ACPA (-) of RA patients. *Rheumatol Int*, ISSN: 1437-160X

of an initial American College of Rheumatology/European League Against Rheumatism consensus conference. *Arthritis Rheum* 61(5): pp. 704-710, ISSN: 0004-

complex in patients with active and inactive rheumatoid arthritis: plasma concentrations and relationship to inflammation. *Clin Chim Acta* 390(1-2): pp. 67-71,

sedimentation rate in patients with rheumatoid arthritis. *J Rheumatol* 24(8): pp.

relationship to vascular disease risk factors and inflammation. *Arthritis Rheum*

disease and acute phase proteins in the course of Behcet disease. *Eur J Ophthalmol*

disease activity, sex, and menopausal status on lipid profiles. *J Rheumatol* 31(9): pp.

fibrinogen cascade in patients with Kawasaki disease. *Pediatr Allergy Immunol* 20(7):

differential diagnostics of rheumatoid arthritis]. *Klin Med (Mosk)* 86(6): pp. 58-61,

phase reactant. *Eur J Biochem* 265(2): pp. 501-523, ISSN: 0014-2956

3591

ISSN: 0009-8981

1477-1485, ISSN: 0315-162X

48(1): pp. 81-89, ISSN: 0004-3591

18(5): pp. 787-791, ISSN: 1120-6721

1746-1753, ISSN: 0315-162X

pp. 699-707, ISSN: 1399-3038

ISSN: 0023-2149

Injury and infection, seemingly unrelated conditions, converge on a common process inflammation, which is mediated partly by innate immune cells including macrophages and monocytes. These innate immune cells are equipped with pattern recognition receptors (such as TLR2, TLR4, and TLR9) (Brightbill *et al.* 1999; Poltorak *et al.* 1998; Hemmi *et al.* 2000), and can recognize both damage- and pathogen-associated molecular patterns (DAMPs, such as HMGB1; and PAMPs, such as endotoxin) (Andersson *et al.* 2000; Chen *et al.* 2009;Krieg 2002;Wang *et al.* 1999;Ivanov *et al.* 2007). In response to various PAMPs or DAMPs, innate immune cells release proinflammatory cytokines (such as TNF, IL-1, IFN-γ or HMGB1 to mount inflammatory responses. If dysregulated, an uncontrolled inflammation may adversely lead to detrimental consequences. To orchestrate the inflammatory response to infection and injury, the liver strategically re-prioritizes the synthesis and systemic release of a group of proteins collectively termed "acute phase proteins" (APPs). For instance, fetuin-A, also called the alpha-2-HS-glycoprotein for the human homologue (Christie *et al.* 1987), has been implicated as an anti-inflammatory protein during injury or infections. In this book chapter, we summarize emerging evidence to support fetuin-A as an acute phase protein capable of attenuating injury- or infection-elicited inflammatory responses.

#### **2. Fetuin-A as a negative or positive APP**

Fetuin-A was first isolated by Pederson more than sixty years ago as a major plasma protein in the fetus (Pedersen 1944).  In adults however, fetuin-A is produced primarily by the liver, and its synthesis is divergently regulated during injury or infection, classifying it as a negative or positive APP.

#### **2.1 Regulators of hepatic Fetuin-A expression**

Although fetuin-A is constitutively expressed in hepatocytes, its expression is negatively regulated by several proinflammatory cytokines. For instance, the fetuin-A expression levels in human HepG2 hepatoma cells were reduced by proinflammatory cytokines such as TNF, IL-1, IL-6, and IFN-γ (Daveau *et al.* 1988;Li *et al.* 2011a). IFN-γ, at concentrations as low as 10-50

Role of Fetuin-A in Injury and Infection 331

Despite its abundance, the functions of fetuin-A remain poorly understood. A wide range of biological functions have been proposed for fetuin-A based on its structural similarities to

Fetuin-A shares sequence similarity to type II TGF-β receptors (Demetriou *et al.* 1996) and insulin receptor tyrosine kinases (Mathews *et al.* 1997;Haasemann *et al.* 1991), and has thus been proposed as an inhibitor of the TGF-β or insulin signaling pathways. After binding to to TGF-β1, fetuin-A prevents TGF-β1 from binding to its receptors, thereby antagonizing TGF-β1-mediated antiproliferative effects (Demetriou *et al.* 1996). Similarly, fetuin-A can also bind to the insulin receptor, and consequently inactivate (rather than activate, as in the case for insulin) the receptor tyrosine kinase (Goustin & Abou-Samra 2010). This may partly explain why higher fetuin-A levels were associated with insulin resistance in some patients

As a glycoprotein, fetuin-A carries two N-linked and three O-linked oligosaccharide chains that terminate with sialic acid residues, and can bind cationic Ca2+ ions. Accordingly, fetuin-A has been proposed as an endogenous inhibitor of pathological mineralization or calcification in soft tissues (Jahnen-Dechent *et al.* 2001;Schinke *et al.* 1996;Szweras *et al.* 2002;Schafer *et al.* 2003;Ketteler *et al.* 2003). Specifically, fetuin-A forms protein-mineral colloids with calcium and phosphate (Heiss *et al.* 2003;Wu *et al.* 2009), thereby preventing uncontrolled mineralization that may otherwise occur under pathological conditions

While investigating the mechanism underlying a cationic molecule spermine-mediated antiinflammatory actions, we serendipitously discovered that macrophages lost their responsiveness to spermine when cultured under low serum conditions (Wang *et al.* 1997). That is, despite the addition of cytokine-suppressing concentrations of spermine, the bacterial lipopolysaccharide (LPS)-induced production of TNF by these serum-starved macrophages was uninhibited. Subsequently, we discovered that these serum-starved macrophages became deprived of fetuin-A that was required for spermine to inhibit TNF production (Wang *et al.* 1997). The involvement of fetuin-A in spermine-mediated immunosuppression was confirmed by adding highly purified fetuin-A or fetuin-specific antibodies, which respectively restored or impaired spermine-mediated TNF inhibition

It is plausible that fetuin-A functions as an opsonin for cationic spermine, and its availability to immune cells may be critical in regulating the innate immune response (Wang & Tracey 1999). Indeed, levels of fetuin-A in macrophage cultures could be altered by LPS stimulation or fetuin-A supplementation (**Figure 1A**). Intriguingly, the exogenously administered fetuin-A was predominantly localized in cytoplasmic punctate structures (**Figure 1B**), which co-localized with vesicles containing an autophagy marker (LC3) - possibly

autophagosomes or amphisomes - in LPS-stimulated macrophages.

**3. Biological functions of Fetuin-A**

**3.1 Inhibitor of insulin or TGF-β Signalling**

with type 2 diabetes (Ix *et al.* 2008).

(Rochette *et al.* 2009).

(Wang *et al.* 1997).

**3.3 Inhibition of inflammation**

**3.2 Inhibition of pathological calcification**

other proteins or physical interactions with biogenic molecules.

ng/ml, reduced fetuin-A expression levels by as much as 50-70% (Li *et al.* 2011a). In contrast, HMGB1 (1 μg/ml), a late proinflammatory mediator of lethal systemic inflammation (Wang *et al.* 1999; Yang *et al.* 2004; Wang *et al.* 2008), elevated fetuin-A expression levels by 2-3 folds in HepG2 cells, suggesting that different cytokines divergently regulate hepatic fetuin-A expression.

#### **2.2 Elevation of Fetuin-A Levels during ischemia**

In patients with cerebral ischemic injury (stroke), plasma fetuin-A levels were paradoxically elevated (Weikert *et al.* 2008;Tuttolomondo *et al.* 2010). The elevation of circulating fetuin-A levels correlated with an increase not only in LDL-cholesterol levels (Tuttolomondo *et al.* 2010) but also in risk of cardiovascular disorders (Weikert *et al.* 2008). Similarly, serum fetuin-A levels were increased up to 10-fold in cattle following traumatic injury (Dziegielewska *et al.* 1992), suggesting fetuin-A as a positive APP during ischemic or traumatic injury. Notably, HMGB1 can be passively leaked from injured cells (Peltz *et al.* 2009), and functions as an early mediator of traumatic or ischemic injury (Zhu *et al.* 2010; Wu *et al.* 2007; Liu *et al.* 2007b; Tsung *et al.* 2005; Tsung *et al.* 2007; Watanabe *et al.* 2005). It is thus plausible that HMGB1 participates in the up-regulation of hepatic fetuin-A expression during injury.

In an animal model of focal cerebral ischemia (i.e., permanent middle cerebral artery occlusion, MCAo), fetuin-A levels in the ischemic brain tissue were also elevated in a timedependent manner, starting between 2-6 h, peaking around 24-48 h, and returning towards base-line at 72 h post MCAo (Wang *et al.* 2010). This time-dependent increase in cerebral fetuin-A levels parallels with the transient elevation of the blood-brain barrier (BBB) permeability (Belayev *et al.* 1996), suggesting that circulating fetuin-A can gain entry across the BBB into the ischemic brain tissue. This possibility was supported by the observation that peripherally (intravenously) administered FITC-labeled fetuin-A was found in the ischemic brain region at 24 h after MCAo (Wang *et al.* 2010).

#### **2.3 Reduction of circulating Fetuin-A levels during infection**

In animal models of endotoxemia and sepsis (induced by cecal ligation and puncture, CLP), circulating fetuin-A levels were decreased in a time-dependent fashion, starting between 2-6 h, reaching a nadir (with maximal reduction by 50-60%) around 24-48 h. Afterwards, fetuin-A levels started to increase, returning towards basal levels approximately 72 h post endotoxemia or sepsis, supporting fetuin-A as a negative APP in animal models of lethal endotoxemia and sepsis (Li *et al.* 2011a). Interestingly, disruption of expression of early proinflammatory cytokines (such as IFN-γ) impaired bacterial endotoxin-mediated down-regulation of fetuin-A expression (Li *et al.* 2011a). It thus appears that early proinflammatory cytokines (such as TNF and IFN-γ) function as negative regulators to reduce circulating fetuin-A levels during an early stage of endotoxemia or sepsis; whereas late-acting proinflammatory mediators (e.g., HMGB1) stimulate fetuin-A expression to restore its circulating levels at a late stage.

In patients with other inflammatory diseases such as pancreatitis (Kusnierz-Cabala *et al.* 2010), chronic kidney diseases (Metry *et al.* 2008), and rheumatoid arthritis (Sato *et al.* 2007), serum fetuin A levels were also decreased by 20-30%. In these patients, circulating fetuin-A levels were not only inversely correlated with levels of inflammatory cytokines (such as IL-6) (Kusnierz-Cabala *et al.* 2010), but also associated with increased mortality rates (Metry *et al.* 2008). Collectively, these observations classify fetuin-A as a negative APP during infection or other inflammatory illness.

ng/ml, reduced fetuin-A expression levels by as much as 50-70% (Li *et al.* 2011a). In contrast, HMGB1 (1 μg/ml), a late proinflammatory mediator of lethal systemic inflammation (Wang *et al.* 1999; Yang *et al.* 2004; Wang *et al.* 2008), elevated fetuin-A expression levels by 2-3 folds in HepG2 cells, suggesting that different cytokines divergently regulate hepatic fetuin-A

In patients with cerebral ischemic injury (stroke), plasma fetuin-A levels were paradoxically elevated (Weikert *et al.* 2008;Tuttolomondo *et al.* 2010). The elevation of circulating fetuin-A levels correlated with an increase not only in LDL-cholesterol levels (Tuttolomondo *et al.* 2010) but also in risk of cardiovascular disorders (Weikert *et al.* 2008). Similarly, serum fetuin-A levels were increased up to 10-fold in cattle following traumatic injury (Dziegielewska *et al.* 1992), suggesting fetuin-A as a positive APP during ischemic or traumatic injury. Notably, HMGB1 can be passively leaked from injured cells (Peltz *et al.* 2009), and functions as an early mediator of traumatic or ischemic injury (Zhu *et al.* 2010; Wu *et al.* 2007; Liu *et al.* 2007b; Tsung *et al.* 2005; Tsung *et al.* 2007; Watanabe *et al.* 2005). It is thus plausible that HMGB1 participates

In an animal model of focal cerebral ischemia (i.e., permanent middle cerebral artery occlusion, MCAo), fetuin-A levels in the ischemic brain tissue were also elevated in a timedependent manner, starting between 2-6 h, peaking around 24-48 h, and returning towards base-line at 72 h post MCAo (Wang *et al.* 2010). This time-dependent increase in cerebral fetuin-A levels parallels with the transient elevation of the blood-brain barrier (BBB) permeability (Belayev *et al.* 1996), suggesting that circulating fetuin-A can gain entry across the BBB into the ischemic brain tissue. This possibility was supported by the observation that peripherally (intravenously) administered FITC-labeled fetuin-A was found in the

In animal models of endotoxemia and sepsis (induced by cecal ligation and puncture, CLP), circulating fetuin-A levels were decreased in a time-dependent fashion, starting between 2-6 h, reaching a nadir (with maximal reduction by 50-60%) around 24-48 h. Afterwards, fetuin-A levels started to increase, returning towards basal levels approximately 72 h post endotoxemia or sepsis, supporting fetuin-A as a negative APP in animal models of lethal endotoxemia and sepsis (Li *et al.* 2011a). Interestingly, disruption of expression of early proinflammatory cytokines (such as IFN-γ) impaired bacterial endotoxin-mediated down-regulation of fetuin-A expression (Li *et al.* 2011a). It thus appears that early proinflammatory cytokines (such as TNF and IFN-γ) function as negative regulators to reduce circulating fetuin-A levels during an early stage of endotoxemia or sepsis; whereas late-acting proinflammatory mediators (e.g., HMGB1)

In patients with other inflammatory diseases such as pancreatitis (Kusnierz-Cabala *et al.* 2010), chronic kidney diseases (Metry *et al.* 2008), and rheumatoid arthritis (Sato *et al.* 2007), serum fetuin A levels were also decreased by 20-30%. In these patients, circulating fetuin-A levels were not only inversely correlated with levels of inflammatory cytokines (such as IL-6) (Kusnierz-Cabala *et al.* 2010), but also associated with increased mortality rates (Metry *et al.* 2008). Collectively, these observations classify fetuin-A as a negative APP during

expression.

**2.2 Elevation of Fetuin-A Levels during ischemia**

in the up-regulation of hepatic fetuin-A expression during injury.

ischemic brain region at 24 h after MCAo (Wang *et al.* 2010).

**2.3 Reduction of circulating Fetuin-A levels during infection**

stimulate fetuin-A expression to restore its circulating levels at a late stage.

infection or other inflammatory illness.

#### **3. Biological functions of Fetuin-A**

Despite its abundance, the functions of fetuin-A remain poorly understood. A wide range of biological functions have been proposed for fetuin-A based on its structural similarities to other proteins or physical interactions with biogenic molecules.

#### **3.1 Inhibitor of insulin or TGF-β Signalling**

Fetuin-A shares sequence similarity to type II TGF-β receptors (Demetriou *et al.* 1996) and insulin receptor tyrosine kinases (Mathews *et al.* 1997;Haasemann *et al.* 1991), and has thus been proposed as an inhibitor of the TGF-β or insulin signaling pathways. After binding to to TGF-β1, fetuin-A prevents TGF-β1 from binding to its receptors, thereby antagonizing TGF-β1-mediated antiproliferative effects (Demetriou *et al.* 1996). Similarly, fetuin-A can also bind to the insulin receptor, and consequently inactivate (rather than activate, as in the case for insulin) the receptor tyrosine kinase (Goustin & Abou-Samra 2010). This may partly explain why higher fetuin-A levels were associated with insulin resistance in some patients with type 2 diabetes (Ix *et al.* 2008).

