MOLECULAR PATHOLOGY DATA: ABOUT THE PATHOGENIC MECHANISMS OF THE INTERSTITIAL LUNG FIBROSIS

POPESCU IULIAN  PhD, MD
The Clinical Department of Radiobiology from the Fundeni Clinical Institute
popdociul@yahoo.com

Alina Halpern  PhD, 3rd Department of "Sf. Stefan" Hospital, Bucharest

 

Definition The history of etiological data

Interstitial lung diseases are a heterogeneous group of disorders, either idiopathic or related to aggressions or inflammatory causes where the place of agression is the lung interstitium.

To this kind belong: the idiopathic interstitial pneumonia, granulomatous disorders and other lung afections.

The most important, most common is the Idiopathic Lung Fibrosis, which has a severe prognostic, similar to lung cancer (1, 2, 3). It is a specific form of interstitial pneumonia, leading to chronic fibrosis of
causes not well known. It is observed in older adults, is limited to the lungs (2) and leads to chronic respiratory failure and death.

The lung fibrosis is the result of an epithelial injury, with a disordered repair, occurring secondary after lung injuries caused by chemotherapy, inhalation of toxins, collagen, vascular maladies or is idiopathic as idiopathic interstitial pneumonia (4, 5). It is characterized by the accumulation of myofibroblasts, a deposit of proteins of the extracellular matrix (6), the release of cytokines, abnormal accumulation of mesenchymal cells and through proliferation (7, 8). The idiopathic pulmonary fibrosis has a histological appearance of interstitial pneumonia. It is characterized by areas of fibrosis, with a number of fibroblasts interposed between areas of normal tissue and is accompanied by the hyperplasia, type II alveolar epithelial cells (AEC II).

The history of pathogenic mechanisms.

It has been suggested the role epithelial necrosis and vascular collapse (9, 10).

The hypothesis of the potential role of epithelial cells (11) (Selman-Prado 2004).

It is currently accepted the assumption that idiopathic pulmonary fibrosis forms following the repeated injury of the alveolar epithelial cells, especially type II cells . The response to these aggresions is associated with a disordered tissue repair and continuous cicatrisation process (12). The repeated injuries make the alveolar epithelial cell to become susceptible to apoptosis. This is proven throug increasing the cellular markers expression of the Protein Surfactant -C and p20caspase-3 in 70-80% of cases (9).

THE MECHANISM OF LUNG FIBROSIS OCCURRENCE

Intra-cellular physiology aspects of the type II alveolar epithelial cells (AECII).

The type II alveolar epithelial cells II sinthesises, secretes, recicles all the components of the lung surfactant, thus reducing the surface tension and enabling the breathing to be carried out under normal transpulmonary pressure. The Lung Surfactant is composed of 90% fat and 10% proteins, as phosphatidylcholine, phosphatidyl dipalmitate (13) .

The Lung Surfactant also produces substances with inborn defense system: defensine, colectine (SP-A and SP-D) and lyzozim that contribute to the defense pathogens (14).

The type II alveolar epithelial cells (AECI) regulate the pulmonary fluid balance and raise the self-recovery characteristics. Cells become similar to STEM cells and progenitor cells (16, 17). The type II alveolar epithelial cells have a proliferation potential (13, 16, 17) and transdifferentiation potential in type I alveolar epithelial cells (18). With all this potential, they suffer from chronic aggression. Explanation:

The SP-C protein has been described within the familial forms of idiopathic pulmonary fibrosis. In these forms, the mutations of SP-C protein are found in the carboxy terminal part (19, 20, 21, 22). It is a protein hydrophobic in interaction with the surfactant lipids. Therefore if mature SP-C were expressed as such in the type II alveolar epithelial cells, this protein would attack the cell and would cause the cell death. But this is normally avoided. Thus, the SP-C protein produced inside the cell originally incorporates the pro-protein at the terminal ends of the amino groups (NH2) and carboxyl (COOH), which then cleaves during transport through the lyzozomal compartment of the type II epithelial alveolar cells (EACI) . At the end of this process, the SP-C protein became mature is co-secreted with the lipid components of the surfactant.

