**5.2 Animal models of COPD**

Animal models of COPD provide the strongest evidence for the roles of proteinases in COPD.

#### *Acute cigarette smoke exposure models*

Exposing mice to smoke for up to 30 days leads to an influx of PMN and macrophages to the lung (68). This is due to direct effects of inhaled smoke on lung capillaries, leading to leakage of thrombin and plasmin into the alveolar space (69,70). These proteinases cleave and activate PAR-1 on macrophages, leading to an increased synthesis of MMP-12 by macrophages (13,14). MMP-12 is responsible for shedding pro-TNF-α from activated macrophages, likely leading to an increase in E-selectin expression on endothelial cells (39). This facilitates transendothelial migration of PMNs. The presence of these increased PMNs and macrophages, releasing serine proteinases, increases lung collagen and elastin breakdown. Delivering human AAT to mice acutely exposed to cigarette smoke prevents PMN influx and ECM destruction. This is probably due to AAT inhibiting both PMN serine proteinase-mediated ECM destruction and thrombin- or plasmin-induced increases in macrophage MMP-12 production (14,71). Further evidence for the role of MMP-12 comes from a study showing that the minor allele of a single-nucleotide polymorphism (SNP) in MMP-12, is associated with a positive effect on lung function in adults who smoke and also a reduced risk of COPD in adult smokers (72).

Diverse Activities for Proteinases in the

*Alveolar septal cell apoptosis models of airspace enlargement* 

**6. Regulation of proteinases in the lung** 

Studies of the NE/

**6.1 Inhibitors of proteinases** 

Pathogenesis of Chronic Obstructive Pulmonary Disease 55

In patients with COPD there is apoptosis of alveolar septal cells (87,88) and leukocytes (89,90), and apoptosis of the endothelial and epithelial cells that make up the alveolar walls. This leads to the development of emphysema. Septal cell apoptosis and airspace enlargement in the absence of overt lung inflammation can be induced rapidly in experimental animals by: 1) pharmacologic blockade of vascular endothelial growth factorreceptors in rodents (91); and 2) transfection of murine alveolar epithelial cells with caspase-3, a pro-apoptotic cysteine proteinase (88). However, increased elastase activity due to acidic proteinases is detected in BAL samples after transfectionof alveolar epithelial cells with caspase-3 (88). Thus, proteinases released from dying structural cells may degrade the lung ECM, thereby acting together with

Proteinases are a significant factor in the pathogenesis of COPD, but do not act in isolation. They interact with other mediators and other pathways and are also regulated by inhibitors.

demonstrated interactions between these two classes of proteinases, with MMP-12 cleaving and inactivating AAT to increase NE-mediated lung injury, and NE cleaving and inactivating TIMP-1 to amplify MMP-12-mediated lung destruction (78). Proteinases also interact with reactive oxygen species (ROS), and ROS production is increased in the lungs of COPD patients. ROS are present in inhaled cigarette smoke itself, or are released by phagocytes activated by inhaled smoke. ROS are known to activate proMMPs *in vitro* and are thought to exacerbate lung inflammation and injury in COPD patients (92). Transgenic mice over-expressing the antioxidant enzyme Cu-Zn superoxide dismutase in the lung are protected from developing chronic lung inflammation, increased lung MMP levels, and emphysema in response to intratracheal instillation of porcine pancreatic elastase, or chronic exposure to cigarette smoke (93). However, mice deficient in a phagocyte-specific

component of the NADPH oxidase, which generates superoxide anions (O2-

greater airspace enlargement in response to cigarette smoke than WT mice (94). This is due to ROS-mediated inactivation of MMPs via oxidative inactivation of residues in the catalytic domain of MMPs (95). Thus, phagocyte-derived O2- (and ROS derived from O2-) in COPD lungs may constrain rather than promote phagocyte MMP-mediated lung injury (94,96). It is noteworthy that clinical trials have failed to demonstrate protective effects of antioxidant supplementation in COPD patients, and this could be linked, in part, to antioxidants

