Human PRCP was sequenced and cloned. It was noted that PRCP might have both exopeptidase and endopeptidase activities PRCP was found to have a modulatory role in the angiotensin II-

PRCP was purified from cell free extracts of Xanthomonas maltophilia, a bacteria species which is normally found in clinical

Overexpression of PRCP on Chinese hamster ovary cells was

Wang and associates described the functional significance of PRCP

Hooley and associates described a C-terminal extension in the PK

Wallingford and associates described that *Prcp*-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet. *Prcp* null mice were also resistant to high-fat diet-induced

Soisson and associates described the crystal structure of human

E112D polymorphism in the prolylcarboxypeptidase gene was found to be associated with blood pressure response to benazepril

Javerzat and associates described overexpression of PRCP during the intermediate phase of the chick chorio-allantoic membrane Recombinant PRCP (rPRCP) metabolized BK1-8 to BK1-7, whereas

PRCP is a 4OHTAM resistance factor in estrogen receptor-positive

PRCPgt/gt mice were found to be hypertensive and prothrombotic

PRCP was found to be active in human cerebrospinal fluid Overexpression of angiotensin type 2 (AT2) receptor in mouse coronary artery endothelial cells was found to increase expression

serine protease domain as a potential substrate for PRCP.

Human PRCP (EC <IP_ADDRESS>) was found in biological fluids

A novel and rapid radioassay for PRCP was developed

PRCP was found to be highly active in macrophages Watson and associates described localization of *Prcp* gene PRCP was found to be an endothelial cell prekallikrein activator Moreira and associates described the extracellular PRCP that

1971 1978

1981 1981 angiotensin III

specimens

reported

obesity

PRCP

activates prekallikrein

in the etiology of preeclampsia

in Chinese hypertensive patients

breast cancer cells

Table 1. Historical Perspective

rPRCP was ineffective in metabolizing BK 1-9

of PRCP, which may contribute to kinin release

appear at sites of inflammation

induced pulmonary vasoconstriction PRCP was found in monkey kidney

was found to contain PRCP

1990

1993

1994

1995 1995

2005

2006

2007

2009

2009

2009

2009

2010

2010 2010

2011

2011

To deal with proper delivery of blood to the tissues as well as many types of insults, including chemical and physical damage as well as infection, the cardiovascular system has evolved an intricate multilayer of specialized biochemical pathways. Multiple feedback mechanisms controlling blood flow with contrasting effects regulate normal vascular function, which have either stimulatory or inhibitory effects. These features enable the cardiovascular system to perform its tasks effectively. Of these regulatory feedback machinery, a few have multiple and redundant control mechanisms that tightly uphold cardiovascular functions. Among these regulatory feedback mechanisms within the mammalian cardiovascular system, we review the renin-angiotensin(Savinetskaia et al., 2008; Hayashi et al., 2010), kallikrein-kinin(Bryant and Shariat-Madar, 2009; Saxena et al., 2011), and pro-opiomelanocortin (POMC) (Wang et al., 2008) systems that communicate with each other. Although within each of these systems are surely a network of molecular interactions with other pathways, their interactions are not either compared or reviewed. However, special attention is given to the role of PRCP in health and disease.

#### **2.1 Renin-angiotensin system (RAS)**

The systemic RAS is a complex, but fairly well understood pathway(Perret-Guillaume et al., 2009; Molteni, 1982; Brown et al., 1983; Oishi et al., 1996; Renshaw et al., 2001; Hettinger et al., 2002; Volzke et al., 2005; Clerk et al., 2005). Although the physiological importance of the RAS for regulation of blood pressure and water homeostasis has been established(Fukuchi et al., 1973; Bing and Nielsen, 1973), emerging evidence suggests that the components of RAS are important players for maintenance and regulation of many physiological and pathophysiological processes, such as angiogenesis (Abali et al., 2002), inflammation (Akishita et al., 2001), tumorigenesis(Ager et al., 2008), thrombosis (Asselbergs et al., 2008; Asselbergs et al., 2007), vascular hypertrophy (Brede et al., 2001), vascular development (Lasaitiene et al., 2006), and remodeling (Akishita et al., 2000; Alcazar et al., 2009). The molecular mechanisms by which the components of RAS exert their pleiotropic effects are redundant in order to ensure an appropriate balance between the stimulatory and inhibitory signals favoring normal blood delivery. More interestingly, there is also a balance between RAS function and its counter-regulatory hormones, in particular plasma kinins, the main effectors of the plasma Kallikrein-Kinin System (KKS) (Shariat-Madar et al., 2002a). The active metabolites of these two systems regulate each other in an antagonistic manner. In view of the opposing effects of these two systems(Bader, 2001), the complex interplay of RAS and KKS with the local microenvironment of endothelial cells lining blood vessels is responsible for delivering nutrients. The RAS and KKS signaling pathways are also responsible for sustaining the high pressure necessary for the blood to reach all of the tissues of the body while maintaining blood pressure within a narrow range.

The RAS encompasses numerous peptides that act in concert in a cascade triggered by low blood volume or a drop in blood pressure cascades or water-fall(Taquini, Jr. and Taquini, 1961; Nicholls and Robertson, 2000; Shariat-Madar and Schmaier, 2004; Ritz, 2005; Oudart, 2005). These peptides exert their actions, which can be autocrine (Chan and Wong, 1996), paracrine (Haulica et al., 2005), or endocrine(Gohlke et al., 1992), acting through specific cell surface receptors on their target cells to control blood pressure and body fluid homeostasis. In a stepwise enzymatic process, the aspartyl protease renin (a rate-limiting enzyme) cleaves circulating angiotensinogen to the inactive angiotensin I (Ang I) (Helmer, 1965). Subsequently,

On the Mechanism of Action of Prolylcarboxypeptidase 259

Although AT2 appears to be cell-type specific with a low expression pattern in adult tissues, it is the dominate receptor in fetal tissues(Akishita et al., 1999; Guan et al., 2008). Experimental evidence in support of AT2-independent G-protein signaling pathway has been demonstrated (Bottari et al., 1991). AT2 receptors are essential for triggering vasodilation and it can also mediate cell death (Gwathmey et al., 2009). Ang II-induced AT2 receptor-dependent vasodilation is mediated by activation of nitric oxide/cyclic guanosine 3',5'-monophosphate (cGMP) signaling cascades. On the other hand, AT2 activation by Ang II may lead to the activation of proapoptotic second messengers, MAPK phosphatase 1 (MKP-1)(Horiuchi et al., 1997), SH2 domain-containing phosphatase 1 (SHP-1)(Feng et al., 2002), and protein phosphatase 2A (PP2A)(Huang et al., 1996) and subsequent inactivation of MAPKs(Bedecs et al., 1997; Horiuchi et al., 1997), and ERK1/2(Horiuchi et al., 1997). While identification and characterization of the molecular switch that controls differential activation of distinct downstream signaling pathways by Ang II are poorly understood, our

In the next step of the RAS cascades, angiotensin converting enzyme 2 (ACE2) and PRCP metabolize Ang II (a prothrombotic risk factor) to angiotensin 1-7 (a vasodilator, antithrombotic factor)(Mallela et al., 2008), highlighting the functional significance of these two enzymes. Notably, ACE2 (Gurley et al., 2006) and PRCP (Adams et al., 2011)null mice do not exhibit excessive elevated blood pressure compared to control littermates, suggesting that these enzymes play a critical role in the maintenance of normal vascular physiology, Figure 1. PRCP also metabolizes angiotensin III (Ang III, Ang2-8) to angiotensin 2-7 (Ang 2- 7)(Mallela et al., 2008). Ang III, like Ang II, is also a pressor agent whose response is mediated by AT1 receptors (Campbell et al., 1979). Ang III is implicated in cell proliferation and matrix accumulation(Ruiz-Ortega et al., 1998). Ang II- and Ang III-induced AT1 receptor stimulation mediate vasoconstriction, mitogenic and hypertrophic effects and inflammatory responses (Ishanov et al., 1998; Schluter and Wenzel, 2008; Marshall et al., 2000). Hydrolysis of Ang III by PRCP leads to a decrease in Ang III-mediated effects, further

pointing out the importance of PRCP in contributing to regulation of blood pressure.

Ang II is a proangiogenic hormone and AT1 mediates its proangiogenic actions(Wilop et al., 2009). The proangiogenic effect of Ang II leads to upregulation of EGF (Friedlander and Terzi, 2006; Lautrette et al., 2005), FGF (Liu et al., 2009), PDGF(Blaschke et al., 2002; Li et al., 2005), IGF-1 (Kajstura et al., 2001; Song et al., 2005(Jia et al., 2011), hepatocytes growth factor(Bataller et al., 2003), and NO(Abassi et al., 1997). Ang II-induced AT1 mediated signaling pathway cause an activation of mitogen-activated protein kinases (MAPK) (Guo et

The MAPK pathway induces cellular protein synthesis, volume regulation, gene expression, and growth (Morrell et al., 1999). In contrast, activation of AT1-independent G-protein signaling pathway by Ang II can also cause endothelial cell proliferation through non-receptor tyrosine kinase Src-mediated increase of NADP oxidase activation, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), P38 MAP kinase, and a decrease in phosphorylation of Src homology 2 (SH2)-containing inositol phosphatase 2 (SHIP2)(Ali et al., 1997b; Feng et al., 2002; Bedecs et al., 1997). These experimental observations suggest that Ang

**2.2 RAS and angiogenesis**

al., 2006; Morrell et al., 1999; Taniyama et al., 2004).

understanding of the downstream events is exponentially mounting.

angiotensin converting enzyme (ACE) cleaves the two C-terminal amino acid residues of Ang I to generate the key effector of RAS, angiotensin II (Ang II, Ang 1-8) under physiological conditions (Figure 1)(Peart, 1975). However, the tonin-, cathepsin G-, tissue plasminogen activator- or chymase-induced Ang II generation is apparently important under pathophysiological conditions (Doggrell and Wanstall, 2004; Doggrell and Wanstall, 2003; Leckie, 2005; Belova, 2000). Recent evidence suggests that Ang I can block a downstream protease, PRCP *in vitro* (Mallela et al., 2008). Up to now, the physiological significant role of Ang I in blocking PRCP is uncertain. Ang II primarily exerts its physiological effects by activating angiotensin type 1 (AT1) receptors (Figure 1) (Chassagne et al., 2000). However, angiotensin type 2 (AT2) receptors also appear to mediate Ang II effects (Figure 1)(Anavekar and Solomon, 2005). Activation of this signaling pathway leads to cardioprotection. Both AT1 and AT2 belong to the G-protein couple receptor (GPCR) superfamily(Ali et al., 1997a). While AT1 can couple to two members of the G family (Gq/11 and Gi) and to G complexes (Okuda et al., 1996), AT2 can couple to Gi and Gs subunits (Kai et al., 1996).

Fig. 1. Metabolites of the renin-angiotensin system influence cardiovascular system. **Ang I**; Angiotensin I, **Ang II**; Angiotensin II, **Ang III**; Angiotensin III, **Ang IV**; Angiotensin IV, Ang1-9; Angiotensin 1-9, Ang1-7; Angiotensin 1-7, **ACE;** Angiotensin converting enzyme, **ACE2;** Angiotensin converting enzyme 2, **AT1**; angiotensin type 1, **AT2**; angiotensin type 2, **AT4**; angiotensin type 4, **PAI-1**; plasminogen activator 1, **TF**; tissue activator, **Mas**; Ang1-7 Mas receptor, **NO**; nitric oxide, **PG**; prostaglandin.

Ang II-induced AT1 receptor-dependent vasoconstriction is mediated by activation of Gq/11, Gi, and G complexes(Kai et al., 1996). The activation of these heterotrimeric G proteins triggers the activation of phospholipase C (PLC)(Griendling et al., 1997) and phospholipase D (PLD)(Griendling et al., 1997; Ushio-Fukai et al., 1999), leading to an increase in intracellular calcium, actin-myosin interactions, and release of leukotrienes.

angiotensin converting enzyme (ACE) cleaves the two C-terminal amino acid residues of Ang I to generate the key effector of RAS, angiotensin II (Ang II, Ang 1-8) under physiological conditions (Figure 1)(Peart, 1975). However, the tonin-, cathepsin G-, tissue plasminogen activator- or chymase-induced Ang II generation is apparently important under pathophysiological conditions (Doggrell and Wanstall, 2004; Doggrell and Wanstall, 2003; Leckie, 2005; Belova, 2000). Recent evidence suggests that Ang I can block a downstream protease, PRCP *in vitro* (Mallela et al., 2008). Up to now, the physiological significant role of Ang I in blocking PRCP is uncertain. Ang II primarily exerts its physiological effects by activating angiotensin type 1 (AT1) receptors (Figure 1) (Chassagne et al., 2000). However, angiotensin type 2 (AT2) receptors also appear to mediate Ang II effects (Figure 1)(Anavekar and Solomon, 2005). Activation of this signaling pathway leads to cardioprotection. Both AT1 and AT2 belong to the G-protein couple receptor (GPCR) superfamily(Ali et al., 1997a). While AT1 can couple to two members of the G family (Gq/11 and Gi) and to G complexes

(Okuda et al., 1996), AT2 can couple to Gi and Gs subunits (Kai et al., 1996).

