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Influence of extracellular matrix components on the expression of integrins and regeneration of adult retinal ganglion cells.

Vecino E, Heller JP, Veiga-Crespo P, Martin KR, Fawcett JW - PLoS ONE (2015)

Bottom Line: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions.We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites.Adult rat RGCs can survive and grow in the presence of different ECM tested.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Histology, University of the Basque Country, UPV/EHU, Leioa, Vizcaya, Spain; John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Purpose: Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites?

Methods: Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against βIII-Tubulin to identify the RGCs, and antibodies against the integrin subunits: αV, α1, α3, α5, β1 or β3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed.

Results: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly α1β1 or α3β1 on L, α1β1 on CI and CIV, and α5β3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK).

Conclusions: Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

No MeSH data available.


Related in: MedlinePlus

Integrin distribution within the retina.The images show the integrin label in green, βIII-Tubulin in red and DAPI staining in blue. Distribution of αV, α1, α3, α5 in A, B,C,D (scale bar in D for all α) and labelling for β1 and β3 in E, F (scale bar in F for both β). Arrowheads point to the retinal pigment epithelium. The α integrins and β1 were mainly located in the retinal ganglion cell layer, endothelium of the vessels located closed to the RGCs wile β3 was found mainly within the inner plexiform layer. The retina layers are indicated in E. Outer segments (OS), outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL).
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pone.0125250.g003: Integrin distribution within the retina.The images show the integrin label in green, βIII-Tubulin in red and DAPI staining in blue. Distribution of αV, α1, α3, α5 in A, B,C,D (scale bar in D for all α) and labelling for β1 and β3 in E, F (scale bar in F for both β). Arrowheads point to the retinal pigment epithelium. The α integrins and β1 were mainly located in the retinal ganglion cell layer, endothelium of the vessels located closed to the RGCs wile β3 was found mainly within the inner plexiform layer. The retina layers are indicated in E. Outer segments (OS), outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL).

Mentions: The integrin subunits that are implicated in the interaction with the substrata studied were also examined in retina sections. The αV, α1, α3, α5, β1 and β3 integrin subunits were present in the RGC layer with different intensities (Fig 3). α5 and β3 staining was intense in the inner plexiform layer of the retina. In addition to the RGCs, α3 was very intensely stained in retinal pigment epithelial cells and in the endothelia of the blood vessels located in the inner most part of the retina. β1 was found in the RGCs and axons wile β3 was located mainly in the inner plexiform layer (IPL) of the retina (Fig 3).


Influence of extracellular matrix components on the expression of integrins and regeneration of adult retinal ganglion cells.

Vecino E, Heller JP, Veiga-Crespo P, Martin KR, Fawcett JW - PLoS ONE (2015)

Integrin distribution within the retina.The images show the integrin label in green, βIII-Tubulin in red and DAPI staining in blue. Distribution of αV, α1, α3, α5 in A, B,C,D (scale bar in D for all α) and labelling for β1 and β3 in E, F (scale bar in F for both β). Arrowheads point to the retinal pigment epithelium. The α integrins and β1 were mainly located in the retinal ganglion cell layer, endothelium of the vessels located closed to the RGCs wile β3 was found mainly within the inner plexiform layer. The retina layers are indicated in E. Outer segments (OS), outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446304&req=5

pone.0125250.g003: Integrin distribution within the retina.The images show the integrin label in green, βIII-Tubulin in red and DAPI staining in blue. Distribution of αV, α1, α3, α5 in A, B,C,D (scale bar in D for all α) and labelling for β1 and β3 in E, F (scale bar in F for both β). Arrowheads point to the retinal pigment epithelium. The α integrins and β1 were mainly located in the retinal ganglion cell layer, endothelium of the vessels located closed to the RGCs wile β3 was found mainly within the inner plexiform layer. The retina layers are indicated in E. Outer segments (OS), outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL).
Mentions: The integrin subunits that are implicated in the interaction with the substrata studied were also examined in retina sections. The αV, α1, α3, α5, β1 and β3 integrin subunits were present in the RGC layer with different intensities (Fig 3). α5 and β3 staining was intense in the inner plexiform layer of the retina. In addition to the RGCs, α3 was very intensely stained in retinal pigment epithelial cells and in the endothelia of the blood vessels located in the inner most part of the retina. β1 was found in the RGCs and axons wile β3 was located mainly in the inner plexiform layer (IPL) of the retina (Fig 3).

Bottom Line: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions.We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites.Adult rat RGCs can survive and grow in the presence of different ECM tested.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Cell Biology and Histology, University of the Basque Country, UPV/EHU, Leioa, Vizcaya, Spain; John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Purpose: Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites?

Methods: Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against βIII-Tubulin to identify the RGCs, and antibodies against the integrin subunits: αV, α1, α3, α5, β1 or β3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed.

Results: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly α1β1 or α3β1 on L, α1β1 on CI and CIV, and α5β3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK).

Conclusions: Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

No MeSH data available.


Related in: MedlinePlus