 

PATHOLOGICAL ASPECTS.

In the case of SP-C mutations they are found in the carboxyl terminal (23). In this situation the carboxyl end is altered as structure and then the protein is wrongfully processed (misfolded). Thus, through the mutation occurred at the level of the SP-C protein – the type II alveolar epithelial cell can not process any longer the protein and this incorrectly processed protein aggregates in cells and co-aggregates with the SP-C proteins remained healthy.

This settling of (misfolded) improperly processed SP-C proteins seems to be the reason for the chronic stress on alveolar epithelial type II cells (AECI) (23) (I-26). Also, the SP-a and SP-C protein mutations

lead to a chronic stress of the Endoplasmic Reticulum, which is the synthesis place of proteins. The telomeres shortening mutations lead to an injury (agression) of the DNA. Both lead to cell apoptosis and further to pulmonary fibrosis (19, 20, 21, 22).

THE ENDOPLASMIC RETICULUM (ER) STRESS

The endoplasmic reticulum (ER) stress is a cyto-protective mechanism. It helps the cell to survive. The ER contains "chaperones" that helps in protein processing (folding). To these belong: BiP - the most important, to which are added further 3 signaling molecules:IRE1, ATF6p90 and PERK.

BiP interacts with the 3 signaling molecules and keeps them in an idle state. If BiP is required to dissociate itself from the signaling molecules in order to help in the processing (folding) of the proteins, then IRE1, ATF6p90 and PERK are activated and produce a redundant signaling process within the cell with the main purpose of helping the cell to improve the processing (folding) of defectively processed (misfolded) proteins in order to rebuild again the homeostasis (24). This process involves the possible ways that affect the synthesis of lipids, overexpression of chaperones, proteasome compounds, oxygen species for antireactive signaling and arachidonic acid metabolism.

If the stress conditions are either overabundant or prolonged, then the cell will be directed to apoptosis through the activation of two main factors: CHOP (C / EBP homologous protein) and ATF4 (activating transcription factor 4)

The pathological mechanism of the SP-C mutations (25, 26).

It was observed that the wild (unmutated) SP-C proteins locate in the lyzozomal compartment, instead the mutated SP-C proteins are located in the endoplasmic reticulum. In cells with mutated SP-C it is noticed a stress response increase of the endoplasmic reticulum. It was also observed that the mutated SP-C cells - under the influence of viruses or proteasome function blocking - rapidly suffers the apoptosis, unlike the wild SP-C protein (unmutated) which do not lead – under the same conditions – to the cell death. In addition to idiopathic lung fibrosis have been observed disturbances in the surfactant transport within the alveolar epithelial cells (EACI) (27). The mature forms of SP-C ready to be excreted are blocked in the lyzozomal compartment and not in the endoplasmic reticulum and Golgi apparatus. The accumulation of mature SP-C proteins causes the swelling of the cell. This determines a reaction of lyzozomal stress by activating D1 cathepsin, glycerol-ceramide and possibly CHOP (9).

 

OTHER FACTORS THAT INTERPOSE IN THE FIBROSIS PROCESS 

 

MYOFIBROBLASTS

Myofibroblasts are components of the fibrosis from various tissues including the lung. It was assumed that they arise through the transdifferentiation of lung resident fibroblasts or fibroblasts migrating from the blood to the areas of fibrosis or areas of aggression with subsequent transdifferentiation.

Also the type II alveolar epithelial cells (AEC II ) aggressed, produce mediators such as PDGF and TNFalfa (28). These mediators lead to the fibroblasts migration to the aggressed areas and there are converted into myofibroblasts (28).

Myofibroblasts can also derive from the type II alveolar -epithelial cells (AEC II) through the Epithelial Mesenchymal Transition (EMT) (29). The Epithelial Mesenchymal Transition is characterized through:

- Loss of the alveolar-epithelial cell polarity.

- Loss of molecules ensuring the cell adhesion, such as: E-cadherin and the occludens -1 area.