#### **3.2 Inhibition of pathological calcification**

As a glycoprotein, fetuin-A carries two N-linked and three O-linked oligosaccharide chains that terminate with sialic acid residues, and can bind cationic Ca2+ ions. Accordingly, fetuin-A has been proposed as an endogenous inhibitor of pathological mineralization or calcification in soft tissues (Jahnen-Dechent *et al.* 2001;Schinke *et al.* 1996;Szweras *et al.* 2002;Schafer *et al.* 2003;Ketteler *et al.* 2003). Specifically, fetuin-A forms protein-mineral colloids with calcium and phosphate (Heiss *et al.* 2003;Wu *et al.* 2009), thereby preventing uncontrolled mineralization that may otherwise occur under pathological conditions (Rochette *et al.* 2009).

#### **3.3 Inhibition of inflammation**

While investigating the mechanism underlying a cationic molecule spermine-mediated antiinflammatory actions, we serendipitously discovered that macrophages lost their responsiveness to spermine when cultured under low serum conditions (Wang *et al.* 1997). That is, despite the addition of cytokine-suppressing concentrations of spermine, the bacterial lipopolysaccharide (LPS)-induced production of TNF by these serum-starved macrophages was uninhibited. Subsequently, we discovered that these serum-starved macrophages became deprived of fetuin-A that was required for spermine to inhibit TNF production (Wang *et al.* 1997). The involvement of fetuin-A in spermine-mediated immunosuppression was confirmed by adding highly purified fetuin-A or fetuin-specific antibodies, which respectively restored or impaired spermine-mediated TNF inhibition (Wang *et al.* 1997).

It is plausible that fetuin-A functions as an opsonin for cationic spermine, and its availability to immune cells may be critical in regulating the innate immune response (Wang & Tracey 1999). Indeed, levels of fetuin-A in macrophage cultures could be altered by LPS stimulation or fetuin-A supplementation (**Figure 1A**). Intriguingly, the exogenously administered fetuin-A was predominantly localized in cytoplasmic punctate structures (**Figure 1B**), which co-localized with vesicles containing an autophagy marker (LC3) - possibly autophagosomes or amphisomes - in LPS-stimulated macrophages.

Role of Fetuin-A in Injury and Infection 333

(Ombrellino *et al.* 2001). In contrast, administration of anti-fetuin-A neutralizing antibodies in combination with carrageenan led to significantly increased paw edema, indicating that

Cerebral ischemia is frequently caused by an obstruction of a cerebral artery. Despite advances in acute and prophylactic therapies, stroke represents the leading cause of longterm disability (500,000-700,000 cases per year), and the third most common cause of death

Cerebral ischemic injury consists of two stages: primary tissue damage in the ischemic core and secondary tissue injury in the surrounding penumbra. The primary injury in the ischemic core is primarily mediated by tissue ion (Ca2+ and Na+) overload (Taylor & Meldrum 1995) and excitotoxicity (Lee *et al.* 1999); whereas the secondary injury in the surrounding penumbra is partly mediated by proinflammatory cytokines (**Figure 2**,

**Primary Early Injury Secondary Late Injury**

Fig. 2. **Cascade of events leading to primary injury in the ischemic core and secondary**

**(Ischemic Core) (Penumbra)**

**Cell Death**

**Expression of Proinflammatory Cytokines**

**Primary Cell Death**

**Leakage of HMGB1 and Others**

**HMGB1 Spillage to the Periphery**

**Activation of Infiltrated Leukocytes**

**Activation of Microglia and Neurons**

fetuin-A plays an important role in counter-regulating inflammatory responses.

**4.2 Cerebral ischemic injury**

Feuerstein *et al.* 1998).

**injury in the surrounding penumbra.**

**Cell Death**

**Elevated Intracellular Ca2+**

**Ca2+ Influx**

(with a mortality rate of 20-25%) in the United States.

**4.2.1 Pathogenesis of cerebral ischemic injury**

**Cerebral Ischemia**

**Failure of Na+ / K+ pumps**

**NMDA Receptors**

**Glutamate Release**

**Neuronal Membrane Depolarization**

When given at higher concentrations (e.g., 3.5 mg/ml), crude fetuin-A (> 98%, Sigma-Aldrich) abrogated endotoxin-induced release of IL-1 and nitric oxide (Dziegielewska *et al.* 1998). Upon purification by gel filtration and ion-exchange chromatography, the highly purified intact fetuin-A could effectively inhibit IFN-γ- or LPS-induced release of HMGB1 (Li *et al.* 2011a), a newly identified late mediator of lethal endotoxemia and sepsis (Wang *et al.* 2008;Wang *et al.* 2009). However, even at the concentrations (e.g., 100 μg/ml) that abrogated LPS-induced HMGB1 release, fetuin-A only partly inhibited LPS-induced TNF secretion, suggesting fetuin-A as an effective inhibitor of HMGB1 release.

Fig. 1. **Exogenous fetuin-A was internalized into cytoplasmic vesicles in macrophage cultures**. A). Supplementation of exogenous fetuin-A prevented endotoxin-induced fetuin-A depletion**.** Murine macrophage-like RAW 264.7 cells were stimulated with LPS (100 ng/ml) in the absence or presence of fetuin-A (100 μg/ml) for 2 h, and cellular fetuin-A levels were determined by Western blotting. The relative fetuin-A levels, as a ratio to β-actin, were expressed as the mean ± SD of three independent experiments. \*, p < 0.05 versus control ("+LPS"); #, p < 0.01 vs control ("+LPS"). B) Exogenous fetuin-A was internalized into LC3 containing cytoplasmic vesicles. GFP-LC3-transfected RAW 264.7 cells were stimulated with LPS (200 ng/ml) in the presence of fetuin-A (100 μg/ml) overnight, and immunostained with fetuin-A-specific antibodies.

#### **4. Therapeutic potential of Fetuin-A in infection or injury**

#### **4.1 Carrageenan-induced paw edema**

In an animal model of carrageenan-induced inflammation, intraperitoneal administration of fetuin-A (5 to 500 mg/kg) dose-dependently attenuated the development of paw edema (Ombrellino *et al.* 2001). The sialic acid moieties of fetuin-A might be required for its antiinflammatory activities. When these sialic acid residues were removed by neuraminidase, the resultant asialofetuin-A failed to potentiate the anti-inflammatory activities of spermine (Wang *et al.* 1997) and failed to attenuate carrageenan-induced TNF production *in vivo*

When given at higher concentrations (e.g., 3.5 mg/ml), crude fetuin-A (> 98%, Sigma-Aldrich) abrogated endotoxin-induced release of IL-1 and nitric oxide (Dziegielewska *et al.* 1998). Upon purification by gel filtration and ion-exchange chromatography, the highly purified intact fetuin-A could effectively inhibit IFN-γ- or LPS-induced release of HMGB1 (Li *et al.* 2011a), a newly identified late mediator of lethal endotoxemia and sepsis (Wang *et al.* 2008;Wang *et al.* 2009). However, even at the concentrations (e.g., 100 μg/ml) that abrogated LPS-induced HMGB1 release, fetuin-A only partly inhibited LPS-induced TNF

**Fetuin-A LC3**

**DAPI Overlay**

Fig. 1. **Exogenous fetuin-A was internalized into cytoplasmic vesicles in macrophage cultures**. A). Supplementation of exogenous fetuin-A prevented endotoxin-induced fetuin-A depletion**.** Murine macrophage-like RAW 264.7 cells were stimulated with LPS (100 ng/ml) in the absence or presence of fetuin-A (100 μg/ml) for 2 h, and cellular fetuin-A levels were determined by Western blotting. The relative fetuin-A levels, as a ratio to β-actin, were expressed as the mean ± SD of three independent experiments. \*, p < 0.05 versus control ("+LPS"); #, p < 0.01 vs control ("+LPS"). B) Exogenous fetuin-A was internalized into LC3 containing cytoplasmic vesicles. GFP-LC3-transfected RAW 264.7 cells were stimulated with LPS (200 ng/ml) in the presence of fetuin-A (100 μg/ml) overnight, and immunostained

In an animal model of carrageenan-induced inflammation, intraperitoneal administration of fetuin-A (5 to 500 mg/kg) dose-dependently attenuated the development of paw edema (Ombrellino *et al.* 2001). The sialic acid moieties of fetuin-A might be required for its antiinflammatory activities. When these sialic acid residues were removed by neuraminidase, the resultant asialofetuin-A failed to potentiate the anti-inflammatory activities of spermine (Wang *et al.* 1997) and failed to attenuate carrageenan-induced TNF production *in vivo*

**4. Therapeutic potential of Fetuin-A in infection or injury**

with fetuin-A-specific antibodies.

0.0 0.2 0.4 0.6 0.8 1.0 1.2

**Fetuin –A / -actin**

**4.1 Carrageenan-induced paw edema**

**LPS** - 100 100 **(ng/ml) Fetuin-A** - - 100 **(g/ml)**

**#**

**A. B.**

\*

secretion, suggesting fetuin-A as an effective inhibitor of HMGB1 release.

(Ombrellino *et al.* 2001). In contrast, administration of anti-fetuin-A neutralizing antibodies in combination with carrageenan led to significantly increased paw edema, indicating that fetuin-A plays an important role in counter-regulating inflammatory responses.

#### **4.2 Cerebral ischemic injury**

Cerebral ischemia is frequently caused by an obstruction of a cerebral artery. Despite advances in acute and prophylactic therapies, stroke represents the leading cause of longterm disability (500,000-700,000 cases per year), and the third most common cause of death (with a mortality rate of 20-25%) in the United States.

#### **4.2.1 Pathogenesis of cerebral ischemic injury**

Cerebral ischemic injury consists of two stages: primary tissue damage in the ischemic core and secondary tissue injury in the surrounding penumbra. The primary injury in the ischemic core is primarily mediated by tissue ion (Ca2+ and Na+) overload (Taylor & Meldrum 1995) and excitotoxicity (Lee *et al.* 1999); whereas the secondary injury in the surrounding penumbra is partly mediated by proinflammatory cytokines (**Figure 2**, Feuerstein *et al.* 1998).

Fig. 2. **Cascade of events leading to primary injury in the ischemic core and secondary injury in the surrounding penumbra.**

Role of Fetuin-A in Injury and Infection 335

During cerebral ischemia, brain spermine levels are decreased (Paschen *et al.* 1992), owing largely to an accompanying increase in the enzymatic activity of brain polyamine oxidase (Ivanova *et al.* 1998). The loss of spermine consequently tilts the balance towards neurotoxicity through activating the NMDA receptor, and increasing susceptibility to

oxidative stress as well as excessive inflammatory response.

Fig. 3. **Divergent roles of spermine in cerebral ischemic injury.**

long-lasting protective effects.

**Toxic Metabolites (e.g., 3-aminopropanal)**

**4.2.3 Peripheral administration of fetuin-A reduced cerebral ischemic injury**

As mentioned earlier, when given peripherally, exogenous fetuin-A gains entry across the BBB into the ischemic brain tissue (**Figure 4**). The time-course of fetuin-A extravasation in the ischemic brain tissue parallels with the time-dependent alteration of the BBB permeability (Belayev *et al.* 1996), which was transiently elevated (5 -25 h post MCAo) followed by a return towards baseline at 72 h post MCAo (Belayev *et al.* 1996). It is possible that the temporal breakdown of the BBB is required for circulating fetuin-A to transiently gain entry into the brain. Consistently, peripheral administration of fetuin-A (50 mg/kg) promoted a dose-dependent protection against cerebral ischemic injury during an early stage of cerebral ischemia (i.e., 24 h post MCAo) (Wang *et al.* 2010). However, the fetuin-Amediated protection was not long-lasting, and gradually diminished at a later stage (e.g., 7 days post MCAo). It is possible that the restore of BBB function at a late stage (3 days after MCAo) limits subsequent fetuin-A extravasation, thereby diminishing fetuin-A-mediated

**Cell Death**

**(-) (+)**

**Elevated Intracellular Ca**

**Ca2+**

**Anti-oxidative Anti-inflammatory**

**NMDA Receptors**

**(-)**

**Spermine**

Low High

**(+)**

**Ca 2+ Influx**

Given the aforementioned pathogenic roles of Ca2+ and spermine in cerebral ischemia (in **section 4.2.1 and 4.2.2**), as well as the capacity of fetuin-A in binding Ca2+ and spermine (in **secton 3.2 and 3.3**) (Suzuki *et al.* 1994;Wang *et al.* 1997), it is plausible that fetuin-A confers protection by caging these toxic cationic molecules (Lee *et al.* 1999;Ivanova *et al.* 1998), thereby depriving them from damaging enzymes (such as Ca2+ -dependent proteases and polyamine oxidase). Furthermore, the fetuin-A-mediated protection is associated with a reduction of ischemia-elicited HMGB1 leakage from the ischemic core, and an inhibition of

#### **<IP_ADDRESS> Primary early injury in the core**

Within seconds to minutes after cerebral ischemia, decreased ATP production leads to failure of the Na+/K+-ATPase pump, disruption of membrane potentials, influx of sodium and calcium, and subsequent release of excitatory amino acids (such as glutamate, **Figure 2**). Engagement of glutamate with the ionotropic N-methyl-D-aspartate receptor (NMDA) leads to Ca2+ influx and activation of damaging proteases (e.g., phospholipase A2, nitric oxide synthase, endonucleases, and calpain) that compromise the functional and structural integrity of neuronal cells within 20-60 minutes (**Figure 2**). Early-stage therapeutics that block ion (Na+ and Ca2+) channels (Taylor & Meldrum 1995) and glutamate receptors (Meldrum 1990) fail in clinical trials, partly because of the impracticalities of administering such drugs at a time when those mechanisms are already activated.  This progressive expansion of cell death in the penumbra (i.e., secondary injury) is mediated by ischemia-elicited inflammatory responses. Within a few hours, microglia and neurons become activated to produce TNF and other cytokines (Kato *et al.* 1996;Botchkina *et al.* 1997). Subsequently, polymorphonuclear cells infiltrate into the ischemic brain tissue within 12-48 hours (Akopov *et al.* 1996), followed by an influx of monocytes and macrophages over a period of one to several days. Together, these centrally- and peripherally-derived cells orchestrate a potentially injurious inflammatory response by overproducing various proinflammatory cytokines (**Figure 2**).

Many pro-inflammatory cytokines (e.g., TNF and IL-1) contribute to cerebral ischemic injury (Buttini *et al.* 1996;Zaremba & Losy 2001), because inhibition of their production (Meistrell *et al.* 1997;Bertorelli *et al.* 1998) or activity (Barone *et al.* 1997;Yang *et al.* 1999) confers protective effects. In addition, an ubiquitous nuclear protein, HMGB1, can be passively released from the ischemic core, and spilled into the surrounding periphery (Qiu *et al.* 2008). In the penumbra, it amplifies a potentially injurious inflammatory response by inducing various cytokines, chemokines, tissue factor and adhesion molecules (Andersson *et al.* 2000;Lv *et al.* 2009;Fiuza *et al.* 2003;Treutiger *et al.* 2003) (**Figure 2**). Indeed, HMGB1-specific neutralizing antibodies and antagonists (e.g., the A box) have been proven protective (Liu *et al.* 2007a;Muhammad *et al.* 2008), supporting a pathogenic role for HMGB1 in ischemic injury.

