Ca2+ sensitization is a phenomenon that allows the ASM to produce more force in response to a given mobilization of intracellular calcium concentration ([Ca2+]i). Ca2+ sensitization seems to rely mainly on a signaling pathway running in parallel to canonical Ca2+ signaling pathways (inositol 3-phosphate receptor (IP3R)-dependent and entry from the extracellular compartment). The pathway is referred to as the Rho-Rho-associated, coiled coil-containing kinase (ROCK) pathway and is activated by certain G protein-coupled receptors (GPCRs) following ligation with their cognate spasmogen (see Figure 4). Rho is a small G (GTPase) protein activated by the exchange of GDP for GTP. The identity of the guanine exchange factor (GEF) that is activated by the GPCR and involved in Rho activation is unclear, and is probably receptor specific. However, one of the downstream signals mediated by Rho that leads to Ca2+ sensitization is well characterized. GTP-bound (active) Rho initially activates ROCK, which then inhibits myosin light chain (MLC) phosphatase (MLCP) both directly, via phosphorylation of the myosin phosphatase-targeting subunit 1 (MYPT1) of MLCP, and indirectly, via CPI-17 (17-kDa PKC-potentiated inhibitory protein of PP1; PP1 stands for protein phosphatase 1, which is the catalytic subunit of the heterotrimeric MLCP). Since the level of MLC phosphorylation depends on a balance between its phosphorylation by MLCK and its dephosphorylation by MLCP, direct or indirect inhibition of MLCP by ROCK causes more MLC phosphorylation for the same degree of MLCK activation. Therefore, when the Rho-ROCK pathway is activated, the same degree of MLCK activation induced by a given sarcoplasmic Ca2+ mobilization leads to more MLC phosphorylation and sequentially more activated cross-bridges and more force.

AHR in animal models of airway inflammation has been attributed to an increased ASMsensitivity to Ca2+ (40, 43). Ca2+ sensitization is more typically triggered by inflammationderived spasmogens (discussed in section 4.3.1), which bind to GPCRs capable of activating the Rho-ROCK pathway. However, non-spasmogenic inflammatory mediators such as TNF have also been shown to potentiate ASM-force by fostering Ca2+ sensitization (107, 172, 191). In addition, the extent of Ca2+ sensitization can be regulated by mediators affecting the levels of expression or activation of key regulatory proteins involved in the Rho-ROCK pathway. For example, IL-13 (41) increases the level of RhoA expression in ASM, which consequently increases the proficiency of Rho-ROCK signaling pathway to induce Ca2+

Fig. 4. Pathways transducing the extracellular signal of the spasmogens from their cognate cell-surface receptor to the contractile apparatus. See text for descriptions and abbreviations. The green and red dots represent Ca2+ and Na+, respectively. The reverse mode of the Na+/Ca2+ exchanger (NCX) is shown. Reproduced with permission from: Bossé and Paré. Airway smooth muscle responsiveness: The origin of airway hyperresponsiveness in asthma? Current Respiratory Medicine Reviews. 7(4): 289-301, 2011.

Fig. 4. Pathways transducing the extracellular signal of the spasmogens from their cognate cell-surface receptor to the contractile apparatus. See text for descriptions and abbreviations. The green and red dots represent Ca2+ and Na+, respectively. The reverse mode of the Na+/Ca2+ exchanger (NCX) is shown. Reproduced with permission from: Bossé and Paré. Airway smooth muscle responsiveness: The origin of airway hyperresponsiveness in

asthma? Current Respiratory Medicine Reviews. 7(4): 289-301, 2011.

sensitization. On the other hand, mediators capable of reducing the expression/activity of intermediate proteins involved in this pathway may have therapeutic potential for diminishing Ca2+ sensitization and AHR. One example is the effect of glucocorticoids in reducing the expression of RhoA, which concomitantly attenuates antigen-induced ASM hyperresponsiveness in a rat model of allergic airway inflammation (40).

In addition to canonical Ca2+ signaling pathways, at least one other Ca2+ signaling pathway contributes to the release of Ca2+ into the sarcoplasm (see Figure 4). This pathway is referred to as the CD38/cyclic adenosine-5'-diphosphate (ADP) ribose (cADPR) signaling pathway and has received significant attention recently in regard to its potential role in AHR. CD38 is a type II transmembrane protein of 45-kDa with dual ecto-enzymatic activity. It possesses an ADP-ribosyl cyclase activity, converting -NAD into cADPR and a cADPR hydrolase activity, converting the cADPR into ADPR. The product of the ADP-ribosyl cyclase activity, cADPR, sensitizes the ryanodine receptor (RyR) for Ca2+ release and, more specifically, potentiates Ca2+-induced Ca2+ release (CICR) (70). Whether cADPR binds directly to RyR or requires other intermediates to open the RyR channel is still a matter of debate (54). What is sure is that cADPR increases the open probability of the RyR, liberating more Ca2+ from the sarcoplasmic reticulum (SR) (196). As is the case for IP3R-dependent Ca2+ release, the CD38 pathway is also activated by binding of specific spasmogens to their cognate receptors (252). Thus, this pathway acts in parallel with the canonical Ca2+ signaling pathways to amplify the mobilization of Ca2+ into the sarcoplasm following spasmogenic stimulation.

The amplitude of Ca2+ release by the CD38/cADPR/RyR pathway, as well as its contribution in ASM-force generation, is influenced by CD38 expression. CD38 expression and/or activity were shown to be upregulated by many of the pro-inflammatory cytokines present in asthmatic airways, such as IFN (229), IFN (53), TNF (53, 229), IL-1 (53) and IL-13 (52). Therefore, in the presence of inflammation, the proficiency of this signaling pathway to liberate the Ca2+ from the internal stores may be enhanced. Consequently, higher force generation would be attained for a given contractile stimulus since more Ca2+ would be released. The influence of inflammatory mediators on the CD38/cADPR/RyR pathway may thus play a role in AHR seen in asthma.

Taken together, these results suggested that the contractile properties of ASM are not fixed but rather plastic; i.e., they can change rapidly in the face of external (inflammatory) cues. So in an inflammatory lung disorder such as asthma, the ASM may be stronger or faster, for example, because of the 'bad' environment in which it is embedded. This increased contractility may only be present *in vivo* when the muscle is exposed to inflammationderived mediators, but might be lost once the muscle is removed from the airways and washed repetitively before being studied *ex vivo.* These results may offer an explanation for the failure of *ex vivo* studies to show an increased strength of ASM from asthmatics (reviewed in (153)). The ability of ASM to rapidly change its contractile capacity in response to inflammatory molecules can also contribute to changes in airway responsiveness observed in response to interventions altering airway inflammation (30, 47, 143, 218). Those are all different ways by which a normal ASM may account for AHR in an inflamed environment. Another way by which inflammation can increase airway responsiveness is by producing spasmogens, which increases ASM-tone.

#### **4.3 Increased ASM-tone**

The responsiveness of asthmatics to bronchodilators, together with more direct evidence (165), indicate that ASM-tone is increased in asthmatic airways. The origin of this augmented ASM-tone is not clear and likely varies between asthmatic individuals. Nonetheless, asthmatic lungs are characterized by the overexpression of inflammationderived spasmogens. These spasmogens trigger sustained ASM contraction and, acting individually or collectively, are a likely cause of increased ASM-tone in asthma. In the present subsection, we discuss the mechanisms by which inflammation-derived spasmogens, together with the attendant increase in ASM-tone, can contribute to AHR.

The rationale is that the amount of airway narrowing in response to a given concentration of a chosen spasmogen (e.g., MCh) is due to the combined effect of this extrinsically delivered spasmogen plus the baseline tone, which is caused by the endogenous spasmogens already present. In other words, it is the cooperative effect of all the spasmogens present at the time of the challenge that determines the total level of ASM activation and, ultimately, the amount of airway narrowing achieved. In addition, one of the causes of AHR can be the contractile synergistic interactions that are often observed when different spasmogens are used in combination. In this regard, a sizable literature exists documenting contractile synergisms between different spasmogens (Table 1). The mechanisms involved in these synergistic interactions have been debated in the last few decades and it is now clear that all the mechanisms that may be involved are not mutually exclusive. The nature of these interplays can give us important clues regarding the role of baseline airway tone in determining the degree of airway responsiveness.

#### **4.3.1 Interactive synergisms between spasmogens**

Initial studies in that area begin in the late 1970s and have started by looking at the combined effect of spasmogens on different measures of lung function in animals (45, 46, 57, 99, 102, 112, 131, 152, 180, 181, 192, 208, 209, 224, 244), as well as in humans (49, 68, 97, 101, 110, 156, 158, 194, 202, 240). Apart from an interactive synergism at the level of ASM contraction, many other factors can account for these *in vivo* interactive synergisms. In fact, although these synergistic interactions can be extremely relevant to the understanding of AHR, the mechanisms involved are sometime impossible to delineate.

First, the interactions between spasmogens were assessed *in vivo* by measuring airway resistance (or conductance) and/or flow parameters (flow rate at different lung volumes, forced expiratory volume in 1 sec (FEV1), etc...). All of these measurements are geometrydependent. As mentioned earlier, airway resistance is inversely proportional to the luminal radius at the 4th power. The potentiation of the effect of one spasmogen by another spasmogen (or an increased baseline tone) might be related to a geometrical effect more than a true synergistic interaction of spasmogens on ASM-force. In other words, an additive effect at the level of muscle activation that may result in an additive effect in terms of ASMforce generation and shortening can be perceived as a synergistic effect when looking at airway resistance.