- The reorganization of the cellular skeleton.

- Acquisition of mesenchymal markers such as fibronectin, alpha-smooth muscle actin.

- The possible acquisition of the migratory phenotype (30).

The main inducer of the Epithelial Mesenchymal Transition in the lung is TGF Beta-1. TGF Beta-1 can cause dramatic changes in the morphology and phenotype of many cell types (29).

Thus, after the extended exposure of the alveolar epithelial cells to the action of TGF-Beta-,1 is produced the Epithelial Mesenchymal Transition (30). It was also noticed that following the biopsies in patients with idiopathic lung fibrosis, the hyperplastic epithelial cells from the fibroblasts foci show both epithelial markers (TTF-1, prosurfactant protein B and C) and mesenchymal markers (N-cadherin, alfa smooth muscle actin (31, 32).

The Epithelial Mesenchymal Transition induced by the administration of TGF Beta-1in the type II alveolar epithelial cells (AEC II) is also driven by the tissue expression and translocation of other mediators, such as the transcription factors SNAI 1 and SNAI 2. The increase in their expression suggests that the epithelial mesenchymal transition mediated by SNAI contributes to the development of the fibroblast pool (1).

It has been proven - in vivo – that the type II alveolar epithelial cells (AEC II) undergo the Epithelial Mesenchymal Transition, suggesting that these cells may serve as source of myofibroblasts in idiopatic lung fibrosis (V).

TGF Beta-1 and TNF alfa-1 induce the Epithelial Mesenchymal Transition to a myofibroblast-like phenotype, this process taking place in alveolar epithelial cells (AEC II).

The type II alveolar epithelial cells (AEC II) are important in the process of fibrosis induction of (33, 34, 35) and the pulmonary fibrosis is closely linked to the aggression of the type II alveolar cells, the inflammation playing a secondary role.

Another source of myofibroblasts are the circulating fibrocytes. These are coming from the bone marrow. They express mesenchymal markers such as: fibronectin, collagen I and collagen III (37). They may also originate on a haematological pathway, such as:CD45 CD37 (36).

The CXCR4-CXCl 12 axis play a role in mobilizing the fibrocytes at the injury place. The chemokine receptor CXCR4 is expressed on the surface of fibrocytes, whereas CXCL 12 is expressed by the type II alveolar epithelial cells ((37, 38, 39).

The primary lesions of the idiopathic lung fibrosis are aggregated by myofibroblasts that promotes a settling of the ECM proteins. The foci of myofibroblasts meet in the subepithelial layers, very close to the areas of injury and repair of the Alveolar Epithelial II cells (40, 41).

THE ALVEOLAR-EPITHELIAL CELLS

The experimental and clinical data show that the injury (aggression) and apoptosis in the alveolar epithelium, having as result the hyperplasia of the type II alveolar epithelial cells (AEC II) are essential data.

The disorder of the II alveolar c epitelial cells (AEC II) programs contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF).

The WNT family include growth factors participating in body development and that diminishes in the case of body degradation (1).

It has noticed that the WNT-Beta Catenin is overexpressed and operative in adults lung with idiopathic pulmonary fibrosis (4, 7, 43).

Also, the WISP-1 (WNT inducible protein-1) is a member of the same family (CCN) of cellular matrix protein secreted and rich in cysteine​​. These are encoded by the WNT target gene and are highly expressed in alveolar epithelial cells (AECI) and mediates the increased proliferation of the alveolar epithelial cells (AEC II) and induce the release of profibrotic markers that MMP7.plasmin, activator inhibitor-1 and SPP in the alveolar cells epithelial (AEC II).

The WISP-1 also induces the epithelial mesenchymal transition (EMT) in the type II alveolar epithelial cells, as well as the production of extracellular matrix proteins by fibroblasts, but without having an effect on the fibroblasts proliferation.