#### **4.2.2 Divergent roles of spermine in cerebral ischemic injury**

Another abundant molecule, spermine, can also be passively released by injured cells (Paschen 1992). At higher (millimolar) concentrations, spermine could be neuroprotective by binding and blocking the NMDA receptor (Araneda *et al.* 1999;Ferchmin *et al.* 2000). In addition, it counter-regulates expression of inflammatory cytokines (Zhang *et al.* 2000;Zhang *et al.* 1997;Zhang *et al.* 1999;Zhu *et al.* 2009) and scavenges free radicals (Ha *et al.* 1998;Adibhatla *et al.* 2002). However, spermine can be enzymatically converted by polyamine oxidases into cytotoxic metabolites (e.g., 3-aminopropanal) (Ivanova *et al.* 1998), which readily spreads and mediates direct cytotoxicities (Ivanova *et al.* 1998). At low (micromolar) concentrations, spermine activates the NMDA receptor (Zubrow *et al.* 2000;Ferchmin *et al.* 2000;Williams 1997), thereby augmenting glutamate-mediated neurotoxicity by overactivating Ca2+ fluxes and disturbances of the calcium homeostasis.

Within seconds to minutes after cerebral ischemia, decreased ATP production leads to failure of the Na+/K+-ATPase pump, disruption of membrane potentials, influx of sodium and calcium, and subsequent release of excitatory amino acids (such as glutamate, **Figure 2**). Engagement of glutamate with the ionotropic N-methyl-D-aspartate receptor (NMDA) leads to Ca2+ influx and activation of damaging proteases (e.g., phospholipase A2, nitric oxide synthase, endonucleases, and calpain) that compromise the functional and structural integrity of neuronal cells within 20-60 minutes (**Figure 2**). Early-stage therapeutics that block ion (Na+ and Ca2+) channels (Taylor & Meldrum 1995) and glutamate receptors (Meldrum 1990) fail in clinical trials, partly because of the impracticalities of administering such drugs at a time when those mechanisms are already activated.  This progressive expansion of cell death in the penumbra (i.e., secondary injury) is mediated by ischemia-elicited inflammatory responses. Within a few hours, microglia and neurons become activated to produce TNF and other cytokines (Kato *et al.* 1996;Botchkina *et al.* 1997). Subsequently, polymorphonuclear cells infiltrate into the ischemic brain tissue within 12-48 hours (Akopov *et al.* 1996), followed by an influx of monocytes and macrophages over a period of one to several days. Together, these centrally- and peripherally-derived cells orchestrate a potentially injurious inflammatory response by

Many pro-inflammatory cytokines (e.g., TNF and IL-1) contribute to cerebral ischemic injury (Buttini *et al.* 1996;Zaremba & Losy 2001), because inhibition of their production (Meistrell *et al.* 1997;Bertorelli *et al.* 1998) or activity (Barone *et al.* 1997;Yang *et al.* 1999) confers protective effects. In addition, an ubiquitous nuclear protein, HMGB1, can be passively released from the ischemic core, and spilled into the surrounding periphery (Qiu *et al.* 2008). In the penumbra, it amplifies a potentially injurious inflammatory response by inducing various cytokines, chemokines, tissue factor and adhesion molecules (Andersson *et al.* 2000;Lv *et al.* 2009;Fiuza *et al.* 2003;Treutiger *et al.* 2003) (**Figure 2**). Indeed, HMGB1-specific neutralizing antibodies and antagonists (e.g., the A box) have been proven protective (Liu *et al.* 2007a;Muhammad *et al.* 2008), supporting a pathogenic role for HMGB1 in ischemic injury.

Another abundant molecule, spermine, can also be passively released by injured cells (Paschen 1992). At higher (millimolar) concentrations, spermine could be neuroprotective by binding and blocking the NMDA receptor (Araneda *et al.* 1999;Ferchmin *et al.* 2000). In addition, it counter-regulates expression of inflammatory cytokines (Zhang *et al.* 2000;Zhang *et al.* 1997;Zhang *et al.* 1999;Zhu *et al.* 2009) and scavenges free radicals (Ha *et al.* 1998;Adibhatla *et al.* 2002). However, spermine can be enzymatically converted by polyamine oxidases into cytotoxic metabolites (e.g., 3-aminopropanal) (Ivanova *et al.* 1998), which readily spreads and mediates direct cytotoxicities (Ivanova *et al.* 1998). At low (micromolar) concentrations, spermine activates the NMDA receptor (Zubrow *et al.* 2000;Ferchmin *et al.* 2000;Williams 1997), thereby augmenting glutamate-mediated neurotoxicity by overactivating Ca2+ fluxes and disturbances of the calcium homeostasis.

prompted the search for downstream targets that also mediate ischemic injury.

**<IP_ADDRESS> Secondary late injury in the penumbral zone**

overproducing various proinflammatory cytokines (**Figure 2**).

**4.2.2 Divergent roles of spermine in cerebral ischemic injury**

**<IP_ADDRESS> Primary early injury in the core**

During cerebral ischemia, brain spermine levels are decreased (Paschen *et al.* 1992), owing largely to an accompanying increase in the enzymatic activity of brain polyamine oxidase (Ivanova *et al.* 1998). The loss of spermine consequently tilts the balance towards neurotoxicity through activating the NMDA receptor, and increasing susceptibility to oxidative stress as well as excessive inflammatory response.

Fig. 3. **Divergent roles of spermine in cerebral ischemic injury.**

#### **4.2.3 Peripheral administration of fetuin-A reduced cerebral ischemic injury**

As mentioned earlier, when given peripherally, exogenous fetuin-A gains entry across the BBB into the ischemic brain tissue (**Figure 4**). The time-course of fetuin-A extravasation in the ischemic brain tissue parallels with the time-dependent alteration of the BBB permeability (Belayev *et al.* 1996), which was transiently elevated (5 -25 h post MCAo) followed by a return towards baseline at 72 h post MCAo (Belayev *et al.* 1996). It is possible that the temporal breakdown of the BBB is required for circulating fetuin-A to transiently gain entry into the brain. Consistently, peripheral administration of fetuin-A (50 mg/kg) promoted a dose-dependent protection against cerebral ischemic injury during an early stage of cerebral ischemia (i.e., 24 h post MCAo) (Wang *et al.* 2010). However, the fetuin-Amediated protection was not long-lasting, and gradually diminished at a later stage (e.g., 7 days post MCAo). It is possible that the restore of BBB function at a late stage (3 days after MCAo) limits subsequent fetuin-A extravasation, thereby diminishing fetuin-A-mediated long-lasting protective effects.

Given the aforementioned pathogenic roles of Ca2+ and spermine in cerebral ischemia (in **section 4.2.1 and 4.2.2**), as well as the capacity of fetuin-A in binding Ca2+ and spermine (in **secton 3.2 and 3.3**) (Suzuki *et al.* 1994;Wang *et al.* 1997), it is plausible that fetuin-A confers protection by caging these toxic cationic molecules (Lee *et al.* 1999;Ivanova *et al.* 1998), thereby depriving them from damaging enzymes (such as Ca2+ -dependent proteases and polyamine oxidase). Furthermore, the fetuin-A-mediated protection is associated with a reduction of ischemia-elicited HMGB1 leakage from the ischemic core, and an inhibition of

Role of Fetuin-A in Injury and Infection 337

functions as a late mediator of endotoxemia and sepsis (Wang *et al.* 1999;Yang *et al.* 2004;Wang *et al.* 2008;Wang *et al.* 2009). In murine models of endotoxemia and sepsis, HMGB1 is first detectable in the circulation eight hours after the onset of diseases, subsequently increasing to plateau levels from 16 to 32 hours (Wang *et al.* 1999;Yang *et al.* 2004) (**Figure 4**). This late appearance of circulating HMGB1 parallels with the onset of animal lethality from endotoxemia or sepsis, and distinguishes itself from TNF and other early proinflammatory cytokines (Wang *et al.* 2001). Therefore, agents capable of selectively attenuating systemic HMGB1 accumulation at a late stage may hold potential in the

In light of the anti-inflammatory activities of spermine *in vitro* (Zhang *et al.* 1997;Zhu *et al.* 2009), we evaluated the effects of spermine on animal survival in animal models of sepsis. Intraperitoneal administration of spermine (1.0 -10 mg/kg, twice daily, for three days) did not protect mice against lethal endotoxemia, but confers a dose-dependent protection against lethal sepsis. This protection was associated with a significant attenuation of systemic accumulation of HMGB1 and other cytokines (e.g., IL-6, KC, MCP-1, MIP-2, TIMP-1, sTNFRI and sTNFRII) (Zhu *et al.* 2009). At a higher dose (100 mg/kg), however, spermine decreased animal survival rate from 58% to 38% at 48 h post CLP, and further decreasing it to 0% at 72 h post CLP. It is possible that spermine is enzymatically converted by polyamine oxidases into cytotoxic metabolites (e.g., 3-aminopropanal), thereby exerting these

To understand the role of fetuin-A in systemic inflammatory diseases, we determined the influence of fetuin-A disruption on endotoxemic and septic lethality. Although fetuin-Adeficient C57BL/6J mice were not more susceptible to cerebral ischemic insult than sex- and body-matched (male, 27-29 g) wild-type C57BL/6J mice (Wang *et al.* 2010), they were more susceptible to lethal endotoxemic or septic insult (Li *et al.* 2011a). It suggests that endogenous fetuin-A occupies an integral role in host defense against lethal systemic

The protective role of fetuin-A was further supported by the observations that supplementation with exogenous fetuin-A (20-100 mg/kg) provided a dose-dependent protection against lethal endotoxemia (Li *et al.* 2011a). In an animal model of sepsis, delayed administration of fetuin-A (20 - 100 mg/kg), beginning 24 h *after* the onset of sepsis and followed by an additional dose at 48 h post CLP, dose-dependently and significantly

Supplementation of fetuin-A was associated with significant reduction of circulating HMGB1 levels, suggesting that fetuin-A confers protection by inhibiting late-acting proinflammatory mediators (Li *et al.* 2011a). The mechanisms underlying fetuin-A-mediated suppression of HMGB1 release may be complex. At the concentrations (100 μg/ml) that fetuin-A attenuated LPS-induced HMGB1 release in macrophage cultures, fetuin-A stimulated autophagy and impaired LPS-induced elevation of cytoplasmic and nuclear HMGB1 levels (Li et al. 2011a). It is presently unknown whether fetuin-A reduces

increased long-term animal survival rates from 45% to 90% (Li *et al.* 2011a).

treatment of lethal sepsis.

inflammation.

**4.3.4 Protective mechanisms**

**4.3.2 Dual roles of spermine in experimental sepsis**

potentially toxic effects when given at higher doses.

**4.3.3 Protective role of Fetuin-A in sepsis**

expression of proinflammatory cytokines (e.g., TNF) in the penumbra (Wang *et al.* 2010) (**Figure 4**), suggesting that fetuin-A confers protection partly by attenuating early inflammatory responses.

Fig. 4. **Protective roles of fetuin-A in cerebral ischemic injury and sepsis.**

#### **4.3 Experimental sepsis**

Sepsis is the most common cause of death in intensive care units, claiming approximately 225,000 victims annually in the U.S. alone. The high mortality of sepsis is in part mediated by bacterial endotoxin , which activates macrophages and monocytes to sequentially release early (e.g., TNF and IL-1) (Dinarello 1996) and late (e.g., HMGB1) proinflammatory cytokines.

#### **4.3.1 Pathogenesis of sepsis**

The pathogenesis of sepsis is partly attributable to dysregulated systemic inflammatory responses that are initiated by early proinflammatory cytokines and sustained by late-acting proinflammatory mediators. For instance, excessive accumulation of early proinflammatory cytokines, including TNF (Tracey *et al.* 1987), interleukin (IL)-1 (Dinarello & Thompson 1991), interferon (IFN)-γ (Heinzel 1990), individually or in combination, contribute to the pathogenesis of lethal systemic inflammation. Because these early cytokines are difficult to target in clinical settings, we searched for other late proinflammatory mediators that may offer a wider therapeutic window.

As aforementioned, HMGB1 is released from activated innate immune cells in response to microbial products (such as endotoxin or CpG-DNA) (Wang *et al.* 1999;Ivanov *et al.* 2007), or host cytokines (e.g., TNF or IFN-γ) (Wang *et al.* 1999;Rendon-Mitchell *et al.* 2003), and

expression of proinflammatory cytokines (e.g., TNF) in the penumbra (Wang *et al.* 2010) (**Figure 4**), suggesting that fetuin-A confers protection partly by attenuating early

**Cerebral Ischemic Injury Endotoxemia and Sepsis**

Fig. 4. **Protective roles of fetuin-A in cerebral ischemic injury and sepsis.**

**Fetuin-A**

**Fetuin-A**

Sepsis is the most common cause of death in intensive care units, claiming approximately 225,000 victims annually in the U.S. alone. The high mortality of sepsis is in part mediated by bacterial endotoxin , which activates macrophages and monocytes to sequentially release early (e.g., TNF and IL-1) (Dinarello 1996) and late (e.g., HMGB1) proinflammatory

**Fetuin-A** *("- APP")*

**Liver** Constitutive Secretion

**TNF, IFN- …** *("Early Mediators")*

**LPS / CLP**

**HMGB1, …** *("Late Mediators")*

**Lethal Systemic Inflammation**

The pathogenesis of sepsis is partly attributable to dysregulated systemic inflammatory responses that are initiated by early proinflammatory cytokines and sustained by late-acting proinflammatory mediators. For instance, excessive accumulation of early proinflammatory cytokines, including TNF (Tracey *et al.* 1987), interleukin (IL)-1 (Dinarello & Thompson 1991), interferon (IFN)-γ (Heinzel 1990), individually or in combination, contribute to the pathogenesis of lethal systemic inflammation. Because these early cytokines are difficult to target in clinical settings, we searched for other late proinflammatory mediators that may

As aforementioned, HMGB1 is released from activated innate immune cells in response to microbial products (such as endotoxin or CpG-DNA) (Wang *et al.* 1999;Ivanov *et al.* 2007), or host cytokines (e.g., TNF or IFN-γ) (Wang *et al.* 1999;Rendon-Mitchell *et al.* 2003), and

inflammatory responses.

**4.3 Experimental sepsis**

**Microglia and Neurons**

**Secondary Ischemic Injury** (Penumbra)

**Cytokines** (e.g., TNF)

**Primary Injury** (Ischemc Core)

**HMGB1 Leakage & Spillage**

**Cerebral Ischemia**

**Infiltrated Leukocytes**

**4.3.1 Pathogenesis of sepsis**

offer a wider therapeutic window.

cytokines.

functions as a late mediator of endotoxemia and sepsis (Wang *et al.* 1999;Yang *et al.* 2004;Wang *et al.* 2008;Wang *et al.* 2009). In murine models of endotoxemia and sepsis, HMGB1 is first detectable in the circulation eight hours after the onset of diseases, subsequently increasing to plateau levels from 16 to 32 hours (Wang *et al.* 1999;Yang *et al.* 2004) (**Figure 4**). This late appearance of circulating HMGB1 parallels with the onset of animal lethality from endotoxemia or sepsis, and distinguishes itself from TNF and other early proinflammatory cytokines (Wang *et al.* 2001). Therefore, agents capable of selectively attenuating systemic HMGB1 accumulation at a late stage may hold potential in the treatment of lethal sepsis.

