**3. Оxidative stress, COPD and heat shock proteins**

Compared to other organs lungs are unique in their exposure to high levels of oxygen. Because of their close contact with the environment the airway epithelium is directly exposed to either exogenous oxidants – (cigarette smoke, airway pollutants), or endogenous ones – generated by phagocytes or other cell types. To keep the balance lungs need efficient adaptive mechanisms that correspond to their physiological functions. If the enzymatic or non-enzymatic antioxidant systems do not provide the corresponding adaptive response oxidative stress occurs.

It is still considered that oxidative stress is one of the triggers, contributing to the enhanced or abnormal inflammatory response, characteristic for COPD patients.

#### **3.1 Oxidative stress, chaperones and epithelial injury in COPD**

The airspace epithelial surface is particularly vulnerable to the effects of oxidative stress. The injury of the epithelium is an important early event, following exposure to cigarette smoke. The noxious effects of the cigarette smoke on human epithelial cell monolayers has been demonstrated by cell detachment, decreased cell adherence and increased cell lysis (Jones, et al, 1980; Lannan S t al, 1994). It is supposed that these effects are in part oxidant mediated since GSH appears to be critical for the maintenance of the epithelial integrity following exposure to smoke. It is demonstrated in studies that the direct exposure to smoke condensates is associated with profound changes in the homeostais of glutathione (GSH) (Li et al, 1994, 1996). Concentration of GSH are significantly decreased after exposure to cigarette smoke condensate. This is due to a decrease of the activity of the enzymes, responsible for the keeping the redox-cycle – glutathioneperoxidase, glucose-6-phosphate dehydrogenase. In addition the depletion of GSH alone induces airway detachment and increases its permeability (Li et al, 1995; Rahman et al, 1995).

Chronic Obstructive Pulmonary Disease - Chaperonopathology 75

both microfilametns, intermediate filaments and microtubules, lead to the phosphorylation of αB-crystalline, underlying the biological importance of this heat shock protein in

Cherneva et al, studied the tissue expression of αB crystalline in 28 COPD patients, 14 with age-related emphysema and 23 smokers without COPD. Immunohistochemistry towards αB crystalline was applied. Results were evaluated semiquantitaively. No nuclear staining was present. Only cytoplasmic staining was observed. In most of the cases there was a homogeneous staining among cells. Two patients with COPD had moderate; 26 had intensive staining. In age-related emphysema 5 patients had weak; 2 had moderate and 7 had intensive staining. In smokers without COPD no staining was detected. The clinical

preserving the integral cell architecture (Launay et al, 2006).

implication of our preliminary results needs further investigation (Fig.1).

Non-COPD smoker Age-related emphysema COPD smoker

**3.2 Oxidative stress, chaperones and neutrophil sequestration in COPD** 

walls of COPD smokers (Kilburn et al, 1975; Hunninghake et al, 1983).

related emphysema and COPD patients.

and in a blinded fashion.

Fig. 1. Tissue expression of αB-crystalline in lung tissues from non-COPD smokers, age-

The levels of αB-crystalline were measured in the fixed lung sections (3-mm thick) by immunohistochemical staining using rabbit polyclonal anti-αB-crystalline antibody (1:500 dilution) with avidin–biotin–peroxidase complex method followed by hematoxylin counter staining. Brown colour and the variability of its intensity represents the presence of αBcrystalline.The assessment of immunostaining intensity was performed semiquantitatively

The oxidant burden in the lungs of smokers can further be augmented by the increased numbers of macrophages (two to four folds) and neutrophils (10-fold). Bronchial biopsies and lung resection studies represent a large number of neutrophils in the lungs and alveolar

The small heat shock proteins – HSP27 and αB crystalline have antioxidant ability and increase cell resistance to oxidative injuries (Arrigo et al, 2001). It is reported (Yan et al, 2002) both in cell cultures and whole animals that their expression correlates to decreased levels of reactive oxygen species (ROS) and nitric oxide (NO•).(Preville et al, 1999; Mehlen et al, 1996) Consequently, in cells exposed to oxidative stress, sHSPs lower the levels of lipid peroxidation (Fridaus et al, 2006; Preville et al, 1998). They maintain the mitochondrial potential and provide the production of ATP, thus corresponding to both increased energy needs for stressed cells on the one hand, and ATP supply for the functional activity of the other chaperones on the other (Paul et al, 2000).

