Inhibitors of lactoperoxidase enzyme.
\r\n\tAll book chapters are produced by forward-thinking specialists in the area of renewable energy and smart grids, with detailed analysis and/or case studies. This book is intended to serve as a reference for graduate students, academics, professionals, and system operators.
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These exhibit antioxidant characteristics and catalyze the oxidation of organic and inorganic substrates with hydrogen peroxide being the electron acceptor [2]. Those enzymes are present in eukaryotes, prokaryotes, and photosynthetic cells [3, 4].
\nLPO is generally found in mammalians such as human [5, 6], bovine [7], buffalo [8], goat [9], sheep [10], llama, cow, camel, and mice milk [6, 11], saliva [12], tears [13], and mammary, salivary, and lachrymal glands [6, 14].
\nPeroxidases are frequently used in the studies of metabolic reactions, enzymatic functions, protein structures [15] and in clinical diagnoses, microanalytic applications, and the food and drug industry [16, 17]. Mammalian POD enzymes are localized in milk, saliva, and tears as lactoperoxidases (LPO) [18] and in leukocytes and platelets as myeloperoxidases [6].
\nProsthetic groups of peroxidase are protoheme and are connected to the apoprotein loosely in contrast to many hemoprote [14]. Firstly, protein portion is synthesized in the organisms bearing peroxidases. However, enzymes are not functionally active. The enzyme gets activated by both apoprotein and hem groups [3, 19]. The general formula of the reaction catalyzed by peroxidases is shown below [3].\n
The H2O2 formed during the metabolism having oxidizing property must be quickly removed. The catalase and peroxidase enzymes exhibiting antioxidant properties play this role in cells [20]. The amount of hydrogen peroxide in the cells is removed by catalase in peroxisomes. In other parts of the cells, the peroxidase enzymes utilize various aromatic components as the substrate to [21] neutralize H2O2 [22].
\nThe characteristics of peroxidase enzyme that is isolated from milk are similar to animal and human peroxidases [23]. It displays 55, 54, and 45% similarity with myeloperoxidase (MPO), eosinophilperoxidase (EPO), and thyroidperoxidase (TPO), respectively. Following xanthine oxidase, lactoperoxidase is the most common enzyme in milk and it is also commonly present in whey which is the liquid remaining after milk has been curdled and strained. Each lactoperoxidase enzyme contains an iron molecule. The conformation of the protein is stabilized by a chelated calcium ion [24–26].
\nThe main function of the LPO enzyme is to catalyze the oxidation of thiocyanate with H2O2 to hypothiocyanite having antimicrobial activity [27, 28].\n
The combined action of these three components, which constitute the ‘lactoperoxidase system’, was defined by Reiter and coworkers [29–31]. The biological significance of the system is protection of the lactating mammary gland and the intestinal tract of newborn infants, thereby providing a natural host defence system against invading microorganisms [23]. The LPO catalyzes the oxidation of some halides (I2 and Br2 but not Cl2) to yield the most oxidizable form of I-, that is, I2 [26].\n
The LPO-H2O2-SCN- system shows bacteriostatic effect and thus prevents bacterial growth and development, whereas the LPO-H2O2-I2 system shows bactericidal potency thus killing bacteria. Both SCN- and I2 have shown strong bactericidal effect when present within the system [32, 33].
\nLactoperoxidase (LPO, E.C. 1.11.1.7) is one of the crucial enzymes in milk with oxidoreductase activity. The peroxidase isolated from milk was given the name lactoperoxidase [23] and was the first enzyme reported to be found in milk [34]. The main function of the enzyme is to catalyze the oxidation of molecules in the presence of hydrogen peroxide and to help production of products with a wide antimicrobial activity. Pseudohalogens, thiocyanates, or halogens should function as second substrates for the enzyme to exhibit such antimicrobial effects [23, 29].
\nThe LPO system shows a significant protective effect in bovine milk. The activation of the system depends on the concentration of the two reactants, thiocyanate and hydrogen peroxide. In the presence of hydrogen peroxide, the system catalyzes the transformation of thiocyanate into hypothiocyanate, which has an antibacterial nature [35–37]. The end products of these compounds are oxidized and hence are safe for human health.
\nMany studies show that this system destroys several bacterial and fungal strains [38–42]. Lactoperoxidase has a broad antifungal activity [43, 44]. Mastitis is a bacterial inflammation in mammals. The effects of different concentrations of thiocyanate-H2O2 medium on several antibacterial and antifungal strains were studied to solve this dairy industry issue [45, 46]. They are capable of reducing bacterial growth by damaging the cell membranes and inhibiting activities of several cytoplasmic enzymes.
\nThe LPO enzyme, a glycoprotein consisting of 8–10% carbohydrate, comprises a chain containing 612 amino acids. It consists of a single polypeptide chain of molecular weight approximately 78 kDa [47]. It is a basic protein containing heme as its prosthetic group with an isoelectric pH value of 9.2 [24–26]. Furthermore, it is very active in acidic pH [48]. It is fairly voluminous as a LPO molecule [49]. The Ca2+ ion stabilizes the enzyme. The Ca2+ ion disappears under pH 5.0 and thus reduces the stability of the enzyme [26].