#### **4.3.2 Dual roles of spermine in experimental sepsis**

In light of the anti-inflammatory activities of spermine *in vitro* (Zhang *et al.* 1997;Zhu *et al.* 2009), we evaluated the effects of spermine on animal survival in animal models of sepsis. Intraperitoneal administration of spermine (1.0 -10 mg/kg, twice daily, for three days) did not protect mice against lethal endotoxemia, but confers a dose-dependent protection against lethal sepsis. This protection was associated with a significant attenuation of systemic accumulation of HMGB1 and other cytokines (e.g., IL-6, KC, MCP-1, MIP-2, TIMP-1, sTNFRI and sTNFRII) (Zhu *et al.* 2009). At a higher dose (100 mg/kg), however, spermine decreased animal survival rate from 58% to 38% at 48 h post CLP, and further decreasing it to 0% at 72 h post CLP. It is possible that spermine is enzymatically converted by polyamine oxidases into cytotoxic metabolites (e.g., 3-aminopropanal), thereby exerting these potentially toxic effects when given at higher doses.

#### **4.3.3 Protective role of Fetuin-A in sepsis**

To understand the role of fetuin-A in systemic inflammatory diseases, we determined the influence of fetuin-A disruption on endotoxemic and septic lethality. Although fetuin-Adeficient C57BL/6J mice were not more susceptible to cerebral ischemic insult than sex- and body-matched (male, 27-29 g) wild-type C57BL/6J mice (Wang *et al.* 2010), they were more susceptible to lethal endotoxemic or septic insult (Li *et al.* 2011a). It suggests that endogenous fetuin-A occupies an integral role in host defense against lethal systemic inflammation.

The protective role of fetuin-A was further supported by the observations that supplementation with exogenous fetuin-A (20-100 mg/kg) provided a dose-dependent protection against lethal endotoxemia (Li *et al.* 2011a). In an animal model of sepsis, delayed administration of fetuin-A (20 - 100 mg/kg), beginning 24 h *after* the onset of sepsis and followed by an additional dose at 48 h post CLP, dose-dependently and significantly increased long-term animal survival rates from 45% to 90% (Li *et al.* 2011a).

#### **4.3.4 Protective mechanisms**

Supplementation of fetuin-A was associated with significant reduction of circulating HMGB1 levels, suggesting that fetuin-A confers protection by inhibiting late-acting proinflammatory mediators (Li *et al.* 2011a). The mechanisms underlying fetuin-A-mediated suppression of HMGB1 release may be complex. At the concentrations (100 μg/ml) that fetuin-A attenuated LPS-induced HMGB1 release in macrophage cultures, fetuin-A stimulated autophagy and impaired LPS-induced elevation of cytoplasmic and nuclear HMGB1 levels (Li et al. 2011a). It is presently unknown whether fetuin-A reduces

Role of Fetuin-A in Injury and Infection 339

Andersson U., Wang H., Palmblad K., Aveberger A.C., Bloom O., Erlandsson-Harris H.,

Araneda R.C., Lan J.Y., Zheng X., Zukin R.S. & Bennett M.V. (1999) Spermine and arcaine block and permeate N-methyl-D-aspartate receptor channels. *Biophys.J* 76, 2899-2911 Barone F.C., Arvin B., White R.F., Miller A., Webb C.L., Willette R.N., Lysko P.G. &

Belayev L., Busto R., Zhao W. & Ginsberg M.D. (1996) Quantitative evaluation of blood-

Bell C.W., Jiang W., Reich C.F. & Pisetsky D.S. (2006) The Extracellular Release of HMGB1 during Apoptotic Cell Death. *Am J Physiol Cell Physiol* 291(6), C1318-1325 Bertorelli R., Adami M., Di Santo E. & Ghezzi P. (1998) MK 801 and dexamethasone reduce

Botchkina G.I., Meistrell M.E., Botchkina I.L. & Tracey K.J. (1997) Expression of TNF and

Brightbill H.D., Libraty D.H., Krutzik S.R., Yang R.B., Belisle J.T., Bleharski J.R., Maitland

Buttini M., Appel K., Sauter A., Gebicke-Haerter P.J. & Boddeke H.W. (1996) Expression of

Chmiela M., Czkwianianc E., Wadstrom T. & Rudnicka W. (1997) Role of Helicobacter pylori surface structures in bacterial interaction with macrophages. *Gut* 40, 20-24 Christie D.L., Dziegielewska K.M., Hill R.M. & Saunders N.R. (1987) Fetuin: the bovine

Daveau M., Christian D., Julen N., Hiron M., Arnaud P. & Lebreton J.P. (1988) The synthesis

Demetriou M., Binkert C., Sukhu B., Tenenbaum H.C. & Dennis J.W. (1996) Fetuin/alpha2-

Dinarello C.A. & Thompson R.C. (1991) Blocking IL-1: interleukin 1 receptor antagonist in

Dubreuil J.D., Giudice G.D. & Rappuoli R. (2002) Helicobacter pylori interactions with host

Dziegielewska K.M. & Andersen N.A. (1998) The fetal glycoprotein, fetuin, counteracts ill-

Dziegielewska K.M., Andersen N.A. & Saunders N.R. (1998) Modification of macrophage

homologue of human alpha 2HS glycoprotein. *FEBS Lett* 214, 45-49

Dinarello C.A. (1996) Biologic basis for interleukin-1 in disease. *Blood* 87, 2095-2147

response to lipopolysaccharide by fetuin. *Immunol Lett* 60, 31-35

Human Monocytes. *J Exp Med* 192, 565-570

brain injury. *Stroke* 28, 1233-1244

the rat brain. *Neurosci Lett* 246, 41-44

HepG2 cells. *FEBS Lett* 241, 191-194

through toll-like receptors. *Science* 285, 732-736

cytokine antagonist. *J Biol Chem* 271, 12755-12761

vivo and in vitro. *Immunol Today* 12, 404-410

*Microbiol Mol Biol Rev* 66, 617-29, table

*Res.* 739, 88-96

3, 765-781

1-16

372-375

Janson A., Kokkola R., Zhang M., Yang H. & Tracey K.J. (2000) High Mobility Group 1 Protein (HMG-1) Stimulates Proinflammatory Cytokine Synthesis in

Feuerstein G.Z. (1997) Tumor necrosis factor-alpha. A mediator of focal ischemic

brain barrier permeability following middle cerebral artery occlusion in rats. *Brain*

both tumor necrosis factor levels and infarct volume after focal cerebral ischemia in

TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia. *Mol Med*

M., Norgard M.V., Plevy S.E., Smale S.T., Brennan P.J., Bloom B.R., Godowski P.J. & Modlin R.L. (1999) Host defense mechanisms triggered by microbial lipoproteins

tumor necrosis factor alpha after focal cerebral ischaemia in the rat. *Neuroscience* 71,

of human alpha-2-HS glycoprotein is down-regulated by cytokines in hepatoma

HS glycoprotein is a transforming growth factor-beta type II receptor mimic and

serum and extracellular matrix proteins: potential role in the infectious process.

effects of the bacterial endotoxin, lipopolysaccharide, in pregnancy. *Biol Neonate.* 74,

cytoplasmic HMGB1 levels by stimulating its degradation in an autophagy-dependent fashion, as what has been shown for other HMGB1 inhibitors such as EGCG, the major catechin of Green tea (*Camellia sinensis*) (Li *et al*. 2011b).

Accumulating evidence has suggested the possibility that fetuin-A functions as a negative regulator of HMGB1 release during lethal systemic inflammation (**Figure 4**). First, the timedependent decrease of circulating fetuin-A levels was accompanied by parallel but opposite changes – a time-dependent increase - of circulating HMGB1 levels in animal models of endotoxemia (Wang *et al.* 1999) or sepsis (Yang *et al.* 2004). Second, disruption of fetuin-A expression led to elevation of serum HMGB1 levels in endotoxemia and sepsis *(Li et al. 2011a)*. Lastly, supplementation of fetuin-A resulted in significant reduction of circulating HMGB1 levels during endotoxemia and sepsis *(Li et al. 2011a)*.

Nevertheless, the current study can not exclude other alternative mechanisms by which fetuin-A confers these protective effects. For instance, fetuin-A may be capable of binding bacteria (Chmiela *et al.* 1997;Dubreuil *et al.* 2002), thereby affecting macrophage-mediated pathogen elimination. Furthermore, fetuin-A may facilitate macrophages-mediated ingestion and elimination of apoptotic neutrophils (Lord 2003;Jersmann *et al.* 2003), thereby preventing secondary necrosis and passive leakage of injurious molecules (e.g., proteases, reactive oxygen species, and HMGB1) (Bell *et al.* 2006).

### **5. Conclusions**

A liver-derived acute phase protein, fetuin-A, appears to be distinctly regulated by different proinflammatory mediators. A previously under-appreciated protective role for fetuin-A in injury and infection has been suggested by recent studies. Fetuin-A is capable of crossing the blood-brain barrier, inhibiting early inflammatory response in animal models of cerebral ischemia, thereby conferring a short-term neuroprotection against ischemic injury. Disruption of fetuin-A expression renders mice significantly more susceptible to lethal endotoxemia or sepsis; whereas repetitive administration of fetuin-A confers a dosedependent and long-lasting protection in animal models of lethal endotoxemia and sepsis. Thus, fetuin-A occupies protective roles against injury- or infection-elicited inflammation.

#### **6. Acknowledgment**

We thank Arvin Jundoria for critical reading of the manuscript. Work in the author's laboratory was supported by the National Institute of General Medical Sciences (R01GM063075 and R01GM070817 to HW) and the National Center of Complementary & Alternative Medicine (R01AT05076 to HW).

#### **7. References**

cytoplasmic HMGB1 levels by stimulating its degradation in an autophagy-dependent fashion, as what has been shown for other HMGB1 inhibitors such as EGCG, the major

Accumulating evidence has suggested the possibility that fetuin-A functions as a negative regulator of HMGB1 release during lethal systemic inflammation (**Figure 4**). First, the timedependent decrease of circulating fetuin-A levels was accompanied by parallel but opposite changes – a time-dependent increase - of circulating HMGB1 levels in animal models of endotoxemia (Wang *et al.* 1999) or sepsis (Yang *et al.* 2004). Second, disruption of fetuin-A expression led to elevation of serum HMGB1 levels in endotoxemia and sepsis *(Li et al. 2011a)*. Lastly, supplementation of fetuin-A resulted in significant reduction of circulating

Nevertheless, the current study can not exclude other alternative mechanisms by which fetuin-A confers these protective effects. For instance, fetuin-A may be capable of binding bacteria (Chmiela *et al.* 1997;Dubreuil *et al.* 2002), thereby affecting macrophage-mediated pathogen elimination. Furthermore, fetuin-A may facilitate macrophages-mediated ingestion and elimination of apoptotic neutrophils (Lord 2003;Jersmann *et al.* 2003), thereby preventing secondary necrosis and passive leakage of injurious molecules (e.g., proteases,

A liver-derived acute phase protein, fetuin-A, appears to be distinctly regulated by different proinflammatory mediators. A previously under-appreciated protective role for fetuin-A in injury and infection has been suggested by recent studies. Fetuin-A is capable of crossing the blood-brain barrier, inhibiting early inflammatory response in animal models of cerebral ischemia, thereby conferring a short-term neuroprotection against ischemic injury. Disruption of fetuin-A expression renders mice significantly more susceptible to lethal endotoxemia or sepsis; whereas repetitive administration of fetuin-A confers a dosedependent and long-lasting protection in animal models of lethal endotoxemia and sepsis. Thus, fetuin-A occupies protective roles against injury- or infection-elicited inflammation.

We thank Arvin Jundoria for critical reading of the manuscript. Work in the author's laboratory was supported by the National Institute of General Medical Sciences (R01GM063075 and R01GM070817 to HW) and the National Center of Complementary &

Adibhatla R.M., Hatcher J.F., Sailor K. & Dempsey R.J. (2002) Polyamines and central

cerebral ischemia in spontaneously hypertensive rats. *Brain Res* 938, 81-86 Akopov S.E., Simonian N.A. & Grigorian G.S. (1996) Dynamics of polymorphonuclear

nervous system injury: spermine and spermidine decrease following transient focal

leukocyte accumulation in acute cerebral infarction and their correlation with brain

catechin of Green tea (*Camellia sinensis*) (Li *et al*. 2011b).

HMGB1 levels during endotoxemia and sepsis *(Li et al. 2011a)*.

reactive oxygen species, and HMGB1) (Bell *et al.* 2006).

**5. Conclusions**

**6. Acknowledgment**

**7. References**

Alternative Medicine (R01AT05076 to HW).

tissue damage. *Stroke* 27, 1739-1743

Role of Fetuin-A in Injury and Infection 341

Ketteler M., Bongartz P., Westenfeld R., Wildberger J.E., Mahnken A.H., Bohm R., Metzger

Kitchener P.D., Dziegielewska K.M., Hutton E.J., Hinrichsen C.F. & Saunders N.R. (1999)

Kitchener P.D., Dziegielewska K.M., Knott G.W., Miller J.M., Nawratil P., Potter A.E. &

Krieg A.M. (2002) CpG motifs in bacterial DNA and their immune effects. *Annu Rev*

Kusnierz-Cabala B., Gurda-Duda A., Panek J., Fedak D., Dumnicka P., Solnica B. & Kulig J.

Lee J.M., Zipfel G.J. & Choi D.W. (1999) The changing landscape of ischaemic brain injury

Li W., Zhu S., Li J., Huang Y., Zhou R., Fan X., Yang H., Gong X., Eissa N.T., Jahnen-Dechent

endotoxin-stimulated macrophages. *Biochem Pharmacol* 81(9), 1152-1163. Liu K., Mori S., Takahashi H.K., Tomono Y., Wake H., Kanke T., Sato Y., Hiraga N., Adachi

Liu K., Mori S., Takahashi H.K., Tomono Y., Wake H., Kanke T., Sato Y., Hiraga N., Adachi

Lord J.M. (2003) A physiological role for alpha2-HS glycoprotein: stimulation of

Lv B., Wang H., Tang Y., Fan Z., Xiao X. & Chen F. (2009) High-mobility group box 1 protein

Mathews S.T., Srinivas P.R., Leon M.A. & Grunberger G. (1997) Bovine fetuin is an inhibitor

Meistrell M.E., Botchkina G.I., Wang H., Di Santo E., Cockroft K.M., Bloom O.,

Meldrum B. (1990) Protection against ischaemic neuronal damage by drugs acting on

Metry G., Stenvinkel P., Qureshi A.R., Carrero J.J., Yilmaz M.I., Barany P., Snaedal S.,

excitatory neurotransmission. *Cerebrovasc Brain Metab Rev* 2, 27-57

prevalent haemodialysis patients. *Eur J Clin Invest* 38, 804-811

macrophage uptake of apoptotic cells. *Clin Sci (Lond)* 105, 267-268

kappaB and Egr-1. *Thromb Haemost* 102, 352-359

of insulin receptor tyrosine kinase. *Life Sci* 61, 1583-1592

damaging cytokine in cerebral ischemia. *Shock* 8, 341-348

dialysis: a cross-sectional study. *Lancet* 361, 827-833

development of the rat. *Int J Dev Neurosci* 17, 21-30

ganglia of perinatal rats. *Int J Dev Neurosci* 15, 717-727

mechanisms. *Nature* 399 Supplement, A7-A14

*Immunol* 20, 709-760

56, 191-195

*J* 21, 3904-3916

*J* 21, 3904-3916

T., Wanner C., Jahnen-Dechent W. & Floege J. (2003) Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on

Fetuin in neurons of the retina and cerebellum during fetal and postnatal

Saunders N.R. (1997) Fetuin expression in the dorsal root ganglia and trigeminal

(2010) Serum fetuin A concentrations in patients with acute pancreatitis. *Clin Lab*

W., Wang P., Tracey K.J., Sama A.E. & Wang H. (2011a) A hepatic protein, fetuin-A, occupies a protective role in lethal systemic inflammation. *PLoS ONE* 6(2), e16945 Li, W., Zhu S., Li J., Assa A., Xu J., Fan S., Eissa N.T., Tracey K.T., Sama A.E., & Wang H.