The antioxidant activity of HSP27 and αB crystalline is performed by the increase of the levels of gluthatione. (Mehlen et al, 1996) They induce the up-regulation of glucose-6 phosphate dehydrogenase – the enzyme that provides the reducing power of the cell, by reducing NADP+ to NADPH(H) + . In addition it is recently observed that HSP27 and αBcrystalline expression decreases iron intracellular levels. Thus they prevent the Fenton reaction and the formation of hydroxyl radical (OH•) (Arrigo et al, 2005; Chen et al, 2006).

Ruicheng Hu et al, 2011 performed proteomic analysis and found that the expression of HSP27 was upregulated in smokers, and this upregulation was particularly marked in COPD smokers. The expression of HSP27 between the groups was confirmed by IHC and Western blotting. Based on their results and other studies that have shown a protective role for HSP27 against oxidative stress and apoptosis, it could be suggested that induction of HSP27 protects the lung cells of smokers and COPD patients against oxidative stress and apoptosis.Their experiments showed that expression of HSP27 was upregulated in the lungs of smokers, and especially smokers with COPD, even though there was no difference in smoking index between smokers with or without COPD. Therefore, it could be suggested that the upregulation of HSP27 expression in smokers is primarily due to oxidative stress and partly due to inflammation, whereas the difference in HSP27 expression between smokers with or without COPD may predominantly be due to inflammation. HSP27 is a multi-functional cytoprotective factor that protects cells from oxidative stress by regulating the activity of several detoxifying enzymes and promoting the degradation of misfolded proteins. HSP27 also protects cells from unfavourable stimuli by playing a role in apoptosis/survival signal transduction pathways (Ito et al, 2003; Bruey et al, 2000). Phosphorylated HSP27 is a ubiquitin-binding protein that binds to 16 polyubiquitin chains and thereby enhances the degradation of ubiquitinated proteins by the 26S proteasome (Jackson et al, 2008) By enhancing the degradation of IκB-α and activating the nuclear factorκB signal transduction pathway, HSP27 promotes cell survival under conditions of stress. (Yu et al, 2008; Kuoyt et al, 1995)

Another function of the sHSPs that triggers the interest towards their participation in the lung epithelial injury is that both of them are responsible for the protection of cytoskeleton.( (Benndorf et al, 1994; Mounier et al, 2002) Small HSPs are involved in the control of cytoskeletal organization during heat and oxidative stress. They maintain the polymerization-depolymerization processes of F-actin and thus are directly responsible for both cell integrity and intracellular contacts. (Jog et al, 2007; Singh et al, 2007; Mairesse et al, 1996). In addition αB-crystalline is a well-known stabilizer of the intermediate filaments and play a major role in cytoskeletal architecture homeostasis (Bennardini et al, 1992). It is demonstrated in epithelial cells that cell signalling pathways, activated by disrupture of

The small heat shock proteins – HSP27 and αB crystalline have antioxidant ability and increase cell resistance to oxidative injuries (Arrigo et al, 2001). It is reported (Yan et al, 2002) both in cell cultures and whole animals that their expression correlates to decreased levels of reactive oxygen species (ROS) and nitric oxide (NO•).(Preville et al, 1999; Mehlen et al, 1996) Consequently, in cells exposed to oxidative stress, sHSPs lower the levels of lipid peroxidation (Fridaus et al, 2006; Preville et al, 1998). They maintain the mitochondrial potential and provide the production of ATP, thus corresponding to both increased energy needs for stressed cells on the one hand, and ATP supply for the functional activity of the