ACE2

**PRCP** ACE 2

**PRCP**

Angiotensinogen

Ang I

Ang IV

APN

Ang II

APA

ACE

AT2 Ang III

Ang 1-9

ACE 2

Ang1-7

Mas

NO, PG

• Vasodilation • Hypotension • Thromboprevention

Fig. 1. Metabolites of the renin-angiotensin system influence cardiovascular system. **Ang I**; Angiotensin I, **Ang II**; Angiotensin II, **Ang III**; Angiotensin III, **Ang IV**; Angiotensin IV, Ang1-9; Angiotensin 1-9, Ang1-7; Angiotensin 1-7, **ACE;** Angiotensin converting enzyme, **ACE2;** Angiotensin converting enzyme 2, **AT1**; angiotensin type 1, **AT2**; angiotensin type 2, **AT4**; angiotensin type 4, **PAI-1**; plasminogen activator 1, **TF**; tissue activator, **Mas**; Ang1-7

Ang II-induced AT1 receptor-dependent vasoconstriction is mediated by activation of Gq/11, Gi, and G complexes(Kai et al., 1996). The activation of these heterotrimeric G proteins triggers the activation of phospholipase C (PLC)(Griendling et al., 1997) and phospholipase D (PLD)(Griendling et al., 1997; Ushio-Fukai et al., 1999), leading to an increase in intracellular calcium, actin-myosin interactions, and release of leukotrienes.

Mas receptor, **NO**; nitric oxide, **PG**; prostaglandin.

AT4

• Vasoconstriction • Blood pressure • Thrombosis

AT1

TF, PAI-1

Although AT2 appears to be cell-type specific with a low expression pattern in adult tissues, it is the dominate receptor in fetal tissues(Akishita et al., 1999; Guan et al., 2008). Experimental evidence in support of AT2-independent G-protein signaling pathway has been demonstrated (Bottari et al., 1991). AT2 receptors are essential for triggering vasodilation and it can also mediate cell death (Gwathmey et al., 2009). Ang II-induced AT2 receptor-dependent vasodilation is mediated by activation of nitric oxide/cyclic guanosine 3',5'-monophosphate (cGMP) signaling cascades. On the other hand, AT2 activation by Ang II may lead to the activation of proapoptotic second messengers, MAPK phosphatase 1 (MKP-1)(Horiuchi et al., 1997), SH2 domain-containing phosphatase 1 (SHP-1)(Feng et al., 2002), and protein phosphatase 2A (PP2A)(Huang et al., 1996) and subsequent inactivation of MAPKs(Bedecs et al., 1997; Horiuchi et al., 1997), and ERK1/2(Horiuchi et al., 1997). While identification and characterization of the molecular switch that controls differential activation of distinct downstream signaling pathways by Ang II are poorly understood, our understanding of the downstream events is exponentially mounting.

In the next step of the RAS cascades, angiotensin converting enzyme 2 (ACE2) and PRCP metabolize Ang II (a prothrombotic risk factor) to angiotensin 1-7 (a vasodilator, antithrombotic factor)(Mallela et al., 2008), highlighting the functional significance of these two enzymes. Notably, ACE2 (Gurley et al., 2006) and PRCP (Adams et al., 2011)null mice do not exhibit excessive elevated blood pressure compared to control littermates, suggesting that these enzymes play a critical role in the maintenance of normal vascular physiology, Figure 1. PRCP also metabolizes angiotensin III (Ang III, Ang2-8) to angiotensin 2-7 (Ang 2- 7)(Mallela et al., 2008). Ang III, like Ang II, is also a pressor agent whose response is mediated by AT1 receptors (Campbell et al., 1979). Ang III is implicated in cell proliferation and matrix accumulation(Ruiz-Ortega et al., 1998). Ang II- and Ang III-induced AT1 receptor stimulation mediate vasoconstriction, mitogenic and hypertrophic effects and inflammatory responses (Ishanov et al., 1998; Schluter and Wenzel, 2008; Marshall et al., 2000). Hydrolysis of Ang III by PRCP leads to a decrease in Ang III-mediated effects, further pointing out the importance of PRCP in contributing to regulation of blood pressure.

#### **2.2 RAS and angiogenesis**

Ang II is a proangiogenic hormone and AT1 mediates its proangiogenic actions(Wilop et al., 2009). The proangiogenic effect of Ang II leads to upregulation of EGF (Friedlander and Terzi, 2006; Lautrette et al., 2005), FGF (Liu et al., 2009), PDGF(Blaschke et al., 2002; Li et al., 2005), IGF-1 (Kajstura et al., 2001; Song et al., 2005(Jia et al., 2011), hepatocytes growth factor(Bataller et al., 2003), and NO(Abassi et al., 1997). Ang II-induced AT1 mediated signaling pathway cause an activation of mitogen-activated protein kinases (MAPK) (Guo et al., 2006; Morrell et al., 1999; Taniyama et al., 2004).

The MAPK pathway induces cellular protein synthesis, volume regulation, gene expression, and growth (Morrell et al., 1999). In contrast, activation of AT1-independent G-protein signaling pathway by Ang II can also cause endothelial cell proliferation through non-receptor tyrosine kinase Src-mediated increase of NADP oxidase activation, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), P38 MAP kinase, and a decrease in phosphorylation of Src homology 2 (SH2)-containing inositol phosphatase 2 (SHIP2)(Ali et al., 1997b; Feng et al., 2002; Bedecs et al., 1997). These experimental observations suggest that Ang

On the Mechanism of Action of Prolylcarboxypeptidase 261

birth, FXI levels are reached to normal adult plasma levels by six months of age. FXI circulates in the plasma as a disulfide bond-linked dimer complexed with HK. It is important for propagation of blood coagulation(Walsh and Griffin, 1981). FXI can be activated to activated FXI (FXIa) by FXIIa-mediated activation on the negatively charged surfaces or thrombinmediated activation on the platelet surface(Baglia et al., 2004), Figure 2. FXIa augments thrombin generation via interaction with a coagulation factor of the extrinsic pathway, factor IX(Baglia and Walsh, 1998; Taketomi et al., 2008). It appears that FXI along with activated platelet are a prerequisite for optimum thrombin generation in which the procoagulant activity

Plasma KKS activation has been demonstrated under physiological conditions and in numerous inflammatory human diseases. Under pathological conditions, conversion of plasma factor XII (FXII) to its active form (FXIIa) is concomitant with the appearance of hematologic responses including coagulation, fibrinolytic, and inflammation. FXIIa initiates propagation of the intrinsic pathway (IP) of blood coagulation via factor XI activation, Fig. **2** (1; Pathway 1). In the presence of HK, FXIIa converts plasma prekallikrein (PK) to kallikrein, Fig. **2** (2; Pathway 2). In a reciprocal feedback mechanism, kallikrein accelerates FXII activation. Kallikrein causes the release of tissue plasminogen activator (tPA) via a

Fig. 2. A schematic representation of factor XII (FXII) activation during pathological plasma proteolytic states. **FXIIa;** activated factor XII, 1; pathway 1, **FXI;** factor XI, **FXIa**; activated factor XI, 2; pathway 2, **HK;** high molecular weight kininogen, **PK;** prekallikrein, **BK;** bradykinin, **tPA;** tissue plasminogen activator, **FDP**; Fibrin degradation Products.

of KKS is so just below the threshold point (Keularts et al., 2001).

bradykinin-dependent pathway.

II is proangiogenic. However, evidence suggests that Ang II may inhibit angiogenesis under certain conditions by activating AT2-receptors, suggesting that AT1 mediates effects that counteract those mediated by AT2 receptors (Chung et al., 1996).

#### **2.3 RAS in cardiovascular disease**

Since Ang II, Ang III, Ang IV, and Ang 1-7 can act systemically and locally to control blood pressure and body fluid homeostasis, components of RAS have become therapeutic targets for diseases such as hypertension(Farmer, 2000), cardiac hypertrophy(Brasier et al., 2000; Billet et al., 2008), congestive heart failure(Volpe and De, 2000; Yang et al., 1998), ischemic heart disease(Burrell et al., 2005), coronary heart disease(Farmer and Torre-Amione, 2001), and renal diseases.

#### **3. Plasma kallikrein kinin system (KKS)**

The primary functions of plasma KKS are to 1) assist hemostasis and limit blood loss, 2) increase capillary permeability and dilate arterioles, 3) promote antiatherogenic and antithrombogenic properties of endothelium by maintaining or increasing antiatherogenic agents including nitric oxide, prostaglandins, and tissue plasminogen activator, and 4) induce acute vascular pain. In addition to its hemodynamic actions, antifibrotic and renoprotective effects of kallikrein have been reported (Pawluczyk et al., 2008). Although the expression and activity of components of plasma KKS are known to be age-dependent (Gordon et al., 1980) for three decades, the mechanism underlying this phenomenon is yet to be unravelled.

The components of KKS, also called the contact activation pathway, consist of three serine zymogens (prekallikrein, factor XII, and factor XI) as well as a kinin and antiangiogenic precursor (high molecular weight kininogen)(Figure 2)(Ratnoff and Saito, 1979). Plasma kallikrein, a serine protease, is synthesized in the liver. It is predominantly secreted by hepatocytes as an inactive molecule called prekallikrein (PK) that circulates in plasma as a heterodimer complex bound to high molecular weight kininogen (HK) with 1:1 molar stoichiometry. PK is a single chain +-globulin that is present in the plasma of humans and of other animal species. About 80-90% of PK is normally in complexed with HK.

HK is a single-chained glycoprotein synthesised by liver and secreted into the circulation (Sueyoshi et al., 1987). HK consists of three domains: an amino acid-terminal heavy chain, a carboxyl-terminal light chain, and bradykinin moiety (Figure 3)(Kleniewski et al., 1988). The heavy chain linked to the light chain by a single disulfide bond (Kleniewski et al., 1988). Domain 6 of HK has a PK and FXI-binding site (Reddigari and Kaplan, 1989). HK binding to cell surface proteins or artificial surfaces is vital for activation of the plasma KKS(Pixley et al., 2011; Joseph et al., 2001; Zhao et al., 2001). The procoagulant activity of HK is dependent on whether it is in complexed with PK or FXI (Gailani and Broze, Jr., 1991). The proteolytic cleavage of HK by kallikrein results in the production of BK and cleaved HK (HKa). HK could be metabolized by activated factor XI into three peptide fragments, yet its physiological significance has been unclear. HK is involved in angiogenesis and in cell adhesion and the matrix of various endothelial cells (Motta et al., 2001).

Unlike PK, factor XI (FXI) is involved in normal hemostasis *in vivo*. FXI synthesis is primarily in the liver. Factor XI activity is age-dependent (Andrew et al., 1987). While its activity is low at

II is proangiogenic. However, evidence suggests that Ang II may inhibit angiogenesis under certain conditions by activating AT2-receptors, suggesting that AT1 mediates effects that

Since Ang II, Ang III, Ang IV, and Ang 1-7 can act systemically and locally to control blood pressure and body fluid homeostasis, components of RAS have become therapeutic targets for diseases such as hypertension(Farmer, 2000), cardiac hypertrophy(Brasier et al., 2000; Billet et al., 2008), congestive heart failure(Volpe and De, 2000; Yang et al., 1998), ischemic heart disease(Burrell et al., 2005), coronary heart disease(Farmer and Torre-Amione, 2001),

The primary functions of plasma KKS are to 1) assist hemostasis and limit blood loss, 2) increase capillary permeability and dilate arterioles, 3) promote antiatherogenic and antithrombogenic properties of endothelium by maintaining or increasing antiatherogenic agents including nitric oxide, prostaglandins, and tissue plasminogen activator, and 4) induce acute vascular pain. In addition to its hemodynamic actions, antifibrotic and renoprotective effects of kallikrein have been reported (Pawluczyk et al., 2008). Although the expression and activity of components of plasma KKS are known to be age-dependent (Gordon et al., 1980) for

The components of KKS, also called the contact activation pathway, consist of three serine zymogens (prekallikrein, factor XII, and factor XI) as well as a kinin and antiangiogenic precursor (high molecular weight kininogen)(Figure 2)(Ratnoff and Saito, 1979). Plasma kallikrein, a serine protease, is synthesized in the liver. It is predominantly secreted by hepatocytes as an inactive molecule called prekallikrein (PK) that circulates in plasma as a heterodimer complex bound to high molecular weight kininogen (HK) with 1:1 molar stoichiometry. PK is a single chain +-globulin that is present in the plasma of humans and of

HK is a single-chained glycoprotein synthesised by liver and secreted into the circulation (Sueyoshi et al., 1987). HK consists of three domains: an amino acid-terminal heavy chain, a carboxyl-terminal light chain, and bradykinin moiety (Figure 3)(Kleniewski et al., 1988). The heavy chain linked to the light chain by a single disulfide bond (Kleniewski et al., 1988). Domain 6 of HK has a PK and FXI-binding site (Reddigari and Kaplan, 1989). HK binding to cell surface proteins or artificial surfaces is vital for activation of the plasma KKS(Pixley et al., 2011; Joseph et al., 2001; Zhao et al., 2001). The procoagulant activity of HK is dependent on whether it is in complexed with PK or FXI (Gailani and Broze, Jr., 1991). The proteolytic cleavage of HK by kallikrein results in the production of BK and cleaved HK (HKa). HK could be metabolized by activated factor XI into three peptide fragments, yet its physiological significance has been unclear. HK is involved in angiogenesis and in cell

Unlike PK, factor XI (FXI) is involved in normal hemostasis *in vivo*. FXI synthesis is primarily in the liver. Factor XI activity is age-dependent (Andrew et al., 1987). While its activity is low at

three decades, the mechanism underlying this phenomenon is yet to be unravelled.

other animal species. About 80-90% of PK is normally in complexed with HK.

adhesion and the matrix of various endothelial cells (Motta et al., 2001).

counteract those mediated by AT2 receptors (Chung et al., 1996).

