between the myocardial cells and blood vessels in the myocardium leading to overall impairment of cardiac function. These results reveal that cardiac fibroblasts and myofibroblasts have been demonstrated to play a key role in reparative fibrosis in the infarcted heart (Díez et al., 2002; Calderone et al., 2006).

Besides of ECM production, cardiac injury causes chronic cardiac fibroblasts and myofibroblasts activation to produce matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to imbalanced collagen/MMP secretion. A number of growth factors, cytokines, and chemokines have been identified that can regulate production of MMPs and TIMPs to maintain ECM homeostasis by cardiac fibroblasts (Moore et al., 2012). In various MMPs that cardiac fibroblasts released, MMP-2 and MMP-9 have been shown to release the ECM-bound latent TGF-β1, thereby inducing collagen synthesis and further contribute to the adverse remodeling (Yu and Stamenkovic, 2000). In MI and unstable angina patients, MMP-2, MMP-9 and TIMP-1 in the serum were significantly elevated compared with healthy controls, suggesting that these MMPs and TIMP-1 and

proinflammatory cytokines could play an important role in the pathophysiology of acute coronary syndrome (Tziakas et al., 2004). In addition, overexpression of MMP-2 led to severe myocardial fibrosis (Bergman et al., 2007) and MMP2-deficient mice showed the reduced myocardial hypertrophy and fibrosis (Matsusaka et al., 2006), while MMP-9 deficiency

partially improved myocardial hypertrophy and fibrosis following pressure overload (Heymans et al., 2005).

1-2. Renin angiotensin system

The renin-angiotensin system (RAS) is a classically hormonal system consists of endocrine, paracrine and intracrine system (Fyhrquist and Saijonmaa, 2008). The manly function of RAS involved the balance of salt and water, blood pressure and natriuresis, it also plays an important local role to regulate regional blood flow and nutrition in several target organs such as heart (Giani et al., 2012; Guimarães et al., 2012), blood vessels (Khakoo et al., 2008), and lungs (Imai et al., 2008; Shrikrishna et al., 2012; Wong et al., 2012). Furthermore, abnormal activation of the RAS is associated with the pathogenesis of cardiovascular and renal diseases such as hypertension (Jan Danser, 2012; Lo et al., 2012), myocardial infarction (Connelly et al., 2011; Burchil et al., 2012) and heart failure (Agarwal et al., 2012; Birner et al., 2012).

In a classical RAS, the glycoprotein angiotensinogen (AGT;

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile) released from the liver is degraded by the enzyme renin that originates in the kidney, generating the inactive angiotensin I (Ang I;

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) (Ferrario and Strawn, 2006). Subsequently, the dipeptide carboxypeptidase, angiotensin-converting enzyme (ACE) hydrolyzes the C-terminal dipeptide His-Leu of decapeptide Ang I to generate octapeptide angiotensin II (Ang II;

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (Kokubu et al, 1979), and the C-terminal peptide Phe of Ang II is metabolised by the carboxypeptidase, ACE2 to produce the vasodilator, angiotensin (1-7) (Ang 1-7; Asp-Arg-Val-Tyr-Ile-His-Pro) (Donoghue et al., 2000; Turner and Hoope, 2002; Rice et al., 2004). Finally, the C-terminal dipeptide His-Pro of Ang 1-7 was been

hydrolyzed by ACE to obtain inactive peptide angiotensin (1-5) (Ang 1-5;

Asp-Arg-Val-Tyr-Ile) (Ferreira et al., 2012).

Ang II is the main regulator of the RAS, has been revealed that stimulate inflammation, cell growth, apoptosis, fibrogenesis, and differentiation to cause tissue repair/remodeling (Ruiz-Ortega and Ortiz, 2005; Mehta and Griendling, 2007). It has a very short half-life and is quickly degraded to Ang III and Ang 1-7, a similar function peptide and an oppose function peptide, respectively (Sun, 2010). Ang II stimulates a wide variety of biological functions in the heart (Zheng et al., 2012), blood vessels (Wehlage et al., 2012), kidneys (Pinheiro et al., 2012), adipose tissue (Kalupahana and Moustaid-Moussa, 2012), pancreas (Lau and Leung, 2011; Chan and Leung, 2011) and brain (Chrissobolis et al., 2012; Vargas et al., 2012) mediated the specific receptors Ang II receptor type 1 (AT1R) and Ang II receptor type 2 (AT2R) (Skeggs et al., 1980; Corvol et al., 1995; Komatsu et al., 2009). The majority

physiological and pathophysiological effects of Ang II are mediated by the AT1R. Compared with those effects through AT1R, Ang II binding to the AT2R generally causes opposite effects such as stimulated bradykinin and nitric oxide to induce a counterregulatory vasodilatation (Horiuchi et al., 1997; Touyz etal., 1999; Sun, 2010).