The pulmonary fibrosis in mice treated with specific antibodies neutralized for WISP reduces the expression of the genes characteristic of fibrosis and cancels the expression of genes related to the Epithelial Mesenchymal Transition (EMT). These changes of expression are associated with the marked attenuation of the lung fibrosis, including the decrease of the collagen deposits, improvement of the lung function and survival. It is thus proven that WISP-1 is a key regulator of the cellular hyperplasia, alveolar epithelial cells (AEC II) and their plasticity and becomes a target for the attenuation of the lung fibrosis (43).

The Fibrosis Mediators

TGF beta-1is a crucial mediator in the development of lung fibrosis.

TGF beta-1 inhibits the epithelial cell proliferation, forwards the epithelial cell migration, stimulates the Epithelial Mesenchymal Transition and increases the cell apoptosis. In addition stimulates the fibroblast proliferation, inhibits the fibroblast apoptosis and stimulates the collagen production.

TGF beta-1 can be activated through the action of integrins, integrins representing the transmembrane heterodimeric protein with 2 alpha and beta integrin subunits. The cyto-skeletal changes activate the alpha and beta integrins, which in turn activates TGF-beta-1 (1).

The thrombin and lipo-phosphatidic acid induce the cyto-skeletal modifications which in turn activate alpha and beta integrins, which further enable TGFbeta-1 (44, 45). The thrombin and lipo-phosphatidic acid are released from thrombocytes, as a result of the injury (aggression). They fix on the PAR-1 epithelial cell surface receptors (protease activated receptor-1 and lipophosphatidic acid receptor-2, and then induce the cytoskeletal changes. The anti-alpha and beta integrin monoclonal antibodies may prevent the acute pulmonary fibrosis and lung injury in vivo (46, 47).

The serotonin inhibits the proliferation of fibroblasts through the receptors 5HTR-2A and 5HTR-2B (48).

It was observed that these 2 receptors are upregulated in the lungs with idiopathic pulmonary fibrosis and non-specific interstitial pneumonia (NSIP). The expression of 5HTR-2A was found specifically in the idiopathic pulmonary fibrosis (IPF). The 5HTR-2A protein is located in fibroblasts, while 5HTR-2B is located in the epithelial cells. The serotonin being a strong vasoconstrictor plays a role in the pathogenesis of the lung hypertension, which is usually noticed in the idiopathic pulmonary fibrosis (49).

The Oxidative Stress.

It is known that in idiopathic pulmonary fibrosis there is an excess of oxidants and a lack of antioxidants factors that contribute to the disease pathogenesis (50). The level of oxidative stress negatively correlates with the lung function and can be used as a marker of disease severity (51). The inflammatory cells in the bronchoalveolar lavage produce reactive oxidant species (ROS), which affect both the lung cells and the cytokines (TGF beta-1) and facilitates the fibrogenesis (50, 52).

The glutathione as antioxidant agent is diminished in the idiopathic pulmonary fibrosis (53) (II, 35). This is partly due to the activation of TGF beta-1, which inhibits the glutathione synthesis (54) (II, 36).

The Apoptosis.

In the normal process of wound healing, the myofibroblasts are eliminated through apoptosis. In the case of myofibroblasts, they have a low sensitivity to apoptosis, while the alveolar-epithelial cells have an increased sensitivity to apoptosis (55) (II, 41). This can be explained by the fact that the patients with idiopathic pulmonary fibrosis have a reduced capacity to produce E2prostaglandin. This leads to a higher sensitivity of the alveolar- epithelial cell to the apoptosis induced by the FAS-ligand, but decreases the fibroblast sensitivity to apoptosis (56). It is also possible that the TGF beta-1 produced by the alveolar epithelial cells to contribute to the increase in the resistance to apoptosis of the myofibroblasts towards apoptosis, through the PI3K/AKT signaling pathways (56).

Autoimmune Issues.

Recent data suggest that autoimmunity could be a possible pathogenic pathway in the idiopathic pulmonary fibrosis. It was noted a neolymphogenesis in the lungs with idiopathic pulmonary fibrosis. In these has been demonstrated the presence of B-cell aggregates, activated T cells, dendritic mature cells. This increases the possibility of an antigenic activity (57, 58).