**2.3 RAS in cardiovascular disease**

**3. Plasma kallikrein kinin system (KKS)**

and renal diseases.

birth, FXI levels are reached to normal adult plasma levels by six months of age. FXI circulates in the plasma as a disulfide bond-linked dimer complexed with HK. It is important for propagation of blood coagulation(Walsh and Griffin, 1981). FXI can be activated to activated FXI (FXIa) by FXIIa-mediated activation on the negatively charged surfaces or thrombinmediated activation on the platelet surface(Baglia et al., 2004), Figure 2. FXIa augments thrombin generation via interaction with a coagulation factor of the extrinsic pathway, factor IX(Baglia and Walsh, 1998; Taketomi et al., 2008). It appears that FXI along with activated platelet are a prerequisite for optimum thrombin generation in which the procoagulant activity of KKS is so just below the threshold point (Keularts et al., 2001).

Plasma KKS activation has been demonstrated under physiological conditions and in numerous inflammatory human diseases. Under pathological conditions, conversion of plasma factor XII (FXII) to its active form (FXIIa) is concomitant with the appearance of hematologic responses including coagulation, fibrinolytic, and inflammation. FXIIa initiates propagation of the intrinsic pathway (IP) of blood coagulation via factor XI activation, Fig. **2** (1; Pathway 1). In the presence of HK, FXIIa converts plasma prekallikrein (PK) to kallikrein, Fig. **2** (2; Pathway 2). In a reciprocal feedback mechanism, kallikrein accelerates FXII activation. Kallikrein causes the release of tissue plasminogen activator (tPA) via a bradykinin-dependent pathway.

Fig. 2. A schematic representation of factor XII (FXII) activation during pathological plasma proteolytic states. **FXIIa;** activated factor XII, 1; pathway 1, **FXI;** factor XI, **FXIa**; activated factor XI, 2; pathway 2, **HK;** high molecular weight kininogen, **PK;** prekallikrein, **BK;** bradykinin, **tPA;** tissue plasminogen activator, **FDP**; Fibrin degradation Products.

On the Mechanism of Action of Prolylcarboxypeptidase 263

Under physiological conditions, PK bound to HK on endothelial cells can lead to PK activation through a FXIIa-independent mechanism, which in turn leads to BK generation. While assembly and activation of the plasma KKS is regulated by PRCP on several cultured cell lines, heat shock protein 90 (HSP90) is the main activator of the plasma PK in mesothelium-derived cell lines (Varano, V et al., 2011). Thus, the plasma KKS participates in both surface-dependent activation of blood clotting and inflammatory process. Further, PRCP participates in cell surface – dependent activation of PK and its dependent pathways. The question remains how PRCP-dependent PK signaling pathway is assembled, activated, and regulated in response to plasma levels of Ang II and/or Ang III. However, in an elegant study, the authors find evidence for a cross-talk among the kinin, PRCP, and angiotensin

Whereas the normally functioning KKS can successfully counteract blood pressure, promote smooth blood flow, modulates neovascularisation and eliminates pathogens by promoting the recruitment of neutrophils to the site of injury, inappropriate responses can lead to extensive tissue damage. KKS has been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, and coagulopathy. Increased BK levels is a hallmark in all of these cases. In some cases, the persistent production of BK due to the deficiency of the blood protein C1-inhibitor, which controls kallikrein and activated FXII, is detrimental to the survival of the patients with hereditary angioedema (HAE). In others, the inability of angiotensin converting enzyme (ACE) to degrade BK leads to elevated BK levels and edema

Various cancers exhibit upregulation of either epidermal growth factor or urokinase receptor or both, which leads to poor prognosis. On studies done on human prostate cancer cells (DU145) the cleaved fragment of HK, HKa and also domain 5 prevented colocalization of uPAR and EGFR, decreased downstream extracellular signal-regulated kinase (ERK) and AKT-mediated signal transduction at EGFR(Wuepper and Cochrane, 1972). This indicates therapeutic potential of HKa and D5 to inhibit metastasis involving migration and invasion

Both HK and LK are proangiogenic as demonstrated by induction of vascularization on chorioallantoic membrane. Monoclonal antibody directed against HK has been shown to inhibit HK-induced neovascularization on chorioallatoic membrane as well as HK-induced fibrosarcoma formation. Domain 5 of HK, also called kininostatin, is antiangiogenic(Colman et al., 2000). There are reports that peptides derived from domain 3 and domain 5 inhibit angiogenesis in Zn2+ independent and dependent manner (Zhang et al., 2002). Antibody

Like HK, BK also is proangiogenic and a reduction in its levels may be associated with impaired neovascularization(Krankel et al., 2008). But HKa is antiangiogenic as it prevents VEGF and FGF2 induced vascularization. HKa through its D5 domain has been shown to inhibit sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) mediated endothelial cell migration by modifying PI3 kinase-Akt signaling (Katkade et al.,

directed against domain 5 also prevents neovascularization(Song et al., 2004).

signaling pathways(Zhu et al., 2010).

in patients on ACE inhibitors.

of human prostate cancer cells.

**3.1 The plasma KKS and cardiovascular diseases**

**3.2 The importance of KKS for angiogenesis**

Fibrinolysis is the process by which plasmin digests fibrin, Fig. **2**. This aids in thrombus (clot) dissolution and limits the promotion of coagulation (clot formation). tPA converts plasma plasminogen to plasmin, which in turn remodels the thrombus. Plasmin breaks down the clot fibrin to fibrin degradation products (FDP). Consequently, FDP blocks thrombin-induced fibrin (clot) formation, Figure 2. Notably, plasmin triggers a positive feedback amplification by accelerating conversion of PK to kallikrein.

Kallikrein acts on HK to generate HKa (cleaved HK) and bradykinin (BK), Figure 3. BKinduced activation of the B2 (B2) receptor leads to the formation of tissue plasminogen activator (tPA, a potent fibrinolytic molecule)(Czokalo-Plichta et al., 2001), Fig. **3**. BK upon activation of its constitutive B2 receptors on endothelial cells leads to an increase in intracellular Ca2+ levels and subsequent production of nitric oxide (NO, a vasodilator) and prostacyclin (PGI2, a platelet activation inhibitor), ultimately leading to vasodilation, increased vascular permeability and edema (Zhao et al., 2001b; Hong, 1980; Palmer et al., 1987). Whereas B2 receptors are constitutively expressed, the expression of bradykinin B 1 (B1) receptors is induced during inflammation (Marceau, 1995). Metabolism of BK by carboxypeptidase N (CPN) in plasma or carboxypeptidase M (CPM) on endothelial cells yields des-Arg9-BK which interacts with B1 receptors to potentiate and/or perpetuate the inflammatory response (Figure 3)(Marceau et al., 1981).

Fig. 3. A schematic representation of PRCP-dependent prekallikrein activation. **HK**; high molecular weight kininogen**, PK;** prekallikrein, **PRCP**; prolylcarboxypeptidase, **K**; kallikrein, **BK1-9**; bradykinin, **HKa**; cleaved HK, **BK**; bradykinin, **NO**; nitric oxide, **PGI2**; prostacyclin, **tPA**; tissue plasminogen activator, **CPN**; carbox peptidase N, **CPM**; Carboxypeptidase M, **BK1-8**; bradykinin 1-8 (des-Arg9-bradykinin), **BK1-7**; bradykinin 1-7.

Fibrinolysis is the process by which plasmin digests fibrin, Fig. **2**. This aids in thrombus (clot) dissolution and limits the promotion of coagulation (clot formation). tPA converts plasma plasminogen to plasmin, which in turn remodels the thrombus. Plasmin breaks down the clot fibrin to fibrin degradation products (FDP). Consequently, FDP blocks thrombin-induced fibrin (clot) formation, Figure 2. Notably, plasmin triggers a positive

Kallikrein acts on HK to generate HKa (cleaved HK) and bradykinin (BK), Figure 3. BKinduced activation of the B2 (B2) receptor leads to the formation of tissue plasminogen activator (tPA, a potent fibrinolytic molecule)(Czokalo-Plichta et al., 2001), Fig. **3**. BK upon activation of its constitutive B2 receptors on endothelial cells leads to an increase in intracellular Ca2+ levels and subsequent production of nitric oxide (NO, a vasodilator) and prostacyclin (PGI2, a platelet activation inhibitor), ultimately leading to vasodilation, increased vascular permeability and edema (Zhao et al., 2001b; Hong, 1980; Palmer et al., 1987). Whereas B2 receptors are constitutively expressed, the expression of bradykinin B 1 (B1) receptors is induced during inflammation (Marceau, 1995). Metabolism of BK by carboxypeptidase N (CPN) in plasma or carboxypeptidase M (CPM) on endothelial cells yields des-Arg9-BK which interacts with B1 receptors to potentiate and/or perpetuate the

Fig. 3. A schematic representation of PRCP-dependent prekallikrein activation. **HK**; high molecular weight kininogen**, PK;** prekallikrein, **PRCP**; prolylcarboxypeptidase, **K**; kallikrein, **BK1-9**; bradykinin, **HKa**; cleaved HK, **BK**; bradykinin, **NO**; nitric oxide, **PGI2**; prostacyclin, **tPA**; tissue plasminogen activator, **CPN**; carbox peptidase N, **CPM**;

Carboxypeptidase M, **BK1-8**; bradykinin 1-8 (des-Arg9-bradykinin), **BK1-7**; bradykinin 1-7.

feedback amplification by accelerating conversion of PK to kallikrein.

inflammatory response (Figure 3)(Marceau et al., 1981).

Under physiological conditions, PK bound to HK on endothelial cells can lead to PK activation through a FXIIa-independent mechanism, which in turn leads to BK generation. While assembly and activation of the plasma KKS is regulated by PRCP on several cultured cell lines, heat shock protein 90 (HSP90) is the main activator of the plasma PK in mesothelium-derived cell lines (Varano, V et al., 2011). Thus, the plasma KKS participates in both surface-dependent activation of blood clotting and inflammatory process. Further, PRCP participates in cell surface – dependent activation of PK and its dependent pathways. The question remains how PRCP-dependent PK signaling pathway is assembled, activated, and regulated in response to plasma levels of Ang II and/or Ang III. However, in an elegant study, the authors find evidence for a cross-talk among the kinin, PRCP, and angiotensin signaling pathways(Zhu et al., 2010).

#### **3.1 The plasma KKS and cardiovascular diseases**

Whereas the normally functioning KKS can successfully counteract blood pressure, promote smooth blood flow, modulates neovascularisation and eliminates pathogens by promoting the recruitment of neutrophils to the site of injury, inappropriate responses can lead to extensive tissue damage. KKS has been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, and coagulopathy. Increased BK levels is a hallmark in all of these cases. In some cases, the persistent production of BK due to the deficiency of the blood protein C1-inhibitor, which controls kallikrein and activated FXII, is detrimental to the survival of the patients with hereditary angioedema (HAE). In others, the inability of angiotensin converting enzyme (ACE) to degrade BK leads to elevated BK levels and edema in patients on ACE inhibitors.

#### **3.2 The importance of KKS for angiogenesis**

Various cancers exhibit upregulation of either epidermal growth factor or urokinase receptor or both, which leads to poor prognosis. On studies done on human prostate cancer cells (DU145) the cleaved fragment of HK, HKa and also domain 5 prevented colocalization of uPAR and EGFR, decreased downstream extracellular signal-regulated kinase (ERK) and AKT-mediated signal transduction at EGFR(Wuepper and Cochrane, 1972). This indicates therapeutic potential of HKa and D5 to inhibit metastasis involving migration and invasion of human prostate cancer cells.

Both HK and LK are proangiogenic as demonstrated by induction of vascularization on chorioallantoic membrane. Monoclonal antibody directed against HK has been shown to inhibit HK-induced neovascularization on chorioallatoic membrane as well as HK-induced fibrosarcoma formation. Domain 5 of HK, also called kininostatin, is antiangiogenic(Colman et al., 2000). There are reports that peptides derived from domain 3 and domain 5 inhibit angiogenesis in Zn2+ independent and dependent manner (Zhang et al., 2002). Antibody directed against domain 5 also prevents neovascularization(Song et al., 2004).