(2011b). EGCG induces autophagy and reduced cytoplasmic HMGB1 levels in

N., Yoshino T. & Nishibori M. (2007a) Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. *FASEB*

N., Yoshino T. & Nishibori M. (2007b) Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. *FASEB*

induces tissue factor expression in vascular endothelial cells via activation of NF-

Vishnubhakat J.M., Ghezzi P. & Tracey K.J. (1997) Tumor necrosis factor is a brain

Heimburger O., Lindholm B. & Suliman M.E. (2008) Low serum fetuin-A concentration predicts poor outcome only in the presence of inflammation in

Dziegielewska K.M., Brown W.M., Gould C.C., Matthews N., Sedgwick J.E. & Saunders N.R. (1992) Fetuin: an acute phase protein in cattle. *J Comp Physiol [B]* 162, 168-171 Dziegielewska K.M., Daikuhara Y., Ohnishi T., Waite M.P., Ek J., Habgood M.D., Lane M.A.,

Ferchmin P.A., Perez D. & Biello M. (2000) Spermine is neuroprotective against anoxia and

Feuerstein G.Z., Wang X. & Barone F.C. (1998) The role of cytokines in the neuropathology

Fiuza C., Bustin M., Talwar S., Tropea M., Gerstenberger E., Shelhamer J.H. & Suffredini

Goustin A.S. & Abou-Samra A.B. (2010) The "thrifty" gene encoding Ahsg/Fetuin-A meets the insulin receptor: Insights into the mechanism of insulin resistance. *Cell Signal*  Ha H.C., Sirisoma N.S., Kuppusamy P., Zweier J.L., Woster P.M. & Casero R.A., Jr. (1998)

Haasemann M., Nawratil P. & Muller-Esterl W. (1991) Rat tyrosine kinase inhibitor shows

Heinzel F.P. (1990) The role of IFN-gamma in the pathology of experimental endotoxemia. *J*

Heiss A., DuChesne A., Denecke B., Grotzinger J., Yamamoto K., Renne T. & Jahnen-

Hemmi H., Takeuchi O., Kawai T., Kaisho T., Sato S., Sanjo H., Matsumoto M., Hoshino K.,

Ivanov S., Dragoi A.M., Wang X., Dallacosta C., Louten J., Musco G., Sitia G., Yap G.S., Wan

endogenous mediator of neuronal and glial cell death. *J Exp Med* 188, 327-340 Ix J.H., Wassel C.L., Kanaya A.M., Vittinghoff E., Johnson K.C., Koster A., Cauley J.A.,

Jahnen-Dechent W., Schafer C., Heiss A. & Grotzinger J. (2001) Systemic inhibition of

Jersmann H.P., Dransfield I. & Hart S.P. (2003) Fetuin/alpha2-HS glycoprotein enhances

Kato H., Kogure K., Liu X.H., Araki T. & Itoyama Y. (1996) Progressive expression of

N-methyl-D-aspartate in hippocampal slices. *Brain Res* 859, 273-279

of stroke and neurotrauma. *Neuroimmunomodulation* 5, 143-159

distribution and origin. *J Comp Neurol* 423, 373-388

endothelial cells. *Blood* 101, 2652-2660

*Natl Acad Sci U S A* 95, 11140-11145

274, 899-902

13333-13341

*Immunol* 145, 2920-2924

DNA. *Nature* 408, 740-745

*Kardiol* 90 Suppl 3, 47-56

*Sci (Lond)* 105, 273-278

mellitus in older persons. *JAMA* 300, 182-188

cerebral ischemia in the rat. *Brain Res.* 734, 203-212

Potter A. & Saunders N.R. (2000) Fetuin in the developing neocortex of the rat:

A.F. (2003) Inflammation-promoting activity of HMGB1 on human microvascular

The natural polyamine spermine functions directly as a free radical scavenger. *Proc*

sequence similarity to human alpha 2-HS glycoprotein and bovine fetuin. *Biochem J*

Dechent W. (2003) Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. *J Biol Chem* 278,

Wagner H., Takeda K. & Akira S. (2000) A Toll-like receptor recognizes bacterial

Y., Biron C.A., Bianchi M.E., Wang H. & Chu W.M. (2007) A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA. *Blood.* 110, 1970-1981 Ivanova S., Botchkina G.I., Al-Abed Y., Meistrell M., Batliwalla F., Dubinsky J.M., Iadecola C.,

Wang H., Gregersen P.K., Eaton J.W. & Tracey K.J. (1998) Cerebral ischemia enhances polyamine oxidation: identification of enzymatically formed 3-aminopropanal as an

Harris T.B., Cummings S.R. & Shlipak M.G. (2008) Fetuin-A and incident diabetes

spontaneous calcification by the serum protein alpha 2-HS glycoprotein/fetuin. *Z*

phagocytosis of apoptotic cells and macropinocytosis by human macrophages. *Clin*

immunomolecules on activated microglia and invading leukocytes following focal

Role of Fetuin-A in Injury and Infection 343

Suzuki M., Shimokawa H., Takagi Y. & Sasaki S. (1994) Calcium-binding properties of fetuin

Szweras M., Liu D., Partridge E.A., Pawling J., Sukhu B., Clokie C., Jahnen-Dechent W.,

Taylor C.P. & Meldrum B.S. (1995) Na+ channels as targets for neuroprotective drugs.

Terkelsen O.B., Jahnen-Dechent W., Nielsen H., Moos T., Fink E., Nawratil P., Muller-Esterl

Tracey K.J., Fong Y., Hesse D.G., Manogue K.R., Lee A.T., Kuo G.C., Lowry S.F. & Cerami A.

Treutiger C.J., Mullins G.E., Johansson A.S., Rouhiainen A., Rauvala H.M., Erlandsson-

Tsung A., Klune J.R., Zhang X., Jeyabalan G., Cao Z., Peng X., Stolz D.B., Geller D.A.,

Tsung A., Sahai R., Tanaka H., Nakao A., Fink M.P., Lotze M.T., Yang H., Li J., Tracey K.J.,

Tuttolomondo A., Di Raimondo D., Di Sciacca R., Casuccio A., Bivona G., Bellia C., Barreca L.,

correlation with clinical and laboratory variables. *Atherosclerosis.* 208, 290-296 Wang H., Bloom O., Zhang M., Vishnubhakat J.M., Ombrellino M., Che J., Frazier A., Yang

Wang H., Li W., Zhu S., Li J., D'Amore J., Ward M.F., Yang H., Wu R., Jahnen-Dechent W.,

Wang H., Liao H., Ochani M., Justiniani M., Lin X., Yang L., Al Abed Y., Wang H., Metz C.,

release and improve survival in experimental sepsis. *Nat Med* 10, 1216-1221 Wang H. & Tracey K.J. (1999) Fetuin opsonizes macrophage-deactivating cations. In: *Update*

Wang H., Ward M.F. & Sama A.E. (2009) Novel HMGB1-inhibiting therapeutic agents for

Wang H., Yang H., Czura C.J., Sama A.E. & Tracey K.J. (2001) HMGB1 as a Late Mediator of Lethal Systemic Inflammation. *Am J Respir Crit Care Med* 164, 1768-1773

after murine liver ischemia-reperfusion. *J Exp Med* 201, 1135-1143

a late mediator of endotoxin lethality in mice. *Science* 285, 248-251

Marshall J.C. & Cohen J.), pp. 155-163. SpringerVerlag Press.

experimental sepsis. *Shock.* 32, 348-357

embryonic and neonatal rat tissues. *Anat Ebryol (Berl)* 197, 125-133

calcium-mediated signaling. *J Exp Med* 204, 2913-2923

Tenenbaum H.C., Swallow C.J., Grynpas M.D. & Dennis J.W. (2002) alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling. *J Biol Chem*

W. & Mollgard K. (1998) Rat fetuin: distribution of protein and mRNA in

(1987) Anti-cachectin/TNF monoclonal antibodies prevent septic shock during

Harris H., Andersson U., Yang H., Tracey K.J., Andersson J. & Palmblad J.E. (2003) High mobility group 1 B-box mediates activation of human endothelium. *J Intern*

Rosengart M.R. & Billiar T.R. (2007) HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and

Geller D.A. & Billiar T.R. (2005) The nuclear factor HMGB1 mediates hepatic injury

Serio A., D'Aguanno G., Ciaccio M., Licata G. & Pinto A. (2010) Fetuin-A and CD40 L plasma levels in acute ischemic stroke: differences in relation to TOAST subtype and

H., Ivanova S., Borovikova L., Manogue K.R., Faist E., Abraham E., Andersson J., Andersson U., Molina P.E., Abumrad N.N., Sama A. & Tracey K.J. (1999) HMG-1 as

Tracey K.J., Wang P. & Sama A.E. (2010) Peripheral administration of fetuin-A attenuates early cerebral ischemic injury in rats. J Cereb Blood Flow Metab 30, 493-

Miller E.J., Tracey K.J. & Ulloa L. (2004) Cholinergic agonists inhibit HMGB1

*in Intensive Care and Emergency Medicine: Immune Response in the Critically Ill* (eds

in fetal bovine serum. *J Exp Zoology* 270, 501-507

277, 19991-19997

*Med* 254, 375-385

504

*Trends Pharmacol Sci* 16, 309-316

lethal bacteraemia. *Nature* 330, 662-664

Muhammad S., Barakat W., Stoyanov S., Murikinati S., Yang H., Tracey K.J., Bendszus M.,

receptor RAGE mediates ischemic brain damage. *J Neurosci* 28, 12023-12031 Ombrellino M., Wang H., Yang H., Zhang M., Vishnubhakat J., Frazier A., Scher L.A.,

Paschen W. (1992) Polyamine metabolism in reversible cerebral ischemia. *Cerebrovasc Brain*

Paschen W., Csiba L., Rohn G. & Bereczki D. (1991) Polyamine metabolism in transient focal

Paschen W., Widmann R. & Weber C. (1992) Changes in regional polyamine profiles in rat

for release of polyamines from injured neurons. *Neurosci Lett* 135, 121-124 Pedersen KO. (1944) Fetuin, a new globulin isolated from serum. *Nature* 154, 575-576

Peltz E.D., Moore E.E., Eckels P.C., Damle S.S., Tsuruta Y., Johnson J.L., Sauaia A., Silliman

Poltorak A., He X., Smirnova I., Liu M.Y., Huffel C.V., Du X., Birdwell D., Alejos E., Silva M.,

Qin S., Wang H., Yuan R., Li H., Ochani M., Ochani K., Rosas-Ballina M., Czura C.J., Huston

Qiu J., Nishimura M., Wang Y., Sims J.R., Qiu S., Savitz S.I., Salomone S. & Moskowitz M.A.

Rendon-Mitchell B., Ochani M., Li J., Han J., Wang H., Yang H., Susarla S., Czura C.,

Rochette C.N., Rosenfeldt S., Heiss A., Narayanan T., Ballauff M. & Jahnen-Dechent W.

Sato H., Kazama J.J., Wada Y., Kuroda T., Narita I., Gejyo F., Gao P. & Yamashita H. (2007)

Schinke T., Amendt C., Trindl A., Poschke O., Muller-Esterl W. & Jahnen-Dechent W. (1996)

(AHSG) in patients with rheumatoid arthritis. *Intern Med* 46, 1685-1691 Schafer C., Heiss A., Schwarz A., Westenfeld R., Ketteler M., Floege J., Muller-Esterl W.,

*Shock* 15, 181-185

*Metab Rev* 4, 59-88

gene. *Science* 282, 2085-2088

*Cereb Blood Flow Metab* 28, 927-938

calcification. *J Clin Invest* 112, 357-366

calcium homeostasis. *J Biol Chem* 271, 20789-20796

*Chembiochem* 10, 735-740

203, 1637-1642

ischemia of rat brain. *Brain Res* 566, 354-357

of mechanical trauma in humans. *Shock* 32, 17-22

Dependent Mechanism. *J Immunol* 170, 3890-3897

Rossetti G., Nawroth P.P., Bierhaus A. & Schwaninger M. (2008) The HMGB1

Friedman S.G. & Tracey K.J. (2001) Fetuin, a negative acute phase protein, attenuates TNF synthesis and the innate inflammatory response to carrageenan.

brains after transient cerebral ischemia (single versus repetitive ischemia): evidence

C.C., Banerjee A. & Abraham E. (2009) HMGB1 is markedly elevated within 6 hours

Galanos C., Freudenberg M., Ricciardi-Castagnoli P., Layton B. & Beutler B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4

J.M., Miller E., Lin X., Sherry B., Kumar A., Larosa G., Newman W., Tracey K.J. & Yang H. (2006) Role of HMGB1 in apoptosis-mediated sepsis lethality. *J Exp Med*

(2008) Early release of HMGB-1 from neurons after the onset of brain ischemia. *J*

Mitchell R.A., Chen G., Sama A.E., Tracey K.J. & Wang H. (2003) IFN-gamma Induces High Mobility Group Box 1 Protein Release Partly Through a TNF-

(2009) A shielding topology stabilizes the early stage protein-mineral complexes of fetuin-A and calcium phosphate: a time-resolved small-angle X-ray study.

Decreased levels of circulating alpha2-Heremans-Schmid glycoprotein/Fetuin-A

Schinke T. & Jahnen-Dechent W. (2003) The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic

The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and

**16**

*USA*

**Neutrophil Gelatinase Associated**

**Lipocalin: Structure, Function and**

Subhankar Chakraborty1, Sukhwinder Kaur1, Zhimin Tong2,

*2The University of Texas MD Anderson Cancer Center, Houston, Texas*

Glycoproteins have a unique position in the pathogenesis of human diseases. Most of the commonly employed protein biomarkers are glycoproteins. Examples include CA19-9 (carbohydrate antigen 19-9) to follow-up patients with pancreatic cancer, CEA (carcinoembryonic antigen) for multiple solid tumors and CA125 (carbohydrate antigen 125) used in the diagnosis, follow-up and therapy of patients with ovarian cancer. Most of these glycoproteins are large molecules. However, there is a family of smaller, secreted glycoproteins (called lipocalins) that are important in the maintenance of health and in combating diseases effectively. A prototype of this family is a small, secreted glycoprotein called Neutrophil gelatinase associated lipocalin or NGAL. In recent years, NGAL has emerged as a novel biomarker in several benign and malignant diseases. Further, studies in cultured cells and in murine models have revealed a pivotal role for this molecule in several physiological processes and pathological conditions. In this Chapter, we review the biology of lipocalins, focusing specifically on NGAL and its role in human health and disease.