Ang 1-7 is an important regulator in the RAS activity acts as an endogenous inhibitor of Ang II. Ang 1-7 binding to the Mas receptor and triggering signal pathways to release of bradykinin (Isa et al., 2011; Gembardt et al., 2012), prostaglandins (Yousif et al., 2012; Costa et al., 2012), and endothelial nitric oxide (Ferrario et al., 2005; Shah et al., 2012) and induce opposite effects to those elicited by Ang II such as apoptosis (Santos et al., 2003; Wang et al., 2012), vasodilation (Savergnini et al., 2010; Pringle et al., 2011), anti-fibrosis (Grobe et al., 2007; Nadu et al., 2008; Ferreira et al., 2010), anti-hypertrophic (Santos et al., 2004; Mercure et al., 2008; Santiago et al., 2010) and anti-proliferative (McCollum et al., 2012; Ni et al., 2012). The schematic representation was present in Fig. 1-2.

Fig. 1-2. Schematic representation of the renin-angiotensin system (RAS) cascade. The significant counterregulatory axes of the RAS are composed by ACE-Ang II-AT1R and ACE2-Ang 1-7-Mas. ACE, angiotensin-converting enzyme; Ang II, angiotensin II; AT1R, Ang II type 1 receptor; AT2R, Ang II type 2 receptor; ACE2, angiotensin-converting enzyme 2; Ang 1-7, angiotensin 1-7; Mas, Ang 1-7 receptor. [Wang et al., 2012]

1-3. Angiotensin converting enzyme II

ACE2 was cloned as a first homolog of human ACE and mapped to the X chromosome by two independent research groups in 2000 (Donoghue et al., 2000; Tipnis et al., 2000). The ACE2 is an 805 amino acid zinc-metallopeptidase and type I integral membrane glycoprotein encoded from 18 exons with a molecular weight of approximately 120 kDa (Turner and Hooper, 2002), it is predominantly observed in the heart, kidneys and testes (Tipnis et al.,

2000) such as cardiomyocytes (Gallagher et al., 2008), luminal surface of tubular epithelial cells (Donoghue et al., 2000; Tipnis et al., 2000) and adult Leydig cells (Douglas et al., 2004).

In addition, ACE2 also had been confined at a lower level in a wide variety of tissues including the brain (Xia and Lazartigues, 2008; Xu et al., 2011), liver (Lambert et al., 2008;

Pereira et al., 2009) and lung (Kuba et al., 2006; Imai et al., 2008).

In molecular structure, the human ace2 gene comprise 18 exons, the first 12 exons of ace2 is similar to the first 11 exons of the ace gene. Moreover, the zinc-binding motif

(HEMGH) of ACE2 is located within exon 9, compared to exon 8 of the ace gene (Donoghue et al., 2000; Tipnis et al., 2000). As like ACE, ACE2 has 2 domains of the amino-terminal catalytic domain and the carboxy-terminal domain, shares 42% sequence identity and 61%

sequence similarity with the catalytic domain of ACE (Donoghue et al., 2000; Tipnis et al., 2000; Douglas et al., 2004). Unlike somatic ACE, ACE2 only contains a single catalytic site with the prototypical zinc-binding HEMGH motif, and functions as a carboxymonopeptidase removing a single C-terminal residue from peptide substrates whereas ACE acts as a

carboxy-dipeptidase (peptidyldipeptidase), removing a C-terminal dipeptide (Clarke and Turner, 2012). In addition, the carboxy-terminal domain of ACE2 shows 48% sequence identity with collectrin, which was a non-catalytic protein that has a critical role in amino acid absorption in the kidney (Danilczyk et al., 2006; Malakauskas et al., 2007), pancreatic

beta-cell proliferation (Akpinar et al., 2005) and insulin exocytosis (Fukui et al., 2005). The molecular structure of ACE, ACE2 and collectrin was present in Fig. 1-3.