Like HK, BK also is proangiogenic and a reduction in its levels may be associated with impaired neovascularization(Krankel et al., 2008). But HKa is antiangiogenic as it prevents VEGF and FGF2 induced vascularization. HKa through its D5 domain has been shown to inhibit sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) mediated endothelial cell migration by modifying PI3 kinase-Akt signaling (Katkade et al.,

On the Mechanism of Action of Prolylcarboxypeptidase 265

also evidence that kinin or B2-receptor inhibitor administration increases renin secretion or decrease plasma renin levels in anesthetized rabbits, respectively(Chiu and Reid, 1997). This data plus the location of B2 receptors in different parts of the kidney and vascular endothelium suggests a role for the plasma KKS in the cardiovascular system (Schricker et

There have been numerous suggestions that kallikrein converts prorenin to renin and a few results suggest that RAS and KKS are interdependent and KKS regulates RAS activity (Sealey et al., 1979). In this report, the plasma prorenin was activated to renin *in vitro* by kallikrein when plasma acidity increased (Sealey et al., 1979). Later, Purdon and colleagues evaluated the effect of kallikrein converting prorenin to renin in plasma deficient in C1 inhibitor (hereditary angioedema) at neutral pH(Purdon et al., 1985). Although PK was completely activated to kallikrein and despite normal prorenin concentration, prorenin was not converted to an active enzyme. Regardless of kallikrein metabolic capability in converting prorenin to renin, studies suggest the presence of a crosstalk between the

In summary, the increased activity of RAS could be responsible both for arterial wall constriction and KKS activation and subsequent BK production to relief vasoconstriction. This explains the previous finding when the pressure is raised in the perfused heart muscle and brain, the flow can be held constant independently of any external neurogenic control(Mosher et al., 1964). The regulation of tissue flow as perfusion pressure changes can be affected by the rate of delivery or removal of BK and Ang II to the perfused region, influencing the state of contraction of the smooth muscle cells of the blocked vessels directly. This signifies the importance of the feedback mechanism between KKS and RAS. These systems may serve as a "on-off" biological switch impacting the risk of thrombosis/bleeding

PRCP has a subtle role in preserving the blood flow through the endothelial lining of blood vessels so that the tissues can maintain cellular function. PRCP exerts this effect through regulation of local levels of autocrine-paracrine hormones such as angiotensin II (Ang II), angiotensin III (Ang III), and bradykinin generation via kallikrein-kinin pathway. Subsequently, the modification of these vasoactive substances influences the secretion or expression of a diverse group of endothelium mediators that are involved in the contraction and relaxation of smooth muscles, regulating blood flow, blood clotting, inhibition of platelet aggregation, and electrolyte homeostasis. It is well-established that dysfunction of

PRCP is a serine peptidase that cleaves peptides at C-terminal amino acids linked to a proline residue. The first evidence that PRCP might play a role in the cardiovascular system was provided by catalytic activity analysis, which showed the metabolism of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7)(Odya et al., 1978b). The mere inactivation of Ang II results in two events that broaden the functional spectrum of PRCP. First, it is a negative regulator of the pressor actions of the renin-angiotensin system (RAS). Second, PRCP

vasoconstricting RAS and the vasodilating KKS(Yokosawa et al., 1979).

endothelium significantly contributes to the development of thrombosis.

and those of low/high blood pressure.

**6.1 Physiology**

**6. Prolylcarboxypeptidase or angiotensinase C**

al., 1995).

2005). HKa acting through uPAR can also bring about apoptosis in endothelial cells, which is due to inhibition of uPA binding to uPAR thus preventing subsequent uPAR internalization and regeneration to the cell surface (Cao et al., 2004). This results in interference with all aspects of angiogenesis such as cell migration, proliferation, and survival.

Histidine proline rich glycoprotein (HPRG) is present in plasma abundantly and might be the predecessor of HK(Koide and Odani, 1987). HPRG is similar in sequence and function to HKa and is shown to bind to tropomyosin and inhibit angiogenesis through apoptosis (Donate et al., 2004).

### **4. Pro-opiomelanocortin (POMC)**

POMC is expressed in numerous mammalian tissues (Tatro et al., 1992; Tatro and Reichlin, 1987). It undergoes extensive posttranslational modification by a family of serine proteases and the prohormone convertases (PCs). The metabolism of POMC results in the generation of alpha-melanocyte stimulating hormone (+-MSH, -MSH1-13) along with -MSH, -MSH, and -endorphin (Shariat-Madar et al., 2010). Although all four of these hormones are important, only -MSH1-13 will be discussed here. The synthesis of +-MSH1-13 from POMC involves numerous specific enzymes in addition to PC1 and PC2. It appears that PRCP plays an important role in regulating -MSH.

#### **5. The interactions among KKS, RAS, and POMC**

The participation of the components of the plasma kallikrein-kinin system in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The availability of transgenic and knock-out mice for B2 and B1 receptors and angiotensinogen and angiotensin II receptors have advanced our understanding about the roles of KKS and specifically these receptors in cardiovascular regulation and inflammatory processes.

Bradykinin (BK) is the natural hypotensive agent that causes vasodilatation by stimulation of endothelial B2 receptors of arteries and arterioles, and the subsequent endothelial release of nitric oxide and prostaglandins(Sharma, 2009). BK influences the kidneys to produce diuresis and natriuresis (Katori and Majima, 2008; Katori and Majima, 2003). The diuretic effect of the exogenous BK administered by the renal artery is by B2 receptors (Boric et al., 1998; Croxatto et al., 1999), whereas the BK–induced natriuresis and increase in renal blood flow are due to both B1 and B2, Figure 3 (Lortie et al., 1992). Tissue injury such as myocardial ischemia and inflammation can cause the expression of B1 receptors on their surfaces (Foucart et al., 1997b; Foucart et al., 1997a). In addition, elevated concentrations of BK have been observed in ischemic myocardium(Burch and DePasquale, 1962; Burch and DePasquale, 1963). Occlusion of the proximal left anterior coronary artery causes a change in BK(Hashimoto et al., 1979; Kimura et al., 1973; Hashimoto et al., 1977). Exposure of the epicardium of anesthetized dogs to BK leads to stimulation of sympathetic afferent fibers protruding into the left ventricle(Uchida and Murao, 1974). Inhibition of the B2 receptor inhibitor resulted in increased kallikrein activity, but not kallikrein mRNA levels, in kidney of adult rats. There is a direct correlation between increased renal interstitial BK levels in dogs in low-sodium balance and the presence of B2 receptors(Siragy et al., 1997). There is

2005). HKa acting through uPAR can also bring about apoptosis in endothelial cells, which is due to inhibition of uPA binding to uPAR thus preventing subsequent uPAR internalization and regeneration to the cell surface (Cao et al., 2004). This results in interference with all aspects of angiogenesis such as cell migration, proliferation, and

Histidine proline rich glycoprotein (HPRG) is present in plasma abundantly and might be the predecessor of HK(Koide and Odani, 1987). HPRG is similar in sequence and function to HKa and is shown to bind to tropomyosin and inhibit angiogenesis through apoptosis

POMC is expressed in numerous mammalian tissues (Tatro et al., 1992; Tatro and Reichlin, 1987). It undergoes extensive posttranslational modification by a family of serine proteases and the prohormone convertases (PCs). The metabolism of POMC results in the generation of alpha-melanocyte stimulating hormone (+-MSH, -MSH1-13) along with -MSH, -MSH, and -endorphin (Shariat-Madar et al., 2010). Although all four of these hormones are important, only -MSH1-13 will be discussed here. The synthesis of +-MSH1-13 from POMC involves numerous specific enzymes in addition to PC1 and PC2. It appears that PRCP plays

The participation of the components of the plasma kallikrein-kinin system in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The availability of transgenic and knock-out mice for B2 and B1 receptors and angiotensinogen and angiotensin II receptors have advanced our understanding about the roles of KKS and specifically these receptors in cardiovascular regulation and inflammatory

Bradykinin (BK) is the natural hypotensive agent that causes vasodilatation by stimulation of endothelial B2 receptors of arteries and arterioles, and the subsequent endothelial release of nitric oxide and prostaglandins(Sharma, 2009). BK influences the kidneys to produce diuresis and natriuresis (Katori and Majima, 2008; Katori and Majima, 2003). The diuretic effect of the exogenous BK administered by the renal artery is by B2 receptors (Boric et al., 1998; Croxatto et al., 1999), whereas the BK–induced natriuresis and increase in renal blood flow are due to both B1 and B2, Figure 3 (Lortie et al., 1992). Tissue injury such as myocardial ischemia and inflammation can cause the expression of B1 receptors on their surfaces (Foucart et al., 1997b; Foucart et al., 1997a). In addition, elevated concentrations of BK have been observed in ischemic myocardium(Burch and DePasquale, 1962; Burch and DePasquale, 1963). Occlusion of the proximal left anterior coronary artery causes a change in BK(Hashimoto et al., 1979; Kimura et al., 1973; Hashimoto et al., 1977). Exposure of the epicardium of anesthetized dogs to BK leads to stimulation of sympathetic afferent fibers protruding into the left ventricle(Uchida and Murao, 1974). Inhibition of the B2 receptor inhibitor resulted in increased kallikrein activity, but not kallikrein mRNA levels, in kidney of adult rats. There is a direct correlation between increased renal interstitial BK levels in dogs in low-sodium balance and the presence of B2 receptors(Siragy et al., 1997). There is

survival.

processes.

(Donate et al., 2004).

**4. Pro-opiomelanocortin (POMC)**

an important role in regulating -MSH.

**5. The interactions among KKS, RAS, and POMC**

also evidence that kinin or B2-receptor inhibitor administration increases renin secretion or decrease plasma renin levels in anesthetized rabbits, respectively(Chiu and Reid, 1997). This data plus the location of B2 receptors in different parts of the kidney and vascular endothelium suggests a role for the plasma KKS in the cardiovascular system (Schricker et al., 1995).

There have been numerous suggestions that kallikrein converts prorenin to renin and a few results suggest that RAS and KKS are interdependent and KKS regulates RAS activity (Sealey et al., 1979). In this report, the plasma prorenin was activated to renin *in vitro* by kallikrein when plasma acidity increased (Sealey et al., 1979). Later, Purdon and colleagues evaluated the effect of kallikrein converting prorenin to renin in plasma deficient in C1 inhibitor (hereditary angioedema) at neutral pH(Purdon et al., 1985). Although PK was completely activated to kallikrein and despite normal prorenin concentration, prorenin was not converted to an active enzyme. Regardless of kallikrein metabolic capability in converting prorenin to renin, studies suggest the presence of a crosstalk between the vasoconstricting RAS and the vasodilating KKS(Yokosawa et al., 1979).

In summary, the increased activity of RAS could be responsible both for arterial wall constriction and KKS activation and subsequent BK production to relief vasoconstriction. This explains the previous finding when the pressure is raised in the perfused heart muscle and brain, the flow can be held constant independently of any external neurogenic control(Mosher et al., 1964). The regulation of tissue flow as perfusion pressure changes can be affected by the rate of delivery or removal of BK and Ang II to the perfused region, influencing the state of contraction of the smooth muscle cells of the blocked vessels directly. This signifies the importance of the feedback mechanism between KKS and RAS. These systems may serve as a "on-off" biological switch impacting the risk of thrombosis/bleeding and those of low/high blood pressure.

#### **6. Prolylcarboxypeptidase or angiotensinase C**

PRCP has a subtle role in preserving the blood flow through the endothelial lining of blood vessels so that the tissues can maintain cellular function. PRCP exerts this effect through regulation of local levels of autocrine-paracrine hormones such as angiotensin II (Ang II), angiotensin III (Ang III), and bradykinin generation via kallikrein-kinin pathway. Subsequently, the modification of these vasoactive substances influences the secretion or expression of a diverse group of endothelium mediators that are involved in the contraction and relaxation of smooth muscles, regulating blood flow, blood clotting, inhibition of platelet aggregation, and electrolyte homeostasis. It is well-established that dysfunction of endothelium significantly contributes to the development of thrombosis.

#### **6.1 Physiology**

PRCP is a serine peptidase that cleaves peptides at C-terminal amino acids linked to a proline residue. The first evidence that PRCP might play a role in the cardiovascular system was provided by catalytic activity analysis, which showed the metabolism of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7)(Odya et al., 1978b). The mere inactivation of Ang II results in two events that broaden the functional spectrum of PRCP. First, it is a negative regulator of the pressor actions of the renin-angiotensin system (RAS). Second, PRCP

On the Mechanism of Action of Prolylcarboxypeptidase 267

Over thirty years ago, Odya et al (Odya et al., 1978d) showed that PRCP can metabolize Ang III to Ang 2-7. Evidence indicates that Ang 2-7 excites some neurons in the paraventricular nuclease (Ambuhl et al., 1992). Differential regulation of Ang 2-7 during ACE inhibitor treatment suggests a cardiovascular role for this peptide (Campbell et al., 1991). Because of the significance of Ang 2-7, AngIII regulation and metabolism are important. Results from several studies show that Ang III is a pressor agent. It exerts its hypertensive effects via AT1 receptors (Felix et al., 1991b; Gammelgaard et al., 2006a). Ang III can enhance renal disease through the overproduction of aldosterone and subsequent development of arterial hypertension and/or to atrial fibrillation (Al-Aloul et al., 2006). Aldosterone which is produced by adrenal cortex maintains blood volume, pressure, and electrolyte balance. It is tightly regulated by renin, an enzyme produced in the juxtaglomerular apparatus of the kidneys. Renin production is elevated by low blood pressure, decreased blood flow to the kidneys or sodium deficiency. In theory, inactivation of Ang III by PRCP might lead to a decrease in blood pressure. However, further studies are needed before claims can be made

It is well known that cells produce and secrete a large variety of proteases either as proenzymes or activated proteases. Although PRCP can be viewed as a proenzyme based on its primary structure, its post-translational regulation has not been characterized. PRCP is distributed throughout endothelial cells, including lysosome, cytoplasm, and plasma membrane. The membrane bound PRCP activates plasma PK to kallikrein in endothelial cells. The activation of PK is characterized by elevated kallikrein expression along with increased local BK production. Kallikrein-induced BK production causes endothelial cells to produce proinflammatory prostaglandins and vasodilatory nitric oxide (Ngo et al., 2009). Thus, the PRCP-dependent PK activation pathway might be considered as an additional