The antioxidant activity of HSP27 and αB crystalline is performed by the increase of the levels of gluthatione. (Mehlen et al, 1996) They induce the up-regulation of glucose-6 phosphate dehydrogenase – the enzyme that provides the reducing power of the cell, by reducing NADP+ to NADPH(H) + . In addition it is recently observed that HSP27 and αBcrystalline expression decreases iron intracellular levels. Thus they prevent the Fenton reaction and the formation of hydroxyl radical (OH•) (Arrigo et al, 2005; Chen et al, 2006). Ruicheng Hu et al, 2011 performed proteomic analysis and found that the expression of HSP27 was upregulated in smokers, and this upregulation was particularly marked in COPD smokers. The expression of HSP27 between the groups was confirmed by IHC and Western blotting. Based on their results and other studies that have shown a protective role for HSP27 against oxidative stress and apoptosis, it could be suggested that induction of HSP27 protects the lung cells of smokers and COPD patients against oxidative stress and apoptosis.Their experiments showed that expression of HSP27 was upregulated in the lungs of smokers, and especially smokers with COPD, even though there was no difference in smoking index between smokers with or without COPD. Therefore, it could be suggested that the upregulation of HSP27 expression in smokers is primarily due to oxidative stress and partly due to inflammation, whereas the difference in HSP27 expression between smokers with or without COPD may predominantly be due to inflammation. HSP27 is a multi-functional cytoprotective factor that protects cells from oxidative stress by regulating the activity of several detoxifying enzymes and promoting the degradation of misfolded proteins. HSP27 also protects cells from unfavourable stimuli by playing a role in apoptosis/survival signal transduction pathways (Ito et al, 2003; Bruey et al, 2000). Phosphorylated HSP27 is a ubiquitin-binding protein that binds to 16 polyubiquitin chains and thereby enhances the degradation of ubiquitinated proteins by the 26S proteasome (Jackson et al, 2008) By enhancing the degradation of IκB-α and activating the nuclear factorκB signal transduction pathway, HSP27 promotes cell survival under conditions of stress.

Another function of the sHSPs that triggers the interest towards their participation in the lung epithelial injury is that both of them are responsible for the protection of cytoskeleton.( (Benndorf et al, 1994; Mounier et al, 2002) Small HSPs are involved in the control of cytoskeletal organization during heat and oxidative stress. They maintain the polymerization-depolymerization processes of F-actin and thus are directly responsible for both cell integrity and intracellular contacts. (Jog et al, 2007; Singh et al, 2007; Mairesse et al, 1996). In addition αB-crystalline is a well-known stabilizer of the intermediate filaments and play a major role in cytoskeletal architecture homeostasis (Bennardini et al, 1992). It is demonstrated in epithelial cells that cell signalling pathways, activated by disrupture of

other chaperones on the other (Paul et al, 2000).

(Yu et al, 2008; Kuoyt et al, 1995)

both microfilametns, intermediate filaments and microtubules, lead to the phosphorylation of αB-crystalline, underlying the biological importance of this heat shock protein in preserving the integral cell architecture (Launay et al, 2006).

Cherneva et al, studied the tissue expression of αB crystalline in 28 COPD patients, 14 with age-related emphysema and 23 smokers without COPD. Immunohistochemistry towards αB crystalline was applied. Results were evaluated semiquantitaively. No nuclear staining was present. Only cytoplasmic staining was observed. In most of the cases there was a homogeneous staining among cells. Two patients with COPD had moderate; 26 had intensive staining. In age-related emphysema 5 patients had weak; 2 had moderate and 7 had intensive staining. In smokers without COPD no staining was detected. The clinical implication of our preliminary results needs further investigation (Fig.1).

Non-COPD smoker Age-related emphysema COPD smoker

Fig. 1. Tissue expression of αB-crystalline in lung tissues from non-COPD smokers, agerelated emphysema and COPD patients.

The levels of αB-crystalline were measured in the fixed lung sections (3-mm thick) by immunohistochemical staining using rabbit polyclonal anti-αB-crystalline antibody (1:500 dilution) with avidin–biotin–peroxidase complex method followed by hematoxylin counter staining. Brown colour and the variability of its intensity represents the presence of αBcrystalline.The assessment of immunostaining intensity was performed semiquantitatively and in a blinded fashion.