Fig. 1-3. Family of enzymes and proteins belonging to the ACE family of proteins. The schematic present the molecular structure of ACE, ACE2 and collectrin. HEMGH is a set of conserved amino acid residues critical for the activity of the zincbinding catalytic site. gACE, germinal angiotensin converting enzyme; sACE: somatic ACE; ACE2, angiotensin converting enzyme II. [Wang et al., 2012]

In RAS, the major substrate of ACE2 are Ang I and Ang II (Donoghue et al., 2000;

Turner and Hooper, 2002; Rice et al., 2004), ACE2 efficiently cleaves a single residue phenylalanine from Ang II to generate Ang 1-7, with about 400-fold higher catalytic efficiency than the conversion of Ang I to Ang 1-9 by removing the C-terminal leucine residue (Vickers et al., 2002). Furthermore, ACE2 is also a multifunctional enzyme as a monocarboxypeptidase to degrade other biological substrates such as vasoactive bradykinin (1–8) (Donoghue et al., 2000), [des-Arg⁹]-bradykinin (Vickers et al., 2002; Warner et al., 2004), Apelin-13 (Kalea and Batlle, 2010), Apelin-17 (Vickers et al., 2002; Oudit and

Penninger, 2011) and Apelin-36 (Kuba et al., 2007). ACE2 hydrolyzed apelin-13 and apelin-36 peptides with high catalytic efficiency (Vickers et al., 2002), and that apelin peptides were mediated APJ receptors to activate the G-protein coupled

seven-transmembrane-domain receptor (GPCR) family predominantly expressed to regulate cardiovascular function and fluid homeostasis in the heart and lungs (Lee et al., 2000; Kleinz and Davenport, 2005; Pitkin et al., 2010).

Ang II-ACE2-Ang 1-7 and apelin-APJ are two important peptide systems with various and fundamental cardiovascular effects, and ACE2 may prevent or supress a variety of vascular and cardiac disorders (Kalea and Batlle, 2010; Oudit and Penninger, 2011; Wang et al., 2012). The major function of ACE2 is to counter-regulate ACE activity by reducing Ang II bioavailability and increasing the vasoprotective/antiproliferative peptide, Ang 1-7 formation.

As a result, ACE2 plays a crucial role in maintaining the balance between the two axes ACE2-Ang 1-7-Mas and ACE-Ang II-AT1R of the RAS, chronic and sustained imbalance may lead to pathophysiology of the cardiovascular, renal, pulmonary and central nervous systems. ACE2 is effectively control fibrosis and structural remodeling in heart (Huentelman et al., 2005; Dong et al., 2012), lung (Shenoy et al., 2010; Rey-Parra et al., 2012) and liver (Paizis et al., 2005; Osterreicher et al., 2009), and extremely beneficial for pulmonary hypertension (Ferreira et al., 2009; Li et al., 2012).

1-4. ACE2 with heart diseases and heart remodeling

In the heart damage such as hypertension, myocardial infarction (MI) and chronic heart failure (CHF) (Cohn et al., 2000), cardiac myocytes were die and been replaced by fibroblasts and collagen to form fibrous tissue, these changes are referred to as “heart remodeling” (Opie et al., 2006). Ang II is a mainly factor of the RAS, activate cardiac fibroblast functions via

AT1R to increase the amount of ECM in the heart (Villarreal et al., 1993; Kim et al., 1995) to induce cardiomyocyte hypertrophy, cardiac remodeling and left ventricular dysfunction (Iwata et al., 2005; De et al., 2006; Whaley-Connell et al., 2007). In the failing heart, the local Ang II concentration is increased and related to the pathological signs of heart failure (Serneri et al., 2001). ACE2 is a significant regulator in RAS to degrade Ang II to suppress the heart

dysfunction that Ang II stimulated (Bikkavilli et al., 2006).

