Applicant

 Name:  PD Dr. Attila Aszodi

 E-mail: attila.aszodi@med.uni-muenchen.de


Integrins in Osteoarthritis and cartilage repair

Beta 1 integrins, a/b receptors that transmit mechano-chemical signals across the cell membrane, are implicated in articular cartilage (AC) development, remodelling and repair. The collagen-binding a10b1 integrin is abundantly expressed on chondrocytes and is present in a sub-population of human mesenchymal stem cells (hMSCs). Our previous studies with genetically modified mice showed that b1 and a10 integrins are required for skeletal development and the lack of the b1 subunit on chondrocytes leads to osteoarthritis (OA)-like changes of the AC. Our recent work identified mechanosensitive signalling pathways downstream of b1 integrin; emphasised the importance of b1 in the mechanical stability of the AC and revealed that priming of a10 in hMSCs enhances chondrogenesis in vitro. To explore a10 integrin in AC degeneration (Aim 1), a10-null and control mice will be investigated in experimental situations reflecting risk factors of OA (aging, injury). The combined role of risk factors and a10-deficiency on the mechanical properties of the AC will be investigated by atomic force microscopy, the impact of a10 and b1 deficient AC on subchondral bone will be assessed by nanoCT and osteoclast/neurotransmitter profiling. To decipher the role of a10 and b1 integrins in mechano-transduction (Aim 2), control and mutant chondrocytes will be mechanically stimulated using an in vitro hip injury model and in chondrocyte-gel plugs. Protein and gene expression profiles including miRNAs will be analyzed upon mechanical stimulation. To challenge AC regeneration (Aim 3), a10 overexpressing hMSCs will be assessed for chondrogenic differentiation and cartilage repair using ex vivo and in vivo repair models.

Figure 1: b1 integrins modulate articular cartilage function

 

A.      Incomplete, mosaic deletion of b1 integrins in the limb bud of mice (b1fl/fl-Prx1crelow) leads to shortened legs (X-ray), partial depletion of the surface expression of b1 integrin subunit on chondrocytes (IHC, immunohistochemistry) and moderate degradation of the articular cartilage (H&E, hematoxylin and eosin staining).

 

B.      Scoring of articular cartilage erosion indicates significantly increased spontaneous OA in one year old knee joint of b1fl/fl-Prx1crelow mice compared to control.

Figure 2: Murine hip injury model

 

A.      Hip cartilage avulsion and subsequent culturing of the explants for gene and protein expression analyses. (fn-femoral neck; hc-hip cartilage).

 

B.      b1 integrins-dependent changes in gene expression following injury of murine hip cartilage. The expression of interleukin 1 alpha (IL1A) and prostaglandin-endoperoxide synthase 2 (PTGS2) are increased, while the expression of nitric oxide synthase 2 (NOS2) and interleukin 1 receptor-like 1 (IL1RL1) are decreased in b1fl/fl-Prx1crelow mice compared with wild type controls.



Figure 3: FGF-2 pre-treatment during monolayer expansion enhances adipose-derived mesenchymal stem cell (ADMSC) chondrogenesis in pellet culture.

 

A.      The experimental design.

 

B.      FGF-2 treatment of monolayer cultured ADMSCs increases the expression of the chondrogenic markers integrin alpha 10 (Itga10) and Sox9 under both normoxic and hypoxic conditions.

 

C.      Western blot demonstrates that FGF-2 elevates a10 protein level in ADMSCs.

 

D.      Safranin Orange staining of pellet sections after stimulation with TGFb1 and BMP2 (TB) shows increased chondrogenesis of FGF-2 pre-treated ADMSCs compared to control (C) and only TGFb1 (T) stimulated pellets.

 

 

E.       Immunohistochemistry demonstrates high collagen II (COL2A1) and aggrecan (AGC1) protein expression in FGF-2 pre-treated pellets upon chondrogenic differentiation in pellet culture.