#### **3.2 Oxidative stress, chaperones and neutrophil sequestration in COPD**

The oxidant burden in the lungs of smokers can further be augmented by the increased numbers of macrophages (two to four folds) and neutrophils (10-fold). Bronchial biopsies and lung resection studies represent a large number of neutrophils in the lungs and alveolar walls of COPD smokers (Kilburn et al, 1975; Hunninghake et al, 1983).

Chronic Obstructive Pulmonary Disease - Chaperonopathology 77

As intracellular chaperones heat shock proteins have anti-apoptotic properties. Gal et al, 2011 showed that cigarette smoke extract stimulates the expression of HSP72 in alveolar epithelial cells, diminishing apoptosis Dimethylarsinic acid exposure also elevated intracellular HSP72 levels, changing the localization of the molecule and suppressing apoptosis of human alveolar cells (Kato et al. 2000). Ruicheng Hu et al, 2011 found that expression of HSP27 and CyPA was upregulated in smokers, and this upregulation was further marked in COPD smokers. HSP27 protects the lung cells of smokers and COPD patients against oxidative stress and apoptosis. HSP27 inhibits apoptosis by stabilising the mitochondrial electric potential and inhibiting the release of cytochrome C (Bruey et al, 2000; Paul et al, 2002). It promotes survival by activating the mitogen-activated protein kinase (MAPK) signal transduction pathway. Stress has been reported to activate the MAPK signal transduction pathway, including p38 MAPK, which induces expression and phosphorylation of HSP27 through MAPK-activated protein kinase 2 (MK2). So HSP27 inhibits both the intrinsic and extrinsic apoptotic pathways. It can inhibit the release of cytochrome-C or Smac-Diablo from mitochondria as well as act downstream of them preventing the formation of apoptosome. It can also act at the level of caspase-3 activation (Arrigo et al, 2007). At the level of the Fas receptor HSP 27 inhibits the extrinsinc signalling pathway by binding to DAXX (Arrigo et al, 2007). HSP27 is responsible with the Akt signalling pathway and also inhibits its activity (Arrigo et al, 2007). Concerning the structural organization of HSP27 in apoptotic cells it seems that its chaperone activity largely correlates with its anti-apoptotic one as far as in apoptotic cells the large oligomers inhibit caspase activation.αB crystalline also excutes protection against a large panel of apoptotic stimuli. It binds proapoptotic Bax, Bcl-xl and p53 polypeptides and prevents their translocation to mitochondria. It also directly inhibits the proteolytic activation of pro-

Chronic obstructive pulmonary disease is a slowly progressive condition, characterized by airflow inflammation, which is largely irreversible. It is suggested that the main etiological factor - cigarette smoking, produces inflammatory response in the lungs of all smokers and

The heat shock response is one of the most evolutionary conserved protective mechanisms in cells. It involves a temporary modification of gene expression. Synthesis of different heat shock proteins helps the organism cope with environmental and physiological stresses. As anti-inflammatory effector the heat shock response modulates signal transduction and gene expression by inhibiting the translocation of transcriptional factor – nuclear factor kappa B to the nucleus and prevents the expression of inflammatory mediators (Wong et al, 1997; Sun et al, 2005; Malhotra and Wong, 2002). Initial observations in animals linked heat shock response to an altered inflammatory response and demonstrate that heat preconditioning confers survival in otherwise lethal endotoxin stress (Snyder et al, 1992; Ensor et al, 1994). Heat conditioned macrophages show decreased secretion of TNF-alpha induced by endotoxins. This decreased secretion was sustained as long as the cells had elevated HSP70 levels. Similar to these studies endotoxin induction of IL-6 was also unchanged in the heat-

caspase 3 (Mao et al, 2004; Liu S et al, 2007).