To circumvent these limitations, others have used *in situ* preparations allowing the assessment of tracheal smooth muscle-isometric force (12, 33, 131) or have measured airway caliber using tantalum bronchography (17). Some of these studies confirmed the synergistic effect of spasmogens on airway narrowing and ASM-force generation. However, these results are also confounded by the acute effect of some spasmogens on vessels and nerves, or by the effect of some spasmogens on airway inflammation and the epithelium. In regard to the vessels, many airway spasmogens also have vasoactive properties. Both vasoconstrictors and vasodilators have significant impact on the degree of airway

augmented ASM-tone is not clear and likely varies between asthmatic individuals. Nonetheless, asthmatic lungs are characterized by the overexpression of inflammationderived spasmogens. These spasmogens trigger sustained ASM contraction and, acting individually or collectively, are a likely cause of increased ASM-tone in asthma. In the present subsection, we discuss the mechanisms by which inflammation-derived spasmogens, together with the attendant increase in ASM-tone, can contribute to AHR. The rationale is that the amount of airway narrowing in response to a given concentration of a chosen spasmogen (e.g., MCh) is due to the combined effect of this extrinsically delivered spasmogen plus the baseline tone, which is caused by the endogenous spasmogens already present. In other words, it is the cooperative effect of all the spasmogens present at the time of the challenge that determines the total level of ASM activation and, ultimately, the amount of airway narrowing achieved. In addition, one of the causes of AHR can be the contractile synergistic interactions that are often observed when different spasmogens are used in combination. In this regard, a sizable literature exists documenting contractile synergisms between different spasmogens (Table 1). The mechanisms involved in these synergistic interactions have been debated in the last few decades and it is now clear that all the mechanisms that may be involved are not mutually exclusive. The nature of these interplays can give us important clues regarding the role of baseline airway tone in

Initial studies in that area begin in the late 1970s and have started by looking at the combined effect of spasmogens on different measures of lung function in animals (45, 46, 57, 99, 102, 112, 131, 152, 180, 181, 192, 208, 209, 224, 244), as well as in humans (49, 68, 97, 101, 110, 156, 158, 194, 202, 240). Apart from an interactive synergism at the level of ASM contraction, many other factors can account for these *in vivo* interactive synergisms. In fact, although these synergistic interactions can be extremely relevant to the understanding of

First, the interactions between spasmogens were assessed *in vivo* by measuring airway resistance (or conductance) and/or flow parameters (flow rate at different lung volumes, forced expiratory volume in 1 sec (FEV1), etc...). All of these measurements are geometrydependent. As mentioned earlier, airway resistance is inversely proportional to the luminal radius at the 4th power. The potentiation of the effect of one spasmogen by another spasmogen (or an increased baseline tone) might be related to a geometrical effect more than a true synergistic interaction of spasmogens on ASM-force. In other words, an additive effect at the level of muscle activation that may result in an additive effect in terms of ASMforce generation and shortening can be perceived as a synergistic effect when looking at

To circumvent these limitations, others have used *in situ* preparations allowing the assessment of tracheal smooth muscle-isometric force (12, 33, 131) or have measured airway caliber using tantalum bronchography (17). Some of these studies confirmed the synergistic effect of spasmogens on airway narrowing and ASM-force generation. However, these results are also confounded by the acute effect of some spasmogens on vessels and nerves, or by the effect of some spasmogens on airway inflammation and the epithelium. In regard to the vessels, many airway spasmogens also have vasoactive properties. Both vasoconstrictors and vasodilators have significant impact on the degree of airway

determining the degree of airway responsiveness.

airway resistance.

**4.3.1 Interactive synergisms between spasmogens**

AHR, the mechanisms involved are sometime impossible to delineate.

Abbreviations in the table: AII, angiotensin II; ACh, acetylcholine; ATP, adenosine triphosphate; BK, bradykinin; CCh, carbachol; Cys-LTs, cysteinyl-leukotrienes; ET-1, endothelin-1; HIST, histamine; 5-HT, 5-hydroxytryptamine (also called serotonine); K+, potassium; LPA, lysophosphatidic acid; LTB4, leukotriene B4; MCh, methacholine; NaF, sodium fluoride; NE, norepinephrine; PA, phosphatidic acid; PAF, platelet activating factor; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2, prostaglandin F2; SM, smooth muscle; SP, substance P; S1P, sphingosine-1-phosphate; TXA2, thromboxane A2.

Table 1. Interactions between spasmogens increasing ASM contractility

responsiveness (151). The potentiating effect of histamine on the responsiveness to muscarinic agonists (33, 131), for example, can likely be due to its vasodilating effect, especially when the drugs are administered intravenously or via the tracheal vasculature. This is because vasodilatation facilitates the delivery of the bronchoactive substance (ACh) to the ASM. Histamine also increases airway epithelium permeability by altering Ecadherin-based adhesions (262). The effect of a second inhaled spasmogen may thus be potentiated by histamine pre-treatment because the second spasmogen will have easier access to the ASM.

In regard to the nerves, many studies have shown synergistic interactions between spasmogens and electrical stimulation of the vagal nerves (17, 33, 57, 88, 114, 137, 217, 258). Similarly, a decrease in responsiveness to spasmogens (other than ACh (88)) was observed following vagotomy (59, 137, 157, 258), cooling of the vagal nerves (58, 88), or treatments with either atropine (59, 219), tetrodotoxin (219) or hexamethonium (59). These results suggest that the basal cholinergic tone synergistically interacts with the exogenously delivered spasmogen. These studies are not included in Table 1 because it is not clear whether the potentiating effect is due to a true synergistic effect at the level of ASM-force generation or due to the ability of the spasmogens to either trigger a vagal reflex (36, 58, 74, 157) or to increase cholinergic neurotransmission and/or synaptic transmission at the

**Mediators** *in vivo ex vivo in vitro*

rings;

strips;

(42)

rings

S1P MCh (122) in guinea pig tracheal

LPA MCh (230), 5-HT (230), SP (230) in

PA MCh (230) in rabbit tracheal rings

Table 1. Interactions between spasmogens increasing ASM contractility

Abbreviations in the table: AII, angiotensin II; ACh, acetylcholine; ATP, adenosine triphosphate; BK, bradykinin; CCh, carbachol; Cys-LTs, cysteinyl-leukotrienes; ET-1, endothelin-1; HIST, histamine; 5-HT, 5-hydroxytryptamine (also called serotonine); K+, potassium; LPA, lysophosphatidic acid; LTB4, leukotriene B4; MCh, methacholine; NaF, sodium fluoride; NE, norepinephrine; PA, phosphatidic acid; PAF, platelet activating factor; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2, prostaglandin F2; SM, smooth muscle; SP, substance P; S1P, sphingosine-1-phosphate; TXA2, thromboxane A2.

responsiveness (151). The potentiating effect of histamine on the responsiveness to muscarinic agonists (33, 131), for example, can likely be due to its vasodilating effect, especially when the drugs are administered intravenously or via the tracheal vasculature. This is because vasodilatation facilitates the delivery of the bronchoactive substance (ACh) to the ASM. Histamine also increases airway epithelium permeability by altering Ecadherin-based adhesions (262). The effect of a second inhaled spasmogen may thus be potentiated by histamine pre-treatment because the second spasmogen will have easier

In regard to the nerves, many studies have shown synergistic interactions between spasmogens and electrical stimulation of the vagal nerves (17, 33, 57, 88, 114, 137, 217, 258). Similarly, a decrease in responsiveness to spasmogens (other than ACh (88)) was observed following vagotomy (59, 137, 157, 258), cooling of the vagal nerves (58, 88), or treatments with either atropine (59, 219), tetrodotoxin (219) or hexamethonium (59). These results suggest that the basal cholinergic tone synergistically interacts with the exogenously delivered spasmogen. These studies are not included in Table 1 because it is not clear whether the potentiating effect is due to a true synergistic effect at the level of ASM-force generation or due to the ability of the spasmogens to either trigger a vagal reflex (36, 58, 74, 157) or to increase cholinergic neurotransmission and/or synaptic transmission at the

tracheal strips;

pig tracheal rings

rabbit tracheal rings;

K+ (215) and CCh (215) in porcine

ACh (48) and HIST (48) in guinea

K+ in bronchial rings from normal but not antigen-challenged mice

> ATP (205) and K+ (205) in bovine tracheal SM cells (205)

MCh (230) in cat tracheal rings; ACh (98) in guinea pig tracheal

ET-1 (195) in bovine bronchial

LTB4 ACh (181) in dogs Cys-LTs HIST (194) and PGD2

access to the ASM.

(194) in humans; PAF (192) in rhesus

monkeys

ganglia (217). These latter phenomena would lead to more ACh release and concomitantly higher ASM activation for the same neural input. On the other hand, the bronchoconstricting effect of spasmogens is known to decrease the vagal tone (13, 210). This is because of their indirect effect on the slowly adapting stretch receptors (SARs), which, upon an enhanced discharge, relax the ASM by reducing parasympathetic tone to the airways. However, other spasmogens, such as bradykinin, would synergize with the effect of these mechanoreceptors via C-fibers activation to initiate the bronconconstricting reflex (150). Together, the influence of the nervous system is intricate and certainly a major confounding factor *in vivo* when one tries to elucidate the synergistic interaction between spasmogens.

Finally, thickening of the airway wall by edema and inflammatory infiltrates, encroachment of the airway lumen by mucus hypersecretion and cellular debris, and reduction in the forces of interdependence between the airway wall and the lung parenchyma by the accumulation of inflammatory exudates in the adventitial layer are all means by which airway inflammation can increase airway responsiveness without affecting ASM-force (reviewed in (26)). Therefore, the hyperresponsiveness observed in response to the second spasmogen, when the first spasmogen is administered hours before the second spasmogen (180), may be due to the inflammation provoked by the initial spasmogen.

To avoid these confounding factors and to investigate whether the synergistic action of two spasmogens on airway responsiveness relies on mechanisms operating at the level of ASM, the only solution is to study the ASM tissue in isolation. Many investigators have focused on this strategy. Most studies were performed using freshly dissected ASM strips or bronchial rings from which isometric force was measured *ex vivo* (12, 32, 38, 48, 71, 87, 98, 122, 135, 156, 173, 174, 184, 185, 195, 203, 204, 215, 230). *In vitro* preparations (i.e., cell culture) have also been used on occasions (205). These reported synergisms are also enumerated inTable 1. The molecular mechanisms involved in contractile synergisms observed between different pairs of spasmogens have been extensively studied. The synergisms involve the co-activation of ASM by two spasmogens acting on two distinct GPCRs. The activation of two distinct GPCRs can lead to intracellular signaling synergisms that ultimately potentiate ASM-force. The more commonly discussed mechanism is Ca2+ sensitization, defined as an increased ASMforce generation in response to a given amount of intracellular Ca2+ mobilization (as discussed in subsection 4.2.3). Many spasmogenic mediators, such as leukotriene C4 (215), isoprostanes (135), ATP (184), endothelin-1 (260), LPA (205), TxA2 (167) and S1P (122), have been shown to increase the responsiveness of the contractile apparatus to Ca2+.