Local skeletal muscle ischemia and acidosis are shown to increase the generation of BK and prostaglandins, the two circulating end-products of the PRCP-dependent pathways, Figure 3 (Piepoli et al., 2001)**.** The increased acidotic response during exercise and inflammatory mediators such as BK and prostacyclin have been shown to cause the abnormal exerciserelated symptoms and autonomic responses in congestive heart failure syndrome (Scott et al., 2003). Emerging evidence suggest that the long-term elevated concentrations of NO and prostacyclin may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease (Kai C.Wollert and Helmut Drexler, 2004). Needless to say, the reduction of endogenous PGI2 synthesis is associated with the occurrence of cyclic reduction of coronary flow(Tada et al., 1984). Thus, PRCP-dependent Ang1-7 and PRCPdependent BK pathways have important biological activities. Altered PRCP expression

PK is markedly elevated during pregnancy(Maki and Soga, 1981). Among proteases that metabolize neuropeptide Y 3-36 (NPY3-36, anorexigenic hormone), plasma kallikrein metabolizes NPY3-36 to NPY3-35(Abid et al., 2009). In addition, specific inhibitor of plasma kallikrein prevents the production of NPY3-35. We find these studies intriguing because elevation of kallikrein should provoke anorectic effect via accelerated inactivation of NPY3- 36. Recent evidence suggests that there is a link between severe prekallikrein deficiency and pregnancy loss (Dasanu and Alexandrescu, 2009). As mentioned earlier, PRCP activates PK to kallikrein. If PRCP is inhibited/down-regulated, kallikrein levels are lower. Thus,

mechanism to counteract the hypertensive activities of both Ang II and Ang III.

levels mediates pathophysiological changes in the cardiovascular system.

of a beneficial effect of PRCP on hypertension.

activity provides a measure of endothelium relaxation due to its ability to initiate nitric oxide generation through Mas receptor activation (Silva et al., 2007). PRCP metabolizes angiotensin III (Ang III) to angiotensin 2-7 (Ang 2-7)(Odya et al., 1978c). Ang III is a pressor agent whose response, like that of Ang II, is mediated by AT1 receptors(Felix et al., 1991a; Gammelgaard et al., 2006b). We described that when the complex of high molecular weight kininogen (HK) and prekallikrein (PK, Fletcher factor) binds to endothelium membrane, PK is rapidly converted to kallikrein by PRCP, Figure 3(Shariat-Madar et al., 2002b). The formed kallikrein then cleaves HK to liberate bradykinin (BK), which leads to vasodilation and subsequent nitric oxide and PGI2 formation by activating constitutive bradykinin B2 and inducible bradykinin B1 receptors (Zhao et al., 2001a; Colman and Schmaier, 1997). BK also causes release of tissue plasminogen activator (tPA, a profibrinolytic factor) to convert plasminogen to plasmin, which inhibits platelet aggregation, thrombus formation and promotes fibrinolysis, Figure 2(Pawluczyk et al., 2006). Thus, PRCP locally regulates nitric oxide-dependent vasodilation and indirectly modulates fibrinolysis within the vascular wall.

#### **6.2 Protein structure and molecular biology**

The human *Prcp* gene has been mapped to 11q14 and the gene is more than 147 kb in length and has 9 exons (Watson, Jr. et al., 1997). More recently, a new *Prcp*-like gene (*Prcp*2) has been discovered. The human Prcp gene family contains at least 2 genes. Both genes share important similarities including significant homology at the nucleotide level. Both genes encode for putative serine proteases. However, the functional role of *Prcp2* gene has not been characterized.

PRCP belongs to the single chain serine peptidase S28 family. It is derived from a larger precursor, prepro-PRCP, whose primary nucleotide structure was first described in a human cDNA clone by Tan et al., 1993. It appears that the precursor is homodimer polypeptides containing a secretory signal sequence. PRCP regulates the activity of bradykinin, Ang II, Ang III, prekallikrein, and -MSH(Odya et al., 1978a; Shariat-Madar et al., 2002c; Moreira et al., 2002; Wallingford et al., 2009), suggesting that PRCP has versatile roles in influencing cardiovascular system. The enzyme is ubiquitously present in all major areas of the vascular bed as well as in the kidney, heart, placenta, and hypothalamus (Jackman et al., 1995b; Skidgel et al., 1981; Rosenblum and Kozarich, 2003; Schmaier, 2003; Shariat-Madar et al., 2010). However, PRCP is highly produced in endothelial cells, which is one cell type involved in the release of vasodilators and antithrombotic mediators.

#### **6.3 PRCP in cardiovascular diseases**

PRCP and angiotensin converting enzyme 2 (ACE2) are exopeptidases, which convert Ang II to Ang 1-7. ACE2 metabolizes AngII at a much faster rate than PRCP (Rice et al., 2004), suggesting that Ang II is a poor substrate for PRCP. Clinical studies have provided reliable evidence that ACE2 is an essential regulator of angiotensin I (Ang I), Ang II and angiotensin-induced cardiac hypertrophy (Huentelman et al., 2005). Increased myocardial levels of Ang II concomitant with a significant decrease in Ang 1-7 in ACE2 deficient hearts has been reported (Kassiri et al., 2009). These data suggest that PRCP may indeed be important in metabolizing Ang II in the absence of ACE2. These observations suggest that PRCP is a redundant catalyst contributing to alternate pathways for Ang II metabolism.

activity provides a measure of endothelium relaxation due to its ability to initiate nitric oxide generation through Mas receptor activation (Silva et al., 2007). PRCP metabolizes angiotensin III (Ang III) to angiotensin 2-7 (Ang 2-7)(Odya et al., 1978c). Ang III is a pressor agent whose response, like that of Ang II, is mediated by AT1 receptors(Felix et al., 1991a; Gammelgaard et al., 2006b). We described that when the complex of high molecular weight kininogen (HK) and prekallikrein (PK, Fletcher factor) binds to endothelium membrane, PK is rapidly converted to kallikrein by PRCP, Figure 3(Shariat-Madar et al., 2002b). The formed kallikrein then cleaves HK to liberate bradykinin (BK), which leads to vasodilation and subsequent nitric oxide and PGI2 formation by activating constitutive bradykinin B2 and inducible bradykinin B1 receptors (Zhao et al., 2001a; Colman and Schmaier, 1997). BK also causes release of tissue plasminogen activator (tPA, a profibrinolytic factor) to convert plasminogen to plasmin, which inhibits platelet aggregation, thrombus formation and promotes fibrinolysis, Figure 2(Pawluczyk et al., 2006). Thus, PRCP locally regulates nitric oxide-dependent vasodilation and indirectly modulates fibrinolysis within the vascular

The human *Prcp* gene has been mapped to 11q14 and the gene is more than 147 kb in length and has 9 exons (Watson, Jr. et al., 1997). More recently, a new *Prcp*-like gene (*Prcp*2) has been discovered. The human Prcp gene family contains at least 2 genes. Both genes share important similarities including significant homology at the nucleotide level. Both genes encode for putative serine proteases. However, the functional role of *Prcp2* gene has not

PRCP belongs to the single chain serine peptidase S28 family. It is derived from a larger precursor, prepro-PRCP, whose primary nucleotide structure was first described in a human cDNA clone by Tan et al., 1993. It appears that the precursor is homodimer polypeptides containing a secretory signal sequence. PRCP regulates the activity of bradykinin, Ang II, Ang III, prekallikrein, and -MSH(Odya et al., 1978a; Shariat-Madar et al., 2002c; Moreira et al., 2002; Wallingford et al., 2009), suggesting that PRCP has versatile roles in influencing cardiovascular system. The enzyme is ubiquitously present in all major areas of the vascular bed as well as in the kidney, heart, placenta, and hypothalamus (Jackman et al., 1995b; Skidgel et al., 1981; Rosenblum and Kozarich, 2003; Schmaier, 2003; Shariat-Madar et al., 2010). However, PRCP is highly produced in endothelial cells, which is one cell type

PRCP and angiotensin converting enzyme 2 (ACE2) are exopeptidases, which convert Ang II to Ang 1-7. ACE2 metabolizes AngII at a much faster rate than PRCP (Rice et al., 2004), suggesting that Ang II is a poor substrate for PRCP. Clinical studies have provided reliable evidence that ACE2 is an essential regulator of angiotensin I (Ang I), Ang II and angiotensin-induced cardiac hypertrophy (Huentelman et al., 2005). Increased myocardial levels of Ang II concomitant with a significant decrease in Ang 1-7 in ACE2 deficient hearts has been reported (Kassiri et al., 2009). These data suggest that PRCP may indeed be important in metabolizing Ang II in the absence of ACE2. These observations suggest that PRCP is a redundant catalyst contributing to alternate pathways for Ang II metabolism.

involved in the release of vasodilators and antithrombotic mediators.

wall.

been characterized.

**6.2 Protein structure and molecular biology**

**6.3 PRCP in cardiovascular diseases**

Over thirty years ago, Odya et al (Odya et al., 1978d) showed that PRCP can metabolize Ang III to Ang 2-7. Evidence indicates that Ang 2-7 excites some neurons in the paraventricular nuclease (Ambuhl et al., 1992). Differential regulation of Ang 2-7 during ACE inhibitor treatment suggests a cardiovascular role for this peptide (Campbell et al., 1991). Because of the significance of Ang 2-7, AngIII regulation and metabolism are important. Results from several studies show that Ang III is a pressor agent. It exerts its hypertensive effects via AT1 receptors (Felix et al., 1991b; Gammelgaard et al., 2006a). Ang III can enhance renal disease through the overproduction of aldosterone and subsequent development of arterial hypertension and/or to atrial fibrillation (Al-Aloul et al., 2006). Aldosterone which is produced by adrenal cortex maintains blood volume, pressure, and electrolyte balance. It is tightly regulated by renin, an enzyme produced in the juxtaglomerular apparatus of the kidneys. Renin production is elevated by low blood pressure, decreased blood flow to the kidneys or sodium deficiency. In theory, inactivation of Ang III by PRCP might lead to a decrease in blood pressure. However, further studies are needed before claims can be made of a beneficial effect of PRCP on hypertension.

It is well known that cells produce and secrete a large variety of proteases either as proenzymes or activated proteases. Although PRCP can be viewed as a proenzyme based on its primary structure, its post-translational regulation has not been characterized. PRCP is distributed throughout endothelial cells, including lysosome, cytoplasm, and plasma membrane. The membrane bound PRCP activates plasma PK to kallikrein in endothelial cells. The activation of PK is characterized by elevated kallikrein expression along with increased local BK production. Kallikrein-induced BK production causes endothelial cells to produce proinflammatory prostaglandins and vasodilatory nitric oxide (Ngo et al., 2009). Thus, the PRCP-dependent PK activation pathway might be considered as an additional mechanism to counteract the hypertensive activities of both Ang II and Ang III.

Local skeletal muscle ischemia and acidosis are shown to increase the generation of BK and prostaglandins, the two circulating end-products of the PRCP-dependent pathways, Figure 3 (Piepoli et al., 2001)**.** The increased acidotic response during exercise and inflammatory mediators such as BK and prostacyclin have been shown to cause the abnormal exerciserelated symptoms and autonomic responses in congestive heart failure syndrome (Scott et al., 2003). Emerging evidence suggest that the long-term elevated concentrations of NO and prostacyclin may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease (Kai C.Wollert and Helmut Drexler, 2004). Needless to say, the reduction of endogenous PGI2 synthesis is associated with the occurrence of cyclic reduction of coronary flow(Tada et al., 1984). Thus, PRCP-dependent Ang1-7 and PRCPdependent BK pathways have important biological activities. Altered PRCP expression levels mediates pathophysiological changes in the cardiovascular system.

PK is markedly elevated during pregnancy(Maki and Soga, 1981). Among proteases that metabolize neuropeptide Y 3-36 (NPY3-36, anorexigenic hormone), plasma kallikrein metabolizes NPY3-36 to NPY3-35(Abid et al., 2009). In addition, specific inhibitor of plasma kallikrein prevents the production of NPY3-35. We find these studies intriguing because elevation of kallikrein should provoke anorectic effect via accelerated inactivation of NPY3- 36. Recent evidence suggests that there is a link between severe prekallikrein deficiency and pregnancy loss (Dasanu and Alexandrescu, 2009). As mentioned earlier, PRCP activates PK to kallikrein. If PRCP is inhibited/down-regulated, kallikrein levels are lower. Thus,

On the Mechanism of Action of Prolylcarboxypeptidase 269

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disruption of PRCP-dependent PK activation may be a risk factor for pregnancy losses. The inhibitors of both kallikrein and PRCP may be contraindicated during pregnancy. However, further investigations are required to define the safety of these inhibitors.