\nThe biocidal activity of the LPO results from the products of the chemical reactions that it catalyzes. Hypothiocyanate, which is the main product of the reaction, interacts with the thiol groups of various proteins, which is critical for the survival of pathogens. The impact of LPO on bacteria results from the oxidation of sulfhydryl. The oxidation of the -SH groups makes the bacterial cytoplasmic membrane lose its ability to transport glucose, potassium ions, amino acids, and peptides [45].
\nThe biological significance of this enzyme results from the fact that it has a natural protection system against the invasion of microorganisms. Besides this antiviral effect, it is reported that it protects animal cells against various damages and peroxidative effects [23, 29–31]. Lactoperoxidase is a significant agent of the defense system against pathogen microorganisms from the digestive system of neonatal babiess. The LPO enzyme functions as a natural compound of the non-immune biological defense system of mammals and it catalyzes the oxidation of the thiocyanate ion into the antibacterial hypothiocyanate [52].
\nAlthough peroxidases have similar catalytic mechanisms, they are distinguished for their ability to oxidize halides and pseudohalides. For example, only myeloperoxidase is able to oxidize bromine, iodine, and chlorine at neutral pH. Lactoperoxidase, on the contrary, can only oxidize iodine and thiocyanate but it either cannot or can hardly oxidize brome under the same conditions (Figure 1) [53].
\nThe activation mechanism of the peroxidase enzyme.
In the first step of this mechanism, the peroxidase enzyme reacts with one equivalent of the peroxide to form an Fe (IV)-containing compound I that is the porphyrin cation radical. In the second step, the cation radical takes a proton of the substrate and reduced (substrate)-Fe (IV) form, the substrate becomes radical to lost a proton. The compound II takes a proton from the substrate and return the first reducing form. Also radicalic substrate which are formed with interacting each other are polymerization [54].
\nThe LPO enzyme is covalently bound to the heme group proteins, with the bond occurring between the hydroxyl group of the heme group and the carboxyl group of the protein [14]. Approximately 10% of the molecule is composed of carbohydrates and the molecule contains five main potential glycosylation regions and 15 semi-cysteine residues [11, 23, 45]. The heme group at the catalytic center is protoporphyrin IX, which is covalently bound to the polypeptide chain along with the disulfide bridge. The iron compound, which is a part of the heme group, constitutes 0.07% of LPO. The calcium ion is tightly bound to the enzyme, which ensures the molecular conformation and structural integrity of the enzyme [55].
\nThe activation of the natural antibacterial system has been adopted for protecting raw milk. This system is defined as the lactoperoxidase/thiocyanate/hydrogen peroxide (LPO) system. The antibacterial effect of the system on milk is based on the oxidation of SCN− ions catalyzed by the lactoperoxidase enzyme in the presence of H2O2. The short-lived components, OSCN- ions, generated in this oxidation reaction have bacteriostatic effect [56, 57]. The system is recommended in developing countries where there are no sufficient cooling facilities for collection of raw milk and their transportation to processing centers [57].
\nLPO oxidized the -SH groups of the enzymes in bacteria such as hexokinase and glyceraldehyde-3-phosphate dehydrogenase and tends to lose biological functions of these enzymes. As a result, the bacterial cytoplasmic membranes are damaged structurally, and glucose, purine, pyrimidine, and amino acid uptake as well as protein, DNA, and RNA synthesis are blocked. Thus, bacteria growth and proliferation is prevented (Figure 2) [58].
\nAntibacterial effect of the lactoperoxidase enzyme.
Milk and milk products area rich sources of mineral, protein, vitamin, nutritional elements [56, 57]. Lactoperoxidase showed antifungal activity in apple juice and salt solution. In vitro findings also show that the LPO/H2O2/halide system has a strong virucidal activity against HIV-1 [59].
\nIn conditions of iodine deficiency, the level of SCN ions in milk plays a vital role in thyroid function. The studies showed that when milk containing 19 ml/L thiocyanate ion and iodine at 0.1 mg/l concentration is consumed, the thyroid functions of patients with iodine deficiency did not have any negative symptoms [60].
\nThe measurement of the activity is based on the oxidation of 2, 2\'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) chromogenic substrate by H2O2 and observation of the increase in the absorbance caused by the resultant colored compound at 412 nm and pH: 6.0 [61]. For determination of LPO activity with spectrophotometric assay, 2.8 mL of ABTS (1 mM) and 0.1 mL of H2O2 (3.2 mM) were pipetted in the spectrophotometer tube of 3 mL. Enzyme solution of 0.1 mL was added and the tube was turned upside down before it was placed in the spectrophotometer. The increase in the absorbance was observed against blank at 412 nm for 3 min and recorded every 60 sec. Phosphate tampon (0.1 M) at pH: 6.0 was used as blank, instead of the enzyme, while all other solutions were used at the same rates. The following formula was used to determine the activity:\n
A | \n: Absorbance (absorbance read at the end of 1 minute) | \n
b | \n: Path length (1 cm) | \n
c | \n: Concentration (μmol/mL) | \n
ε | \n: Extinction coefficient (32400 M−1 × cm−1) | \n
Df | \n: Dilution coefficient | \n
V | \n: Velocity of the reaction (μmol/mL.min.) | \n
One LPO unit (EU) is defined as the amount of LPO that catalyzes the oxidation of l μmol of substrate (ABTS) per min at 20°C [52].
