Primary cell culture of rat chondrocytes and hypertrophic differentiation protocol

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Gene and protein structure of SSAO

For many years now a lot of work has been devoted to cloning AO. SSAO was cloned for the first time in 1998 from human gut smooth muscle and since then, in many other tissues in different species.
Mu et al., (1994) cloned bovine liver amine oxidase and they reported the nucleotide sequence of bovine serum amine oxidase and its predicted protein sequence. Moreover, Høgdall et al., (1998) reported that the nucleotide sequence of a bovine lung cDNA was similar but not identical to the cDNA sequence of copper-containing amine oxidase from liver described by Mu et al., in 1994. This result was explained by different mechanisms of gene expression ensuring tissue-specific expression of the different forms.
Cloning of the human SSAO was first reported in the placenta (Zhang et al., 1996). The studies of Imamura et al., (Imamura et al., 1997 and 1998) on the human retinal amine oxidase gene (AOC2) revealed its localization on chromosome 17, in 17q21.
In 1998, Smith et al., cloned SSAO/vascular adhesion molecule 1 (VAP-1) from human gut smooth muscle. The protein obtained from the open-reading frame in the VAP-1 cDNA encoded a 763-amino acid protein of 84.6kD.
In 1998, Cronin et al., reported two alternatively spliced transcripts of human copper-containing AO pseudogene in liver and placenta.
In the same year, Bono et al., (1998) cloned and characterized the mouse kidney VAP-1. The amplified 800-bp fragment has shown 80% amino acid identity to human hVAP-1. Moreover, they determined the nucleotide sequence, structural organization and chromosomal localization in the genome of a gene encoding for mouse VAP-1. The mVAP-1 gene is composed of four exons separated by three introns and encodes a protein with high identity to copper-containing amine oxidases. The active site is composed of two important histidines that coordinate a copper atom located in exon 1. Moreover, exon 1 includes 8 putative glycosylation sites which are probably essential for adhesive properties of the enzyme (Bono et al., 1998). The main features of the gene are presented in figure I-3.

Soluble forms of SSAO/VAP-1

Several works have already confirmed the presence of a soluble form of SSAO (sSSAO). This soluble form of SSAO has been found in man, presumably resulting from the proteolytic cleavage of membrane-bound hVAP-1 (Abella et al., 2004; Stolen et al., 2004a). This hypothesis is supported by the sequence of the membrane domain of SSAO/VAP-1 with the intracellular NH2-terminal end characterized by a region containing 23 predominantly hydrophobic amino acids. The amino acid residue at position 19 was proposed as the cleavage site (Smith et al., 1998). In 2005, Jakobsson et al., produced and crystallized an N-terminally truncated, human sSSAO containing 29 residues. The sources of sSSAO were suggested to be the endothelial cells of high endothelial venules, adipocytes (Stolen et al., 2004a) and vascular smooth muscle cells (Göktürk et al., 2003). The level of sSSAO is low in human and rodents plasma compared to cow or pig. It increases in inflammatory diseases like atherosclerosis.

TPQ and enzymatic reaction mechanisms

Essential for the deamination reaction carried out by SSAO, is the topa-quinone (TPQ) cofactor. SSAO uses a tyrosine residue as a redox cofactor, that is post-translationally modified to 2, 4, 5-trihydroxyphenylalanine quinone (TPQ) (Janes et al., 1990; Klinman, 1996; McGuirl et al., 1999). As for the TPQ precursor, a residue of tyrosine situated in the active site with a consensus sequence (Asn-Tyr-Asp/Glu) was indicated (Mu et al., 1992). During the post-translational modification leading to the topa-quinone formation, a functional copper binding site, which is preserved through evolution, and oxygen play an essential role.
The enzymatic reaction can be divided into two half-reactions (Fig. I-7). First, the reduction of TPQ by the substrate takes place. The primary amine group of the substrate is trapped in a covalent bond to the TPQ residue and a Schiff base is formed. Then hydrolysis takes place and an aldehyde is released. The reduced cofactor is attached to aminoquinol-Cu(II). In the second oxidative half-reaction, cupper participates in an electron transfer between TPQ-aminoquinol and molecular oxygen. The TPQ is re-oxidated by molecular oxygen with a concomitant release of hydrogen peroxide and ammonia (Jalkanen et al., 2001; Dawkes et al., 2001; Klinman et al., 2003; Dubois et al., 2005).