The activation of POMC is characterized by elevated -MSH1-13 production. -MSH1-13 regulates inflammation and food intake, suggesting that it may contribute to tissue damage production. Recently, experimental evidence indicated that PRCP metabolizes -MSH1-13 to -MSH1-12 (Wallingford et al., 2009). Mice deficient for PRCP present high blood pressure (Adams G.N et al., 2009), a sustained long-term reduction in food intake, and increased energy expenditure (Wallingford et al., 2009). In humans, hypertension is considered to be a multifactorial disorder. *Prcp* gene and its variants contribute to hypertension (Wang et al., 2006c; Zhang et al., 2009; Zhu et al., 2010). Rutaecarpine stimulates expression of PRCP in the circulation and small arteries in renovascular hypertensive rats (Qin et al., 2009). Since PRCP variant promotes disease progression (Zhang et al., 2009) in Chinese hypertensive patients and risk of preeclampsia in black and non-black women (Wang et al., 2006b), it is a candidate for pharmacological intervention.

#### **7. Conclusions and future directions**

In conclusion, PRCP is one of the conserved proteases throughout evolution in vertebrates. In-vitro and in-vivo studies of PRCP, this enzyme serves to generate nitric oxide and prostaglandins. Notably, bradykinin and bradykinin 1-8 are important mediators of pain (Argent et al., 1954) and endotoxin-induced vascular permeability {Ueno, 1995 783 /id}. BK production is increased in ischemic myocardial tissue (Dell'Italia and Oparil, 1999). They are involved in the control of local blood flow. Although PRCP is less efficient than ACE2 induced NO and prostaglandin generation under pathophysiological conditions, this redundant mechanism has presumably evolved for ischemic remodeling and smooth blood flow (Figure 1 and Figure 3). Overactivation of this mechanism may contribute to myocardial tissue damage or endothelial dysfunction. It inactivates BK1-8, suggesting that PRCP may also reduce marked inflammatory response. It appears that PRCP is fine-tuned to discriminate between physiological and pathophysiological changes. Since cardiomyocytes are unable to proliferate (Lagrand et al., 2000), inactivation of BK1-8 by PRCP may contribute to prevention of tissue damage. Further studies are warranted to evaluate the role of PRCP in cardiovascular system, in particular the heart.

#### **8. Acknowledgment**

This work was supported by National Institute of Health [NCRR/NIH P20RR021929] to ZSM.

#### **9. References**

disruption of PRCP-dependent PK activation may be a risk factor for pregnancy losses. The inhibitors of both kallikrein and PRCP may be contraindicated during pregnancy. However,

The activation of POMC is characterized by elevated -MSH1-13 production. -MSH1-13 regulates inflammation and food intake, suggesting that it may contribute to tissue damage production. Recently, experimental evidence indicated that PRCP metabolizes -MSH1-13 to -MSH1-12 (Wallingford et al., 2009). Mice deficient for PRCP present high blood pressure (Adams G.N et al., 2009), a sustained long-term reduction in food intake, and increased energy expenditure (Wallingford et al., 2009). In humans, hypertension is considered to be a multifactorial disorder. *Prcp* gene and its variants contribute to hypertension (Wang et al., 2006c; Zhang et al., 2009; Zhu et al., 2010). Rutaecarpine stimulates expression of PRCP in the circulation and small arteries in renovascular hypertensive rats (Qin et al., 2009). Since PRCP variant promotes disease progression (Zhang et al., 2009) in Chinese hypertensive patients and risk of preeclampsia in black and non-black women (Wang et al., 2006b), it is a

In conclusion, PRCP is one of the conserved proteases throughout evolution in vertebrates. In-vitro and in-vivo studies of PRCP, this enzyme serves to generate nitric oxide and prostaglandins. Notably, bradykinin and bradykinin 1-8 are important mediators of pain (Argent et al., 1954) and endotoxin-induced vascular permeability {Ueno, 1995 783 /id}. BK production is increased in ischemic myocardial tissue (Dell'Italia and Oparil, 1999). They are involved in the control of local blood flow. Although PRCP is less efficient than ACE2 induced NO and prostaglandin generation under pathophysiological conditions, this redundant mechanism has presumably evolved for ischemic remodeling and smooth blood flow (Figure 1 and Figure 3). Overactivation of this mechanism may contribute to myocardial tissue damage or endothelial dysfunction. It inactivates BK1-8, suggesting that PRCP may also reduce marked inflammatory response. It appears that PRCP is fine-tuned to discriminate between physiological and pathophysiological changes. Since cardiomyocytes are unable to proliferate (Lagrand et al., 2000), inactivation of BK1-8 by PRCP may contribute to prevention of tissue damage. Further studies are warranted to evaluate the role of PRCP

This work was supported by National Institute of Health [NCRR/NIH P20RR021929] to ZSM.

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further investigations are required to define the safety of these inhibitors.

candidate for pharmacological intervention.

**7. Conclusions and future directions**

in cardiovascular system, in particular the heart.

**8. Acknowledgment**

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down' by small interfering RNA transfection induces a profibrotic phenotype in rat

blockade: clinical evidence before the ONTARGET study. J. Hypertens. *27 Suppl 2*,

Colman, R.W. (2011). Interaction of high-molecular-weight kininogen with endothelial cell binding proteins suPAR, gC1qR and cytokeratin 1 determined by

weight kininogen recognizes its prekallikrein binding site and inhibits its coagulant

Fluorescent multiplex polymerase chain reaction analysis of four genes associated with inpaired fibrinolysis and myocardial infarction. Blood Coagul. Fibrinolysis *12*, 245-251. Ritz, E. (2005). The role of the kidney in cardiovascular medicine. Eur. J. Intern. Med. *16*,

of rennin-angiotensin-aldosterone system genes and NO synthase gene to the

renin gene expression by nitric oxide is counteracted by tonic inhibition through

plasma prorenin activation by Hageman factor-dependent conversion of plasma

Prolylcarboxypeptidase (PRCP) as a new target for obesity treatment. Diabetes

of prolylcarboxypeptidase as an endothelial cell prekallikrein activator. J Biol.

prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese

angiotensin systems in blood pressure regulation in sepsis. J. Endotoxin. Res. *10*, 3-13.

**14**

*Poland*

**Adolescent Obesity Predicts**

Jarosaw Derejczyk1, Barbara Kapci\$ska2, Ewa Sadowska-Kr#pa2, Olga St#pie\$-Wyrobiec1, El'bieta Kimsa2 and Katarzyna Kempa2

Rapid increase in the prevalence of obesity among children and adolescents has become a major worldwide health issue. Several large epidemiological studies have demonstrated that childhood and adolescent obesity is a significant independent predictor of metabolic disorders, such as hypertension, dyslipidemia and insulin resistance, which have a major impact on the premature development of atherosclerosis and cardiovascular morbidity and mortality in adulthood (Bibbins-Domingo et al., 2007; Dietz & Robinson, 2005; Franks et al., 2010). The cluster of metabolic disorders including abdominal obesity, insulin resistance, dyslipidemia and elevated blood pressure has been defined as metabolic syndrome (Alberti et al., 2006; Aggoun, 2007, Han & Lean, 2006). It is now generally accepted that overweight or obese children and adolescents are at increased risk for some or all of the metabolic syndrome (MS) features (Carnethon et al., 2004; Franks et al., 2010; Magnussen et al., 2010; Zimmet et al., 2007). The data from the Third National Health and Nutrition Examination Survey (NHANESIII, 1988-1994) have demonstrated that about 4 % of the whole population and almost 30% of the overweight or obese 12 to 19-y-old adolescents met the criteria of MS (Cook et al., 2003). The early occurrence of the MS in childhood and at the pubertal age was also found to have a major impact on the development of atherosclerosis, a life-time risk of cardiovascular disease (Aggoun, 2007), and an increased rate of premature death (Franks et al., 2010; Nieto, 1992). Excessive body weight in childhood and adolescence is considered a strong predictor of adult obesity (Wang et al. 2008) and obesity-related health consequences including diabetes and heart disease (Carnethon et al., 2004; Must et al., 1992; Morrison et al. 2007) regardless of whether the parents were obese. Notably, parental obesity was found to double the risk of

being obese in adulthood (Shengxu et al., 2003; Whitaker et al., 1997).

would help to implement prevention programs targeted at risk groups.

The main reason for undertaking the present study was to evaluate the prevalence of obesity among 15-y-old adolescents and to estimate the risk for adverse health outcomes in this population sample. Another goal was to find out relatively simple biochemical markers that could be suitable for the early diagnosis of metabolic disorders and cardiovascular risk, and

**1. Introduction**

**Cardiovascular Risk**

*1John Paul II Geriatric Hospital in Katowice, 2Department of Physiological and Medical Sciences,*

*Academy of Physical Education in Katowice*

### **Adolescent Obesity Predicts Cardiovascular Risk**

Jarosaw Derejczyk1, Barbara Kapci\$ska2, Ewa Sadowska-Kr#pa2,

Olga St#pie\$-Wyrobiec1, El'bieta Kimsa2 and Katarzyna Kempa2 *1John Paul II Geriatric Hospital in Katowice, 2Department of Physiological and Medical Sciences, Academy of Physical Education in Katowice Poland*

#### **1. Introduction**

274 Recent Advances in Cardiovascular Risk Factors

Volpe, M. and De, P.P. (2000). Angiotensin II AT2 subtype receptors: an emerging target for

Volzke, H., Kleine, V., Robinson, D.M., Grimm, R., Hertwig, S., Schwahn, C., Eckel, L., and

Wallingford, N., Perroud, B., Gao, Q., Coppola, A., Gyengesi, E., Liu, Z.W., Gao, X.B.,

intake by inactivating alpha-MSH in rodents. J. Clin. Invest *119*, 2291-2303. Walsh, P.N. and Griffin, J.H. (1981). Platelet-coagulant protein interactions in contact

Wang, L., Feng, Y., Zhang, Y., Zhou, H., Jiang, S., Niu, T., Wei, L.J., Xu, X., Xu, X., and Wang,

Wang, S.X., Fan, Z.C., and Tao, Y.X. (2008). Functions of acidic transmembrane residues in

Watson, B., Jr., Nowak, N.J., Myracle, A.D., Shows, T.B., and Warnock, D.G. (1997). The

Wilop, S., von, H.S., Crysandt, M., Esser, A., Osieka, R., and Jost, E. (2009). Impact of

Yang, B., Li, D., Phillips, M.I., Mehta, P., and Mehta, J.L. (1998). Myocardial angiotensin II receptor expression and ischemia-reperfusion injury. Vasc. Med. *3*, 121-130. Yokosawa, N., Takahashi, N., Inagami, T., and Page, D.L. (1979). Isolation of completely

Zhang Yan, Hong Xiu-mei, Hou-xun, Li Jian-ping, Huo Yong, and Xu Xi-ping (2009). E112D

Zhao, X., Southwick, K., Cardasis, H.L., Du, Y., Lassman, M.E., Xie, D., El-Sherbeini, M.,

Zhao, Y., Qiu, Q., Mahdi, F., Shariat-Madar, Z., Rojkjaer, R., and Schmaier, A.H. (2001).

Zhu, L., Carretero, O.A., Liao, T.D., Harding, P., Li, H., Sumners, C., and Yang, X.P. (2010).

a candidate gene for essential hypertension. Genomics *44*, 365-367.

between renin and kallikrein. Biochim. Biophys. Acta *569*, 211-219.

Rettig, R. (2005). Renin-angiotensin system and haemostasis gene polymorphisms and outcome after coronary artery bypass graft surgery. Int. J. Cardiol. *98*, 133-139.

Diament, A., Haus, K.A., Shariat-Madar, Z., Mahdi, F., Wardlaw, S.L., Schmaier, A.H., Warden, C.H., and Diano, S. (2009). Prolylcarboxypeptidase regulates food

X. (2006). Prolylcarboxypeptidase gene, chronic hypertension, and risk of

human melanocortin-3 receptor binding and activation. Biochem. Pharmacol. *76*,

human angiotensinase C gene (HUMPCP) maps to 11q14 within 700 kb of D11S901:

angiotensin I converting enzyme inhibitors and angiotensin II type 1 receptor blockers on survival in patients with advanced non-small-cell lung cancer undergoing first-line platinum-based chemotherapy. J. Cancer Res. Clin. Oncol. *135*,

inactive plasma prorenin and its activation by kallikreins. A possible new link

polymorphism in the prolylcarboxypeptidase gene is associated with blood pressure response to benazepril in Chinese hypertensive patients. Chinese Medical

Geissler, W.M., Pryor, K.D., Verras, A., Garcia-Calvo, M., Shen, D.M., Yates, N.A., Pinto, S., and Hendrickon, R.C. (2010). Peptidomic profiling of human cerebrospinal fluid identifies YPRPIHPA as a novel substrate for

Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation. Am. J. Physiol Heart Circ. Physiol *280*,

Role of prolylcarboxypeptidase in angiotensin II type 2 receptor-mediated bradykinin release in mouse coronary artery endothelial cells. Hypertension *56*,

cardiovascular therapy. Ital. Heart J. *1*, 96-103.

activation. Ann. N. Y. Acad. Sci. *370*, 241-252.

520-530.

1429-1435.

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H1821-H1829.

384-390.

prolylcarboxypeptidase. Proteomics.

preeclampsia. Am. J. Obstet. Gynecol. *195*, 162-171.