\n2,2’-azino-bis(3-etilbenztiazolin-6-sülfonik asit (ABTS) [62], p-phenylenediamine [63], pyrogallol, guaiacol, catechol, phenols, aromatic amines, ascorbates, epinephrine, and tetramethylbenzidine [6, 14, 47, 52, 61].
\nTo date, several chromatographic methods have been reported about purification and characterization of the LPO enzyme from bovine milk [14]. For example, CM-Cellulose [64], CM-Sephadex ion-exchange chromatography [46, 64], Sephadex G-100 gel filtration chromatography [6, 64], hydrophobic affinity chromatography on Phenyl-Sepharose CL-4B [5], and Toyopearl-SP cation exchange chromatography [64] are among the methods used in the purification of LPO enzyme from bovine milk. LPO was purified in one stage using the affinity technique and sulfanilamide was used as the ligand [65].
\nThe Km and Vmax values are suitable parameters for kinetic studies. Km is a substrate concentration at which half of the enzyme active sites are filled. Vmax is an expression of the catalytic activity of the enzyme.
\nTo find Km and Vmax values for LPO, activity was measured at 20°C, 412 nm, at pH 6.0, for five different substrate concentrations. For this purpose, generally 0.2–1.5 mL volume from the stock solution of the substrates was used. The total volume was made up to 2.8 mL with a buffer solution and then 0.1 mL enzyme and 0.1 mL H2O2 were added. The Km and Vmax values were calculated from the Lineweaver-Burk graph [65, 66].
\nThe Ki and IC50 values show the inhibitory effect on enzyme. The Ki and IC50 values depend on the inhibitory mechanism. IC50 is the inhibitor concentration required for 50% inhibition and Ki value is the constant.
\nTo investigate the inhibitory effects of some inhibitors on LPO and to determine the IC50 values, the LPO activity was measured in the presence of five different concentrations of inhibitor. For example, the experimental procedure for inhibitor: A control sample without inhibitor was taken as 100% and an activity-[Inhibitor] plot was drawn. To determine the Ki, three different concentrations were used for x inhibitor. ABTS was also used as a substrate at five different concentrations. Lineweaver-Burk plots (1/V-1/[S]) were obtained for inhibitor; the Ki and the inhibition type were calculated from these plots [65].
\nThe inhibitors of the LPO enzyme were identified in studies on the enzyme [52, 67]. Non-selective monoamine reuptake inhibitors consist of opipramol, lofepramime, dibenzepin, protriptyline, melitracen, butriptyline, dimetacrine, dosulepin, and quinipramine; selective serotonin reuptake inhibitors include alaproclate and etoperidone; non-selective monoamine oxidase inhibitors comprise moclobemide, toloxatone, and isocarboxazid; and other antidepressants are viloxazine, minaprine, bifemelane, oxaflozane, and medifoxamine (Table 1).
\n\nMany inhibitors such as sulfanilamides [65], propofol and derivatives [68, 69], some anesthetic drugs [70], some bacteria [46], some phenolic acid compounds and phenolics [71, 77], avermectins [73], adrenaline, melatonin, serotonin and norepinephrine [45, 73, 74], fungi and bacteria [58], antibiotics [75], hydrazines [52], and some thiocarbamide compounds [67] are assayed and reported as a LPO inhibitor in the literature.
\nInhibitor | \nIC50 | \nKi | \nInhibition type | \nPublication | \n
---|---|---|---|---|
L-Adrenaline | \n34.5 mM | \n2.26 mM | \nNoncompetitive | \nSisecioğlu et al. [74] | \n
Ceftazidime pentahydrate | \n0.048 mM | \n0.018 ±0.0035 mM | \nCompetitive | \nSisecioğlu et al. [76] | \n
Prednisolone | \n0.053 mM | \n0.019 ±0.0005 mM | \nCompetitive | \n\n |
Amikacin sulfate | \n0.26 mM | \n0.04 ±0.015 mM | \nCompetitive | \n\n |
Ceftriaxone sodium | \n0.29 mM | \n0.10 ±0.055 mM | \nCompetitive | \n\n |
Teicoplanin | \n1.016 mM | \n0.13 ±0.022 mM | \nCompetitive | \n\n |
Melatonin | \n1.46 mM | \n0.82 ±0.28 | \nCompetitive | \nSisecioğlu et al. [75] | \n
Serotonin | \n1.29 mM | \n0.26 ±0.04 | \nCompetitive | \n\n |
Norepinephrine | \n67.2 mM | \n62 mM | \nNoncompetitive | \nSisecioğlu et al. 2010b | \n
L-Ascorbic acid (Vitamin Q) | \n2.03 mM | \n0.508 ±0.257 mM | \nCompetitive | \nSisecioğlu et al. [43] | \n
Menadione sodium Bisulfate (Vitamin K3), | \n0.025 mM, | \n0.0107 ±0.0044 mM, | \nCompetitive | \n\n |
Folic acid | \n0.0925 mM | \n0.0218 ±0.0019 mM | \nCompetitive | \n\n |
2,6-Dimethylphenol | \n836.67 nM | \n4442 nM | \n\n | Koksal et al. 2014 | \n
2,6-Di-Tbutylphenol | \n10 nM | \n9 nM | \nCompetitive | \n\n |
Di(2,6-Dimethylphenol) | \n6.86 nM | \n0.53 nM. | \nCompetitive | \n\n |
Di(2,6-Di-Tbutylphenol) | \n185 nM | \n48.33 nM | \nCompetitive | \n\n |
Di(2,6-Diisopropylphenol) | \n154 nM | \n19.33 nM | \nCompetitive | \n\n |