However, these synergisms in terms of force generation can also be related to an additive effect in terms of ASM activation, especially when the concentrations of spasmogens used are low. This is because the dose-response curve to any spasmogen is sigmoidal, regardless of whether the response is measured in terms of ASM-force or shortening. That means that initially, a threshold concentration is needed before any response can be measured, but then there is a progressive increase toward a steeper, relatively linear slope followed by a progressive decrease in slope to reach a plateau. When two different spasmogens are used in combination, their additive effect on ASM-activation could be erroneously interpreted as being synergistic because sub-threshold concentrations of two spasmogens could have a measurable effect on ASM-force or shortening. Similarly, if one uses a concentration just above the response threshold, the subsequent addition of a different or the same spasmogen will have more effect simply because it is now acting on the steeper part of the doseresponse curve. In fact, many of the previously documented examples of 'synergism' performed either *in vivo* (110, 112, 131, 158, 244) or *ex vivo* (38, 71, 87, 135) were involving low concentrations of spasmogens, and were unable to demonstrate a synergism at higher concentrations for at least some of the reported spasmogenic interactions (71, 87, 131). So the level of basal tone, even if low, can seemingly synergize with the extrinsically delivered spamogen to cause AHR simply by an additive effect at the level of ASM activation

Collectively, the force potentiation of one spasmogen (or baseline tone) on another spasmogen can be due to many factors such as: 1-use of sub-threshold or barely active concentrations of spasmogens, which simply act additively in terms of ASM activation but will be perceived as being synergistic in terms of ASM-force, ASM shortening, airway narrowing or airway resistance; 2-use of geometry-dependent measurements such as airway resistance; so that the effect of the second spasmogen is augmented by the reduced airway caliber caused by the first spasmogen; 3-a synergistic effect due to the contribution of vascular, neural or inflammatory effects when tested *in vivo* or *in situ*, which can be observed when the spasmogens used have vasoactive and inflammatory activities or when they can trigger either a vagal reflex and/or an increase in the efferent cholinergic traffic; and 4-a true synergistic effect on myosin cross-bridge cycling due to the convergence of intracellular signaling pathways that are activated by the ligation of two distinct GPCRs (such as Ca2+ sensitization).

#### **4.3.2 Force adaptation**

There is an additional, more subtle way by which the tone can contribute to AHR. Recent studies from our laboratory have demonstrated that a short period (~30 min) of increased ASM-tone augments ASM strength over time (20, 21). Specifically, ASM was exposed to ACh and ASM-force generation in response to EFS was monitored both before and after the induction of the ACh-induced tone. The first EFS in the presence of tone was given 1 min post ACh administration and then at 5-min intervals thereafter. Addition of ACh immediately increased the total force (ACh-induced tone + EFS-induced force), suggesting that ASM generates more force in response to two sub-maximal stimuli (ACh + EFS) compared with EFS alone. More surprisingly, while the EFS-induced force decreased immediately following the administration of ACh, it recovered significantly over time. Since the tone produced by the exogenous ACh remained relatively constant, the total force increased over time. We termed this phenomenon 'force adaptation' (20). The potential significance of force adaptation on airway narrowing based on a computational model has been demonstrated recently (Bossé *et al*., in press in the journal of Respiratory Physiology & Neurobiology).

Force adaptation is distinct from the synergisms in force that were attributed to the convergence of intracellular signaling pathways described above, which occur as a result of activation of two distinct GPCRs. This form of synergism cannot occur when ACh is combined with activation of the ASM by EFS, which was the experimental setting that was used to describe force adaptation. This is because EFS triggers ASM contraction by releasing ACh from the nerve endings (145), which, in turn, binds on the same M3 receptor as the exogenously administered ACh. In fact, all studies investigating the acute effect of a muscarinic agonist on EFS-induced force have failed to show a synergistic effect (57, 114). Therefore, despite abundant literature reporting synergistic effects between spasmogens on ASM strength, force adaptation is a phenomenon with no precedent.

The molecular mechanisms involved in force adaptation are also likely to be different from those described above because it occurs over minutes. This time-course would be too fast for

performed either *in vivo* (110, 112, 131, 158, 244) or *ex vivo* (38, 71, 87, 135) were involving low concentrations of spasmogens, and were unable to demonstrate a synergism at higher concentrations for at least some of the reported spasmogenic interactions (71, 87, 131). So the level of basal tone, even if low, can seemingly synergize with the extrinsically delivered

Collectively, the force potentiation of one spasmogen (or baseline tone) on another spasmogen can be due to many factors such as: 1-use of sub-threshold or barely active concentrations of spasmogens, which simply act additively in terms of ASM activation but will be perceived as being synergistic in terms of ASM-force, ASM shortening, airway narrowing or airway resistance; 2-use of geometry-dependent measurements such as airway resistance; so that the effect of the second spasmogen is augmented by the reduced airway caliber caused by the first spasmogen; 3-a synergistic effect due to the contribution of vascular, neural or inflammatory effects when tested *in vivo* or *in situ*, which can be observed when the spasmogens used have vasoactive and inflammatory activities or when they can trigger either a vagal reflex and/or an increase in the efferent cholinergic traffic; and 4-a true synergistic effect on myosin cross-bridge cycling due to the convergence of intracellular signaling pathways that are activated by the ligation of two distinct GPCRs

There is an additional, more subtle way by which the tone can contribute to AHR. Recent studies from our laboratory have demonstrated that a short period (~30 min) of increased ASM-tone augments ASM strength over time (20, 21). Specifically, ASM was exposed to ACh and ASM-force generation in response to EFS was monitored both before and after the induction of the ACh-induced tone. The first EFS in the presence of tone was given 1 min post ACh administration and then at 5-min intervals thereafter. Addition of ACh immediately increased the total force (ACh-induced tone + EFS-induced force), suggesting that ASM generates more force in response to two sub-maximal stimuli (ACh + EFS) compared with EFS alone. More surprisingly, while the EFS-induced force decreased immediately following the administration of ACh, it recovered significantly over time. Since the tone produced by the exogenous ACh remained relatively constant, the total force increased over time. We termed this phenomenon 'force adaptation' (20). The potential significance of force adaptation on airway narrowing based on a computational model has been demonstrated recently (Bossé *et al*., in press in the journal of Respiratory Physiology &

Force adaptation is distinct from the synergisms in force that were attributed to the convergence of intracellular signaling pathways described above, which occur as a result of activation of two distinct GPCRs. This form of synergism cannot occur when ACh is combined with activation of the ASM by EFS, which was the experimental setting that was used to describe force adaptation. This is because EFS triggers ASM contraction by releasing ACh from the nerve endings (145), which, in turn, binds on the same M3 receptor as the exogenously administered ACh. In fact, all studies investigating the acute effect of a muscarinic agonist on EFS-induced force have failed to show a synergistic effect (57, 114). Therefore, despite abundant literature reporting synergistic effects between spasmogens on

The molecular mechanisms involved in force adaptation are also likely to be different from those described above because it occurs over minutes. This time-course would be too fast for

ASM strength, force adaptation is a phenomenon with no precedent.

spamogen to cause AHR simply by an additive effect at the level of ASM activation

(such as Ca2+ sensitization).

**4.3.2 Force adaptation**

Neurobiology).

*de novo* gene expression; so it is not likely due to transcriptional activation of genes involved in ASM contraction induced by the initial tone. On the other hand, if it was due to biochemical events such as increased phosphorylation of MLC due MLCP deactivation leading to Ca2+ sensitization (as described in subsection 4.2.3), the potentiating effect would be very fast and seen at the first EFS following ACh administration (1 min). Taken together, the kinetic of force adaptation is too slow to be explained by fast biochemical reactions, such as protein phosphorylation, but too fast for *de novo* gene expression. We thus suggest that this time-dependent increase in ASM-force is due to post-translational mechanisms. All the necessary machinery responsible for this increased ASM-force might be present within the cell prior to activation, but it is only upon stimulation (i.e., induction of tone) that this protein machinery re-organizes to optimize generation of force. Processes such as actin (264) or myosin (213) filamentogenesis and/or re-arrangement of the cytoskeleton and its connectivity with the contractile apparatus, the plasmalemma and the ECM (264) are likely possibilities. However, these remain pure speculations as the mechanisms underlying force adaptation have never been investigated.

#### **4.3.3 Increased tone on airway wall stiffness**

Apart from ASM-force, increased ASM-tone can also impact other ASM contractile properties. For instance, stiffness is greatly enhanced upon ASM activation. By increasing ASM stiffness, an increased tone would reduce the strains experienced by the airway wall due to the cyclical stresses of breathing. Interestingly, Noble and coworkers (178) have demonstrated that the amount of airway wall strain induced by a DI is proportional to its bronchodilating effect (178). They also showed that the main factor limiting the strain was the magnitude of ASMtone, which was controlled by delivering different concentrations of ACh. These observations suggested that the ASM needs not only to be stressed but to be stretched for a DI to be effective in reducing ASM contractility. They concluded by saying that a stiffer airway caused by an increased ASM-tone reduces the strain induced by the dynamic load associated with DI and, for this reason, reduces the bronchodilating effect of a DI.

Taken all together, these observations suggest that baseline tone is of crucial importance in determining the degree of airway responsiveness to an inhalational challenge with a spasmogen. The increased tone observed in asthma can be from inflammatory and/or vagal origin. In both cases, this increased tone acts additively with the extrinsically delivered spamogen in terms of ASM activation, force generation, ASM shortening and airway diameter narrowing. This can also be translated into a synergistic effect when geometrydependent measurements are taken, such as airway cross-sectional area of the lumen or airflow resistance (or other measurements relying on it, such as peak expiratory flow (PEF), FEV1, …). In cases where the increased tone is mediated by inflammation-derived spasmogens, the potentiating effect of increased tone on airway responsiveness can also be due to a vascular effect, a neural effect, an inflammatory effect and/or an increase in ASMforce caused by either Ca2+ sensitization or force adaptation. Finally, augmented tone also increases ASM stiffness and, concomitantly, airway wall stiffness, which can further enhance airway responsiveness by many ways as discussed in subsections 2.2 and 2.3.

#### **4.4 Heterogeneity**

To assess airway responsiveness airway resistance is measured at the mouth using the forced oscillation technique or, alternatively, flow parameters are measured using standard spirometry. These measurements take into account airway resistance but also lung tissue and chest wall resistance. Since chest wall resistance does not change during bronchoprovocation, and lung tissue resistance does not change by much, the progressive increase of resistance occurring in response to increasing doses of a spasmogen is thought to reflect changes of airway caliber. In other words, the change of resistance along the course of a challenge is viewed as being due to airway narrowing caused by ASM shortening. However, even this change of resistance is intricate. This is because this resistance represents represents the combined resistance of all generations of airways arranged in series and in parallel. Therefore, simply looking at the factors affecting resistance in a single airway, as we have done since the beginning of this chapter, may not be sufficient to understand the full nature of airway responsiveness.

#### **4.4.1 Mathematical link between airway narrowing heterogeneity and airway responsiveness**

The link between heterogeneity and AHR was initially though as being simply due to geometric considerations. As aforementioned, the relationship between airway luminal radius and resistance to airflow is not linear. A decrease in radius causes an exponential increase in airway resistance. For this reason, even if the total cross sectional area within a given airway generation is the same, inhomogeneous airway caliber increases airway resistance. In other words, the increase in resistance caused by narrowing of some airways is not compensated for by the decrease in resistance caused by dilation of other airways. Airway narrowing heterogeneity upon a bronchoprovocative challenge with a spasmogen will have the same consequence. So that the increases in resistance caused by augmented narrowing in some airways are not compensated for by the attenuated increases in resistance caused by reduced narrowing happening in other airways.