Proteinase inhibitors are present in the extracellular matrix. To maintain their action, proteinases need to circumvent these inhibitors through inactivation of the proteinase

Serpins can be cleaved and inactivated by MMPs (98-102), NE (103,104), cathepsin B (105), and bacterial proteinases (106). Serine proteinases cleave and inactivate TIMPs (107).

mice chronically exposed to cigarette smoke

), develop

septal cell apoptosis to cause loss of alveolar units and airspace enlargement.

and MMP-12/

inducing reductions in ROS-mediated inactivation of MMPs (97).

inhibitor, evading them and / or overwhelming them.

**6.2 Inactivation of proteinase inhibitors** 

#### *Chronic smoke exposure models*

When wild type (WT) mice are exposed to cigarette smoke for 3-6 months they develop pulmonary changes of airspace enlargement, inflammation and small airway subepithelial fibrosis, making this a good model to investigate the role of proteinases in COPD (73,74).

Work with proteinase deficient mice has confirmed the role of MMP-12 and NE in chronic inflammation and airspace enlargement and MMP-9 and possibly MMP-12 in sub-epithelial fibrosis. MMP-12 deficient mice (MMP-12-/-mice) when chronically exposed to cigarette smoke show no increase in macrophages and no airspace enlargement, and so are completely protected from the changes seen in the wild type model (73). In the absence of MMP-12 mediated elastin degradation, the remaining elastin fragments attract monocytes (75). T lymphocytes also play a role in these processes with CD8+ T-cell-deficient (CD8-/-) showing a blunted response to smoke exposure and protection from emphysema (76). This is mediated by a CD8+ T cell product, interferon gamma (IFN-) inducible protein 10 (IP-10), which induces production of MMP-12 and degradation of the lung ECM. Carrying this through to the human disease, there has been demonstration of increased Th1 cells associated with increased levels of IP-10 and MMP-12 in lung tissue from human COPD patients (77).

NE-/- mice are 60% protected from airspace enlargement and have decreased influx of PMN and monocytes into the lung compared to smoke-exposed WT mice [(78); Fig. 3]. NE likely contributes to airspace enlargement directly by degrading elastin and other ECM protein components of the alveolar walls (78).

There is also a direct action of cigarette smoke on the pulmonary airways. When rodent airways are exposed acutely to cigarette smoke, increases in growth factor and collagen production are detectable within 2 hours, and before inflammation occurs in the airway walls (79). This suggests that smoke directly promotes small airway subepithelial fibrosis and that smoke-induced inflammation and proteinase production are unnecessary for this process. However, in guinea pigs chronically exposed to cigarette smoke for up to 6 months, inflammatory cell MMPs amplify this process, since delivering a synthetic dual inhibitor of MMPs-9 and -12 to these animals significantly reduces small airway fibrosis (80). The use of MMP inhibitors in human COPD patients remains to be explored.

#### *Transgenic murine models*

These models are used to investigate over-expression of various proteinases, in contrast to the study of a deficiency of a protein in the knock-out murine models. Transgenic mice overexpressing MMP-1 in the lung develop enlarged airspaces (81), which may either reflect abnormal alveolar development or destruction of mature interstitial collagens by MMP-1. Adult transgenic mice over-expressing a Th1 cytokine (IFN-), a Th2 cytokine (IL-13), or a cytokine with Th1 and Th2 activities (IL-18) in airway epithelial cells spontaneously develop obvious lung inflammation, increased lung levels of MMPs and cysteine proteinases, and airspace enlargement (82-84). In mice over-expressing IL-13, the metalloproteinases MMPs - 9 and -12 play critical roles in promoting airspace enlargement, with MMP-12 also promoting inflammation and driving the increased expression of other MMPs in the lung (85). In transgenic mice over-expressing IFN-, cathepsin S stimulates lung epithelial apoptosis, lung inflammation, and airspace enlargement (86).

When wild type (WT) mice are exposed to cigarette smoke for 3-6 months they develop pulmonary changes of airspace enlargement, inflammation and small airway subepithelial fibrosis, making this a good model to investigate the role of proteinases in COPD (73,74).