 Most protein families are classified on the basis of similarity in amino acid sequences, domain architecture and three-dimensional protein structure. The lipocalins however are a unique exception in that the percentage of sequence identity among different members of the lipocalin family is sometimes even lower than the minimum (20%) identity required to call it a reliable alignment (Flower *et al.*, 2000; Flower, 1996; Flower, 1995; Flower, 1994). Despite this lack of a sequence similarity, the lipocalins share three short stretches of amino acid sequences (or motifs) that are part of the structural conservation among different lipocalin sequences. These motifs, which are aptly termed as "structurally conserved regions or SCRs" are used to classify this large family into two main sub-families-

**1. Introduction**

**2. The lipocalin family**

**Role in Human Pathogenesis**

Surinder K. Batra1 and Sushovan Guha2 *1University of Nebraska Medical Center, Omaha, NE*

## **Neutrophil Gelatinase Associated Lipocalin: Structure, Function and Role in Human Pathogenesis**

Subhankar Chakraborty1, Sukhwinder Kaur1, Zhimin Tong2, Surinder K. Batra1 and Sushovan Guha2 *1University of Nebraska Medical Center, Omaha, NE 2The University of Texas MD Anderson Cancer Center, Houston, Texas*

*USA*

#### **1. Introduction**

344 Acute Phase Proteins – Regulation and Functions of Acute Phase Proteins

Wang H., Zhang M., Soda K., Sama A. & Tracey K.J. (1997) Fetuin protects the fetus from

Wang H., Zhu S., Zhou R., Li W. & Sama A.E. (2008) Therapeutic potential of HMGB1-

Watanabe T., Kubota S., Nagaya M., Ozaki S., Nagafuchi H., Akashi K., Taira Y., Tsukikawa

Weikert C., Stefan N., Schulze M.B., Pischon T., Berger K., Joost H.G., Haring H.U., Boeing

Williams K. (1997) Modulation and block of ion channels: a new biology of polyamines. *Cell*

Wu C.Y., Martel J., Young D. & Young J.D. (2009) Fetuin-A/albumin-mineral complexes

Wu H., Chen G., Wyburn K.R., Yin J., Bertolino P., Eris J.M., Alexander S.I., Sharland A.F. &

Yang G.Y., Gong C., Qin Z., Liu X.H. & Lorris B.A. (1999) Tumor necrosis factor alpha

Zhang M., Borovikova L.V., Wang H., Metz C. & Tracey K.J. (1999) Spermine inhibition of

Zhang M., Caragine T., Wang H., Cohen P.S., Botchkina G., Soda K., Bianchi M., Ulrich P.,

Zhang M., Wang H. & Tracey K.J. (2000) Regulation of macrophage activation and

Zhu S., Ashok M., Li J., Li W., Yang H., Wang P., Tracey K.J., Sama A.E. & Wang H. (2009a)

Zhu S., Li W., Ward M.F., Sama A.E. & Wang H. (2010) High mobility group box 1 protein

Zubrow A.B., Numagami Y., Fritz K.I., Mishra O.P. & Delivoria-Papadopoulos M. (2000)

monocyte activation and inflammation. *Mol Med* 5, 595-605

that restrains the immune response. *J Exp Med* 185, 1759-1768

inflammatory markers. *Mol Med* 15, 275-282

*Inflamm.Allergy Drug Targets.* 9, 60-72

piglets. *Brain Res* 854, 11-18

S., Oowada S. & Nakano S. (2005) The role of HMGB-1 on the development of necrosis during hepatic ischemia and hepatic ischemia/reperfusion injury in mice. *J*

H. & Fritsche A. (2008) Plasma fetuin-a levels and the risk of myocardial infarction

resembling serum calcium granules and putative nanobacteria: demonstration of a

Chadban S.J. (2007) TLR4 activation mediates kidney ischemia/reperfusion injury.

expression produces increased blood-brain barrier permeability following temporary focal cerebral ischemia in mice. *Brain Res Mol Brain Res* 69, 135-143 Yang H., Ochani M., Li J., Qiang X., Tanovic M., Harris H.E., Susarla S.M., Ulloa L., Wang

H., DiRaimo R., Czura C.J., Wang H., Roth J., Warren H.S., Fink M.P., Fenton M.J., Andersson U. & Tracey K.J. (2004) Reversing established sepsis with antagonists of endogenous high-mobility group box 1. *Proc Natl Acad Sci U S A* 101, 296-301 Zaremba J. & Losy J. (2001) Early TNF-alpha levels correlate with ischaemic stroke severity.

Cerami A., Sherry B. & Tracey K.J. (1997) Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism

inflammation by spermine: a new chapter in an old story. *Crit Care Med* 28, N60-

Spermine protects mice against lethal sepsis partly by attenuating surrogate

as a potential drug target for infection- and injury-elicited inflammation.

Spermine dependent activation of the N-methyl-D-aspartate receptor and the effect of nitric oxide synthase inhibition during hypoxia in the cerebral cortex of newborn

targeting agents in sepsis. *Expert Rev Mol Med* 10:e32.

and ischemic stroke. *Circulation* 118, 2555-2562

dual inhibition-seeding concept. *PLoS.One.* 4, e8058

TNF [letter]. *Lancet* 350, 861-862

*Surg Res* 124, 59-66

*J.Clin.Invest* 117, 2847-2859

*Acta Neurol Scand* 104(5), 288-295

*Signal* 9, 1-13

N66

Glycoproteins have a unique position in the pathogenesis of human diseases. Most of the commonly employed protein biomarkers are glycoproteins. Examples include CA19-9 (carbohydrate antigen 19-9) to follow-up patients with pancreatic cancer, CEA (carcinoembryonic antigen) for multiple solid tumors and CA125 (carbohydrate antigen 125) used in the diagnosis, follow-up and therapy of patients with ovarian cancer. Most of these glycoproteins are large molecules. However, there is a family of smaller, secreted glycoproteins (called lipocalins) that are important in the maintenance of health and in combating diseases effectively. A prototype of this family is a small, secreted glycoprotein called Neutrophil gelatinase associated lipocalin or NGAL. In recent years, NGAL has emerged as a novel biomarker in several benign and malignant diseases. Further, studies in cultured cells and in murine models have revealed a pivotal role for this molecule in several physiological processes and pathological conditions. In this Chapter, we review the biology of lipocalins, focusing specifically on NGAL and its role in human health and disease.

#### **2.  Most protein families are classified on the basis of similarity in amino acid sequences, domain architecture and three-dimensional protein structure. The lipocalins however are a unique exception in that the percentage of sequence identity among different members of the lipocalin family is sometimes even lower than the minimum (20%) identity required to call it a reliable alignment (Flower *et al.*, 2000; Flower, 1996; Flower, 1995; Flower, 1994). Despite this lack of a sequence similarity, the lipocalins share three short stretches of amino acid sequences (or motifs) that are part of the structural conservation among different lipocalin sequences. These motifs, which are aptly termed as "structurally conserved regions or SCRs" are used to classify this large family into two main sub-families-

Neutrophil Gelatinase Associated Lipocalin:

is indicated by the black boxed region.

biological activity of the interacting protein (A1M).

Structure, Function and Role in Human Pathogenesis 347

(RBP), stabilize the interactor (NGAL), protect normal cells from damage (C8γ) or alter the

Fig. 1. Schematic representation of the lipocalin fold. The characteristic feature of lipocalins is the "lipocalin fold" which comprises of an N-terminal 3-10 helix followed by eight beta sheets (A-I) arranged in an antiparallel orientation. The eighth beta sheet is connected to an alpha helix (denoted as α1), which is in turn connected to a C-terminal beta sheet. The beta sheets are connected by loops (L1-L7). Loops L1,L3,L5 and L7 form the open end of the molecule (i.e. the opening to the ligand binding site of NGAL). The portion of the lipocalin fold that are structurally conserved between different lipocalins is indicated by the blue boxed regions while the region that shows significant conservation in amino acid sequence

Functionally, the lipocalins are generally well known as transport proteins that exist in the extracellular space. The prototypical lipocalin involved in transport of cargo in the body is RBP which binds to and transports retinol (Vitamin A) from the liver to various organs. Binding to RBP not only protects retinol from being excreted by the kidneys but also from being oxidized within the body. Further, it also permits the transport of a relatively insoluble retinol in the form of a soluble complex to the tissues to mediate its effects. Other functions attributed to lipocalins including regulation of cell ageing, survival and differentiation (probasin and purpurin), modulation of immune response (NGAL, AGP, PP14 and AIM), prostaglandin synthesis (glutathione-independent PGD2 synthase),

the "kernel" and the "outlier" lipocalins (**Table 1**). The kernel lipocalins contain all three motifs while the outlier lipocalins contain only one or two of these (but not all three) SCRs.

Table 1. List of known Kernel and Outlier Lipocalin proteins

The secondary and tertiary structure of the lipocalin family members is very similar and is characterized by the presence of a "lipocalin fold"- a symmetrical structure comprised almost entirely of β-pleated sheets closed at the two ends by two α helices (**Figure 1)**. There are eight antiparallel β sheets linked to each other by seven short loops. The N-terminal region of the protein forms the ligand binding cavity which is closed by a conserved 310 helix. The cup-shaped cavity of the lipocalins enclosed within the β-pleated sheets is well adapted for binding to a wide array of hydrophobic ligands- for instance, while retinol binding protein (RBP) binds primarily to its endogenous ligand retinol, it can also bind to several other hydrophobic ligands including β-ionone, β-carotene, cholesterol, terpenoids and long chain esters of retinol and retinoic acid.

Many lipocalins have also been shown to bind to specific receptors including RBP, alpha1 microglobulin, major urinary protein (MUP) and Odorant-binding protein (OBP). A third property of lipocalins is their tendency to form complexes with soluble macromolecules. Some of the well-known protein-protein complexes involving the lipocalin family members include the complex of RBP with transthyretin, NGAL with human neutrophil gelatinase, C8γ with C8α as part of the membrane attack complex (MAC) and α1-microglobulin (A1M) with immunoglobulin A (IgA). These associations may either serve to transport proteins

the "kernel" and the "outlier" lipocalins (**Table 1**). The kernel lipocalins contain all three motifs while the outlier lipocalins contain only one or two of these (but not all three) SCRs.

**Abbreviation Protein name**

Alpha 1M Αlpha 1-microglobulin ApoD Apolipoprotein D A2U α2micro-Globulin BBP Bilin binding protein Blg β1-Lactoglobulin

CPP Choroid plexus protein

PGDS Prostaglandin D synthase PP14 Pregnancy protein 14

AAAG Αlpha 1-Acid glycoprotein

OBP Odorant binding protein

VEGP von Ebner's-gland protein Table 1. List of known Kernel and Outlier Lipocalin proteins

ACC α-Crustacyanin MUP Major Urinary Protein

PURP Purpurin Lazarillo

Aphrodisin

and long chain esters of retinol and retinoic acid.

Probasin

**Outlier Lipocalins**

CRABP2 Cellular Retinoic acid binding protein

NGAL Neutrophil gelatinase associated Lipocalin

The secondary and tertiary structure of the lipocalin family members is very similar and is characterized by the presence of a "lipocalin fold"- a symmetrical structure comprised almost entirely of β-pleated sheets closed at the two ends by two α helices (**Figure 1)**. There are eight antiparallel β sheets linked to each other by seven short loops. The N-terminal region of the protein forms the ligand binding cavity which is closed by a conserved 310 helix. The cup-shaped cavity of the lipocalins enclosed within the β-pleated sheets is well adapted for binding to a wide array of hydrophobic ligands- for instance, while retinol binding protein (RBP) binds primarily to its endogenous ligand retinol, it can also bind to several other hydrophobic ligands including β-ionone, β-carotene, cholesterol, terpenoids

Many lipocalins have also been shown to bind to specific receptors including RBP, alpha1 microglobulin, major urinary protein (MUP) and Odorant-binding protein (OBP). A third property of lipocalins is their tendency to form complexes with soluble macromolecules. Some of the well-known protein-protein complexes involving the lipocalin family members include the complex of RBP with transthyretin, NGAL with human neutrophil gelatinase, C8γ with C8α as part of the membrane attack complex (MAC) and α1-microglobulin (A1M) with immunoglobulin A (IgA). These associations may either serve to transport proteins

**Kernel Lipocalins**

C8γ C8γ

(RBP), stabilize the interactor (NGAL), protect normal cells from damage (C8γ) or alter the biological activity of the interacting protein (A1M).

Fig. 1. Schematic representation of the lipocalin fold. The characteristic feature of lipocalins is the "lipocalin fold" which comprises of an N-terminal 3-10 helix followed by eight beta sheets (A-I) arranged in an antiparallel orientation. The eighth beta sheet is connected to an alpha helix (denoted as α1), which is in turn connected to a C-terminal beta sheet. The beta sheets are connected by loops (L1-L7). Loops L1,L3,L5 and L7 form the open end of the molecule (i.e. the opening to the ligand binding site of NGAL). The portion of the lipocalin fold that are structurally conserved between different lipocalins is indicated by the blue boxed regions while the region that shows significant conservation in amino acid sequence is indicated by the black boxed region.

Functionally, the lipocalins are generally well known as transport proteins that exist in the extracellular space. The prototypical lipocalin involved in transport of cargo in the body is RBP which binds to and transports retinol (Vitamin A) from the liver to various organs. Binding to RBP not only protects retinol from being excreted by the kidneys but also from being oxidized within the body. Further, it also permits the transport of a relatively insoluble retinol in the form of a soluble complex to the tissues to mediate its effects. Other functions attributed to lipocalins including regulation of cell ageing, survival and differentiation (probasin and purpurin), modulation of immune response (NGAL, AGP, PP14 and AIM), prostaglandin synthesis (glutathione-independent PGD2 synthase),

Neutrophil Gelatinase Associated Lipocalin:

Structure, Function and Role in Human Pathogenesis 349

Fig. 2. Domain architecture of human NGAL and its homologues. Human NGAL and its homologues are characterized by a high degree of similarity in their domain architecture. All NGAL homologues (with the exception of the horse and wild hog) contain an N-

involved in the transport of hydrophobic ligands (Source:

http://www.ebi.ac.uk/interpro/).

terminal signal peptide which is cleaved prior to secretion of the protein. This is followed by the lipocalin domain (IPR000566 Lipocln\_cytosolic\_FA-bd) which makes up most of the length of the protein. This domain which has the consensus sequence [DENG] - {A} - [DENQGSTARK] - x(0,2) - [DENQARK] - [LIVFY] - {CP} - G - {C} - W - [FYWLRH] - {D} - [LIVMTA] is characteristic of proteins belonging to the lipocalin family. Structurally, the lipocalin domain is an eight stranded antiparallel beta barrel enclosing a ligand binding site which can bind to small hydrophobic molecules (e.g. steroids, retinoids, bilins and lipids). The rabbit and horse NGAL proteins also contain a second domain called the "lipocalin-2" domain (Accession number: IPR013208 Lipocalin\_2).  We will examine the role of a member of the lipocalin family (NGAL) that has been extensively studied particularly as a potential marker for diagnosis and prognosis in several human diseases. We will investigate its biology, functions and clinical applications both in benign and malignant diseases.

#### **3. Neutrophil gelatinase associated lipocalin (NGAL)-genomic organization and protein structure**

Neutrophil gelatinase associated lipocalin (NGAL) also known as migration stimulating factor inhibitor (MSFI), human neutrophil lipocalin (HNL), alpha-1 microglobulin related protein, siderocalin or uterocalin is a 198 amino acid glycoprotein encoded by a gene located on the chromosome locus 3p11. It was first isolated from mouse kidney cells infected with a simian virus (SV-40) (Hraba-Renevey *et al.*, 1989). Triebel and colleagues identified a novel association between NGAL and the gelatinolytic enzyme MMP-9 (matrix metalloproteinase 9 or gelatinase B) known to degrade several components of the basement membrane including type I gelatin and collagen types I, IV, V and XI (Triebel *et al.*, 1992). Human NGAL protein (or LCN2) is represented in upper case, while the murine and rat homologues are usually represented in lower case (Ngal or Lcn2).