Sulphanilamide | \n0.84 nM | \n3.57 nM | \nCompetitive | \nAtasever et al. [65] | \n
Emamectin benzoate | \n4.33 μM | \n6.82±2.60 μM | \nCompetitive | \nKoksal et al. [73] | \n
Eprinomectin | \n16.90 μM | \n4.80±1.95 μM | \nCompetitive | \n\n |
Moxidectin-Vetranal | \n99.00 μM | \n61.31±9.89 μM | \nCompetitive | \n\n |
Abamectin | \n138.60 μM | \n103.73±34.03 μM | \nCompetitive | \n\n |
Doramectin | \n173.20 μM | \n80.14±29.38 μM | \nCompetitive | \n\n |
Ivermectin | \n231.00 μM | \n519.97±47.62 μM | \nNoncompetitive | \n\n |
Caffeic acid | \n393.61 nM | \n430.033±79.04 nM | \nCompetitive | \nGulcin et al. [72] | \n
Ketamine | \n0.29 mM | \n0.019 ± 0.031 | \nNoncompetitive | \nOzdemir et al. [70] | \n
Bupivacaine | \n0.155 mM | \n0.015 ±0.021 mM | \nNoncompetitive | \n\n |
Inhibitors of lactoperoxidase enzyme.
LPO is the second most abundant whey enzyme in bovine milk, [31, 77] and its concentration is approximately 30 mg/L [23]. The peroxidase activity in cow milk is 20 times richer than in human milk and contains 1.2–19.4 units/mL LPO [78]. The mean LPO enzyme activity varies in different species, for example, 1.4 units/mL in cow, 0.34–2.38 units/mL in lamp, 1.5–4.45 units/mL in goat, 0.794 units/mL in buffalo, 22.0 units/mL in pig, and 0.06–0.97 units/mL in human [78].
\nThere is a growing interest in the purification of LPO with increasing applications. The LPO system’s natural biological functions are preferred against antimicrobial chemicals. Lactoperoxidase has many fields of application. It is widely used especially in milk-processing facilities in the milk industry [79]. LPO is used in milk and cheese to reduce the microflora [23]. The LPO enzyme derived from various animal sources has a significant role in the suppression of bacterial growth and helps bacterial inhibition. Inhibition of bacterial growth by the bovine LPO is attributed to the peroxidase system, which contains H2O2 and thiocyanate [9]. The antimicrobial effect of the LPO system occurs naturally in milk. LPO has a bacteriostatic effect on gram-positive and gram-negative bacteria. The antibacterial studies on the LPO enzyme purified from camel milk show that the LPO-thiocyanate and peroxide system leads to significant inhibition of pathogenic bacteria.
\nMost popular applications of the system include food production for preservation of raw milk, pasteurized milk, and cheese milk during storage and/or transportation to the processing plants. The system can be used to avoid the suppression of acidity in yoghurt. In the absence of refrigeration, LPO system is preferable [80] and the system can be used to extend the shelf-life of pasteurized, raw, and cheese milk [79, 81]. This is used for the preservation of emulsions and cosmetics.
\nThe LPO system can ensure an extensive spectrum of antimicrobial properties against bacteria and yeasts when it is composed of LPO, H2O2, SCN-, and I2. Hence, it is preferred in oral care products and cosmetic preservation [82, 83]. The system is used against fish pathogenic bacteria in aquaculture with strong bacterial effects.
\nThe renin-angiotensin-aldosterone system (RAAS) has long been discovered, since 1898 by Tigerstedt and Berman, but still possesses many mysteries to be solved. The RAAS has a major impact in controlling blood pressure and fluid balance.
The most important result of the activation of this system is the generation of angiotensin II (ANG II), which is a biologically active hormone that is produced through sequential cleavage of peptides derived from the initial substrate: the molecule of angiotensinogen. ANG II induces its actions after binding to different types of receptors and will induce a wide spectrum of biological reactions that can impact almost every system: the immune system, vessels, brain, heart and kidney.
The main role of RAAS is to maintain the circulatory homeostasis, by maintaining the fluid volume. Abnormal activation of RAAS will determine hypertension and cardiac hypertrophy that may lead to heart failure. This is the reason why the pharmacological inhibition of this system has proven to induce such a beneficial effect in cardiovascular diseases such as hypertension and congestive heart failure.
Renin is produced by the epithelioid cells of the juxtaglomerular cells under the form of a precursor named preprorenin. Afterwards it may be released as prorenin or may be processed to active renin, which will be stored in granules. The release of renin granules is the rate-limiting step of the renin-angiotensinogen-aldosterone cascade.
The next step consists in the release of angiotensinogen from the liver, which will be metabolized by renin, thus liberating angiotensin I (ANG I) [1, 2].