In the heart, ACE2 had been certified a dramatic decrease with aging (Xie et al., 2006) and expressed in coronary microcirculation (Donoghue et al., 2000), macrophages (Burrell et al., 2005), myofibroblasts (Guy et al., 2008), cardiofibroblasts (Zhong et al., 2010), and cardiomyocytes (Gallagher et al., 2008). ACE2 polymorphism was also been reported that four single nucleotide polymorphisms were associated with higher left ventricular mass index, higher septal wall thickness and increased odds ratio for left ventricular hypertrophy (Lieb et al., 2006). In addition, ACE and ACE2 immunoreactivity were higher in cardiac tissue of patients with ischemic heart failure compared to normal subjects (Burrell et al., 2005), the ACE2 activity was also increased in failing human heart ventricles obtained from patients with either idiopathic dilated cardio-myopathy or primary pulmonary hypertension (Zisman et al., 2003). These results appear that ACE2 plays a significant role in cardiac diseases.

In MI rats, cardiac ACE2 mRNA expression and activity were decreased after 2-4 weeks ligation of the left coronary artery (Karram et al., 2005), increased at 4 weeks (Burrell et al., 2005) and down-regulation at 8 weeks post-MI (Ocaranza et al., 2006), these results suggest that regulation of cardiac ACE2 expression and activity varies depending on disease state and time point at which measurements are obtained.

ACE2 overexpression protects the heart from Ang II-induced hypertrophy (ez-Freire et al., 2006), MI (Der et al., 2008), fibrosis (Huentelman et al., 2005), and also improved left ventricular remodeling after experimental MI (D´ıez-Freire et al., 2006). In MI rat,

overexpression of ACE2 inhibited the development of early atherosclerotic lesions by suppressing the growth of vascular smooth muscle cells (Rentzsch et al., 2008) and ameliorated left ventricular remodeling and dysfunction (Zhao et al., 2010). In addition, cardiac fibroblasts infected with ACE2 lentivirus decreased the collagen production that acute hypoxic exposure induced (Grobe et al., 2007).

Loss of ACE2 worsened the pathological remodeling and progressive reduction in LV contractile function to cause systolic dysfunction and heart failure (Crackower et al., 2002;

Yamamoto et al., 2006; Bodiga et al., 2011). In ACE2 null mice, transverse aortic constriction or exacerbated pressure overload stimulated cardiac Ang II and AT1R activation increased.

This result reduced cardiac contractility and induced cardiac dysfunction and heart

remodeling (Gurley et al., 2006; Yamamoto et al., 2006). Furthermore, the response of Ang II stimulated via hypoxia-activation was greater in cardiomyocytes isolated from ACE2^−/y mice than isolated from WT mice (Keidar et al., 2007). In the absence of ACE2, p47^phox NADPH oxidase subunit plays a critical role to activate myocardial NAPDH oxidase system to

increase superoxide and activate MMPs leading to the severe adverse myocardial remodeling and dysfunction in ACE2 KO mice (Bodiga et al., 2011). Almost reports appear that ACE2 plays a protector in the heart and loss of ACE2 severely impaired cardiac function was probably related to the Ang II accumulation. The significant references were listed in Appendix 8-1.

1-5. Matrix metalloproteinases

The tissue fibrosis is caused by excessive accumulation of extracellular matrix (ECM) components, especially types I and III collagen, in various pathological manifestation diseases (LeRoy et al., 1974; Uitto et al., 1979). The balance of ECM components is maintained by

matrix metalloproteinases (MMPs) and MMPs inhibitors, tissue inhibitors of

metalloproteinases (TIMPs) (Clutterbuck et al., 2009). MMPs are essential components for various normal biological processes such as embryonic development, morphogenesis,

reproduction tissue resorption and remodeling (Szarvas et al., 2011), they also implicated in a number of key pathologic processes including inflammation, fibrosis, arthritis, pulmonary diseases and cancer (Amălinei et al., 2010), because of the abnormally ECM deposition that imbalance MMPs and the TIMPs caused.

MMPs had been discovered by Gross and Lapiere in 1962, are a group of Zn²⁺ and calcium dependent endopeptidases of common significant peptide chain sections, however glycosylated in different amount and different locations (Sternlicht and Werb 2001). MMPs comprise a large family of protease and share several similarities in terms of their structure, regulation and function (Nagase and Woessner, 1999; Bode and Maskos, 2001). Up to now, 28 types of MMPs have been identified, and they are further divided into six major subfamilies based on structure and substrate specificity, including collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and other MMPs (Table 1-1; Vargová et al., 2012).