The consequences of baseline heterogeneity on baseline resistance and of non-homogeneous airway narrowing on airway responsiveness were predicted based on computational models. Bates (14) was one of the first to address the issue of heterogeneity. He developed a stochastic model of the airway tree in which the values of airway radii within every airway generation were chosen randomly according to a probability distribution function. The mean values were based on realistic values (morphometric data of human lungs). He found that, while the mean values were kept the same, increasing the standard deviations progressively leads to greater airway resistance. This suggested that the simple presence of heterogeneity increases airway resistance. The model further predicted that, upon ASM activation, both baseline airway caliber heterogeneity and ASM shortening heterogeneity increase airway responsiveness. Obviously, this is only true if one assumes that the flow is not redistributed into more patent airways.

More sophisticated computational models have then been developed and brought additional insights about the origin of this increased resistance evoked by baseline or airway narrowing heterogeneity. The main contributor to this increased resistance comes from nearly closed peripheral airways, especially when resistance is measured at frequency near the breathing frequency (138). These airway closures (or near closed) ultimately lead to ventilation defects and attendant flow limitation. They might be the link between ventilation heterogeneity and AHR.

#### **4.4.2 Non-uniform ventilation with air trapping and AHR**

Using the forced oscillation technique, several groups noticed that the frequencydependence of resistance and elastance was increased at baseline in asthmatics (139) and

and chest wall resistance. Since chest wall resistance does not change during bronchoprovocation, and lung tissue resistance does not change by much, the progressive increase of resistance occurring in response to increasing doses of a spasmogen is thought to reflect changes of airway caliber. In other words, the change of resistance along the course of a challenge is viewed as being due to airway narrowing caused by ASM shortening. However, even this change of resistance is intricate. This is because this resistance represents represents the combined resistance of all generations of airways arranged in series and in parallel. Therefore, simply looking at the factors affecting resistance in a single airway, as we have done since the beginning of this chapter, may not be sufficient to

**4.4.1 Mathematical link between airway narrowing heterogeneity and airway**

resistance caused by reduced narrowing happening in other airways.

The link between heterogeneity and AHR was initially though as being simply due to geometric considerations. As aforementioned, the relationship between airway luminal radius and resistance to airflow is not linear. A decrease in radius causes an exponential increase in airway resistance. For this reason, even if the total cross sectional area within a given airway generation is the same, inhomogeneous airway caliber increases airway resistance. In other words, the increase in resistance caused by narrowing of some airways is not compensated for by the decrease in resistance caused by dilation of other airways. Airway narrowing heterogeneity upon a bronchoprovocative challenge with a spasmogen will have the same consequence. So that the increases in resistance caused by augmented narrowing in some airways are not compensated for by the attenuated increases in

The consequences of baseline heterogeneity on baseline resistance and of non-homogeneous airway narrowing on airway responsiveness were predicted based on computational models. Bates (14) was one of the first to address the issue of heterogeneity. He developed a stochastic model of the airway tree in which the values of airway radii within every airway generation were chosen randomly according to a probability distribution function. The mean values were based on realistic values (morphometric data of human lungs). He found that, while the mean values were kept the same, increasing the standard deviations progressively leads to greater airway resistance. This suggested that the simple presence of heterogeneity increases airway resistance. The model further predicted that, upon ASM activation, both baseline airway caliber heterogeneity and ASM shortening heterogeneity increase airway responsiveness. Obviously, this is only true if one assumes that the flow is

More sophisticated computational models have then been developed and brought additional insights about the origin of this increased resistance evoked by baseline or airway narrowing heterogeneity. The main contributor to this increased resistance comes from nearly closed peripheral airways, especially when resistance is measured at frequency near the breathing frequency (138). These airway closures (or near closed) ultimately lead to ventilation defects and attendant flow limitation. They might be the link between ventilation

Using the forced oscillation technique, several groups noticed that the frequencydependence of resistance and elastance was increased at baseline in asthmatics (139) and

understand the full nature of airway responsiveness.

not redistributed into more patent airways.

**4.4.2 Non-uniform ventilation with air trapping and AHR**

heterogeneity and AHR.

**responsiveness**

upon MCh challenge in both asthmatics and non-asthmatics (111, 139). In accordance to the computational models discussed above, this is indicative of a heterogenous pattern of constriction that includes randomly distributed airway closures or near closures (138). These observations are supported by ventilation imaging studies such as hyperpolarized 3He MRI (39, 207, 234), single-photon emission computed tomography (116) and positron emission tomography (169). These studies showed that asthmatics have increased ventilation heterogeneity at baseline and, even if heterogeneous airway response to a spamogen is also observed in non-asthmatics, the level of ventilation heterogeneity achieved is greater in asthmatics (39, 207, 234). This heterogeneous pattern of ventilation is characterized by relatively large zone of non-ventilated area, which supports the presence of airway closure. These ventilations defects represent zones of air trapping, which are characteristic of obstructive diseases and known to contribute to airflow limitation. Ventilation heterogeneity can thus be one of the sources of AHR seen in asthmatics. This is consistent with recent reports showing that baseline ventilation heterogeneity measured by nitrogen washouts correlates with the severity of AHR in both younger (60) and older (96) asthmatic subjects. The increase in ventilation heterogeneity in response to a spasmogenic challenge may simply be due to an amplifying effect over the baseline heterogeneity. However, upon

induced bronchoconstriction, airway narrowing heterogeneity can also contribute to ventilation heterogenetity. King and coworkers (115) have used high resolution computed tomography (HRCT) to measure the heterogeneity of airway narrowing in response to MCh challenge in human subjects. The heterogeneity was measured by the standard deviations of the changes in luminal airway caliber caused by MCh. They showed that in airways of 2 mm of diameter that the variability of airway narrowing was greater than the measure of repeatability (the variability in the changes in luminal airway caliber when two pre-MCh scans were compared), suggesting that airways narrow heterogeneously. They also shown that for the same level of bronchoconstriction achieved, asthmatic airways narrowed more heterogeneously than non-asthmatics. Together, these observations suggest that the larger and more numerous ventilation defects observed in asthmatics during a spasmogenic challenge are due to both greater baseline heterogeneity and greater airway narrowing heterogeneity.

#### **4.4.3 Mechanistic link between airway narrowing heterogeneity and airway responsiveness**

As aforementioned, the emergence of ventilation defects during bronchoconstriction is essentially the link between ventilation heterogeneity and AHR. In other words, simple heterogeneity without airway closure (or near closure) does not seem to cause AHR. It is true that for the same mean level of bronchoconstriction, heterogeneity would increase resistance but only if the flow is still evenly distributed among the different airways. However, during heterogeneous constriction, the flow is more likely to be redirected into more conductant (patent) airways and correspondingly less flow would be redirected into more resistant (constricted) airways. Consequently, the overall resistance would rather decrease with heterogeneity. On the other hand, when the airways are closed (or nearly), the volume of air subtended by these airways is trapped and will not be able to be expelled (at least not as fast) upon maximal expiratory flow maneuvers and that would be the cause of AHR.

It is also unlikely that the ventilation defects are due to random closure of single peripheral airways. Based on the size of these ventilation defects, they are more likely due to clustering of constricted peripheral airways. In a computational model developed by Venegas and coworkers (236, 253), it was shown that airway closure occurs in cluster. In their model, the bronchial tree branches dichotomously, where every bifurcation is composed of one parent airway and two daughter airways with slight heterogeneity between them. During bronchoconstriction, the flow is redistributed according to divergence of patency among daughter airways. The model predicted that all the airways shorten uniformly when the level of ASM activation is low. However, passed a certain threshold of ASM activation, daughter airways develop a dichotomic response. Whereas one constricts excessively the other one dilates. This is because the initial heterogeneity between the two daughter airways fosters the redistribution of flow into the more conductant airway. The insufflation pressure thus rises in the hyperventilated areas subtended by this airway, which increases the load impeding ASM shortening. In fact, the model predicted that this airway dilates despite the rise in ASM activation. On contrary, diminution of flow in the other daughter airway causes regional lung deflation and loss of parenchymal tethering recoil. The load impeding muscle shortening thus decreases and, for the same level of ASM activation, more narrowing occurs. Because of axial interdependence of pathways in series, all the smaller airways downstream of the excessively closed airways are also affected. The size of the ventilation defects depends on the first airway generation afflicted by this dichotomic response. Since more ASM activation is required to close larger airways, this dichotomic response begins in more peripheral airways. But as the ASM activation progressively rises, the model predicted that larger and larger airways are affected and larger and larger patches of lung become non-ventilated.

Taken together, Venegas and colleagues suggested that only a slight degree of heterogeneity at baseline can trigger a self-perpetuating feedback loop when a certain level of ASM activation is achieved; such that the redistribution of flow in slightly more patent airways, makes the other airways and their downstream pathways unstable because of the loss of elastic recoil. This ultimately leads to clustering of peripheral airway constriction and the emergence of large ventilation defects, which, in turn, cause flow limitation. The chase for the factors determining this baseline heterogeneity, which was heretofore ignored, is now on.

#### **4.4.4 Factors potentially involved in determining baseline heterogeneity**

The amplifying effect described by Venegas and coworkers (236), which can lead to large ventilation defects and attendant AHR, relies on subtle structural and/or functional changes that were already present prior the inhalation of the spasmogen. Therefore, the identification of factors responsible for this baseline airway wall heterogeneity is of major interest.

Remodeling can certainly impact heterogeneity and can potentially discriminate the different levels of heterogeneity observed between asthmatics and non-asthmatics. Remodeling occurs non-uniformly throughout the tracheobronchial tree. The occurrence of epithelium desquamation or inflammatory infiltrates for example is patchy in nature. The extent of subepithelial fibrosis, ASM enlargement, and other structural changes present in asthmatic lungs could also vary at different locations in the lung. Together, these disparities alter the mechanical properties of the airway wall non-uniformly along the bronchial tree. It can also modify airway geometry and sometimes lead to the formation of bottlenecks, a characteristic feature found in terminal bronchioles of chronic obstructive pulmonary disease (COPD) patients (McDonough *et al*., provisionally accepted in the New England Journal of Medicine). The effect of a bottleneck is treacherous because not only it increases the resistance to airflow in an airway that otherwise may look normal but also because it reduces ventilation in the parenchymal tissue subtended by this airway. This ultimately fosters the development of a ventilation defect due to a decrease in the force of airway-parenchymal interdependence. Greater ventilation defects at baseline seen in asthmatics can thus be attributed to remodeling.