Angiotensinogen is included in the superfamily of Serpin A8 proteins. Serpin is a large and diverse superfamily of protease inhibitors and related proteins. It is released into the blood stream after removal of the 33-amino acid signal peptide and will remain in the circulation for approximately 5 h. However, it remains still unclear how much intact angiotensinogen versus catabolized angiotensinogen [the so-called des-(Ang I)-Agt] is present in the circulation. There is few data in the literature describing the proportions of intact Agt versus des-(Ang I)-Agt in the circulation. Other studies have suggested that des-(Ang I)-Agt may induce angiogenesis. Even though the liver is the main source of angiotensinogen synthesis, there are some other sources for this enzyme: the brain, heart, kidney, lung, adrenal gland, adipose tissue, blood vessels and digestive tube. An independent tissue regulation of angiotensinogen levels has also been proven [3, 4, 5, 6].
ANG I will be transformed in ANG II due to angiotensin-converting enzyme (ACE), which is released from the endothelial cells. The angiotensinogen-converting enzyme is a dicarboxypeptidase that cleaves two amino acids from ANG I, thus generating ANG II (Figure 1).
The renin-angiotensin-aldosterone cascade [3].
There have been described two different types of ACE: somatic and testicular, which are both a result of the alternative splicing of a single gene. The role of ACE in forming ANG II from ANG I is primordial for its biological functions as ANG II is the major effector molecule of the RAAS. The ACE acts on other biologically active peptides, not only on ANG I, one of the most representative being bradykinin [7, 8, 9].
Bradykinin will be transformed into an inactive peptide through the action of ACE. This biological pathway for bradykinin metabolism is very significant in vivo. The ancient term for ACE is kininase II. As bradykinin promotes vasodilatation and induces a natriuretic effect, the pharmacological inhibition of ACE will diminish the kininase activity and will subsequently lower the blood pressure. The cleavage of ANG II by angiotensin-converting enzyme type 2 produces the heptapeptide angiotensin 1-7 (Ang 1-7). This peptide binds to the Mas receptor (MasR) and induces downstream vasodilatation, which has an opposing effect of the hypertensive action of AT1R signalling. The cardioprotective properties of Ang 1-7 can diminish or reverse heart failure and hypertensive cardiac remodelling [10] (Figure 2).
ANG II formation [11].
ACE2 is a monocarboxypeptidase that transforms ANG I into Ang 1-9 which is a nonapeptide, and ANG II in Ang 1-7 (a heptapeptide). The discovery of these molecules unravels a distinct enzymatic pathway for ANG I and ANG II catabolism that will have an antagonist role to RAAS activation.
Ang 1-7 is a biologically active peptide that induces a wide range of effects, many of them being antagonist to those caused to Ang II. An endogenous orphan receptor, Mas (MasR), was identified in 2003; afterwards this receptor was proved to be Ang 1-7 receptor. It also protects cardiovascular function as it enhances vasodilatation via elevated release of NO and bradykinin, as well as diminishing the production of reactive oxygen species (ROS). The major effects of Ang 1-7 are those induced by MasR activation. These effects counterbalance the ones induced by ANG II and include the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-endothelial nitric oxide synthase (eNOS) pathway, the inhibition of protein kinase C (PKC)-p38 MAPK pathways and the inhibition of collagen expression to limit cardiac fibrosis [12].
Also Ang 1-7 therapies have shown cardioprotective effects in preclinical models of non-ischemic and ischemic cardiomyopathy as it inhibited cardiomyocyte growth in vitro and diminished the ventricular hypertrophy induced by myocardial infarction in vivo. Ang 1-7 diminished the myocardial levels of pro-inflammatory cytokines (TNFα and IL-6), thus having a beneficial effect in cardiac inflammation.
Recent studies demonstrated that recombinant human ACE2 generated Ang 1-7 and Ang 1-9, while recombinant murine ACE2 generated predominantly Ang 1-7.
Ang 1-9 has also proved to be beneficial as it acts on AT2R, thus leading to cardio protection. Therefore, the ACE2/Ang 1-7/MasR and ACE2/Ang 1-9/AT2R axes are now considered to be physiological antagonists that inhibit the RAAS [10, 12, 13].
ANG II can act on two receptors: angiotensin type 1 and angiotensin type 2 (AT1R and AT2R). The effect of AT1R consists in increased sodium retention, promotes vasoconstriction (mostly on the efferent arteriole), induces sympathetic nervous system activity, determines thirst and promotes the release of aldosterone from the glomerular zone of the suprarenal glands (Figure 3).
The RAAS activation [13].
The action on AT2R stimulation are antagonist to the one on AT1R as it induces vasodilatation and inhibits the inflammatory and fibrotic processes. This receptor is prevalent in the foetus; therefore, it has an important role in normal ontogenesis but has a minor role in adults [13, 14, 15, 16, 17] (Figure 4).
The actions of angiotensin receptors [11].
Aldosterone secretion results from the AT1R stimulation and is the final step of the RAAS. It is an important regulator of the hydric balance and of sodium and potassium exchange. The strongest inducers in aldosterone secretion are ANG II and the increased extracellular concentration of potassium, the synthesis of aldosterone being induced by CYP11B2 gene expression. Aldosterone will activate the mineralocorticoid receptor, leading to increased reabsorption of natrium and water in kidneys and promoting potassium excretion (Figure 3).