Protein expression of SSAO in different tissues

Over the past forty years, the presence of SSAO has been reported in many diverse tissues. The first major source of SSAO was in large arteries. VAP-1 was found in the media layer of large vessels such as the aorta (Salmi et al., 1993). It was demonstrated in bovine and rabbit aorta (Lyles, 1996), rat aorta (Gubisne-Haberle et al., 2004) and next in human vasculature (Lewinsohn, 1984). Further studies associated a SSAO presence in the aorta with smooth muscle cells layer (VSMC) (Lewinsohn, 1984; Lyles et al., 1985; Precious et al., 1988). SSAO activity measured in VSMC from pig and rat cultured in vitro confirmed this association (Hysmith et al., 1987; Blicharski et al., 1990). Moreover, several studies have reported that SSAO is mainly localized in the plasma membrane of cells in rat aorta (Wibo et al., 1980), in pig aortic SMC (Hysmith et al., 1988a; 1988b) and in human tonsil VSMC (Jaakkola et al., 1999). SSAO was also shown to be present in non-vascular smooth muscle (Lewinsohn, 1981) and mouse small intestine (Gubisne-Haberle et al., 2004).
Recently we can find reports about SSAO/VAP-1 expression in foetal tissues. Already during the early stage of human foetal development (7 weeks) SSAO is detectable in the smooth muscle layer of the aortic and gut wall, smooth muscle and fat cells in different organs. VAP-1 expression in primary lymphoid organs was seen in thymus and in secondary lymphoid organs, the spleen was positive for VAP-1 presence. Moreover, a study has confirmed the active form of VAP-1 in foetal tissues (Salmi et al., 2006).
In 2008, Valente et al., showed that SSAO/VAP-1 is expressed during mouse embryonic development. Expression of VAP-1 is already detected at embryonic day 9 (ED9) in the myocardial progenitor cells of the ventricular wall of the heart. Then at ED14, VAP-1 was found in the heart, aorta, pulmonary vessels and vessels and arteries of the lung. In later stages of embryonic development, VAP-1 was detected also in the sensory organs, smooth muscle tissue and skeletal elements.
Another major source of SSAO is white and brown adipose tissues. SSAO was detected in rat adipocytes (Barrand et al., 1982 and 1984a; Raimondi et al., 1991), and localized in plasma membranes of rat brown adipose tissue (Barrand et al., 1984b).
Salmi et al., (1993) have reported high expression of SSAO/VAP-1 in endothelial cells of high endothelial venules (HEV).
For the first time the presence of SSAO activity in non-vascularized tissues such as rat articular cartilage was reported in 1987 by Lyles and Berti (1987) and in odontoblast by Norqvist and Oreland (1981, 1982).

The role of SSAO in different cells/tissues

SSAO is expressed in many tissues, extensively in adipocytes and in the vasculature, particularly in smooth muscle cells. SSAO has been found in endothelial cells of high endothelial venules of peripheral lymph nodes. An increase in the soluble form of SSAO was found in serum of patients with inflammatory diseases like atherosclerosis or inflammatory liver disease. There exist many reports about the role of SSAO in these tissues.

Adipocytes (glucose uptake and cell differentiation)