However, ventilation heterogeneity is mainly attributable to the fact that airways narrow heterogeneously. So, it is upon bronchoconstriction that the functional consequences of these disparities (localized remodeling changes) along the length of a pathway are exacerbated. For example, remodeling heterogeneity can affect locally the initial geometry, which has a huge influence on airway responsiveness (as discussed in subsection 4.1.3). It can also modify locally the after-load impeding ASM shortening. Another example would be the patchiness of ASM mass enlargement, which can potentially amplify regional differences in airway narrowing upon ASM activation.

Gain in contractile properties (e.g., force-generating capacity) due to ASM behaviors could also be patchy within the lungs. For example, acquisition of supplemental force by length adaptation can occur in an area where the airway caliber is smaller because of a localized decrease in the force of airway-parenchymal interdependence. The baseline ASM-tone also likely differs in different areas of the lung. Its magnitude may be spatially correlated with zones of inflammation, where inflammation-derived spasmogens are produced/released. That would also lead to force adaptation and the attendant gain in force-generating capacity in those areas. All these factors can affect ASM contractile properties in a localized manner and contribute to narrowing heterogeneity and AHR.

Hence, remodeling and inflammatory changes seen in asthmatic lungs can give rise to a greater baseline level of airway wall heterogeneity. It can also cause inhomogeneity of narrowing upon a bronchoprovocative challenge with a spasmogen by changing the load impeding ASM shortening or by fostering the development of increased ASM contractility. Together with the strong interplay between the parenchymal tethering and airway patency, this increased level of heterogeneity can be sufficient to trigger a self-perpetuating loop leading to patches of hypo- or non-ventilated area and the appearance of AHR in asthmatics.

## **5. Conclusion**

36 Lung Diseases – Selected State of the Art Reviews

of constricted peripheral airways. In a computational model developed by Venegas and coworkers (236, 253), it was shown that airway closure occurs in cluster. In their model, the bronchial tree branches dichotomously, where every bifurcation is composed of one parent airway and two daughter airways with slight heterogeneity between them. During bronchoconstriction, the flow is redistributed according to divergence of patency among daughter airways. The model predicted that all the airways shorten uniformly when the level of ASM activation is low. However, passed a certain threshold of ASM activation, daughter airways develop a dichotomic response. Whereas one constricts excessively the other one dilates. This is because the initial heterogeneity between the two daughter airways fosters the redistribution of flow into the more conductant airway. The insufflation pressure thus rises in the hyperventilated areas subtended by this airway, which increases the load impeding ASM shortening. In fact, the model predicted that this airway dilates despite the rise in ASM activation. On contrary, diminution of flow in the other daughter airway causes regional lung deflation and loss of parenchymal tethering recoil. The load impeding muscle shortening thus decreases and, for the same level of ASM activation, more narrowing occurs. Because of axial interdependence of pathways in series, all the smaller airways downstream of the excessively closed airways are also affected. The size of the ventilation defects depends on the first airway generation afflicted by this dichotomic response. Since more ASM activation is required to close larger airways, this dichotomic response begins in more peripheral airways. But as the ASM activation progressively rises, the model predicted that larger and larger airways are affected and larger and larger patches of lung become

Taken together, Venegas and colleagues suggested that only a slight degree of heterogeneity at baseline can trigger a self-perpetuating feedback loop when a certain level of ASM activation is achieved; such that the redistribution of flow in slightly more patent airways, makes the other airways and their downstream pathways unstable because of the loss of elastic recoil. This ultimately leads to clustering of peripheral airway constriction and the emergence of large ventilation defects, which, in turn, cause flow limitation. The chase for the factors determining

The amplifying effect described by Venegas and coworkers (236), which can lead to large ventilation defects and attendant AHR, relies on subtle structural and/or functional changes that were already present prior the inhalation of the spasmogen. Therefore, the identification

Remodeling can certainly impact heterogeneity and can potentially discriminate the different levels of heterogeneity observed between asthmatics and non-asthmatics. Remodeling occurs non-uniformly throughout the tracheobronchial tree. The occurrence of epithelium desquamation or inflammatory infiltrates for example is patchy in nature. The extent of subepithelial fibrosis, ASM enlargement, and other structural changes present in asthmatic lungs could also vary at different locations in the lung. Together, these disparities alter the mechanical properties of the airway wall non-uniformly along the bronchial tree. It can also modify airway geometry and sometimes lead to the formation of bottlenecks, a characteristic feature found in terminal bronchioles of chronic obstructive pulmonary disease (COPD) patients (McDonough *et al*., provisionally accepted in the New England Journal of Medicine). The effect of a bottleneck is treacherous because not only it increases the resistance

this baseline heterogeneity, which was heretofore ignored, is now on.

**4.4.4 Factors potentially involved in determining baseline heterogeneity**

of factors responsible for this baseline airway wall heterogeneity is of major interest.

non-ventilated.

Despite abundant evidence implicating the ASM as the culprit of AHR in asthma, the latest genetic studies are relatively weak to support the role of asthma susceptibility gene risk variants in causing ASM dysfunction. The more reliable genetic analyses performed to date have instead suggested that the well-recognized genetic predisposition to suffer from asthma may be related to polymorphisms in genes that are predominately involved in immunology and/or epithelial integrity and functions. Even if we cannot rule out the contribution of genetic alterations affecting the ASM in AHR at the present time, the bulk of evidence rather suggests that if alterations in ASM mechanics contribute to AHR, these alterations are acquired as a result of inflammatory mediators and/or extracellular matrix remodeling that are present in asthmatic lungs. For example, lung defects can foster the appearance of normal ASM behaviors that render the muscle hypercontractile, such as length adaptation, force adaptation and changes in ASM cell's phenotype. These ASM behaviors are the testimony that he contractile properties of the ASM are not fixed but rather plastic (adaptable).

On the other hand, alterations in ASM contractile properties are not necessarily a prerequisite to suffer from AHR. Many lung defects are sufficient alone (i.e., without the need of increased ASM contractility) to cause AHR. These include but are not restricted to: 1-alterations in epithelial integrity, which increase the bioavailability of the inhaled spasmogen onto the ASM; 2-increased ASM-tone (due to the preponderance of inflammation-derived spasmogens, an augmented vagal input and/or decreased expression of relaxant factors), which acts additively with the extrinsically-delivered spasmogen in terms of ASM activation and ASM shortening, and synergistically in terms of narrowing of the cross-sectional area of the lumen and airway resistance; 3-obstruction of the vessels irrigating the ASM, which prevents clearance of the spasmogens into the circulation and concomitantly sequesters the spasmogens in the vicinity of ASM; 4-increased mass of the material inner the ASM layer, which increases airway narrowing for any given degree of ASM shortening; 5-decreased ASM-load (due to adventitial thickening, detachment of the parenchymal tethers from the outer edge of the airway wall, decrease in lung recoil, reduction in lung volume, diminution of the radial constraint limiting ASM bulging during shortening, other changes in ECM constitution which may change the mechanical properties of the airway wall to make it easier to compress by affecting, or not, its pattern of folding), which increases ASM shortening for any given degree of ASM activation; and 6-ventilation heterogeneity with airway closure, which affects flow measurements (such as FEV1, PEF and others) because of air trapping. For all of these reasons, some asthmatics can be hypperresponsive even when their ASM operates as normal. In conclusion, we think that the ASM is often 'blamed' for the AHR seen in asthma simply because of its unequivocal role in airway responsiveness. We would like to propose that without further or more convincing proofs to incriminate ASM in AHR, this obeisant tissue should still be considered 'innocent'.

#### **6. References**

need of increased ASM contractility) to cause AHR. These include but are not restricted to: 1-alterations in epithelial integrity, which increase the bioavailability of the inhaled spasmogen onto the ASM; 2-increased ASM-tone (due to the preponderance of inflammation-derived spasmogens, an augmented vagal input and/or decreased expression of relaxant factors), which acts additively with the extrinsically-delivered spasmogen in terms of ASM activation and ASM shortening, and synergistically in terms of narrowing of the cross-sectional area of the lumen and airway resistance; 3-obstruction of the vessels irrigating the ASM, which prevents clearance of the spasmogens into the circulation and concomitantly sequesters the spasmogens in the vicinity of ASM; 4-increased mass of the material inner the ASM layer, which increases airway narrowing for any given degree of ASM shortening; 5-decreased ASM-load (due to adventitial thickening, detachment of the parenchymal tethers from the outer edge of the airway wall, decrease in lung recoil, reduction in lung volume, diminution of the radial constraint limiting ASM bulging during shortening, other changes in ECM constitution which may change the mechanical properties of the airway wall to make it easier to compress by affecting, or not, its pattern of folding), which increases ASM shortening for any given degree of ASM activation; and 6-ventilation heterogeneity with airway closure, which affects flow measurements (such as FEV1, PEF and others) because of air trapping. For all of these reasons, some asthmatics can be hypperresponsive even when their ASM operates as normal. In conclusion, we think that the ASM is often 'blamed' for the AHR seen in asthma simply because of its unequivocal role in airway responsiveness. We would like to propose that without further or more convincing proofs to incriminate ASM in AHR, this obeisant tissue should still be

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## **Polymerization and Oxidation of Alpha-1-Antitrypsin in Pathogenesis of Emphysema**

Aleksandra Topic1 and Dragica Radojkovic2

*1University of Belgrade, Faculty of Pharmacy, Department of Medical Biochemistry, 2University of Belgrade, Institute of Molecular Genetics and Genetic Enginieering, Belgrade Serbia*

#### **1. Introduction**

54 Lung Diseases – Selected State of the Art Reviews

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The last two decades efforts have been made in investigation of genes that encode proteins involved in pathogenesis of emphysema and chronic obstructive pulmonary disease (COPD). So far, SERPINA1 gene which encodes protein alpha-1-antitrypsin (A1AT) is the only defined genetic risk factor associated with early development of emphysema.

The A1AT is dominant protein of 1 electrophoretic fraction of serum proteins, whose main physiological role is to inhibit neutrophil elastase (NE) in the lower respiratory tract, and protect pulmonary connective tissue from NE released from triggered neutrophiles. Neutrophil elastase is serine protease that degrades elastin of the alveolar walls as well other structural proteins of a variety of tissues.

Hereditary alpha-1-antitrypsin deficiency (A1ATD) is associated with retention of mutant A1AT polymers in hepatocytes which leads to decrease of circulating A1AT with less than 15% of normal level in A1ATD homozygotes. Since the integrity of lung alveoli is maintained by proper circulating level of A1AT, severe deficiency of this protein was identified as genetic risk factor for emphysema and COPD. Clinical manifestation of emphysema in patients with A1ATD occurs in 3th decade in smokers and in the 5th decade in non-smokers (Larsson, 1978; Janus et al., 1985) and requires replacement therapy with purified A1AT pooled from donor plasma.