**4. Inflammation, COPD and heat shock proteins** 

those who develop COPD have an abnormal or enhanced inflammation

**4.1 Intracellular heat shock proteins – Antinflammatory molecules** 

Neutrophils are first recruited and then sequestered due to size difference between neutrophils and pulmonary capillary vessels (MacNee, 1993). Radiolabelled studies have shown that lungs normally contain a large number of non-circulating neutrophils, which are retained or moving slowly across lung capillary bed (Selby et al, 1991). In comparison to erythrocytes, neutrophils are usually retained and the number of retained cells correlates to their capacity to adapt to the physilologically narrower diameter of the lung capillaries. The less deformable the cells the greater the sequestration of these cells in the pulmonary circulation. The deposition of the cells in microcirculation allows them to interact longer with the endothelium, to adhere to endothelial cells, or transmigrate in the interstitium and alveolar airspaces and respond to the inflammatory cytokines or infections. Thus any conditions that make the neutrophils less deformable create a predisposition to their sequestration in the lung capillary bed.

Studies in humans show that neutrophils and red blood cells are transiently sequesterd in lung capillary bed during smoking and return to the general circulation after smoking cessation. In vitro experiments show that cells exposed to smoke are less flexible. A similar result can be demonstrated in vivo in patients that are actively smoking – their cells lose flexibility. Decreased neutrophil deformability occurs owing to the assembly of the cytoskeleton – polymerization of microfilaments (F-actin), resulting in cell stiffening. It has been suggested that since each puff of cigarette smoke contains > 1016 oxidant molecules the effect of cigarette smoking is probably oxidantly mediated. This is confirmed by the fact that the decrease of neutrophil deformability is accompanied by the depletion of GSH (Drost et al, 1992). In addition oxidants affect it by altering cellular cytoskeleton through the polymerization of actin.

The control of F-actin cytoskeleton is mediated by the oligomers of HSP27. It is responsible not only for chemotaxis and cytoskeleton reorganization during migration, but also for processes engaged in exocytosis. It is recently observed that this small heat shock protein regulates neutrophil chemotaxis and exocytosis through the control of actin reorganization. (Jog et al, 2007; Singh et al, 2007)

The role of small heat shock proteins in the maintenance of neutrophil cytoskeleton and their reorganization under oxidative stress and inflammation is not investigated in COPD patients. It could however be speculated that the biological role of this chaperones related to the assembly and dynamics of the cell architecture makes them a target for future research. Since the deposition of neutrophils is triggered by oxidative stress it would be curious to compare the levels of expression of these small heat shock molecules in both COPD smokers, COPD non-smokers and healthy smokers.

#### **3.3 Oxidative stress, chaperones and apoptosis in COPD**

It has been proposed recently that COPD is associated with the loss of alveolar endothelial cells as well as lung epithelial cells and that apoptosis can be an essential element of emphysema. Apoptosis in emphysematous lungs is more commonly observed than in nonsmoker's lungs. Peripheral blood leukocytes and lymphocytes of patients with COPD also show increased rates of apoptosis (Tuder et al, 2003; Kasahara et al, 2001). Studies have reported that cigarette smoke induces apoptosis of lung structural cells by oxidative/endoplasmic reticulum stress.When the lungs are insulted with oxidants, consumption of intracellular reductants is increased resulting in aberrant protein folding.

Neutrophils are first recruited and then sequestered due to size difference between neutrophils and pulmonary capillary vessels (MacNee, 1993). Radiolabelled studies have shown that lungs normally contain a large number of non-circulating neutrophils, which are retained or moving slowly across lung capillary bed (Selby et al, 1991). In comparison to erythrocytes, neutrophils are usually retained and the number of retained cells correlates to their capacity to adapt to the physilologically narrower diameter of the lung capillaries. The less deformable the cells the greater the sequestration of these cells in the pulmonary circulation. The deposition of the cells in microcirculation allows them to interact longer with the endothelium, to adhere to endothelial cells, or transmigrate in the interstitium and alveolar airspaces and respond to the inflammatory cytokines or infections. Thus any conditions that make the neutrophils less deformable create a predisposition to their