Table 1-1. Types of different matrix metalloproteinases and their substrate specificity

Subgroups MMPs Name Substrate

Collagenases

MMP-1 Collagenase-1

Collagen I, II, III, VII, VIII, X, and gelatin

MMP-8 Collagenase-2

Collagen II, IV, IX, X, and gelatin, α-casein, β-casein

MMP-14 MT1-MMP Gelatin, fibronectin and laminin

MMP-15 MT2-MMP Gelatin, fibronectin and laminin

MMP-16 MT3-MMP Gelatin, fibronectin and laminin

MMP-17 MT4-MMP Fibrinogen and fibrin

MMP-24 MT5-MMP Gelatin, fibronectin and laminin

MMP-25 MT6-MMP Gelatin

Other MMPs

MMP-12 Metalloelastase Collagen IV, elastin and gelatin

MMP-19 RASI-1 Collagen I, IV and gelatin

MMP-20 Enamelysin Collagen I, IV, and gelatin

MMP-23 CA-MMP Gelatin

MMP-26 Matrilysin-2, endometase Collagen IV and gelatin

MMP-28 Epilysin Gelatin

[Swarnakar et al., 2011]

The major structure of all MMPs consists of three domains: N-terminal hydrophobic signal sequence, a propeptide domain region and a catalytic domain (Nagase, 1997; Visse and Nagase, 2003). The N-terminal hydrophobic signal sequence decides the MMPs which been released out or maintained in the cell membrane. For example, membrane-type MMPs utilize transmembrane and cytosolic domains anchoring them to the cell membrane (Nagase and Woessner 1999; Stoker and Bode, 1995). The function of the propeptide domain is to maintain latency of the MMPs until a signal for activation is given. Catalytic domain contains two ions of zinc and at least one ion of calcium bound on various amino acid residues. The catalytic domain of all MMPs contains the consensus motif HExGHxxGxxH and three histidines that coordinate with the zinc ion in the active center. Second ion of zinc and calcium are bound in inactive part of catalytic domain with high affinity, but their role remains still unknown (van Wart and Hansen-Birkedal 1990; Nagase and Woessner 1999). The molecular structure of different type of MMPs was present in Fig. 1-4.

Fig. 1-4. Family of enzymes and proteins belonging to the MMPs family of proteins. The schematic present the mainly molecular structure of collagenases, stromelysins, gelatinases, matrylysins and membrane type MMPs. S, cysteine switch; FU, intracellular furin-like serine proteinases; F, collagen-binding type II repeats of fibronectin; Zn²⁺, zinc-binding site; H, hemopexin domain. [Vargová et al., 2012]

The ECM decreased by MMPs is mainly impressed by TIMPs, MMPs and TIMPs play a critical role in maintaining the balance between ECM deposition and degradation in

physiological processes (Hulboy et al., 1997; Vu and Werb, 2000). Four TIMPs, TIMP-1, -2, -3, and -4, have been identified (Cruz-Munoz and Khokha, 2008), these TIMPs are secreted by a variety of cell lines such as smooth muscle cells and macrophages. TIMPs also involved in the process of inflammation and fibrosis, their activity is increased by PDGF and TGF-β and either increased or decreased by different ILs (Jones et al., 2003). In addition, evidences suggest that fibrotic livers have high expression of the TIMP-1 and TIMP-2, and thus the

combination of low expression of MMPs and high TIMPs may prevent the degradation of the fibrillar collagens.

1-6. ACE2 and gelatinase (MMP-2 and MMP-9)

ACE2 is a newly identified component of RAS and plays a negative regulator of Ang II in the RAS. Most of published papers reveal that Ang II could break the balance of MMPs expression in heart and induce heart remodeling (Brassard et al., 2005; Yaghooti et al., 2011), but the relative between ACE2 and MMPs are still unknown. In 2009, Kassiri’s group utilized the left anterior descending artery ligation and ACE2 KO mice to investigate the role of ACE2 in MI (Kassiri et al., 2009). In wild-type mice, ACE2 was persistent increased in the infarct zone of heart, ACE2-deficient was increased interferon-γ, interleukin-6, phosphorylation of ERK1/2 and JNK1/2 signaling pathways and MMP-2 and MMP-9 levels in response to MI.

Loss of ACE2 also associated with the increased expression and phosphorylation of p47^phox, Ang II levels, NADPH oxidase activity, and superoxide generation, which could lead to

在文檔中血管收縮素對心臟細胞的第二型血管收縮素轉化酶表現調節與第二型基質金屬蛋白酶表現之影響 (頁 15-32)