Genetic epidemiologic studies show that A1ATD may affect 1 in about 1,500 individuals in Europe (De Serres, 2002). Approximately 3.4 million individuals of all racial subgroups are affected by A1ATD worldwide (De Serres, 2002).

Liver disease in early childhood is second clinically significant consequence resulting from retention of mutant A1AT polymers in hepatocytes (Eriksson, 1986; Sveger, 1976). Clinically it is presents as neonatal cholestasis which may progress to juvenile chirosis or slowly progress to the liver disease in adults (Mahadeva and Lomas, 1998).

In the early sixties of the last century, Laurell and Eriksson discovered that the absence of the electrophoretic 1-globulin pattern of serum is associated with A1AT deficiency (Laurell and Eriksson, 1963). At the same time was discovered an association between A1ATD and emphysema in relatively young patients in fourth decade of life (Eriksson, 1964; Lieberman, 1969). These observations suggested a significant role of A1ATD in pathogenesis of emphysema. The proteinase-antiproteinase hypothesis, established by Janoff (Janoff, 1985) still remains central in our understanding of the pathogenesis of lung disease. According to this hypothesis, emphysema in A1ATD arises from an imbalance of neutrophil elastase and A1AT as antielastase, which leads to inappropriate antielastase defense and the relatively excessive activity of neutrophil elastase and consequent degradation of elastin and other extracellular matrix components of the lower respiratory tract.

However, only 1% of patients with COPD are A1ATD (Lieberman, et al., 1986), indicating that A1ATD alone is not sufficient to induce emphysema (Silverman, et al., 1989). The additional factor which may induce emphysema in A1ATD is inflammation, when elastin repair mechanisms are overwhelmed by a massive attack of elastase from triggered neutrophils and cigarette smoke. Studies of the genetic and environmental factors have shown a difference in the reduction of pulmonary function in A1ATD, indicating that additional genetic factors (modifier genes) may influence the pulmonary function in A1ATD subjects (Silverman et al., 1990). Also, the single-nucleotide polymorphisms (SNPs) were identified in the six haplotypes of the SERPINA1 gene, which controls synthesis of A1AT (Chappell, et al., 2006). Several environmental factors that accelerate the onset of symptoms in A1ATD patients, such as personal and second hand exposure to tobacco smoke in childhood, respiratory infections (Mayer et al., 2006), and higher exposures to ozone (Wood et al., 2009) have been also identified.

In addition to the low circulating levels of A1AT in hereditary A1ATD, the risk of emphysema includes reduced antielastase activity. Functional inactivation of A1AT by oxidants present in cigarette smoke could impair antielastase defence in lower respiratory tract, and represent acquired A1AT inactivation. Furthermore, in hereditary A1ATD smoking could impair the function of A1AT both quantitatively and qualitatively.

## **2. Alpha-1-antitrypsin**

Alpha-1-antitrypsine (A1AT) is the archetype of the serpin family of proteins. SERPINs (SER*ine* P*roteinase* IN*hibitors*) are the superfamily of structurally related proteins that control many physiological processes. A1AT is a highly polymorphic, acute-phase glycoprotein, synthesised in hepatocytes (Koj et al., 1978) and subsequently secreted into the plasma. Hepatic synthesis of this acute phase protein by SERPINA1 gene is under control of different cytokines, such as interleukin-1 (IL-1), tumour necrosis factors (TNF) and most effectively the interleukin-6 family of cytokines (interleukin-6, leukaemia inhibitory factor, oncostatin M) (Richards and Gauldie, 1991).  Extra hepatic synthesis of A1AT is important in preventing tissue damage in the site of inflammation or injury. For instance, synthesis of A1AT in monocytes is up-regulated by inflammatory mediators such as IL-1 and TNF in lung tissue (Knoell et al., 1998). Serum level of A1AT is elevated in inflammation, trauma, and pregnancy.

Healthy individuals produce 34 mg of A1AT per kilogram of body weight per day (Jones, 1978). Normal reference interval for antigenic concentration of serum A1AT measured by nephelometry is 15-40 M (0.83-2.20 g/L) (ATS/ERS Statement, 2003). The threshold level of 11 M (0.59 g/L) provides relevant antielastase protection of lower respiratory tract (WHO Meeting, 1996.). As a relatively small protein (52 kD), the mature A1AT is capable to diffuse into many organs. The concentration of A1AT in organs is lower than in plasma. Thus, Olsen et al. (Olsen et al., 1975) reported that A1AT level in a bronchoalveolar lavage fluid of non-smokers is 7% of serum level, with a higher value (11%) in smokers. Also, total amount of A1AT in the lavage fluid of smokers was significantly greater than in non-smokers. The same authors reported about five times higher A1AT concentration in pulmonary alveolar macrophages in smokers in comparison to non-smokers. All these results suggest an increased A1AT concentration in the air spaces of the cigarette smokers.

#### **2.1 Structure and function of alpha-1-antitrypsin**

56 Lung Diseases – Selected State of the Art Reviews

1969). These observations suggested a significant role of A1ATD in pathogenesis of emphysema. The proteinase-antiproteinase hypothesis, established by Janoff (Janoff, 1985) still remains central in our understanding of the pathogenesis of lung disease. According to this hypothesis, emphysema in A1ATD arises from an imbalance of neutrophil elastase and A1AT as antielastase, which leads to inappropriate antielastase defense and the relatively excessive activity of neutrophil elastase and consequent degradation of elastin and other

However, only 1% of patients with COPD are A1ATD (Lieberman, et al., 1986), indicating that A1ATD alone is not sufficient to induce emphysema (Silverman, et al., 1989). The additional factor which may induce emphysema in A1ATD is inflammation, when elastin repair mechanisms are overwhelmed by a massive attack of elastase from triggered neutrophils and cigarette smoke. Studies of the genetic and environmental factors have shown a difference in the reduction of pulmonary function in A1ATD, indicating that additional genetic factors (modifier genes) may influence the pulmonary function in A1ATD subjects (Silverman et al., 1990). Also, the single-nucleotide polymorphisms (SNPs) were identified in the six haplotypes of the SERPINA1 gene, which controls synthesis of A1AT (Chappell, et al., 2006). Several environmental factors that accelerate the onset of symptoms in A1ATD patients, such as personal and second hand exposure to tobacco smoke in childhood, respiratory infections (Mayer et al., 2006), and higher exposures to ozone (Wood

In addition to the low circulating levels of A1AT in hereditary A1ATD, the risk of emphysema includes reduced antielastase activity. Functional inactivation of A1AT by oxidants present in cigarette smoke could impair antielastase defence in lower respiratory tract, and represent acquired A1AT inactivation. Furthermore, in hereditary A1ATD

Alpha-1-antitrypsine (A1AT) is the archetype of the serpin family of proteins. SERPINs (SER*ine* P*roteinase* IN*hibitors*) are the superfamily of structurally related proteins that control many physiological processes. A1AT is a highly polymorphic, acute-phase glycoprotein, synthesised in hepatocytes (Koj et al., 1978) and subsequently secreted into the plasma. Hepatic synthesis of this acute phase protein by SERPINA1 gene is under control of different cytokines, such as interleukin-1 (IL-1), tumour necrosis factors (TNF) and most effectively the interleukin-6 family of cytokines (interleukin-6, leukaemia inhibitory factor, oncostatin M) (Richards and Gauldie, 1991).  Extra hepatic synthesis of A1AT is important in preventing tissue damage in the site of inflammation or injury. For instance, synthesis of A1AT in monocytes is up-regulated by inflammatory mediators such as IL-1 and TNF in lung tissue (Knoell et al., 1998). Serum

Healthy individuals produce 34 mg of A1AT per kilogram of body weight per day (Jones, 1978). Normal reference interval for antigenic concentration of serum A1AT measured by nephelometry is 15-40 M (0.83-2.20 g/L) (ATS/ERS Statement, 2003). The threshold level of 11 M (0.59 g/L) provides relevant antielastase protection of lower respiratory tract (WHO Meeting, 1996.). As a relatively small protein (52 kD), the mature A1AT is capable to diffuse into many organs. The concentration of A1AT in organs is lower than in plasma. Thus,

smoking could impair the function of A1AT both quantitatively and qualitatively.

level of A1AT is elevated in inflammation, trauma, and pregnancy.

extracellular matrix components of the lower respiratory tract.

et al., 2009) have been also identified.

**2. Alpha-1-antitrypsin**

The mature A1AT protein is a single chain composed of 394 amino acids. The main characteristics of the protein are: Met358 residue at the active site, isoelectric point ranging from 4.4 to 4.6, and a total molecular weight of 52 kDa. Crystalographic analysis of the mature protein reveals that A1AT is a globular protein with tree N asparigynil-linked carbohydrate side chains on the external surface of the one end of the molecule (Loebermann et al., 1984). The side chains are composed of N-acetylglukosamine, mannose, galactose and sialic acid and they are N-linked to amino acids Asn46, Asn83 and Asn247. These carbohidrate side chains are on the outside surface of one-half of the elongated structure. The difference in carbohydrate side chains at position of Asn83 is responsible for the two major bands of A1AT when serum focused at pH 4-4.9 on thin-layer polyacrilamide gel. The internal structure of A1AT is highly ordered with 30 percent -helices and 40 percent -pleated sheets. There are nine -helices (AI) and three -sheets (AI).

Similar to other inhibitory serpins, A1AT is "suicide" or "single use" inhibitor that employs a unique and extensive conformational change in the process of inhibition of target proteases (Figure 1.). The hallmark of serpins is the reactive centre loop (RCL) that presents the key P1-P'1 methionine–serine bond as a pseudosubstrate for the cognate proteinase, neutrophil elastase (Johnson and Travis, 1978). The reactive centre loop of A1AT is highly stressed external loop protruding from the molecule with Met358-Ser359 in the active center. Inhibitory process begins by docking of the serpin and the protease, and formation of Michaelis complex. Like the other inhibitory serpins, the structure of the RCL is crucial for the ability of the inhibitor to undergo a "stressed to relaxed" (S→R) conformational change. The active A1AT is in metastable or "stressed form", which is essential for inhibition of proteases. During the process of inhibition, A1AT is like mousetrap with spring-like shift from a metastable to a hyperstable state (Hunington et al., 2000; Carrell and Lomas, 2002). After the formation of Michaelis complex there are two possible different ending of the reaction. One is inactivation of protease, where serpin has undergone the S → R transition, and the protease hangs distorted at the base of the molecule. The other possibility is A1AT substrate-like behavior, where RCL forms the fourth -sheet, providing the opportunity for the protease to escape the conformational trap, leaving active protease and inactive cleaved serpin. Thus, *in vivo*, A1AT can exist in: native inhibitory conformation with an exposed RCL, latent conformation with a partially inserted RCL and non-inhibitory conformation.