Studies in humans show that neutrophils and red blood cells are transiently sequesterd in lung capillary bed during smoking and return to the general circulation after smoking cessation. In vitro experiments show that cells exposed to smoke are less flexible. A similar result can be demonstrated in vivo in patients that are actively smoking – their cells lose flexibility. Decreased neutrophil deformability occurs owing to the assembly of the cytoskeleton – polymerization of microfilaments (F-actin), resulting in cell stiffening. It has been suggested that since each puff of cigarette smoke contains > 1016 oxidant molecules the effect of cigarette smoking is probably oxidantly mediated. This is confirmed by the fact that the decrease of neutrophil deformability is accompanied by the depletion of GSH (Drost et al, 1992). In addition oxidants affect it by altering cellular cytoskeleton through the

The control of F-actin cytoskeleton is mediated by the oligomers of HSP27. It is responsible not only for chemotaxis and cytoskeleton reorganization during migration, but also for processes engaged in exocytosis. It is recently observed that this small heat shock protein regulates neutrophil chemotaxis and exocytosis through the control of actin reorganization.

The role of small heat shock proteins in the maintenance of neutrophil cytoskeleton and their reorganization under oxidative stress and inflammation is not investigated in COPD patients. It could however be speculated that the biological role of this chaperones related to the assembly and dynamics of the cell architecture makes them a target for future research. Since the deposition of neutrophils is triggered by oxidative stress it would be curious to compare the levels of expression of these small heat shock molecules in both COPD

It has been proposed recently that COPD is associated with the loss of alveolar endothelial cells as well as lung epithelial cells and that apoptosis can be an essential element of emphysema. Apoptosis in emphysematous lungs is more commonly observed than in nonsmoker's lungs. Peripheral blood leukocytes and lymphocytes of patients with COPD also show increased rates of apoptosis (Tuder et al, 2003; Kasahara et al, 2001). Studies have reported that cigarette smoke induces apoptosis of lung structural cells by oxidative/endoplasmic reticulum stress.When the lungs are insulted with oxidants, consumption of intracellular reductants is increased resulting in aberrant protein folding.

sequestration in the lung capillary bed.

polymerization of actin.

(Jog et al, 2007; Singh et al, 2007)

smokers, COPD non-smokers and healthy smokers.

**3.3 Oxidative stress, chaperones and apoptosis in COPD** 

As intracellular chaperones heat shock proteins have anti-apoptotic properties. Gal et al, 2011 showed that cigarette smoke extract stimulates the expression of HSP72 in alveolar epithelial cells, diminishing apoptosis Dimethylarsinic acid exposure also elevated intracellular HSP72 levels, changing the localization of the molecule and suppressing apoptosis of human alveolar cells (Kato et al. 2000). Ruicheng Hu et al, 2011 found that expression of HSP27 and CyPA was upregulated in smokers, and this upregulation was further marked in COPD smokers. HSP27 protects the lung cells of smokers and COPD patients against oxidative stress and apoptosis. HSP27 inhibits apoptosis by stabilising the mitochondrial electric potential and inhibiting the release of cytochrome C (Bruey et al, 2000; Paul et al, 2002). It promotes survival by activating the mitogen-activated protein kinase (MAPK) signal transduction pathway. Stress has been reported to activate the MAPK signal transduction pathway, including p38 MAPK, which induces expression and phosphorylation of HSP27 through MAPK-activated protein kinase 2 (MK2). So HSP27 inhibits both the intrinsic and extrinsic apoptotic pathways. It can inhibit the release of cytochrome-C or Smac-Diablo from mitochondria as well as act downstream of them preventing the formation of apoptosome. It can also act at the level of caspase-3 activation (Arrigo et al, 2007). At the level of the Fas receptor HSP 27 inhibits the extrinsinc signalling pathway by binding to DAXX (Arrigo et al, 2007). HSP27 is responsible with the Akt signalling pathway and also inhibits its activity (Arrigo et al, 2007). Concerning the structural organization of HSP27 in apoptotic cells it seems that its chaperone activity largely correlates with its anti-apoptotic one as far as in apoptotic cells the large oligomers inhibit caspase activation.αB crystalline also excutes protection against a large panel of apoptotic stimuli. It binds proapoptotic Bax, Bcl-xl and p53 polypeptides and prevents their translocation to mitochondria. It also directly inhibits the proteolytic activation of procaspase 3 (Mao et al, 2004; Liu S et al, 2007).