Structurally, human NGAL contains a 20-amino acid signal peptide at the N-terminal end of the protein followed by the "lipocalin" domain. This domain which is responsible for binding of lipocalins to their ligands, is structurally comprised of an eight stranded β barrel with its loops running in an antiparallel direction. A comparison of the amino acid sequence between NGAL homologues in different species reveals that human NGAL is highly similar to the homologue present in chimpanzees (98% identity) but shows much less identity to the mouse and rat homologues (62% and 63% identity respectively). Despite the limited identity of their amino acid sequences, the mouse and human homologue are remarkably similar in their domain architecture (**Figure 2**) and three-dimensional structure. This feature, i.e. marked structural identity in the absence of significant sequence identity is a common feature of the lipocalin-family of proteins and underlies the conserved function of the lipocalin domain. Studies carried out on mouse Ngal (also called mLcn2) have revealed that it can bind hydrophobic ligands like retinol, cholesterol oleate and oleic acid (Chu *et al.*, 1998). NMR (nuclear magnetic resonance) studies have revealed that the NGAL molecule contains eight antiparallel β strands that form a barrel shaped structure (Coles *et al.*, 1999). Three β bulges present in this barrel (one formed by the 1st and two by the 6th β strand) have been suggested to contribute to the ligand binding site of NGAL. Hydrophobic residues (Tryptophan, Valine and Phenylalanine) present at the base of this barrel-like structure have been shown to be involved in direct binding to the ligand. A patch of positively charged amino acid residues (Lysine and Arginine) present near the mouth of the barrel and projecting into the open end of the molecule has also been suggested to be also important in binding to ligands. Further, a negatively charged "pit" present at the base of the barrel formed by the amino acids Aspartate and Glutamate and a nearby unpaired Cysteine residue have been suggested to be crucial for binding of NGAL to the gelatinase MMP-9. Based on whether NGAL is free or bound to a ligand, it is termed as "apo" or "holo" NGAL respectively. The conformational change between these two forms of the protein is affected by a conformation change occurring at the open end of the NGAL protein.

coloration (insecticyanin, BBP and crusticyanin), smell (OBP), taste (VEGP) and as sex hormones or pheromones (MUP and Aphrodisin) (Flower *et al.*, 2000; Flower, 1996; Flower, 1995; Flower, 1994). We will examine the role of a member of the lipocalin family (NGAL) that has been extensively studied particularly as a potential marker for diagnosis and prognosis in several human diseases. We will investigate its biology, functions and clinical

**3. Neutrophil gelatinase associated lipocalin (NGAL)-genomic organization**

Neutrophil gelatinase associated lipocalin (NGAL) also known as migration stimulating factor inhibitor (MSFI), human neutrophil lipocalin (HNL), alpha-1 microglobulin related protein, siderocalin or uterocalin is a 198 amino acid glycoprotein encoded by a gene located on the chromosome locus 3p11. It was first isolated from mouse kidney cells infected with a simian virus (SV-40) (Hraba-Renevey *et al.*, 1989). Triebel and colleagues identified a novel association between NGAL and the gelatinolytic enzyme MMP-9 (matrix metalloproteinase 9 or gelatinase B) known to degrade several components of the basement membrane including type I gelatin and collagen types I, IV, V and XI (Triebel *et al.*, 1992). Human NGAL protein (or LCN2) is represented in upper case, while the murine and rat

Structurally, human NGAL contains a 20-amino acid signal peptide at the N-terminal end of the protein followed by the "lipocalin" domain. This domain which is responsible for binding of lipocalins to their ligands, is structurally comprised of an eight stranded β barrel with its loops running in an antiparallel direction. A comparison of the amino acid sequence between NGAL homologues in different species reveals that human NGAL is highly similar to the homologue present in chimpanzees (98% identity) but shows much less identity to the mouse and rat homologues (62% and 63% identity respectively). Despite the limited identity of their amino acid sequences, the mouse and human homologue are remarkably similar in their domain architecture (**Figure 2**) and three-dimensional structure. This feature, i.e. marked structural identity in the absence of significant sequence identity is a common feature of the lipocalin-family of proteins and underlies the conserved function of the lipocalin domain. Studies carried out on mouse Ngal (also called mLcn2) have revealed that it can bind hydrophobic ligands like retinol, cholesterol oleate and oleic acid (Chu *et al.*, 1998). NMR (nuclear magnetic resonance) studies have revealed that the NGAL molecule contains eight antiparallel β strands that form a barrel shaped structure (Coles *et al.*, 1999). Three β bulges present in this barrel (one formed by the 1st and two by the 6th β strand) have been suggested to contribute to the ligand binding site of NGAL. Hydrophobic residues (Tryptophan, Valine and Phenylalanine) present at the base of this barrel-like structure have been shown to be involved in direct binding to the ligand. A patch of positively charged amino acid residues (Lysine and Arginine) present near the mouth of the barrel and projecting into the open end of the molecule has also been suggested to be also important in binding to ligands. Further, a negatively charged "pit" present at the base of the barrel formed by the amino acids Aspartate and Glutamate and a nearby unpaired Cysteine residue have been suggested to be crucial for binding of NGAL to the gelatinase MMP-9. Based on whether NGAL is free or bound to a ligand, it is termed as "apo" or "holo" NGAL respectively. The conformational change between these two forms of the protein is affected

applications both in benign and malignant diseases.

homologues are usually represented in lower case (Ngal or Lcn2).

by a conformation change occurring at the open end of the NGAL protein.

**and protein structure**

Fig. 2. Domain architecture of human NGAL and its homologues. Human NGAL and its homologues are characterized by a high degree of similarity in their domain architecture. All NGAL homologues (with the exception of the horse and wild hog) contain an Nterminal signal peptide which is cleaved prior to secretion of the protein. This is followed by the lipocalin domain (IPR000566 Lipocln\_cytosolic\_FA-bd) which makes up most of the length of the protein. This domain which has the consensus sequence [DENG] - {A} - [DENQGSTARK] - x(0,2) - [DENQARK] - [LIVFY] - {CP} - G - {C} - W - [FYWLRH] - {D} - [LIVMTA] is characteristic of proteins belonging to the lipocalin family. Structurally, the lipocalin domain is an eight stranded antiparallel beta barrel enclosing a ligand binding site which can bind to small hydrophobic molecules (e.g. steroids, retinoids, bilins and lipids). The rabbit and horse NGAL proteins also contain a second domain called the "lipocalin-2" domain (Accession number: IPR013208 Lipocalin\_2). This domain is present in a small number of proteins (calycin, apolipoprotein D and Lipocalin 2) and is believed to be involved in the transport of hydrophobic ligands (Source: http://www.ebi.ac.uk/interpro/).

Neutrophil Gelatinase Associated Lipocalin:

(Marques *et al.*, 2008).

Structure, Function and Role in Human Pathogenesis 351

with MAP, plasma NGAL levels are significantly elevated (nearly 15-fold) within 48 hours (of onset of symptoms) in patients with SAP (Chakraborty *et al.*, 2010). The NGAL levels showed a tendency to decline when measured in blood samples collected at later time points (i.e. 72 hrs, 96 hrs and 120 hrs). To get a better idea of the time course of the rise in NGAL levels in the blood, we induced either mild or severe acute pancreatitis in mice and followed the change in plasma Ngal levels over time. We found that Ngal levels in mice rose early (within 6 hours following induction of AP) and remained elevated in those with SAP while

Meningitis, particularly that caused by gram negative bacteria can cause a systemic inflammatory response syndrome (SIRS) associated with sepsis and significant mortality. A key mediator of this inflammatory response is lipopolysaccharide (LPS), a structural biomolecule present in the outer wall of gram negative bacteria. LPS is a potent stimulator of the immune response and a central player in septic shock in patients with gram negative bacterial infections. Ngal mRNA levels were found to be significantly induced in the choroid plexus (a structure in the ventricles of the brain that produces cerebrospinal fluid) within 12 hours following intraperitoneal injection of LPS while the levels of circulating Ngal (in the cerebrospinal fluid) was strongly induced within 6 hours after injection of LPS

A significant increase in the level of circulating Ngal was also noted in rats induced to develop features of autoimmune myocarditis by injection of purified porcine myosin (Ding *et al.*, 2010). However, compared to the acute inflammations described earlier, the elevation in Ngal in the rats was significantly delayed, being first evident 9 days following immunization with the foreign protein. A significant increase in NGAL mRNA levels has also been reported in the heart tissues from patients with myocarditis (Ding *et al.*, 2010). Psoriasis, a chronic inflammatory disease of the skin is characterized by the formation of plaques covered by variable amount of scales. NGAL mRNA levels were shown to be significantly upregulated (nearly 10-fold) in the psoriatic lesions (Nomura *et al.*, 2003; Mallbris *et al.*, 2002). Further, NGAL levels were also upregulated in lesions that like psoriasis, exhibited a dysregulation in the process of keratinocyte differentiation (termed as parakeratosis). These diseases include pityriasis rubra pilaris, porokeratosis and chronic (not acute) eczema. Significantly, NGAL expression (from the affected areas of skin) disappeared once the psoriatic lesions had healed with treatment, suggesting that NGAL may play a role

NGAL levels were significantly elevated in gum (i.e. alveolar mucosa) tissue from patients affected with localized juvenile periodontitis (LJP), a chronic inflammatory disease of the gums affecting young adults and associated with severe destruction of both soft tissues and bone (Van Dyke *et al.*, 1985). In addition to bacterial infections, NGAL, particularly circulating levels are significantly altered in certain viral infections. For instance, circulating NGAL levels were significantly lower in patients infected with the human immune deficiency virus (HIV) who had not received any therapy (i.e. treatment naïve) compared to non-HIV controls. Significantly, treatment of these patients with the highly active antiretroviral therapy (HAART) was associated with a progressive increase in circulating NGAL levels (Landro *et al.*, 2008). In vitro studies using neutrophils isolated from the three groups of patient revealed that in healthy and HAART-treated HIV positive patients, the neutrophils released NGAL normally into the culture medium. However, in untreated HIV positive patients, this process was defective suggesting that the release of NGAL from

in the pathogenesis of diseases with dysregulated epidermal differentiation.

trending down to levels in control mice (no pancreatitis) and in those with MAP.

Recent data on the ligand binding pocket of NGAL has revealed that it is significantly larger and more polar than a similar pocket in other lipocalin proteins (Goetz *et al.*, 2000). This has led to the speculation that NGAL may bind to large, macromolecular and relatively less hydrophobic ligands including mammalian proteins. However, the identity of one or more of these mammalian ligands for NGAL remains to be elucidated. However, NGAL has been shown to bind to bacterial proteins. Specifically, it binds to iron-chelating proteins released by both gram negative bacteria (i.e. enterobacteria) and mycobacteria (e.g. *Mycobacterium tuberculosis*) (Nairz *et al.*, 2009; Holmes *et al.*, 2005). These proteins, termed as enterochelin and carboxymycobactin (CMB) respectively have been shown to bind to the ligand binding cavity of NGAL. The latter (i.e. CMBs) however, forms a better fit into the ligand binding pocket of NGAL. This has been in part attributed to the larger number of hydrophobic residues in CMBs which permits the formation of a larger number of hydrogen bonds and Van Der Walls interactions with the ligand binding cavity of NGAL.

#### **3. Expression of NGAL in normal tissues**

Although first discovered as a component of the late granules of neutrophils, NGAL is also strongly expressed in several normal adult human tissues including the non-neoplastic breast ducts, kidney, liver, lungs, trachea, small intestine, bone marrow, thymus, prostate, adipose tissue and macrophages. A weak to no expression of NGAL is observed in the normal pancreas, endometrial glands, and the thymus and peripheral blood leucocytes. NGAL is however completely absent from the normal brain, heart, skeletal muscle, spleen, testes, ovary and colon (Le, V *et al.*, 1997; Seth *et al.*, 2002; Moreno-Navarrete *et al.*, 2010; Cowland *et al.*, 2003; Furutani *et al.*, 1998; Cowland and Borregaard, 1997).

Limited studies done in human fetal tissues reveal that NGAL is expressed in trophoblast cells of the placenta, cartilage forming cells (chondrocytes) and in the epithelial cells present in the developing lung and small intestine. NGAL expression has also been reported in the epidermis of the fetal skin beginning around the 20th week of gestation. With increasing age of the fetus, the expression spreads to the lower layers of the skin and becomes progressively concentrated around the hair follicles (Mallbris *et al.*, 2002).

#### **4. Altered expression of NGAL in benign and malignant diseases**

#### **4.1 Inflammatory diseases**

The expression of NGAL is altered in several benign disease conditions including inflammatory and metabolic disorders. Inflammatory conditions associated with an increase in NGAL expression include pancreatitis, meningitis, myocarditis, psoriasis and periodontitis.

Acute pancreatitis (AP) is a reversible, acute inflammation of the pancreas that affects nearly 210,000 patients every year in the United States alone (Banks and Freeman, 2006). While nearly 85% of these patients have a mild form of the illness (termed as mild acute pancreatitis or MAP) and make an uneventful recovery, about 15%-20% of them develop features of severe acute pancreatitis (SAP) (Carroll *et al.*, 2007). SAP is associated with significant morbidity and mortality primarily owing to multi-organ failure (MOF, usually in the first week following onset of disease) and infected pancreatic necrosis (2 weeks or later after onset of disease). We have reported that compared to healthy individuals and those

Recent data on the ligand binding pocket of NGAL has revealed that it is significantly larger and more polar than a similar pocket in other lipocalin proteins (Goetz *et al.*, 2000). This has led to the speculation that NGAL may bind to large, macromolecular and relatively less hydrophobic ligands including mammalian proteins. However, the identity of one or more of these mammalian ligands for NGAL remains to be elucidated. However, NGAL has been shown to bind to bacterial proteins. Specifically, it binds to iron-chelating proteins released by both gram negative bacteria (i.e. enterobacteria) and mycobacteria (e.g. *Mycobacterium tuberculosis*) (Nairz *et al.*, 2009; Holmes *et al.*, 2005). These proteins, termed as enterochelin and carboxymycobactin (CMB) respectively have been shown to bind to the ligand binding cavity of NGAL. The latter (i.e. CMBs) however, forms a better fit into the ligand binding pocket of NGAL. This has been in part attributed to the larger number of hydrophobic residues in CMBs which permits the formation of a larger number of hydrogen bonds and

Although first discovered as a component of the late granules of neutrophils, NGAL is also strongly expressed in several normal adult human tissues including the non-neoplastic breast ducts, kidney, liver, lungs, trachea, small intestine, bone marrow, thymus, prostate, adipose tissue and macrophages. A weak to no expression of NGAL is observed in the normal pancreas, endometrial glands, and the thymus and peripheral blood leucocytes. NGAL is however completely absent from the normal brain, heart, skeletal muscle, spleen, testes, ovary and colon (Le, V *et al.*, 1997; Seth *et al.*, 2002; Moreno-Navarrete *et al.*, 2010;

Limited studies done in human fetal tissues reveal that NGAL is expressed in trophoblast cells of the placenta, cartilage forming cells (chondrocytes) and in the epithelial cells present in the developing lung and small intestine. NGAL expression has also been reported in the epidermis of the fetal skin beginning around the 20th week of gestation. With increasing age of the fetus, the expression spreads to the lower layers of the skin and becomes

The expression of NGAL is altered in several benign disease conditions including inflammatory and metabolic disorders. Inflammatory conditions associated with an increase in NGAL expression include pancreatitis, meningitis, myocarditis, psoriasis and

Acute pancreatitis (AP) is a reversible, acute inflammation of the pancreas that affects nearly 210,000 patients every year in the United States alone (Banks and Freeman, 2006). While nearly 85% of these patients have a mild form of the illness (termed as mild acute pancreatitis or MAP) and make an uneventful recovery, about 15%-20% of them develop features of severe acute pancreatitis (SAP) (Carroll *et al.*, 2007). SAP is associated with significant morbidity and mortality primarily owing to multi-organ failure (MOF, usually in the first week following onset of disease) and infected pancreatic necrosis (2 weeks or later after onset of disease). We have reported that compared to healthy individuals and those

Van Der Walls interactions with the ligand binding cavity of NGAL.