Rapid increase in the prevalence of obesity among children and adolescents has become a major worldwide health issue. Several large epidemiological studies have demonstrated that childhood and adolescent obesity is a significant independent predictor of metabolic disorders, such as hypertension, dyslipidemia and insulin resistance, which have a major impact on the premature development of atherosclerosis and cardiovascular morbidity and mortality in adulthood (Bibbins-Domingo et al., 2007; Dietz & Robinson, 2005; Franks et al., 2010). The cluster of metabolic disorders including abdominal obesity, insulin resistance, dyslipidemia and elevated blood pressure has been defined as metabolic syndrome (Alberti et al., 2006; Aggoun, 2007, Han & Lean, 2006). It is now generally accepted that overweight or obese children and adolescents are at increased risk for some or all of the metabolic syndrome (MS) features (Carnethon et al., 2004; Franks et al., 2010; Magnussen et al., 2010; Zimmet et al., 2007). The data from the Third National Health and Nutrition Examination Survey (NHANESIII, 1988-1994) have demonstrated that about 4 % of the whole population and almost 30% of the overweight or obese 12 to 19-y-old adolescents met the criteria of MS (Cook et al., 2003). The early occurrence of the MS in childhood and at the pubertal age was also found to have a major impact on the development of atherosclerosis, a life-time risk of cardiovascular disease (Aggoun, 2007), and an increased rate of premature death (Franks et al., 2010; Nieto, 1992). Excessive body weight in childhood and adolescence is considered a strong predictor of adult obesity (Wang et al. 2008) and obesity-related health consequences including diabetes and heart disease (Carnethon et al., 2004; Must et al., 1992; Morrison et al. 2007) regardless of whether the parents were obese. Notably, parental obesity was found to double the risk of being obese in adulthood (Shengxu et al., 2003; Whitaker et al., 1997).

The main reason for undertaking the present study was to evaluate the prevalence of obesity among 15-y-old adolescents and to estimate the risk for adverse health outcomes in this population sample. Another goal was to find out relatively simple biochemical markers that could be suitable for the early diagnosis of metabolic disorders and cardiovascular risk, and would help to implement prevention programs targeted at risk groups.

Adolescent Obesity Predicts Cardiovascular Risk 277

Concentrations of serum glucose, total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides (TG) were assessed by the enzymatic methods using commercially available diagnostic kits (BioMaxima cat.no. 1-033-0400, 1-023-0400, 1-029-0200, 1-053-0400, respectively). Concentrations of low-density lipoprotein cholesterol (LDL-C) were calculated from TC, HDL-C, and TG using the Friedewald formula ( Friedewald et al., 1972). Serum insulin was measured by the immunoradiometric method (Insulin IRMA IM3210, Immunotech SA, France), sensitivity was 2.0 mIU/ml, and autoantibodies against oxidized LDL (oLAb) in serum samples were evaluated using ELISA assay (oLAb-ELISA, BI-20032;

To evaluate risk for vascular disease , the lipid ratios (TC/HDL-C, LDL-C/HDL-C, TG/HDL-C) and atherogenic index of plasma [AIP= log10(TG/HDL-C) with TG and HDL-C expressed in molar concentrations] (Dobiášová & Frohlich, 2001, 2004, 2011) were calculated. The homeostatic model assessment (HOMA) was used to estimate insulin resistance according to the method described by Matthews (1998). Insulin resistance score (HOMA-IR) was computed using the following formula: HOMA-IR=glucose (mmol/L)\*insulin (mIU/L)/22.5, with the cut-off point for adolescents less than or equal to 3.16 , as suggested by Keskin et al. (2005). Moreover, in 231 individuals (121 boys and 110 girls) serum adiponectin was measured by an immunoenzymatic method (Human Adiponectin ELISA, BioVendor, cat. no. RD195023100), and serum insulin-like growth factor (IGF-1) was evaluated in 80 adolescents (40 boys and 40 girls) using a commercially available diagnostic kit (IGF-I ELISA, Immunodiagnostic Systems GmbH, sensitivity 3.1

All statistical analyses were performed with STATISTICA 7.1 software (StatSoft, Tulsa, OK, USA). The data reported as means ± SD were tested for homogeneity of variances by using the Levene test, then two-way ANOVA was performed to analyze the effect of gender and the degree of overweight/obesity on the studied variables, followed where appropriate, by the Tukey post-hoc comparisons. Spearman's rank order correlation analysis was performed to assess relationships between selected variables. Additionally, Pearson's Chi-square test of independence was used to evaluate the impact of the degree of overweight/obesity on SBP, serum triglycerides and surrogate markers of insulin resistance (HOMA-IR, TG/HDL-C). To predict the relative contribution of selected metabolic variables to prevalence of over normal SBP, insulin resistance (as assessed by HOMA-IR) and atherogenic potential of plasma (as assessed by AIP) a stepwise multiple regression analysis was applied. The level of

Physical characteristics of individual groups of the participants are presented in Table 1. In the evaluation of between-group differences, the data obtained in 113 adolescents (55 boys and 58 girls) with underweight, in 100 individuals (54 boys and 46 girls) with a tendency to overweight or overweight, and in 113 obese subjects (57 boys and 56 girls) were compared to the data obtained in 179 adolescents (98 boys and 81 girls) with normal weight. Mean

Biomedica GmbH, Wien, Austria) according to Tatzber & Esterbauer (1995).

/g/L, reference range for 12 to 15 y old subjects 142-525 /g/L).

significance of P<0.05 was chosen for all statistical comparisons.

**2.2 Analytical procedures**

**2.3 Statistics**

**3. Results**

### **2. Methods**

#### **2.1 Population sample**

The data were obtained from 505 adolescents, middle school pupils (264 boys and 241 girls) aged 15 years, who were enrolled into a cross- sectional health screening study undertaken by the municipal health care authorities of Katowice (Poland) - in collaboration with our group. In the morning, after an overnight fast, the participants reported to the hospital to undergo a brief general medical examination, including assessment of anthropometric parameters, measurement of blood pressure, and collection of venous blood samples for biochemical analyses. The participants, boys and girls separately, were classified into one of the four groups, according to their Body Mass Index (BMI) score, using the age and gender-specific cutoff points of the BMI percentiles established for 15-years old Polish youth by Palczewska & Nied&wiecka (2002): group A of the underweight (<25 c), group B of the normal weight (25- 75c), group C of the overweight (75-97 c), and group D of the obese (>97 c) individuals (Table 1). Criterion for diagnosis of obesity at BMI 97th percentile was consistent with the WHO references for children 5 to 19 years old (available at http://www.who.growthref). Informed consent was obtained from all adolescents and their parents prior to participation in the study, the protocol of which was approved by the local Ethics Committee.

Table 1. Anthropometric characteristics, and systolic (SBP) and diastolic (DBP) blood pressure of 15-year-old Polish adolescents. Significance of differences *vs.* normal weight individuals: \*p<0.05, \*\*\* p<0.001

#### **2.2 Analytical procedures**

276 Recent Advances in Cardiovascular Risk Factors

The data were obtained from 505 adolescents, middle school pupils (264 boys and 241 girls) aged 15 years, who were enrolled into a cross- sectional health screening study undertaken by the municipal health care authorities of Katowice (Poland) - in collaboration with our group. In the morning, after an overnight fast, the participants reported to the hospital to undergo a brief general medical examination, including assessment of anthropometric parameters, measurement of blood pressure, and collection of venous blood samples for biochemical analyses. The participants, boys and girls separately, were classified into one of the four groups, according to their Body Mass Index (BMI) score, using the age and gender-specific cutoff points of the BMI percentiles established for 15-years old Polish youth by Palczewska & Nied&wiecka (2002): group A of the underweight (<25 c), group B of the normal weight (25- 75c), group C of the overweight (75-97 c), and group D of the obese (>97 c) individuals (Table 1). Criterion for diagnosis of obesity at BMI 97th percentile was consistent with the WHO references for children 5 to 19 years old (available at http://www.who.growthref). Informed consent was obtained from all adolescents and their parents prior to participation in the study,

the protocol of which was approved by the local Ethics Committee.

Underweight (<25 c) X±SD

Variable Percentile ranges (c)

Normal weight (25-75 c) X±SD

No. of cases N=55 N=98 N=54 N=57 Body height, m 1.65\*±0.08 1.71±0.07 1.69±0.09 1.70±0.08 Body mass, kg 45.9\*\*\*±5.2 57.7±6.0 70.3\*\*\*±7.93 82.7\*\*\*±11.6

No. of cases N=58 N=81 N=46 N=56 Body height, m 1.65±0.08 1.66±0.08 1.65±0.09 1.64±0.08 Body mass, kg 46.1\*\*\*±5.1 54.1±6.5 67.2\*\*\*±7.8 79.4\*\*\*±11.8

Table 1. Anthropometric characteristics, and systolic (SBP) and diastolic (DBP) blood pressure of 15-year-old Polish adolescents. Significance of differences *vs.* normal weight

BMI 16.7\*\*\*±0.9 19.7±1.1 24.6\*\*\*±0.9 28.6\*\*\*±3.4 SBP, mm Hg 118.8±10.6 124.9±14.3 132.8\*±14.8 135.7\*\*±15.2 DBP, mm Hg 71.3±8.5 71.6±11.1 73.4±9.2 73.7±10.2

BMI 17,0\*\*\*±0.9 19.6±1.2 24.7\*\*\*±0.8 29.3\*\*\*±3.2 SBP, mm Hg 118.7±12.9 124.8±12.4 124.7±15.7 132.5\*±15.3 DBP, mm Hg 69.4±0.08 74.4±9.2 72.9±11.1 74.8±11.1

Marginally overweight (75-90 c) and overweight (90-97 c) X±SD

Obese (>97 c) X±SD

**2. Methods**

Gender

Boys

Girls

individuals: \*p<0.05, \*\*\* p<0.001

**2.1 Population sample**

Concentrations of serum glucose, total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides (TG) were assessed by the enzymatic methods using commercially available diagnostic kits (BioMaxima cat.no. 1-033-0400, 1-023-0400, 1-029-0200, 1-053-0400, respectively). Concentrations of low-density lipoprotein cholesterol (LDL-C) were calculated from TC, HDL-C, and TG using the Friedewald formula ( Friedewald et al., 1972). Serum insulin was measured by the immunoradiometric method (Insulin IRMA IM3210, Immunotech SA, France), sensitivity was 2.0 mIU/ml, and autoantibodies against oxidized LDL (oLAb) in serum samples were evaluated using ELISA assay (oLAb-ELISA, BI-20032; Biomedica GmbH, Wien, Austria) according to Tatzber & Esterbauer (1995).

To evaluate risk for vascular disease , the lipid ratios (TC/HDL-C, LDL-C/HDL-C, TG/HDL-C) and atherogenic index of plasma [AIP= log10(TG/HDL-C) with TG and HDL-C expressed in molar concentrations] (Dobiášová & Frohlich, 2001, 2004, 2011) were calculated. The homeostatic model assessment (HOMA) was used to estimate insulin resistance according to the method described by Matthews (1998). Insulin resistance score (HOMA-IR) was computed using the following formula: HOMA-IR=glucose (mmol/L)\*insulin (mIU/L)/22.5, with the cut-off point for adolescents less than or equal to 3.16 , as suggested by Keskin et al. (2005). Moreover, in 231 individuals (121 boys and 110 girls) serum adiponectin was measured by an immunoenzymatic method (Human Adiponectin ELISA, BioVendor, cat. no. RD195023100), and serum insulin-like growth factor (IGF-1) was evaluated in 80 adolescents (40 boys and 40 girls) using a commercially available diagnostic kit (IGF-I ELISA, Immunodiagnostic Systems GmbH, sensitivity 3.1 /g/L, reference range for 12 to 15 y old subjects 142-525 /g/L).

#### **2.3 Statistics**

All statistical analyses were performed with STATISTICA 7.1 software (StatSoft, Tulsa, OK, USA). The data reported as means ± SD were tested for homogeneity of variances by using the Levene test, then two-way ANOVA was performed to analyze the effect of gender and the degree of overweight/obesity on the studied variables, followed where appropriate, by the Tukey post-hoc comparisons. Spearman's rank order correlation analysis was performed to assess relationships between selected variables. Additionally, Pearson's Chi-square test of independence was used to evaluate the impact of the degree of overweight/obesity on SBP, serum triglycerides and surrogate markers of insulin resistance (HOMA-IR, TG/HDL-C). To predict the relative contribution of selected metabolic variables to prevalence of over normal SBP, insulin resistance (as assessed by HOMA-IR) and atherogenic potential of plasma (as assessed by AIP) a stepwise multiple regression analysis was applied. The level of significance of P<0.05 was chosen for all statistical comparisons.

#### **3. Results**

Physical characteristics of individual groups of the participants are presented in Table 1. In the evaluation of between-group differences, the data obtained in 113 adolescents (55 boys and 58 girls) with underweight, in 100 individuals (54 boys and 46 girls) with a tendency to overweight or overweight, and in 113 obese subjects (57 boys and 56 girls) were compared to the data obtained in 179 adolescents (98 boys and 81 girls) with normal weight.  Serum biochemical parameters in 15-year-old Polish adolescents. Significance of differences *vs.* normal weight individuals: \*p<0.05, \*\* p<0.005, NS-non significant

14.2\*\*±10.1 8.3±4.7 9.0±4.5 11.3±5.5 P<0.01

No. of cases N=58 N=81 N=46 N=56 N=241

Normal weight (25-75 c) X±SD

Percentile ranges (c)

Marginally overweight (75-90 c) and overweight (90-97 c) X±SD

Obese (>97 c) X±SD

Main effect: degree of overweight P value

Gender

Boys

Girls

Variables

Cholesterol (TC),

Adiponectin,

IGF-1, ng/mL

Cholesterol (TC),

Adiponectin, ng/mL (N=110)

IGF-1, ng/mL

Underweight (<25 c) X±SD

values and percentiles of BMI were similar among the groups of boys and girls with various degree of overweight, while the mean values of BMI in both groups of normal-weight teens were very close to the 50 percentile of BMI distribution for 15 year old boys and girls (Palczewska & Nied&wiecka, 2001; WHO Growth reference data for 5 to 19 years, available at http://www.who.growthref).