Non-inhibitory conformation of A1AT occurs in certain circumstances: when A1AT is in complex with neutrophil elastase, when the reactive center loop of A1AT is cleaved by nontarget proteinases, when reactive oxygen species oxidized A1AT, and when A1ATD variants form polymers.

Moreover, A1AT non-inhibitory conformations show other biological effects. For instance, oxidized A1AT and the cleaved peptide fragment of A1AT stimulate monocyte activation, and A1AT-elastase complexes and polymeric A1AT are chemotactic for neutrophils (Banda et al., 1988; Dabbagh et al., 2001; Moraga and Janciauskiene, 2000; Moraga et al., 2001).

Fig. 1. Structure of alpha-1antitrypsin and reaction with target protease (modified from Law, et al., 2006)

#### **2.2 Physiological roles of alpha-1-antitrypsin**

The main physiological role of A1AT is protection of lower respiratory tract by inhibiting proteases released from triggered neutrophiles including neutrophil elastase, cathepsin G, and proteinase-3 (Carrell, 1986). The target proteases of A1AT derive from azurophilic granules of polymorphonuclear neutrophils which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at the sites of inflammation. This protective role of A1AT occurs primarily extracellular. A1AT enters the lung from the circulation by passive diffusion (Stockley, 1984). Besides direct inhibition of NE, there are evidences that A1AT exhibits anti-inflammatory properties to suppress cigarette smoke induced production of tumor necrosis factor (TNF) and matrix metalloproteinase 12 (MMP12) by alveolar macrophages, and subsequent inflammatory cell infiltration (Churg et al., 2007). Furthermore, studies have shown that native A1AT modulates function of immune cells, such as neutrophils (Bergin et al., 2010), monocytes (Janciauskiene et al., 2007), and T cells (Lu et al., 2006). *Ex vivo* and *in vitro* experiments have shown that endogenous A1AT in blood contributes to the suppression of proinflammatory cytokine synthesis (Pott et al., 2009). Thus, A1AT is an endogenous inhibitor of proinflammatory cytokine production in whole blood, and may participate in innate immune response to an inflammation-inducing stimulus. The recently discovered role of A1AT in prevention of emphysema is the inhibition of lung endothelial cell apoptosis due to inactivation of intracellular caspase-3 (Petrache et al., 2006a; Petrache et al., 2006b). Antiapoptotic role of A1AT in the lung *in vivo* and *in vitro* in micro vascular endothelial cells is associated with intracellular presence of A1AT. Lung endothelial cells don't produce A1AT and they take it. Sohrab et al. (Sohrab et al., 2009) showed that clathrin-mediated endocytosis predominantly regulates A1AT intracellular function in the lung endothelium, and might represent an important determinant of the serpin's protection against development of cigarette smoke-induced emphysema. The uptake is severely affected by exposure to cigarette smoke extract *in vitro* and *in vivo*, probably directly influencing clathrin-mediated endocytosis. Furthermore, polymers of A1AT exhibit a marked decrease in lung endothelial cell uptake. Inhibition of A1AT uptake by cigarette smoke may further weaken the A1AT protective role in the lung.

In the last decade other physiological roles of A1AT have been discovered, such as roles in atherogenesis (Talmud et al., 2003), angiogenesis (Huang et al., 2004), fibroblast proliferation, and procollagen synthesis (Dabbagh et al., 2001).

#### **2.3 Genetics of alpha-1-antitrypsin**

58 Lung Diseases – Selected State of the Art Reviews

Fig. 1. Structure of alpha-1antitrypsin and reaction with target protease (modified from Law,

The main physiological role of A1AT is protection of lower respiratory tract by inhibiting proteases released from triggered neutrophiles including neutrophil elastase, cathepsin G, and proteinase-3 (Carrell, 1986). The target proteases of A1AT derive from azurophilic granules of polymorphonuclear neutrophils which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at the sites of inflammation. This protective role of A1AT occurs primarily extracellular. A1AT enters the lung from the circulation by passive diffusion (Stockley, 1984). Besides direct inhibition of NE, there are evidences that A1AT exhibits anti-inflammatory properties to suppress cigarette smoke induced production of tumor necrosis factor (TNF) and matrix metalloproteinase 12 (MMP12) by alveolar macrophages, and subsequent inflammatory cell infiltration (Churg et al., 2007). Furthermore, studies have shown that native A1AT modulates function of immune cells, such as neutrophils (Bergin et al., 2010), monocytes (Janciauskiene et al., 2007), and T cells (Lu et al., 2006). *Ex vivo* and *in vitro* experiments have shown that endogenous A1AT in blood contributes to the suppression of proinflammatory cytokine synthesis (Pott et al., 2009). Thus, A1AT is an endogenous inhibitor of proinflammatory cytokine production in whole blood, and may participate in innate immune response to an inflammation-inducing stimulus. The recently discovered role of A1AT in prevention of emphysema is the inhibition of lung endothelial cell apoptosis due to inactivation of intracellular caspase-3 (Petrache et al., 2006a; Petrache et al., 2006b). Antiapoptotic role of A1AT in the lung *in vivo* and *in vitro* in micro vascular endothelial cells is associated with intracellular presence of A1AT. Lung endothelial cells don't produce

et al., 2006)

**2.2 Physiological roles of alpha-1-antitrypsin**

The alpha-1-antitrypsin is encoded by SERPINA1 gene (serpin peptidase inhibitor, clade A) located in proteinase inhibitor (Pi) locus on the long arm of chromosome 14q32.1 (Schroeder et al., 1985; Billingsley et al., 1993). The Pi locus is 12.2 kb long and consists of 4 coding exons, 3 non-coding exons and 6 introns (Figure 2.). At the 5 region of the SERPINA1 gene there are three non-protein coding exons (IA, IB, IC) which control gene transcription. Exons referred as exons II-V are coding and containing the sequence information that defines the protein itself. The start codon (ATG) for translational of the mRNA and the signal peptide are in exon II, and the stop codon (TAA) is in exon V, followed by the polyadenilatyon signal (ATTAA). The carbohydrate attachmnent site (Asn46, Asn83, Asn247) are coded for in exon II and III. The region coding for the reactive loop with the active inhibitory centre Met358 is within exon V.

Following transcription, A1AT mRNA is translated on ribosome bound to the the rough endoplasmatic reticulum, producing a preprotein of 418 amino acids. The signal peptide of 24 residues is removed during secretion into the cisterne of the rough endoplasmatic reticulum where the protein is glycosylated with high-mannose type carbohydrates, and folds into appropriate globular tree-dimensional configuration. Complete protein maturation is accomplished within the Golgi apparatus and protein is secreted.

Fig. 2. Structure of the SERPINA1 gene (Crystal et al., 1989)

Hepatocytes and monocytes have two different promotors (Perlino et al., 1987) that operate via different mechanisms. The SERPINA1 gene in macrophages is transcribed from a macrophags-specific promoter located about 2,000 bp upstream of the hepatocyte-specific promoter. Transcription from the two SERPINA1 promoters is mutually exclusive; the macrophage promoter is silent in hepatocytes, and the hepatocyte promoter is silent in macrophages. In macrophages, two distinct mRNAs are generated by alternative splicing. In addition, Hafeez et al. (Hafeez et al., 1992) demonstrated that the SERPINA1 gene has 3 monocyte-specific transcriptional initiation sites upstream from a single hepatocyte-specific transcriptional initiation site. Macrophages use these sites during basal and modulated expression. Hepatoma cells use the hepatocyte-specific transcriptional initiation site during basal and modulated expression, but also switch to transcription from the upstream macrophage transcriptional initiation sites during modulation by the acute phase mediator interleukin-6 (IL-6).

#### **2.4 Polymorphism of alpha-1-antitrypsin**

The A1AT coding gene SERPINA1 is a highly polimorphic, with more than 125 SNPs reported in public SNP databases (Entrez SNP). Protein variants of A1AT are classified by the Pi (P*rotese* i*nhibitor*) system and each variant is identified by migration on agarose gel electrophoresis. These differences in migration relate to variations in protein charge resulting from amino acid alterations (Fagerhol and Laurell, 1970; Cox, 1978). Isoelectric focusing in the narrow range of pH (4.2-4.9) has enabled identification of more A1AT variants then in agarose gel electrophoresis. The alleles were given symbols according to the relative electrophoretic mobility of the allele product, so anodal variants are marked with the first letters, and cathodal with last letters. All A1AT variants are categorized according to the serum level and functional activity as normal, deficient, null and dysfunctional.

#### **2.4.1 Normal A1AT variants**

Normal A1AT variants have normal serum level and functional activity to inhibit neutrophil elastase. More than 95% of normal variants are the "common" M1 (Ala213), M1 (Val213), M2 and M3. Among Caucasians, M1 (Val213) is the most common, and M1 (Ala213), M2 and M3 are less frequent. The "rare" normal variants with frequencies less than 1% are: M4, Balhambra, F, PStAlbans and XChirstchurch. Usually, rare variants are named by the birthplace of the oldest individual tested in pedegree. PiM homozygotes and heterozygotes are characterized by normal serum level of A1AT (20-50 M) and normal functional activity (ATS/ERS Statement, 2003).

#### **2.4.2 Deficient A1AT variants**

Deficient variants are associated with lower serum level of A1AT than normal variants. Several mutations associated with A1ATD have been identified, and the most common are Z and S alleles. Rare A1ATD variants are MMalton, MMineralSprings, MNichinan, MProcida, PLowell, SIiyama and others.

Gene-mapping studies have shown that the PiZ allele probably arose in Northern Europe (Cox et al., 1985). Age estimates of A1AT variants based on microsatellite variation, suggest that the Z deficiency allele appeared 107 to 135 generations ago and could have been spread in neolithic times. Frequency of the Z allele shows a large variation in Caucasians, but is rare or absent in Asians and Africans (De Croo et al., 1991; Hutchison, 1998.).

The PiS deficiency allele has an older 279-generation to 470-generation age and from its high incidence on the Iberia peninsula it has been suggested that it could have originated in this region (Seixas et al., 2001).