It has been observed that AOC3 gene expression increases during adipogenesis (Moldes et al., 1999; Subra et al., 2003). In adipocytes, it has been reported previously that SSAO played a role in glucose transport. Glucose transport is stimulated in rat adipocytes treated with benzylamine in the presence of vanadate. Nevertheless, this effect is totally abolished with a SSAO inhibitor. In addition, benzylamine promoted also the translocation of GLUT4 to the plasma membrane in rat adipocytes (Enrique-Tarancon et al., 1998; Morris et al., 1997). The oxidation of tyramine – another SSAO substrate, in the presence of vanadate synergistically stimulated glucose transport in adipocytes by peroxovanadate formation. Moreover, studies have shown that peroxovanadate which is a powerful insulin-mimetic compound can be generated by exo-, but also endogenous hydrogen peroxide, the product of SSAO enzymatic action (Marti et al., 1998). The same group has shown that in vivo administration of benzylamine and vanadate decreased plasma glucose levels in rats, suggesting an effect on glucose deposition in the whole animal body (Enrique-Tarancon et al., 2000).
Morin et al., (2001), have confirmed that SSAO activity is localized on the human adipocyte membranes and reported its insulin-like interaction with glucose and lipid metabolism. In vitro, SSAO-dependent oxidation of amines stimulates glucose transport and inhibits lipolysis (Iglesias-Osma et al., 2005). Adipocyte treatment with TNF-alpha which increases obesity-linked insulin resistance and decreases insulin-stimulated glucose transport, causes a decrease in SSAO expression in those cells (Mercier et al., 2003).
Yu et al., (2004), have confirmed that inhibition of SSAO decreases glucose transport in adipocytes obtained from SSAO inhibitor pre-treated mice. The weight gain in obese diabetic KKAy mice treated with a selective SSAO inhibitor was significantly decreased more so than in control mice that were not treated with the SSAO inhibitor. In addition, SSAO-mediated oxidation of methylamine and aminoacetone induces glucose uptake by adipocytes and as a result causes a transient reduction of blood glucose.
The effect of glucose uptake inducted by administration of SSAO substrates is totally absent in adipocytes from AOC3 KO mice. This observation proves that SSAO-dependent oxidation and production of peroxide hydrogen is necessary to modulate an insulin-like effect of the amines (Bour et al., 2007; Stolen et al., 2005).
In addition, Iglesias-Osma et al., (2004 and 2005) have shown for the first time that methylamine increases in vivo glucose disposal, and exhibits an antihyperglycaemic action which is not mediated by a clear effect on insulin secretion.
Moreover, SSAO is involved in terminal differentiation and development of adipocytes. In vitro pre-adipocytes exposed to a SSAO substrate like methylamine increase cell differentiation depending on treatment time and dose of the substrate. Moreover, this effect is abolished by catalase, what indicates that SSAO activity accelerates cell differentiation via hydrogen peroxide production (Mercier et al., 2001; Fontana E et al., 2001; Carpéné et al., 2006).

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Vascular smooth muscle cells

It has been shown that SSAO is strongly expressed in vascular smooth muscle cells (VSMC) and El Hadri has suggested in 2002, that this amine oxidase can have a role in the cell differentiation process. It was reported that SSAO mRNA expression and its enzymatic activity increased during VSMC differentiation. Moreover, the activation of SSAO by methylamine regulates glucose uptake in differentiated VSMCs and increases glucose transporter 1 (GLUT1) expression at the cell surface. Likewise, SSAO activation stimulates the glucose transport in adipocytes and its inhibition abolishes this process. This effect has been found during VSMC treatment with methylamine. Addition of catalase to the culture medium results in an inhibition of glucose transport through a diminution of the membrane expression of GLUT1. However, SSAO-dependent VSMC differentiation was not confirmed in this study.
Increased aldehyde and H2O2 production by SSAO in the presence of MA was shown to induce cytotoxicity of VSMC. In the presence of MA, cells underwent apoptosis by caspase-3 stimulation. However, the number of caspase-3 positive cells was significantly decreased when SSAO activity was inhibited with semicarbazide. Moreover, SSAO catalytic activity in the presence of MA induced vascular cell death by p53 phosphorylation. In addition, the increased VSMC apoptosis was associated with increased aldehyde formation and not with H2O2 (Hernandez et al., 2006, Sole et al., 2008).

Table of contents :