Work with proteinase deficient mice has confirmed the role of MMP-12 and NE in chronic inflammation and airspace enlargement and MMP-9 and possibly MMP-12 in sub-epithelial fibrosis. MMP-12 deficient mice (MMP-12-/-mice) when chronically exposed to cigarette smoke show no increase in macrophages and no airspace enlargement, and so are completely protected from the changes seen in the wild type model (73). In the absence of MMP-12 mediated elastin degradation, the remaining elastin fragments attract monocytes (75). T lymphocytes also play a role in these processes with CD8+ T-cell-deficient (CD8-/-) showing a blunted response to smoke exposure and protection from emphysema (76). This is mediated by a CD8+ T cell product, interferon gamma (IFN-) inducible protein 10 (IP-10), which induces production of MMP-12 and degradation of the lung ECM. Carrying this through to the human disease, there has been demonstration of increased Th1 cells associated with increased levels of IP-10 and MMP-12 in lung tissue from human COPD

NE-/- mice are 60% protected from airspace enlargement and have decreased influx of PMN and monocytes into the lung compared to smoke-exposed WT mice [(78); Fig. 3]. NE likely contributes to airspace enlargement directly by degrading elastin and other ECM protein

There is also a direct action of cigarette smoke on the pulmonary airways. When rodent airways are exposed acutely to cigarette smoke, increases in growth factor and collagen production are detectable within 2 hours, and before inflammation occurs in the airway walls (79). This suggests that smoke directly promotes small airway subepithelial fibrosis and that smoke-induced inflammation and proteinase production are unnecessary for this process. However, in guinea pigs chronically exposed to cigarette smoke for up to 6 months, inflammatory cell MMPs amplify this process, since delivering a synthetic dual inhibitor of MMPs-9 and -12 to these animals significantly reduces small airway fibrosis (80). The use of

These models are used to investigate over-expression of various proteinases, in contrast to the study of a deficiency of a protein in the knock-out murine models. Transgenic mice overexpressing MMP-1 in the lung develop enlarged airspaces (81), which may either reflect abnormal alveolar development or destruction of mature interstitial collagens by MMP-1. Adult transgenic mice over-expressing a Th1 cytokine (IFN-), a Th2 cytokine (IL-13), or a cytokine with Th1 and Th2 activities (IL-18) in airway epithelial cells spontaneously develop obvious lung inflammation, increased lung levels of MMPs and cysteine proteinases, and airspace enlargement (82-84). In mice over-expressing IL-13, the metalloproteinases MMPs - 9 and -12 play critical roles in promoting airspace enlargement, with MMP-12 also promoting inflammation and driving the increased expression of other MMPs in the lung (85). In transgenic mice over-expressing IFN-, cathepsin S stimulates lung epithelial

MMP inhibitors in human COPD patients remains to be explored.

apoptosis, lung inflammation, and airspace enlargement (86).

*Chronic smoke exposure models* 

patients (77).

components of the alveolar walls (78).

*Transgenic murine models* 

#### *Alveolar septal cell apoptosis models of airspace enlargement*

In patients with COPD there is apoptosis of alveolar septal cells (87,88) and leukocytes (89,90), and apoptosis of the endothelial and epithelial cells that make up the alveolar walls. This leads to the development of emphysema. Septal cell apoptosis and airspace enlargement in the absence of overt lung inflammation can be induced rapidly in experimental animals by: 1) pharmacologic blockade of vascular endothelial growth factorreceptors in rodents (91); and 2) transfection of murine alveolar epithelial cells with caspase-3, a pro-apoptotic cysteine proteinase (88). However, increased elastase activity due to acidic proteinases is detected in BAL samples after transfectionof alveolar epithelial cells with caspase-3 (88). Thus, proteinases released from dying structural cells may degrade the lung ECM, thereby acting together with septal cell apoptosis to cause loss of alveolar units and airspace enlargement.