#### **<IP_ADDRESS> Z variant**

60 Lung Diseases – Selected State of the Art Reviews

Hepatocytes and monocytes have two different promotors (Perlino et al., 1987) that operate via different mechanisms. The SERPINA1 gene in macrophages is transcribed from a macrophags-specific promoter located about 2,000 bp upstream of the hepatocyte-specific promoter. Transcription from the two SERPINA1 promoters is mutually exclusive; the macrophage promoter is silent in hepatocytes, and the hepatocyte promoter is silent in macrophages. In macrophages, two distinct mRNAs are generated by alternative splicing. In addition, Hafeez et al. (Hafeez et al., 1992) demonstrated that the SERPINA1 gene has 3 monocyte-specific transcriptional initiation sites upstream from a single hepatocyte-specific transcriptional initiation site. Macrophages use these sites during basal and modulated expression. Hepatoma cells use the hepatocyte-specific transcriptional initiation site during basal and modulated expression, but also switch to transcription from the upstream macrophage transcriptional initiation sites during modulation by the acute phase mediator

The A1AT coding gene SERPINA1 is a highly polimorphic, with more than 125 SNPs reported in public SNP databases (Entrez SNP). Protein variants of A1AT are classified by the Pi (P*rotese* i*nhibitor*) system and each variant is identified by migration on agarose gel electrophoresis. These differences in migration relate to variations in protein charge resulting from amino acid alterations (Fagerhol and Laurell, 1970; Cox, 1978). Isoelectric focusing in the narrow range of pH (4.2-4.9) has enabled identification of more A1AT variants then in agarose gel electrophoresis. The alleles were given symbols according to the relative electrophoretic mobility of the allele product, so anodal variants are marked with the first letters, and cathodal with last letters. All A1AT variants are categorized according to the serum level and functional activity as normal, deficient, null and dysfunctional.

Normal A1AT variants have normal serum level and functional activity to inhibit neutrophil elastase. More than 95% of normal variants are the "common" M1 (Ala213), M1 (Val213), M2 and M3. Among Caucasians, M1 (Val213) is the most common, and M1 (Ala213), M2 and M3 are less frequent. The "rare" normal variants with frequencies less than 1% are: M4, Balhambra, F, PStAlbans and XChirstchurch. Usually, rare variants are named by the birthplace of the oldest individual tested in pedegree. PiM homozygotes and heterozygotes are characterized by normal serum level of A1AT (20-50 M) and normal functional activity (ATS/ERS

Deficient variants are associated with lower serum level of A1AT than normal variants. Several mutations associated with A1ATD have been identified, and the most common are Z and S alleles. Rare A1ATD variants are MMalton, MMineralSprings, MNichinan, MProcida, PLowell, SIiyama

Gene-mapping studies have shown that the PiZ allele probably arose in Northern Europe (Cox et al., 1985). Age estimates of A1AT variants based on microsatellite variation, suggest that the Z deficiency allele appeared 107 to 135 generations ago and could have been spread in neolithic times. Frequency of the Z allele shows a large variation in Caucasians, but is rare

or absent in Asians and Africans (De Croo et al., 1991; Hutchison, 1998.).

interleukin-6 (IL-6).

**2.4.1 Normal A1AT variants**

**2.4.2 Deficient A1AT variants**

Statement, 2003).

and others.

**2.4 Polymorphism of alpha-1-antitrypsin**

The Z variant represents a "classic"A1ATD variant and derives from M1 (Ala213) following a point mutation which is the same for the all Z individuals who are probably descendants of a single ancestral progenitor. The result of mutation is the substitution of a *G*AG that codes for Glu342 with an *A*AG that code for Lys342 (Nukiwa et al., 1987). The Z mutation perturbs the folding (Yu et al., 1995.) and structure of the protein (Lomas et al., 1993a). It distorts the relationship between the reactive centre loop and -sheet A, and the consequent perturbation in structure allows opening of -sheet A to favor partial loop insertion and formation of an unstable intermediate (M\*) (Figure 3.). Then the patent -sheet A accepts the loop of another A1AT molecule to form a dimer (D) which then extends to form chains of loop-sheet polymer (P) (Lomas, et al., 1992; Elliott et al., 1996a; Lomas, 2000).

Fig. 3. Formation of Z polymers (Lomas, 2005)

Process of polymerization depends on concentration and temperature. Abnormality in posttranslational modification of protein causes accumulation of the A1AT polymers in the cisterna of the rough endoplsmatic reticulum with a drastic reduction in secretion rates. The abnormal protein accumulates in hepatocytes and forms inclusion bodies (aggregates) that are positive to diastase-resistant periodic acid Schiff (PAS-D) staining and visible on microscopy. The retained A1AT polymers are cytotoxic for hepatocytes and can cause a diverse liver damages, ranging from neonatal hepatitis to juvenile cirrhosis, and hepatocellular carcinoma in adults (Eriksson, et al., 1986). As a consequence of polymer accumulation, hepatocytes of PiZZ homozygote secrete only 10–15 % of normal quantity. PiMZ heterozygotes have about 50 % of normal A1AT circulating level.

Besides low circulation level, the Z protein also less efficiently inhibits elastase (Ogushi, et al., 1987). Consequently, in PiZZ individuals, quantitative and qualitative defects of A1AT lead to early-onset COPD including emphysema and chronic bronchitis.

A similar form of loop-sheet polymers *in vivo* with hepatic inclusions and plasma deficiency was found in two other variants, MMalton (Phe52 deleted) (Lomas et al., 1995) and SIiyama (Ser53→Phe) (Lomas et al., 1993b.), that are common in Sardinia and Japan, respectively.

A1AT polymers were also detected in bronchoalveolar lavage fluid (BALF) from PiZ homozygotes with emphysema. This conformational transition may further reduce the levels of functional proteinase inhibitor in the lungs, and consequently exacerbate lung tissue damage (Elliott et al., 1998).

Studies of Parmar et al. (Parmar et al., 2002) and Mulgrew et al. (Mulgrew et al., 2004) showed that Z A1AT locally produced on the epithelial surface of the lung polymerizes and A1AT polymers demonstrate proapoptotic and proinflammatory effects. These studies also revealed that unlike M A1AT protein, Z A1AT protein polymerized at body temperature, and in addition of being an ineffective antiprotease inhibitor, might become a strong neutrophil chemoattractant, thus representing an ongoing source of inflammation in the lungs of individuals with A1ATD. Thus, polymerization of locally produced ZA1AT is a contributory factor to the lung inflammation experienced by those with A1AT deficiency and that standard antiprotease therapies may not address this problem. Other studies reported that even PiZZ patients with near-normal lung function had high concentrations of neutrophils on respiratory epithelial surfaces (Rouhani et al., 2000). Neutrophil burden in PiZZ and in PiMZ (on a lesser extent) is attributed to leukotriene B4 or IL-8 released from neutrophils or epithelial cells (Woolhouse et al., 2002; Malerba et al., 2006). Neutrophil accumulation in the lung of PiZZ deficient individuals is multifactorial and chemoattraction due to polymerized Z protein represents another potential cause of neutrophil dominated inflammation. These findings suggest a novel mechanism in pathogenesis of emphysema associated with Z antitrypsin deficiency.

Lomas (Lomas, 2006) highlighted a possible role of Z mutant in systemic response to infection. In the case of invasion of pathogens, organism initiates a systemic inflammatory response that results in increased secretion of Z A1AT as acute phase protein, by hepatocytes. Factors such as: elevation of body temperature, increased concentration of mutant Z A1AT, and lower pH at the site of bacterial invasion of lung (Stockley and Burnett, 1979) favor polymerization of mutant Z A1AT. Polymers possess chemotactic properties, which in turn amplify inflammatory response and enhance the recruitment of neutrophils. Excessive burden of neutrophils may cause increase of proinflamatory and proxidative factors.

Therefore, a rational approach in therapy of A1ATD would be to inhibit the polymerization of the Z protein (intracellularly and extracellularly), accompanied by standard augmentation therapy. It is clear that in addition to increased level of A1AT above a putative therapeutic threshold, it is necessary to increase the secretion of active nonpolymerized form of Z protein. This approach could potentially ameliorate the liver disease, and defend respiratory epithelial surface, providing antielastase protection and avoiding the proinflammatory effects of polymerized Z A1AT.

Currently, there is some progress in development of synthetic peptide designed to selectively inhibit Z polymerisation (Mahadeva et al., 2002; Parfrey et al., 2004; Chang et al., 2006; Mallya et al., 2007; Chang et al., 2009).

#### **<IP_ADDRESS> S variant**

In contrast to the Z allele, S causes only mild plasma deficiency. The genetic sequence of the S variant derives from M1 (Val213) as a result of a mutation which cause substitution of G*A*A that codes for Glu264 with an G*T*A that codes for Val264 (Owen et al., 1976; Yoshida et al., 1977).

The single mutation of the S variant leads to spontaneous polymer formation, but slower than the Z variant, without affecting the ability to inhibit neutrophil elastase (Elliott, et al*.,*

1996b; Mahadeva et al., 1999; Dafforn et al., 1999). The slower polymerization causes less retention of S variant in the liver, and hence the plasma levels are 60% of the normal M allele. Thus, carriers of S allele (PiSS, PiSZ and PiMS) have levels of 52%, 32% and 75% of normal level, respectively. Furthermore, Z A1AT forms heteropolymers with S A1AT (Mahadeva et al., 1999), which explains cases of hepatic cirrhosis in PiSZ patients (Cruz et al., 1975; Campra et al., 1973; Craig et al., 1975.). Also, PiSZ smokers are at significant risk of the development of COPD, while in nonsmoking individuals the PiSZ phenotype may confer little or no risk to develop COPD (Turino et al., 1996).

#### **<IP_ADDRESS> Null A1AT variants**

62 Lung Diseases – Selected State of the Art Reviews

A1AT polymers were also detected in bronchoalveolar lavage fluid (BALF) from PiZ homozygotes with emphysema. This conformational transition may further reduce the levels of functional proteinase inhibitor in the lungs, and consequently exacerbate lung

Studies of Parmar et al. (Parmar et al., 2002) and Mulgrew et al. (Mulgrew et al., 2004) showed that Z A1AT locally produced on the epithelial surface of the lung polymerizes and A1AT polymers demonstrate proapoptotic and proinflammatory effects. These studies also revealed that unlike M A1AT protein, Z A1AT protein polymerized at body temperature, and in addition of being an ineffective antiprotease inhibitor, might become a strong neutrophil chemoattractant, thus representing an ongoing source of inflammation in the lungs of individuals with A1ATD. Thus, polymerization of locally produced ZA1AT is a contributory factor to the lung inflammation experienced by those with A1AT deficiency and that standard antiprotease therapies may not address this problem. Other studies reported that even PiZZ patients with near-normal lung function had high concentrations of neutrophils on respiratory epithelial surfaces (Rouhani et al., 2000). Neutrophil burden in PiZZ and in PiMZ (on a lesser extent) is attributed to leukotriene B4 or IL-8 released from neutrophils or epithelial cells (Woolhouse et al., 2002; Malerba et al., 2006). Neutrophil accumulation in the lung of PiZZ deficient individuals is multifactorial and chemoattraction due to polymerized Z protein represents another potential cause of neutrophil dominated inflammation. These findings suggest a novel mechanism in pathogenesis of emphysema