I. Introduction
1. Amine oxidases
1.1. Amine oxidases
1.1.1. FAD-containing amine oxidases
1.1.2. TPQ-containing amine oxidases DAO Lysyl oxidase
1.2. The semicarbazide-sensitive amine oxidase
1.2.1. Gene and protein structure of SSAO Cloning of the gene Protein domains and active sites Soluble forms of SSAO/VAP-1
1.2.2. TPQ and enzymatic reaction mechanisms
1.3. Enzymatic reaction of SSAO
1.3.1. Substrates (MA, BZM)
1.3.2. Products of SSAO reaction
Hydrogen peroxide
1.3.3. Inhibitors
1.4. Protein expression of SSAO in different tissues
1.5. The role of SSAO in different cells/tissues
1.5.1. Adipocytes (glucose uptake and cell differentiation)
1.5.2. Vascular smooth muscle cells
1.5.3. Endothelial cells of high endothelial venules
1.5.4. Chondrocytes
1.6. Expression and activity of SSAO in pathological conditions
Other diseases associated with SSAO
2. Cartilage
2.1. Physiology of cartilage
2.2. Articular cartilage function
2.3. Structure of the hyaline cartilage
2.3.1. Chondrocytes
2.3.2. Extracellular matrix (ECM) components Collagens Proteoglycans
2.4. Cartilage formation
2.4.1. Long bone development
2.4.2. Markers of chondrocyte differentiation
Maturation and hypertrophy
2.4.3. Transcriptional factors of chondrogenesis Sox9 Runx2
2.5. Diseases of joints and cartilage degradation
2.5.1. Osteoarthritis (OA)
2.5.3. Animal models of cartilage diseases
3. Vascular Part
3.1. General structure and function of blood vessels
3.1.1. Vein
3.1.2. Artery
3.1.3. Capillaries
3.2. Structure of an aorta
3.2.1. Intima EC
3.2.2. Media Vascular smooth muscle cells vSMC differentiation and phenotype changing ECM
3.2.3. Adventitia
3.3. Remodeling of the arterial wall
3.4. Diseases of the vascular system
3.4.1. Atherosclerosis – Inflammatory disease of arterial blood vessels Endothelial cell dysfunction Cell types involved in atherosclerosis Atherosclerotic plaque characterization Animal models of atherosclerosis – ApoE-/- mice
II. Working hypothesis and objectives
III. Materials and methods
1. Models
1.1. Human cartilage
1.2. Animals
1.2.1. Normal rats
1.2.2. Models of cartilage degradation: MIA
1.2.3. WT and knock out SSAO-/- mice
1.2.4. Model of atherosclerosis: ApoE-/- SSAO-/ -double knock out mice
1.3. Cells: models of hypertrophic differentiation
1.3.1. Primary cell culture of rat chondrocytes and hypertrophic differentiation protocol
1.3.2. Progressive passage of rat chondrocytes
1.3.3. ATDC5
2. Common methods
2.1. Histology and Immunohistochemistry
2.2. Western blot
2.3. RNA isolation
2.4. RT- and real time qPCR
3. Specific methods
3.1. SSAO activity
3.1.1. Homogenates preparation for SSAO activity measurement
3.1.2. SSAO activity measurement
3.2. Measurement of 2-Deoxy-D-glucose uptake
3.3. MTT
3.4. Atherosclerotic lesion quantification in aorta
3.5. Atherosclerotic plaque quantification in the aortic sinus
3.6. Mesurement of cytokines
3.7. Mice genotyping
3.8. Experiments performed in collaboration
3.9. Statistical analysis
IV. Cartilage and chondrocyte differentiation
1. Hypothesis
2. Objectives and strategies
3. Results
3.1. The presence of SSAO in rat cartilage
3.1.1. SSAO expression in healthy cartilage of Wistar rats.
3.1.2. SSAO is present in the rat growth plate
3.2. The role of SSAO in cartilage
3.2.1. Model of chondrocyte terminal differentiation ATDC5 Primary rat chondrocytes culture
3.2.2. Expression and enzyme activity of SSAO SSAO in ATDC5 SSAO expression and activity during primary chondrocyte differentiation.
3.2.3. Effect of SSAO inhibition on terminal chondrocyte differentiation
3.2.4. The role of SSAO in terminal chondrocyte differentiation
3.3. The presence of SSAO in human OA cartilage
3.3.1. SSAO in human more and less diseased cartilage
3.3.2. Expression and activity of SSAO in human more and less diseased cartilage
3.4. Presence of SSAO in cartilage of the MIA rat model of cartilage degradation
4. Discussion
5. Conclusions
6. Perspectives
V. Vascular and atherosclerosis studies
1. Hypothesis
2. Objectives and Strategies
3. Results
3.1. Role of SSAO in plaque development: Invalidation of SSAO in ApoE-/- mice
3.1.1. Lipid profile in ApoE-/- and ApoE-/-SSAO-/- mice
3.1.2. Kinetics of disease development
3.1.3. Phenotype of atherosclerosis plaques and media α-actin expression in media of aortic sinus Lymphocyte infiltration in media of aortic sinus Monocyte/macrophage presence in media of aortic sinus Collagen content in the media and the plaque of the aortic sinus
3.1.4. Exploration of pro- and anti-inflammatory profiles – in spleen – in aorta
3.1.5. Exploration of the VSMC phenotype
3.1.6. Role of SSAO in cellular trafficking in ApoE-/-SSAO-/- mice vs ApoE-/- mice – in mice under HFD
4. Discussion
5. Conclusion
6. Perspectives
VI. General discussion
VII. References


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