Moreover, the cells TCD4+ produce, either cytokines , which induce the production of autoantibodies by the B cells or mediators like IL-10, TGFbeta-1, TNF alpha, which facilitates the fibrogenesis (1,47)

It has been suggested that Periplakin (a small protein located in desmosomes) may be a target for autoantibodies (59). The desmosomes have a role in increasing the alveolar epithelium. The presence of autoantibodies against Periplakin in the serum and bronchoalveolar lavage in patients with idiopathic pulmonary fibrosis is associated with the disease severity. Periplakin autoantibodies have been found in 40% of these cases and none in the control group.

The coagulation cascade.

It was acknowledged that the activation of the coagulation cascade has profibrotic effects (28, 60). The thrombin can influence the deposition of connective tissue proteins and the fibrosis development by stimulating the collagen production (60) (II, 7 (10), 49). This is possible by using the action of PAR-1, which induces a proteolytic activity. Both thrombin and PAR-1 promote the fibrosis development by overregulating the expression of Fibroblast Connective Tissue Growth Factor (61).

The coagulation X factor plays a part in the pathogenesis of idiopathic pulmonary fibrosis. It is activated by means of the coagulation factor VII together with the fibroblast connective tissue growth factor. Thus the X factor may activate the genetic pathways either through the activation of TGFbeta-1, which is the main fibrogenic cytokine, either by inducing the fibroblast differentiation in myofibroblast by using the PAR-1 signaling pathway. Targeting the X factor of the coagulation could be a treatment method for the idiopathic pulmonary fibrosis (62) (II/3).

MicroRNA

These are post-transcriptional gene regulators and have multiple roles in the cell differentiation, proliferation and repair (1). MicroRNA let-7d, which is expressed in normal lung epithelial cells is subregulated in idiopathic pulmonary fibrosis. During this time its target molecule is the very mobile group AT-hook2 and this is a known regulator of the Epithelial Mesenchymal Transition and is overexpressed in idiopathic pulmonary fibrosis (63).

Role of biomarkers

There have been suggested various biomarkers as predictors of the disease severity and its progression.

Thus we have:

1) The serum level of KL6 (a glyco-protein similar to mucin with high molecular weight (64) was associated with the decrease of survival in idiopathic pulmonary fibrosis.

2) The surfactant proteine A and D are high in idiopathic pulmonary fibrosis and may predict the survival (65, 66, 67)

3) Matrix metalloproteinases alo participate in the pathogenesis of the lung fibrosis. Patients with high levels in serum and bronchoalveolar lavage fluid - Matrix metalloproteinases MMP 3, 7, 8, 9 have a severe prognostic (68).

4) The circulating fibrocytes are progenitors of mesenchymal cells. They are found increased in patients with idiopathic pulmonary fibrosis and during the exacerbations and may independently predict the disease mortality.

5) The oxidative stress markers are found high in the breath condensate from patients with idiopathic pulmonary fibrosis and H2O2 may be associated with the disease severity (69)

6) The serum level of the CCL-18 chemokine produced by the alveolar macrophages may independently predict the mortality in idiopathic pulmonary fibrosis (70).

7) Neutrophils in bronchoalveolar lavage is a marker of disease severity and alteration in the lung function (71).

8) The periostin (a protein of the extracellular matrix (EMC) is involved in the fibrosis process. It is highly expressed in patients with idiopathic pulmonary fibrosis and nonspecific interstitial fibrotic pneumonia. It may be used as a marker in order to distinguish the fibrotic interstitial pneumonia of non-fibrotic interstitial pneumonia (72).

The mechanisms leading to the injury of the alveolar epithelial cells type II to pulmonary fibrosis.

There are 3 theories on this issue:

1) The first that was involved is the epithelial mesenchymal transition, where the epithelial cells undergo a transdifferentiation in fibroblasts and their consecutive activation.