Besides the systemic RAAS, a local system found in many tissues, including the heart has been described. The local RAAS functions in a dual manner: independently and in correlation with systemic RAAS components. This second pathway of metabolisation present in the heart consists in ANG II synthesis by the endopeptidase chymase. Chymase is a serine protease found in a type of granulocytes called mast cells, which are located in the heart interstitium. Mast cells contain granules that are filled with cytokines and proteases (including chymase). The degranulation releases chymase during the inflammatory process via a classically mediated ligand-dependent pathway. Chymase actions are similar to ACE but with a much higher catalytic activity in transforming ANG I into ANG II. Therefore, chymase may be in fact a major ANG II-forming enzyme in the human heart. Its role may be linked to many pathological processes, such as hypertension, atherosclerosis, vascular proliferation, development of cardiomyopathies, myocardial infarction and heart failure, as well as cardiac fibrosis [18, 19, 20, 21].
A third pathway consists in the action of ACE2 (monocarboxypeptidase angiotensin-converting enzyme 2), which is similar to ACE in <50% of amino acidic constitution and therefore is unresponsive to the treatment with ACE inhibitors. This enzyme transforms ANG I into Ang 1-9 and ANG II into Ang 1-7, thus promoting the synthesis of cardiorenal-protective peptides, through diminishment of ANG I and ANG II levels. This pathway may lead to a key role in modulating ANG II actions in cardiovascular disease [22].
The implication of gene alterations in the ethiopathogeny of different diseases is an important study direction in the later years. The RAAS is not only involved in the cardiovascular homeostasy but also in the development of the coronary artery disease. It seems that angiotensin-converting enzyme gene mutations have the key role in this process.
ACE gene is found on the long arm of chromosome 17, position 23 (17q23), and include 26 exons and 25 introns (Figure 5) [23].
The ACE gene location [29].
There are several possible mutations of this gene which initiate cardiovascular disease. The most common one is represented by deletions and insertions in the 16th intron (presence or absence of the 287-bp Alu repeat sequence), resulting in three possible genotypes, identified by the length of the fragments: II (490 bp), ID (490, 190 bp) and DD (190 bp) (Figure 6).
ACE genotypes: II (490 bp), ID (490, 190 bp) and DD (190 bp) [30].
Mutations are determined by collecting 2 ml of blood from the patients, extracting the DNA from the leukocytes, followed by amplification of the genetic material, agarose gel electrophoresis in order to separate the fragments and UV light identification of coloured fragments according to their length.
Those genotypes interact with the conventional environmental factors and influence the severity of the coronary artery disease. Most of the studies have demonstrated that the D allele is a risk factor, while the I allele is a protective factor. This hypothesis was first formulated by Cambien et al., in the study published in Circulation in 1994 [24]. They postulated that D allele is correlated with high plasma levels of ACE and that genotypes ID and DD increase the predisposition for myocardial infarction. Since then, this theory was largely debated. Guney et al. performed a study on 203 patients with acute coronary syndrome or positive functional tests, selected based on the severity of the stenosis (>70%), as revealed by the coronary angiography. The control group was formed of 140 patients with nonsignificant coronary stenosis. Results showed a higher prevalence of the D allele in the patient group than the control group (p = 0.002), in patients with hyperlipidaemia (p = 0.009) and smoking habit (p = 0.004), and a higher number of diseased vessel (two/three vessel diseases) (p = 0.002). The conclusion drawn was that D allele interacts with conventional risk factor and determines the degree of severity, since patients with D allele have higher plasma levels of ACE and are therefore more exposed to angiotensin II.
Dhar et al., in a study on 217 patients with coronary disease compared to 255 control patients (with negative Treadmill test), also found the D allele as an independent risk factor in coronary vessel disease (p < 0.05). Most of the ID and DD patients were heavy smokers, diabetic and dyslipidemic, suggesting the interaction between D allele and risk factors. Another association pointed out by other studies is that D allele is more frequent in obese patients. The high levels of angiotensin II might lead to disturbances in macronutrient oxidation, causing fat deposits and weight gain [25, 26]. Despite the fact that most of the studies focusing on this mutation were positive, results are still controversial, since there are a few studies which showed negative results. Poorgholi et al. concluded from their 1050 CAD patients’ study that I/D polymorphism is not a predisposant factor [27]. Fujimura et al. also found no association between ACE gene polymorphism and CAD in his study on 1840 patients (947 ischemic patients and 893 healthy controls) [28]. Although in some populations, there were no positive correlations between D allele and coronary disease; this may be due to geographical differences and ethnical differences. In general, D allele seems to be a predisposing factor, and probably the severity of the ischemic heart disease (infarction or unstable angina, one/two/three vessel diseases) depends on the interactions between this mutation and the other risk factors: hypertension, diabetes mellitus, obesity, smoking habit and dyslipidaemia. In most studies, patients with DD genotype were hospitalized for myocardial infarction and had multiple vessel disease and a high exposure to the conventional cardiovascular risk factors [31, 32]. The role of D allele in atherosclerosis was generally studied by measuring and analysing the carotid artery intima-media thickness (IMT). Results were statistically significant for high-risk patients, rather than low-risk patients. Therefore, D variant carriers have an increased risk of atherosclerosis, but only if they are also exposed to other genetic or environmental risk factors [33].