Cowland *et al.*, 2003; Furutani *et al.*, 1998; Cowland and Borregaard, 1997).

progressively concentrated around the hair follicles (Mallbris *et al.*, 2002).

**4. Altered expression of NGAL in benign and malignant diseases**

**3. Expression of NGAL in normal tissues**

**4.1 Inflammatory diseases**

periodontitis.

with MAP, plasma NGAL levels are significantly elevated (nearly 15-fold) within 48 hours (of onset of symptoms) in patients with SAP (Chakraborty *et al.*, 2010). The NGAL levels showed a tendency to decline when measured in blood samples collected at later time points (i.e. 72 hrs, 96 hrs and 120 hrs). To get a better idea of the time course of the rise in NGAL levels in the blood, we induced either mild or severe acute pancreatitis in mice and followed the change in plasma Ngal levels over time. We found that Ngal levels in mice rose early (within 6 hours following induction of AP) and remained elevated in those with SAP while trending down to levels in control mice (no pancreatitis) and in those with MAP.

Meningitis, particularly that caused by gram negative bacteria can cause a systemic inflammatory response syndrome (SIRS) associated with sepsis and significant mortality. A key mediator of this inflammatory response is lipopolysaccharide (LPS), a structural biomolecule present in the outer wall of gram negative bacteria. LPS is a potent stimulator of the immune response and a central player in septic shock in patients with gram negative bacterial infections. Ngal mRNA levels were found to be significantly induced in the choroid plexus (a structure in the ventricles of the brain that produces cerebrospinal fluid) within 12 hours following intraperitoneal injection of LPS while the levels of circulating Ngal (in the cerebrospinal fluid) was strongly induced within 6 hours after injection of LPS (Marques *et al.*, 2008).

A significant increase in the level of circulating Ngal was also noted in rats induced to develop features of autoimmune myocarditis by injection of purified porcine myosin (Ding *et al.*, 2010). However, compared to the acute inflammations described earlier, the elevation in Ngal in the rats was significantly delayed, being first evident 9 days following immunization with the foreign protein. A significant increase in NGAL mRNA levels has also been reported in the heart tissues from patients with myocarditis (Ding *et al.*, 2010).

Psoriasis, a chronic inflammatory disease of the skin is characterized by the formation of plaques covered by variable amount of scales. NGAL mRNA levels were shown to be significantly upregulated (nearly 10-fold) in the psoriatic lesions (Nomura *et al.*, 2003; Mallbris *et al.*, 2002). Further, NGAL levels were also upregulated in lesions that like psoriasis, exhibited a dysregulation in the process of keratinocyte differentiation (termed as parakeratosis). These diseases include pityriasis rubra pilaris, porokeratosis and chronic (not acute) eczema. Significantly, NGAL expression (from the affected areas of skin) disappeared once the psoriatic lesions had healed with treatment, suggesting that NGAL may play a role in the pathogenesis of diseases with dysregulated epidermal differentiation.

NGAL levels were significantly elevated in gum (i.e. alveolar mucosa) tissue from patients affected with localized juvenile periodontitis (LJP), a chronic inflammatory disease of the gums affecting young adults and associated with severe destruction of both soft tissues and bone (Van Dyke *et al.*, 1985). In addition to bacterial infections, NGAL, particularly circulating levels are significantly altered in certain viral infections. For instance, circulating NGAL levels were significantly lower in patients infected with the human immune deficiency virus (HIV) who had not received any therapy (i.e. treatment naïve) compared to non-HIV controls. Significantly, treatment of these patients with the highly active antiretroviral therapy (HAART) was associated with a progressive increase in circulating NGAL levels (Landro *et al.*, 2008). In vitro studies using neutrophils isolated from the three groups of patient revealed that in healthy and HAART-treated HIV positive patients, the neutrophils released NGAL normally into the culture medium. However, in untreated HIV positive patients, this process was defective suggesting that the release of NGAL from

Neutrophil Gelatinase Associated Lipocalin:

**4.3 Malignant diseases**

Structure, Function and Role in Human Pathogenesis 353

both the cause and the extent of renal damage. For instance, following ischemic injury, NGAL levels in the kidney tissue rises by nearly 10-fold within only 3 hours (Mishra *et al.*, 2003). Further, the rise appears to be sustained, being evident for several days following the initial insult. This pattern, an early rise and persistence, makes NGAL a highly sensitive marker of early AKI. Like in CKD, the kidney is the major source of NGAL release in AKI. However, other sources of NGAL that have been suggested include organs that synthesize and release mediators of an acute inflammatory response (e.g. the liver) and immune cells that contain NGAL in their granules (neutrophils and macrophages) (Hvidberg *et al.*, 2005).

The expression of NGAL is significantly increased in several solid and hematological tumors and been shown to correlate with both tumor characteristics and disease outcome. Solid tumors that express high levels of NGAL arise in a variety of organs including the digestive (e.g. esophagus, stomach, liver, bile ducts and pancreas), respiratory (lungs), endocrine (thyroid gland and ovaries), reproductive (breast and endometrium) organs and even in the skin.

Pancreatic cancer (PC) is the most lethal of all malignant solid tumors in humans (Chakraborty *et al.*, 2011). Several groups including ours have now shown that while NGAL is either completely absent or weakly expressed in the normal pancreatic ducts, a strong expression of NGAL is seen in the malignant cells (Moniaux *et al.*, 2008; Furutani *et al.*, 1998; Tong *et al.*, 2008; Tong *et al.*, 2011). We also observed that the expression of NGAL first appears in pre-malignant lesions (termed as Pancreatic intraepithelial neoplasia or PanINs), with a progressive increase from low to high grade dysplasia. In invasive pancreatic cancer, NGAL expression was significantly higher in well-differentiated PC while poorly differentiated cancer cells showed no expression of the protein (Moniaux *et al.*, 2008). Functional studies to investigate the role of NGAL in PC (discussed later) suggest that it plays a role in the progression of this deadly malignancy. Further, quantitative measurement of NGAL levels in plasma revealed that there was a significant increase in the levels of NGAL in the plasma compared to healthy individuals. These results suggest that NGAL might be potentially useful as a novel biomarker for the diagnosis of PC. Similar to PC, NGAL was weakly expressed by the normal liver cells but strongly expressed in hepatocellular carcinoma (Lee *et al.*, 2011). In gastric cancer however, NGAL expression remains controversial with two groups reporting opposing results- one reporting overexpression and the other a significant downregulation of NGAL in pre-malignant and

Ngal, the mouse homologue of human NGAL was strongly upregulated in mice that overexpress Insulin like growth factor (IGF-2), a major growth promoting hormone that acts during *in utero* development. These mice developed lung tumors spontaneously which

Among the endocrine tumors, papillary, follicular and anaplastic thyroid cancers all express NGAL (Iannetti *et al.*, 2008). Notably, the expression of NGAL increases with the loss of tumor differentiation. While NGAL is not detected in the normal ovarian follicles and weakly expressed in benign ovarian tumors, a strong expression of NGAL is noted in borderline and grade 1 (well differentiated) malignant ovarian tumors (Lim *et al.*, 2007). Interestingly, its expression decreased in grade 2 (moderately differentiated) and 3 (poorly

Of the malignancies arising from reproductive organs, ductal carcinoma of the breast and endometrial cancer express high levels of NGAL (Miyamoto *et al.*, 2011; Bauer *et al.*, 2008).

malignant gastric tissues (Alpizar *et al.*, 2009; Wang *et al.*, 2010).

showed a significant upregulation of Ngal mRNA.

differentiated) ovarian cancer (Cho and Kim, 2009).

neutrophils is inhibited by HIV infection and can be restored following therapy. What proteins of the virus mediate this inhibition, and if there is a prognostic significance of the restoration of NGAL levels following HAART therapy remain unanswered questions.

#### **4.2 Metabolic diseases**

Metabolic diseases associated with a dysregulation in NGAL expression include obesity, pre-eclampsia and kidney disease.

Obesity, defined as a body mass index (BMI) of ≥30kg/m2 is now recognized to be associated with low grade chronic inflammation and insulin resistance (Pi-Sunyer, 2002). Studies have shown that NGAL levels are significantly elevated in the adipose tissues of both overweight mice and obese human subjects (Wang *et al.*, 2007). Studies in healthy human subjects have revealed that insulin can significantly increase the level of circulating NGAL in the blood (Tan *et al.*, 2009). Similar results have emerged from studies in pregnant women with gestational diabetes who had significantly higher levels of circulating NGAL than those without this complication of pregnancy (D'Anna *et al.*, 2009a).

Another pregnancy associated complication associated with a rise in circulating NGAL levels is pre-eclampsia. This condition, characterized by the development of hypertension in or after the 20th week of pregnancy is associated with significant maternal and fetal complications. The elevated levels of NGAL in pre-eclampsia were positively correlated with both systolic and diastolic blood pressure and presence of proteinuria but showed a significant negative correlation with maternal age, pre-pregnancy BMI and weight (D'Anna *et al.*, 2008; D'Anna *et al.*, 2009b). Other conditions where NGAL levels are significantly elevated include atherosclerosis (Anwaar *et al.*, 1998; Elneihoum *et al.*, 1997; te Boekhorst *et al.*, 2011) and hepatitis (Bu *et al.*, 2006). Both hyperlipidemia and chronic alcohol ingestion are associated with significant metabolic perturbations and the aforementioned correlative studies suggest that NGAL has a role to play in the pathogenesis of these diseases. With the development of animal models to study the function of NGAL (discussed later), it is expected that the mechanisms by which NGAL modulates metabolic pathways in health and disease will become better delineated in the near future.

The kidney is essential for our survival as it performs the crucial function of maintaining the fluid and mineral balance in our body. This in turn maintains the osmolarity of inter and intracellular fluids crucial for cellular metabolism. Both acute and chronic damage to the kidneys causes metabolic perturbations associated with a significant increase in the expression of NGAL.

Chronic kidney disease (CKD) is a disease characterized by a progressive decline in renal function and associated with significant morbidity. Studies in a mouse model that mimics the pathophysiology of CKD in humans revealed that NGAL is the most highly upregulated gene in the kidneys of mice with severe CKD (Viau *et al.*, 2010). It appears that the damaged kidneys synthesize a significantly higher amount of NGAL than usual, particularly in the proximal convoluted tubules (PCT) and the ascending limb of the loop of Henle. NGAL being a secreted protein is then released into the ultrafiltrate and subsequently excreted in urine. The level of Ngal expression in the diseased kidneys correlated positively with the severity of renal lesions (in mice) suggesting that Ngal may promote renal damage in CKD (Viau *et al.*, 2010). A similar observation was also noted in an animal model of autosomal polycystic kidney disease (ADPKD), another cause of CKD in humans (Lau *et al.*, 2000).

NGAL levels are elevated in acute kidney injury (AKI) resulting from a wide variety of insults to the kidney ranging from ischemia to toxins. This rise occurs early and depends on both the cause and the extent of renal damage. For instance, following ischemic injury, NGAL levels in the kidney tissue rises by nearly 10-fold within only 3 hours (Mishra *et al.*, 2003). Further, the rise appears to be sustained, being evident for several days following the initial insult. This pattern, an early rise and persistence, makes NGAL a highly sensitive marker of early AKI. Like in CKD, the kidney is the major source of NGAL release in AKI. However, other sources of NGAL that have been suggested include organs that synthesize and release mediators of an acute inflammatory response (e.g. the liver) and immune cells that contain NGAL in their granules (neutrophils and macrophages) (Hvidberg *et al.*, 2005).

#### **4.3 Malignant diseases**

352 Acute Phase Proteins – Regulation and Functions of Acute Phase Proteins

neutrophils is inhibited by HIV infection and can be restored following therapy. What proteins of the virus mediate this inhibition, and if there is a prognostic significance of the restoration of NGAL levels following HAART therapy remain unanswered questions.

Metabolic diseases associated with a dysregulation in NGAL expression include obesity,

Obesity, defined as a body mass index (BMI) of ≥30kg/m2 is now recognized to be associated with low grade chronic inflammation and insulin resistance (Pi-Sunyer, 2002). Studies have shown that NGAL levels are significantly elevated in the adipose tissues of both overweight mice and obese human subjects (Wang *et al.*, 2007). Studies in healthy human subjects have revealed that insulin can significantly increase the level of circulating NGAL in the blood (Tan *et al.*, 2009). Similar results have emerged from studies in pregnant women with gestational diabetes who had significantly higher levels of circulating NGAL

Another pregnancy associated complication associated with a rise in circulating NGAL levels is pre-eclampsia. This condition, characterized by the development of hypertension in or after the 20th week of pregnancy is associated with significant maternal and fetal complications. The elevated levels of NGAL in pre-eclampsia were positively correlated with both systolic and diastolic blood pressure and presence of proteinuria but showed a significant negative correlation with maternal age, pre-pregnancy BMI and weight (D'Anna *et al.*, 2008; D'Anna *et al.*, 2009b). Other conditions where NGAL levels are significantly elevated include atherosclerosis (Anwaar *et al.*, 1998; Elneihoum *et al.*, 1997; te Boekhorst *et al.*, 2011) and hepatitis (Bu *et al.*, 2006). Both hyperlipidemia and chronic alcohol ingestion are associated with significant metabolic perturbations and the aforementioned correlative studies suggest that NGAL has a role to play in the pathogenesis of these diseases. With the development of animal models to study the function of NGAL (discussed later), it is expected that the mechanisms by which NGAL modulates metabolic pathways in health

The kidney is essential for our survival as it performs the crucial function of maintaining the fluid and mineral balance in our body. This in turn maintains the osmolarity of inter and intracellular fluids crucial for cellular metabolism. Both acute and chronic damage to the kidneys causes metabolic perturbations associated with a significant increase in the

Chronic kidney disease (CKD) is a disease characterized by a progressive decline in renal function and associated with significant morbidity. Studies in a mouse model that mimics the pathophysiology of CKD in humans revealed that NGAL is the most highly upregulated gene in the kidneys of mice with severe CKD (Viau *et al.*, 2010). It appears that the damaged kidneys synthesize a significantly higher amount of NGAL than usual, particularly in the proximal convoluted tubules (PCT) and the ascending limb of the loop of Henle. NGAL being a secreted protein is then released into the ultrafiltrate and subsequently excreted in urine. The level of Ngal expression in the diseased kidneys correlated positively with the severity of renal lesions (in mice) suggesting that Ngal may promote renal damage in CKD (Viau *et al.*, 2010). A similar observation was also noted in an animal model of autosomal polycystic kidney disease (ADPKD), another cause of CKD in humans (Lau *et al.*, 2000). NGAL levels are elevated in acute kidney injury (AKI) resulting from a wide variety of insults to the kidney ranging from ischemia to toxins. This rise occurs early and depends on

than those without this complication of pregnancy (D'Anna *et al.*, 2009a).

and disease will become better delineated in the near future.

**4.2 Metabolic diseases**

expression of NGAL.

pre-eclampsia and kidney disease.

The expression of NGAL is significantly increased in several solid and hematological tumors and been shown to correlate with both tumor characteristics and disease outcome. Solid tumors that express high levels of NGAL arise in a variety of organs including the digestive (e.g. esophagus, stomach, liver, bile ducts and pancreas), respiratory (lungs), endocrine (thyroid gland and ovaries), reproductive (breast and endometrium) organs and even in the skin.