2) The second theory states that the death of alveolar epithelial cells of type II (AEC II) leads to the control loss of the mesenchymal cells. As a result they will proliferate and produce more collagen. The E2 prostaglandin is a key factor leading to the fibroblasts differentiation and their proliferation (73). In idiopathic pulmonary fibrosis, the PG level (E2) is low. The fibroblasts lack of control leads to excessive epithelial apoptosis. In addition, the type II alveolar epithelial cells aggressed release profibrotic products like TGFbeta1, TNFalfa, CTGDF, tissue factor and factors VII and factor X of the coagulation, resulting in the activation of mesenchymal cells ( (74, 75, 76, 77).

3) The third theory holds that the death of the alveolar epithelial cells leads to the release of factors attracting the circulating fibrocytes, which then invade the lung and locally are increased the fibroblasts deposits (77). Also in the acute exacerbations of the idiopathic pulmonary fibrosis increases the number of circulating fibrocytes (73) (I, 40).

In conclusion it is not yet known the place occupied by each of these 3 theories in the mechanism of transition to pulmonary fibrosis, each of them leading to fibroproliferation, raise of the fibroblasts deposit and to collagen accumulation.

The treatment.

Among the substances tested to obtain special therapeutic results:

1) Pirfenidone, which has antifibrotic and antiinflammatory activity. It lowers fibroblasts proliferation and reduces collagen formation (79, 80). In bleomycin-induced fibrosis it reduces the fibrosis extension and the number and size of fibroblasts response. It also diminishes the forced vital capacity decrease and leads to the increase of the duration of PFS (progression free survival) (81). In the clinical trials it was shown that the natural course of the disease slowly decreases but it cannot lead to a complete cessation of the natural course of the disease (82).

2) BIBF 1120, which is a triple kinase inhibitor, aiming at the receptors of the fibroblast growth factor, vascular endothelial growth factor and platelet derived growth factor. Its efficiency will be clinically proven (83) (

3) N-acetyl-cysteine (NAC) has antioxidant characteristics, reduces the lung function decline and in the experiment - in vitro - contributes to the intracellular reserve recovery of the reactive oxidative species (ROS) induced by TGFbeta-1, thus preventing the Epithelial mesenchymal transition (84).

4) HGF (the hepatocyte growth factor) has an inhibitory effect - in vitro – of the idiopathic pulmonary transition through the induction of SMAD-7, which is a inhibitor of the TGFbeta- 1 signaling (85).

5) The combination of steroids, azathioprine, and N-acetyl-cysteine has moderate beneficial effects in the easy and soft forms of the idiopathic pulmonary fibrosis (84, 85).

6) Other substances which have given good results

- Etanercept, acting against TGFbeta-1, TNFalfa, PG (E2)

- Bosentan, acting on Endothelin

- Imatinibmesylat, acting on PDGF (9) .

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GLOSSARY

Myofibroblast. It is a cell located between fibroblasts and smooth muscle cell.

ECM (extra celular matrix). It is the extracellular part of a tissue, which is usually the support for cell. Contributes to rearranging of cells one to another. It consists of proteins and glycozaminoglycan.

Protein Surfactant (SP). There are 4 members-SP:A and SP-D, being hydrofile, SP-B and SP-C are hydrofobe.

SP-A. It is the most abundant and intervenes in the immune system.

SP-D. It is the largest protein. Intervenes in the immune system and regulates the surfactant balance.

SP-B. It is hydrofobe. Is necessary for the lung function.

SP-C. It is hydrofobe. The surfactant protein is the smallest, but very abundant. It acts as a lever for moving the lipids. It has a stabilizing effect on the compressed surfactant.

pCaspase-3. It is related to apoptosis. It cleaves the vital proteins.

Pro-protein.

Defensines. Proteins active against microbes, fungi, viruses. The immune cells contain defensins, which are used to kill the phagocytosed bacteria.

Colectines. They are soluble receptors belonging to the Collagen family. SP-A and SP-D are colectines. They are involved in the immune system.

Chapperoni. These are proteins that assist in the protein-processing in order to complement the three dimensions (folding). They also assist the unprocessed proteins (unfolding) in molecular biology.