The association between ACE gene mutations and hypertension is explained by high plasmatic levels of ACE and a consequently increased quantity of ANG II in D-allele carriers. However, the predisposition for preeclampsia in D-allele women is less understood, and it is just a hypothesis still under debate. Preeclampsia implies high blood pressure and proteinuria after 20 weeks of pregnancy. It is the consequence of many risk factors, both genetic and environmental, and the RAAS may play a key role, since inactivating this system leads to a failure in achieving the hypervolemic status needed during pregnancy [34]. The studies on pregnant women are rare and often very small, making the available data on this issue very inconsistent. Some evidence suggests that it may come as a result of an abnormal regulation of the RAAS system and high intrauterine artery resistance [35]. Abedin et al., in a study on 296 patients, found no association between I/D polymorphisms but demonstrated a positive correlation with another ACE gene mutation, ACE rs4343, which was significantly present in the case group versus control group (p = 0.0001) [36]. On the other hand, a large meta-analysis including 45 studies found that DD and ID pregnant women have a higher risk of pregnancy-induced hypertension than II patients (74% D allele in the case group versus 56% D allele in the control group) [37]. Still, the large heterogeneity of the included patients lowers the force of the obtained results.
Another line of study in this field is represented by the involvement of RAAS system in the development of hypertrophic cardiomyopathy. This condition requires an anterior interventricular septum larger than 13 mm or a posterior interventricular septum larger than 15 mm, in the absence of hypertension and valvular disease. In most cases, hypertrophy is asymmetric. The implication of sarcomere protein gene mutations in the physiopathology of this disease is widely acknowledged. Later studies bring into attention a new puzzle piece in this genetic disorder, suggesting that ACE gene may also be involved, since the inhibition of ACE reduces cardiac hypertrophy and remodellation post-myocardial infarction [38, 39]. Some studies suggest that ACE gene mutations explain the intrafamilial phenotype differences in hypertrophic cardiomyopathy, which is considered a monogenic disease [40].
Genetic background often explains the different response to medication of patients with the same medical conditions.
“Sodium sensitive” hypertensive patients have certain physiopathological particularities: enhanced activity of the sympathetic nervous system, reduced renal excretion of sodium, hypersensitivity to vasoconstrictive hormones (adrenaline, ANG II) and alterations of the counteracting atrial natriuretic peptide (ANP) [41]. The connection between salt and hypertension is still under investigation, and it has not been fully explained [42], although it is clear that for some hypertensive patients, the mean blood pressure is higher when salt ingestion is increased. Studies focusing on determining the frequency of sodium sensitivity in hypertensive patients revealed the following distribution: 51% high sodium sensitivity, 33% intermediate sensitivity and 16% sodium resistance [43]. Patients with D allele display a higher sensitivity to dietary sodium intake, and therefore the risk of developing hypertension in those patients is increased, when high-sodium/low-potassium diet is associated. However, when the diet is low in sodium, the risk between presence and absence of D allele seems to be the same [44]. ACE inhibitors represent a gold standard therapy for cardiovascular and renal disease. But almost half of the hypertensive patients respond to ACE inhibitors alone. Most patients require multiple drug associations. Previous studies analysed the role of ACE gene polymorphisms in modulating treatment response in different populations around the world, and the results are still inconsistent. Heidari et al. in their study on 72 newly diagnosed hypertensive patients revealed a stronger response to enalapril and lisinopril in the DD genotype than ID (p = 0.03) and II (p = 0.001) [45]. Arnett et al. demonstrated a greater response to hydrochlorothiazide in hypertensive women during a 4-week study [46]. Results are still controversial. Other studies found no relationship between RAAS inhibitory treatment and genetic polymorphisms. For example, the study performed by Millions et al. did not link the D allele with a supplementary reduction of blood pressure during the treatment with ACE inhibitors [47]. Despite the results of some important trials stating that DD patients have a better response to ACE inhibitors, those results turn out non-reproducible [48]. Moreover, the pharmacogenomic analysis from PROGRESS study (5685 patients) based on the neuroprotective effect of perindopril after stroke revealed no implication of the ACE polymorphism on the end points [49]. There is also some evidence pointing out a different response to hydrochlorothiazide according to this polymorphism and sex: diuretic treatment was more efficient in II women and DD men [50]. Others suggested the synergism between II, ID and a-adducin Gly/Trp as an even better predictor of response to diuretic treatment [50]. The importance of the polymorphism in salt-sensitive population, the size of the study population and the ethnic variability may partially account for those differences. The differences in the response to ACE inhibitors between Caucasians and Africans are overwhelming [51], and this definitely represents a solid motivation for further investigations in this field. As for the response to beta blockers, a very important piece in the hypertension puzzle, it seems that ACE gene mutations might also be involved. A randomized controlled trial in hypertensive patients treated with atenolol demonstrated a more important blood pressure decrease in patients with an AGT M 235 T or G-6A genotype. But those results were not reproducible [48].
Pharmacogenomics is a new medical field trying to achieve personalized treatments according to our genetic package, in an attempt to obtain better results in the treatment-resistant patients. Given that RAAS is a key system in cardiovascular haemodynamic, discovering mutations that explain individual differences in efficacy and toxicity of various drug categories, as well as designing treatment strategies taking genetic data in consideration, will certainly be a revolutionary breakthrough in the future, both for hypertension and coronary artery disease.