The readings of systolic and diastolic blood pressure (SBP and DBP) in all groups of adolescents, stratified according to gender and degree of overweight, were compared with the data of the updated blood pressure charts for children and adolescents according to the child's age and height percentile. For the purposes of this study, the following criteria were adopted. The adolescents were classified as normotensive if their systolic BP was <90th percentile, i.e. <130 mm Hg. The measured SBP >90th percentile, which could indicate prehypertension or hypertension, was classified as "over normal". These criteria were conform to the recommendations of the National High Blood Pressure Education Program The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, (2004), and to the guidelines of the IDF 2007 Consensus Statement (Zimmet et al., 2007; Alberti et al.,2006, 2007). There were numerous cases, in particular in overweight or obese boys and in obese girls, when SBP exceeded 136 mm Hg, the level suggesting occurrence of significant hypertension according to blood pressure criteria established by the Task Force report (Blood pressure charts for children and adolescents by age and height percentile*,* 2005; Update on the 1987 Task Force Report on High Blood Pressure in Children and Adolescents. A Working Group report from the National High Blood Pressure Education Program, 1996). Notably, more than a half of obese teens, especially girls, had over normal systolic BP.

Results of biochemical tests obtained in all groups stratified by gender and the degree of overweight are presented in Table 2. Except for a very limited number of cases, fasting blood glucose level, in all groups of boys and girls, did not exceed the upper limit of the normal range (100 mg/dL), while fasting insulin concentration tended toward higher values with growing degree of overweight to reach, in several cases, levels exceeding the upper limit of the reference range of the method used (2,6 – 22 µU/mL) (Table 2, Fig.1). As expected, the prevalence of insulin resistance, as assessed by the HOMA-IR index, increased significantly with the degree of overweight as measured by BMI. In the prevailing number of cases, the fasting serum levels of total cholesterol, HDL-C, LDL-C and calculated lipid ratios (TC /HDL-C, LDL-C / HDL-C) were within the specific reference ranges, while the concentration of TG and the TG/HDL-C ratio were found to reach the markedly higher values only in both groups of obese individuals (Table 2, Fig.1). Except for serum HDL-C, which was higher (p<0.05) in girls, there were no significant inter-gender differences for the remaining tested variables. There was a clear tendency toward higher levels of SBP, insulin, HOMA-IR , triglycerides, and TG/HDL-C ratio with increased degree of overweight ( Fig.1) in adolescent boys and girls, and most of these measures were affected significantly by the degree of overweight (Table 2). It is also worth to note that the degree of overweight/obesity significantly affected the atherogenic index of plasma (AIP), which adopted the lowest (most favorable) levels in the underweight groups, and then increased progressively with rising body mass index (Table 2, Fig.1). No between-group differences were found in titers of autoantibodies against oxidized LDL (oLAb), while individual oLAb titers were very variable, reaching over normal levels (>600 mU/mL) (Pincemail et al., 2000) in several subjects from each group. Similarly, no between-group differences were observed

values and percentiles of BMI were similar among the groups of boys and girls with various degree of overweight, while the mean values of BMI in both groups of normal-weight teens were very close to the 50 percentile of BMI distribution for 15 year old boys and girls (Palczewska & Nied&wiecka, 2001; WHO Growth reference data for 5 to 19 years, available

The readings of systolic and diastolic blood pressure (SBP and DBP) in all groups of adolescents, stratified according to gender and degree of overweight, were compared with the data of the updated blood pressure charts for children and adolescents according to the child's age and height percentile. For the purposes of this study, the following criteria were adopted. The adolescents were classified as normotensive if their systolic BP was <90th percentile, i.e. <130 mm Hg. The measured SBP >90th percentile, which could indicate prehypertension or hypertension, was classified as "over normal". These criteria were conform to the recommendations of the National High Blood Pressure Education Program The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, (2004), and to the guidelines of the IDF 2007 Consensus Statement (Zimmet et al., 2007; Alberti et al.,2006, 2007). There were numerous cases, in particular in overweight or obese boys and in obese girls, when SBP exceeded 136 mm Hg, the level suggesting occurrence of significant hypertension according to blood pressure criteria established by the Task Force report (Blood pressure charts for children and adolescents by age and height percentile*,* 2005; Update on the 1987 Task Force Report on High Blood Pressure in Children and Adolescents. A Working Group report from the National High Blood Pressure Education Program, 1996). Notably, more than a half of obese

Results of biochemical tests obtained in all groups stratified by gender and the degree of overweight are presented in Table 2. Except for a very limited number of cases, fasting blood glucose level, in all groups of boys and girls, did not exceed the upper limit of the normal range (100 mg/dL), while fasting insulin concentration tended toward higher values with growing degree of overweight to reach, in several cases, levels exceeding the upper limit of the reference range of the method used (2,6 – 22 µU/mL) (Table 2, Fig.1). As expected, the prevalence of insulin resistance, as assessed by the HOMA-IR index, increased significantly with the degree of overweight as measured by BMI. In the prevailing number of cases, the fasting serum levels of total cholesterol, HDL-C, LDL-C and calculated lipid ratios (TC /HDL-C, LDL-C / HDL-C) were within the specific reference ranges, while the concentration of TG and the TG/HDL-C ratio were found to reach the markedly higher values only in both groups of obese individuals (Table 2, Fig.1). Except for serum HDL-C, which was higher (p<0.05) in girls, there were no significant inter-gender differences for the remaining tested variables. There was a clear tendency toward higher levels of SBP, insulin, HOMA-IR , triglycerides, and TG/HDL-C ratio with increased degree of overweight ( Fig.1) in adolescent boys and girls, and most of these measures were affected significantly by the degree of overweight (Table 2). It is also worth to note that the degree of overweight/obesity significantly affected the atherogenic index of plasma (AIP), which adopted the lowest (most favorable) levels in the underweight groups, and then increased progressively with rising body mass index (Table 2, Fig.1). No between-group differences were found in titers of autoantibodies against oxidized LDL (oLAb), while individual oLAb titers were very variable, reaching over normal levels (>600 mU/mL) (Pincemail et al., 2000) in several subjects from each group. Similarly, no between-group differences were observed

at http://www.who.growthref).

teens, especially girls, had over normal systolic BP.

Table 2. Serum biochemical parameters in 15-year-old Polish adolescents. Significance of differences *vs.* normal weight individuals: \*p<0.05, \*\* p<0.005, NS-non significant

Adolescent Obesity Predicts Cardiovascular Risk 281

The analysis carried out to assess the prevalence ( %) of adolescents with over normal values of SBP (>130 mm Hg), serum triglycerides (> 150 mg/dL) (Zimmet et al., 2007), HOMA-IR (>3.16) (Keskin et al., 2005), and TG/HDL-C ratio (>3.0) (McLaughlin et al., 2003; Hannon et al., 2006) revealed a rising number of individuals fulfilling these criteria with an increasing degree of overweight (Table 3). Noteworthy, Pearson's chi-square test showed significant increases in the prevalence (%) of individuals with over normal levels of these parameters

> Normal weight (25-75 c)

B+G 34.5 13.4 31.1 40.0 56.9 48.5 <10-5 B 36.2 13.0 32.3 50.0 52.8 24.1 <10-4 G 32.8 13.8 29.6 28.3 60.7 30.1 <10-5

B+G 22.5 8.0 19.6 25.5 39.3 33.2 <10-5 B 21.7 10.9 20.1 22.6 33.3 8.4 <0.05 G 23.4 5.2 18.5 28.9 45.5 27.5 <10-5

B+G 10.7 6.2 6.7 7.0 24.8 30.3 <10-5 B 9.5 7.3 3.1 7.4 24.6 20.4 <0.0005 G 12.0 5.2 11.1 6.5 25.0 12.9 <0.005

B+G 6.3 4.4 3.4 5.0 14.2 15.3 <0.005 B 7.2 5.5 2.1 7.4 17.5 13.3 <0.005 G 5.4 3.5 5.0 2.2 10.7 4.5 NS

Table 3. Prevalence (%) of 15 year-old boys (B) and girls (G) with over normal values of

In order to assess the impact of the degree of overweight on selected biochemical parameters and biomarkers of atherogenic risk, the Spearman rank correlation coefficients were computed, and selected statistically significant associations are summarized in Tables 4 and 5. As expected, the BMI and serum triglycerides correlated positively with systolic and diastolic blood pressure, insulin, and HOMA-IR. Serum adiponectin correlated negatively with lipid ratios (TG/HDL-C, TC/HDL-C, LDL-C/HDL-C), while it was positively associated with HDL-C. The oLAb titers correlated positively with serum LDL-C

The important finding was that the atherogenic index of plasma (AIP) was positively correlated with the BMI, systolic and diastolic blood pressure, TG, total cholesterol, common lipid ratios (LDL-C/HDL-C, TC/HDL-C), insulin, HOMA-IR, but negatively associated with HDL-C and adiponectin (Table 5). Although these relationships do not infer casual

systolic blood pressure (SBP), HOMA-IR, triglycerides (TG), and TG/HDL-C.

Marginally overweight (75-90 c) and overweight (90-97 c)

Obese (>97c)

Pearson's Chi-square (df=3)

\*2 P value

Prevalence (%) of individuals with over normal values of SBP, HOMA-IR, TG, and TG/HDL-C ratio

> Underweight (<25 c)

with raising BMI.

SBP >130 mmHg

TG >150 mg/dL

C > 3.0

HOMA-IR >3,16

TG/HDL-

and LDL-C/HDL-C ratio (Table 4).

Group: Boys (B) and/or girls (G)

in serum IGF-1, there was only a slight tendency toward higher IGF-1 levels in boys and the lowest values in underweight individuals, although both effects did not reach statistical significance. Serum adiponectin in both groups of overweight or obese individuals was close to the levels recorded in normal weight teens, but the highest concentrations of this adipokine, significantly (p<0.05) different from the values recorded in normal weight individuals, were found in the underweight groups (Table 2, Fig1).

Fig. 1. The impact of the degree of overweight/obesity on systolic blood pressure (SBP), HOMA-IR, serum concentration of triglycerides (TG), TG/HDL ratio, insulin and adiponectin levels, and atherogenic index of plasma (AIP) in 15-year-old Polish adolescents with underweight (A), normal weight (B), overweight (C) and obesity (D). Significance of differences *vs.* individuals with normal weight: \*p<0.05,\*\* p<0.01, \*\*\*p<0.001

in serum IGF-1, there was only a slight tendency toward higher IGF-1 levels in boys and the lowest values in underweight individuals, although both effects did not reach statistical significance. Serum adiponectin in both groups of overweight or obese individuals was close to the levels recorded in normal weight teens, but the highest concentrations of this adipokine, significantly (p<0.05) different from the values recorded in normal weight

Fig. 1. The impact of the degree of overweight/obesity on systolic blood pressure (SBP), HOMA-IR, serum concentration of triglycerides (TG), TG/HDL ratio, insulin and

differences *vs.* individuals with normal weight: \*p<0.05,\*\* p<0.01, \*\*\*p<0.001

8,9 9,5 10,2

**ABCD**

14,2

\*

1,83

16,9 19,7

\*\*\*

**HOMA**

**BMI, kg/m2**

**Adiponectin, ng/mL**

**1 1,5 2 2,5 3 3,5 4**

**TG / HDL**

adiponectin levels, and atherogenic index of plasma (AIP) in 15-year-old Polish adolescents with underweight (A), normal weight (B), overweight (C) and obesity (D). Significance of

**-0,2**

**-0,15**

**-0,1**

**-0,05**

**AIP**

**0**

**Insulin, mU/L**

2,41

\*

**TG, mg/dl**

**SBP, mm Hg**

\*

\*

29

\*\*\*

**-0,086**

**-0,058 -0,051**

8,9 12,1 14,5 17,2

**ABCD**

106,9 <sup>111</sup> 114,6

**ABCD**

118,7 126,8 129,1 134,0

**ABCD**

**ABCD**

**-0,008**

131,1

\*\*

\*\*\*

\*\*

individuals, were found in the underweight groups (Table 2, Fig1).

24,6

\*\*\*

2,49 3,08 3,49

**ABCD**

1,95 2,06 2,1

**ABCD**

**ABCD**

The analysis carried out to assess the prevalence ( %) of adolescents with over normal values of SBP (>130 mm Hg), serum triglycerides (> 150 mg/dL) (Zimmet et al., 2007), HOMA-IR (>3.16) (Keskin et al., 2005), and TG/HDL-C ratio (>3.0) (McLaughlin et al., 2003; Hannon et al., 2006) revealed a rising number of individuals fulfilling these criteria with an increasing degree of overweight (Table 3). Noteworthy, Pearson's chi-square test showed significant increases in the prevalence (%) of individuals with over normal levels of these parameters with raising BMI.