Folding. is the process by which the protein molecules assume the shape, conformation.

Folding protein. is the physical process through which a polypeptide folds into its three-dimensional structure and characteristics.

IRE-1 (inositol requiring-1), APF6p90 (activating transcription factor 6), PERK (dRNA activated transcription factor protein kinase-like ER kinase).

The endoplasmic reticulum stress activates a set of signaling pathways, which are collectively called the UPR (unfolded protein response) namely unprocessed protein (unfolded without the three dimensions appearance). This response is made by the 3 signaling pathways (IRE-1, ATF6p90, PERK). They further the survival by reducing the raw (unfolded) protein molecules or incorrectly (misfolded) processed.

TF4 (transcrition factor4) sau CREB2. More stress conditions (hypoxia, anoxia, endoplasmic reticulum stress) initiate a response pathway on the unprocessed (unfolded) protein lead to an increased synthesis of ATF4.

Catepsin is a human protein encoded by the gene CTSD, which degrades the proteins.

Catepsin D is an aspartyl protease.

Glycerol-ceramide. Half of the isolated phospholipids are the phosphosphingolipid. Two important phosphosphingolipids were characterized as ceramide- phosphoryl etanolamine and ceramide-phosphoryl-glycerol.

PDGF (platelet derived growth factor). It is a protein that plays an important role in angiogenesis. It is synthesized, deposited and released following the activation of thrombocytes can also be produced by the smooth muscle cells, activated macrophages, endothelial cells. 

TNF alpha. It is a cytokine involved in the systemic inflammation. Stimulates the acute phase reaction. It is produced by the activated macrophages, induce the cell apoptosis, inhibits the tumorigenesis.

Occludens-1 zone. ZO-1 aligns the cytoplasmic face of strong (narrow) junctions.

Alfa smooth muscle actin. Is a human protein codified by the ACTA2gene located in the area 10q22-q24. It is a marker of myofibroblasts. Is involved in the cell motility.

E-Cadherin. It is a tumor suppressor gene having a critical role in maintaining the adherent junctions in the cell contact areas. The loss of these adhesion properties leads to the transformation of benign lesions in metastatic cancer.

N-Cadherin. Is a member of the Cadherin gene family, which mediates the cell adhesion dependent on the calcium ions. The increased expression of N-cadherin leads to a phenotype similar to the Epithelial mesenchymal transition. The cancer cells with the high expression of N-cadherin result in invasion and metastasis through increasing the cell motility.

Fibronectin. It is a glycoprotein with high molecular weight which links to the integrins. It can also limks to the components of the extracellular matrix, such as fibrin and collagen. Plays a major role in the cell adhesion, cell growth, migration and differentiation. The alteration of its expression is observed both in cancer and in fibrosis.

TGF beta-1 (transforming growth factor-1). Is a polypeptide member of the TGF-beta cytokines superfamily. Controlls the cell proliferation and differentiation. It is a cytokine with a role in immunity, cancer and fibrosis. Induces the apoptosis and has crucial role in resuming the cell cycle. TGF beta-1 mutations lead to lack of control cell.

Colagen I şi Colagen III. Collagen is a protein common in animals and humans. There are 3 types of collagen (I, II, III) and are the foundation of some tissues. The type I collagen is found in skin, bone, teeth, tendon, ligament and interstitial tissue. The type IV collagen is found in phagocytes, microbes, blood vessels.

CXCR4. It is a protein codified by the CXCR4 gene. Is an alphachemokine that is a specific receptor of the Stromal Derived Factor-1 (SDF-1) also named as CXCL12, which has a very potent activity for lymphocytes. It is important for hematopoietic stem cells. The CXCR4 expression is low or absent in many healthy tissues but is increased in various cancers and is linked to metastasis in tissues with high concentrations of CXCL12 such as the lung. 

SDF-1 (CXCL12). It is a small chemokine. It enables the leukocytes and is induced by the proinflammatory stimuli. It is a powerful chemostatic for lymphocytes. It is important in angiogenesis.

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