The blockade of RAAS has become a central therapeutic strategy for patients with cardiac pathology. The inhibition of RAAS system is realized with modulating drugs such as ACEi, angiotensin receptor blockers (ARBs) and mineralocorticoid receptor antagonists (MRA) [52, 53].
The direct renin inhibition is obtained upstream of the action of the converting enzyme and has been well-known for decades. This type of blockade prevents the generation of ANG I and subsequently ANG II. The most used drug is named aliskiren; it has been evaluated in two clinical heart failure trials: ASTRONAUT and ATMOSPHERE. These trials proved that aliskiren failed to reduce cardiovascular death or heart failure hospitalization, but it induced a significantly more important decrease from baseline in NT-proBNP levels [54].
One of the latest advances in RAAS is represented by the development of innovative AT1 receptor blockers which have a dual action, going beyond simple antagonism of the binding of ANG II. Similar to the classic ARBs, these molecules will act on the superfamily of G-protein-coupled receptors (GPCRs). When GPCRs are activated by an agonist, they will determine intracellular dissociation of a heterotrimeric G protein into G and G subunits. The result consists in inducing second messenger-mediated cellular responses. Other groups of proteins which induce specific signalling pathways in a manner that is independent of the protein G are represented by the 13-arrestins [52, 54].
Even though efficient blockade of RAAS system can be obtained at different levels, the cornerstone therapy remains the use of angiotensin-converting enzyme inhibitors (ACEi), ARBs and MRA. RAAS blockade combined with natriuretic peptides augmentation is considered to be a revolutionary treatment in heart failure patients.
The renin-angiotensin-aldosterone system and the natriuretic peptides have a yin/yang relationship that has a great potential in inducing beneficial effects, by influencing both systems. The desired effects of the RAAS blockade can be enhanced by augmenting the natriuretic peptides activity. Even though the monotherapy with neprilysin inhibitors has failed to prove efficiency in heart failure patients, the association with a RAAS blocker has overcome the disappointing results, and even more, it has proven to be one of the most promising therapies in patients with different types of heart failure [55, 56, 57].
The renin-angiotensin-aldosterone system is a key piece in the puzzle of cardiovascular homeostasis and disease. Therapeutical targeting of this system at different levels represents an important medical breakthrough. The observation that patients with the same condition and exposure to risk factors may have different disease evolutions and responses to treatment led to genetic studies and to the discovery of the importance of ACE D allele in the determinism of coronary artery disease, arterial hypertension and hypertrophic cardiomyopathy. Therefore, developing treatment strategies according to the specific genetic pattern of the patient is the next step in medical evolution.
The authors declare no conflict of interest.
The company was founded in Vienna in 2004 by Alex Lazinica and Vedran Kordic, two PhD students researching robotics. While completing our PhDs, we found it difficult to access the research we needed. So, we decided to create a new Open Access publisher. A better one, where researchers like us could find the information they needed easily. The result is IntechOpen, an Open Access publisher that puts the academic needs of the researchers before the business interests of publishers.
",metaTitle:"Our story",metaDescription:"The company was founded in Vienna in 2004 by Alex Lazinica and Vedran Kordic, two PhD students researching robotics. While completing our PhDs, we found it difficult to access the research we needed. So, we decided to create a new Open Access publisher. A better one, where researchers like us could find the information they needed easily. The result is IntechOpen, an Open Access publisher that puts the academic needs of the researchers before the business interests of publishers.",metaKeywords:null,canonicalURL:"/page/our-story",contentRaw:'[{"type":"htmlEditorComponent","content":"We started by publishing journals and books from the fields of science we were most familiar with - AI, robotics, manufacturing and operations research. Through our growing network of institutions and authors, we soon expanded into related fields like environmental engineering, nanotechnology, computer science, renewable energy and electrical engineering, Today, we are the world’s largest Open Access publisher of scientific research, with over 4,200 books and 54,000 scientific works including peer-reviewed content from more than 116,000 scientists spanning 161 countries. Our authors range from globally-renowned Nobel Prize winners to up-and-coming researchers at the cutting edge of scientific discovery.
\\n\\nIn the same year that IntechOpen was founded, we launched what was at the time the first ever Open Access, peer-reviewed journal in its field: the International Journal of Advanced Robotic Systems (IJARS).
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\\n\\nWe started by publishing journals and books from the fields of science we were most familiar with - AI, robotics, manufacturing and operations research. Through our growing network of institutions and authors, we soon expanded into related fields like environmental engineering, nanotechnology, computer science, renewable energy and electrical engineering, Today, we are the world’s largest Open Access publisher of scientific research, with over 4,200 books and 54,000 scientific works including peer-reviewed content from more than 116,000 scientists spanning 161 countries. Our authors range from globally-renowned Nobel Prize winners to up-and-coming researchers at the cutting edge of scientific discovery.
\n\nIn the same year that IntechOpen was founded, we launched what was at the time the first ever Open Access, peer-reviewed journal in its field: the International Journal of Advanced Robotic Systems (IJARS).
\n\n2004
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\n\n2006
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