Limits...
Comparative expression analysis of aquaporin-5 (AQP5) in keratoconic and healthy corneas.

Garfias Y, Navas A, Pérez-Cano HJ, Quevedo J, Villalvazo L, Zenteno JC - Mol. Vis. (2008)

Bottom Line: AQP5 mRNA was expressed with no significant differences between KC and non-KC tissues.Moreover, AQP5 protein expression analysis did not reveal differences in protein levels and/or cell location among KC and non-KC tissues.Our results do not support a role for AQP5 in KC etiopathogeny or as a disease marker.

View Article: PubMed Central - PubMed

Affiliation: Research Unit, Institute of Ophthalmology Conde de Valenciana, Mexico City, Mexico. yogarfias@institutodeoftalmologia.org

ABSTRACT

Purpose: Keratoconus (KC) is a common progressive corneal disease characterized by excessive stromal thinning, central or paracentral conical protrusion, and disruptions in Bowman's layer. The etiology of KC is largely unknown, and a combination of genetic and environmental factors is believed to play a role in the origin of the disease. Recently, the absence of transcripts of the water channel, aquaporin-5 (AQP5), was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR) in KC tissues and was proposed as a possible marker for KC. In this study, we sought to evaluate AQP5 mRNA and protein expression in KC and non-KC corneal tissues using a combination of techniques.

Methods: A total of 69 samples of corneal tissue were analyzed including 39 corneal buttons from patients with advanced KC, 16 samples of non-KC corneal epithelium belonging to patients who underwent surface refractive surgery, 12 sclerocorneal rims obtained from healthy donor subjects, and two healthy corneal buttons. Determination of AQP5 transcript and protein expression patterns was performed by means of real time RT-PCR, immunohistochemistry, immunocytochemistry, and flow cytometry methods. Cell culture was performed to identify AQP5 protein expression in KC epithelial cells.

Results: AQP5 mRNA was expressed with no significant differences between KC and non-KC tissues. Moreover, AQP5 protein expression analysis did not reveal differences in protein levels and/or cell location among KC and non-KC tissues. Interestingly, AQP5 expression continues for up to 21 days in the isolated KC corneal epithelial cells.

Conclusions: Our results do not support a role for AQP5 in KC etiopathogeny or as a disease marker. Genetic background differences or a distinct pathogenetic KC cascade specific to the analyzed population could account for the dissimilarities observed in KC-related AQP5 expression.

Show MeSH

Related in: MedlinePlus

AQP5 real time polymerase chain reaction comparative analysis in keratoconus and non-keratoconus corneal tissue. PCR products from both samples KC and non-KC corneal tissue are shown. Curves A and B correspond to the PGK-1 gene amplification from healthy and KC corneal tissue, respectively. Curves C and D correspond to AQP5 amplification from healthy and KC corneal tissue, respectively. The x-axis represents the number of cycles, and the y-axis represents normalized fluorescence. The graph is a representative example of five separate assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2358923&req=5

f2: AQP5 real time polymerase chain reaction comparative analysis in keratoconus and non-keratoconus corneal tissue. PCR products from both samples KC and non-KC corneal tissue are shown. Curves A and B correspond to the PGK-1 gene amplification from healthy and KC corneal tissue, respectively. Curves C and D correspond to AQP5 amplification from healthy and KC corneal tissue, respectively. The x-axis represents the number of cycles, and the y-axis represents normalized fluorescence. The graph is a representative example of five separate assays.

Mentions: Band intensities of PCR products were compared by densitometric analysis (Figure 1). No statistically significant differences (p>0.05) were observed between KC (n=10) and non-KC corneal tissue (n=14). To determine if differences in the amplification reaction due to distinct initial RNA concentration existed, real time PCR was performed, normalizing with a constitutive PGK-1 gene. The average relative increment of samples from both groups ranged between 1.01 and 1.05 with no statistically significant differences (p>0.05; Figure 2).


Comparative expression analysis of aquaporin-5 (AQP5) in keratoconic and healthy corneas.

Garfias Y, Navas A, Pérez-Cano HJ, Quevedo J, Villalvazo L, Zenteno JC - Mol. Vis. (2008)

AQP5 real time polymerase chain reaction comparative analysis in keratoconus and non-keratoconus corneal tissue. PCR products from both samples KC and non-KC corneal tissue are shown. Curves A and B correspond to the PGK-1 gene amplification from healthy and KC corneal tissue, respectively. Curves C and D correspond to AQP5 amplification from healthy and KC corneal tissue, respectively. The x-axis represents the number of cycles, and the y-axis represents normalized fluorescence. The graph is a representative example of five separate assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2358923&req=5

f2: AQP5 real time polymerase chain reaction comparative analysis in keratoconus and non-keratoconus corneal tissue. PCR products from both samples KC and non-KC corneal tissue are shown. Curves A and B correspond to the PGK-1 gene amplification from healthy and KC corneal tissue, respectively. Curves C and D correspond to AQP5 amplification from healthy and KC corneal tissue, respectively. The x-axis represents the number of cycles, and the y-axis represents normalized fluorescence. The graph is a representative example of five separate assays.
Mentions: Band intensities of PCR products were compared by densitometric analysis (Figure 1). No statistically significant differences (p>0.05) were observed between KC (n=10) and non-KC corneal tissue (n=14). To determine if differences in the amplification reaction due to distinct initial RNA concentration existed, real time PCR was performed, normalizing with a constitutive PGK-1 gene. The average relative increment of samples from both groups ranged between 1.01 and 1.05 with no statistically significant differences (p>0.05; Figure 2).

Bottom Line: AQP5 mRNA was expressed with no significant differences between KC and non-KC tissues.Moreover, AQP5 protein expression analysis did not reveal differences in protein levels and/or cell location among KC and non-KC tissues.Our results do not support a role for AQP5 in KC etiopathogeny or as a disease marker.

View Article: PubMed Central - PubMed

Affiliation: Research Unit, Institute of Ophthalmology Conde de Valenciana, Mexico City, Mexico. yogarfias@institutodeoftalmologia.org

ABSTRACT

Purpose: Keratoconus (KC) is a common progressive corneal disease characterized by excessive stromal thinning, central or paracentral conical protrusion, and disruptions in Bowman's layer. The etiology of KC is largely unknown, and a combination of genetic and environmental factors is believed to play a role in the origin of the disease. Recently, the absence of transcripts of the water channel, aquaporin-5 (AQP5), was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR) in KC tissues and was proposed as a possible marker for KC. In this study, we sought to evaluate AQP5 mRNA and protein expression in KC and non-KC corneal tissues using a combination of techniques.

Methods: A total of 69 samples of corneal tissue were analyzed including 39 corneal buttons from patients with advanced KC, 16 samples of non-KC corneal epithelium belonging to patients who underwent surface refractive surgery, 12 sclerocorneal rims obtained from healthy donor subjects, and two healthy corneal buttons. Determination of AQP5 transcript and protein expression patterns was performed by means of real time RT-PCR, immunohistochemistry, immunocytochemistry, and flow cytometry methods. Cell culture was performed to identify AQP5 protein expression in KC epithelial cells.

Results: AQP5 mRNA was expressed with no significant differences between KC and non-KC tissues. Moreover, AQP5 protein expression analysis did not reveal differences in protein levels and/or cell location among KC and non-KC tissues. Interestingly, AQP5 expression continues for up to 21 days in the isolated KC corneal epithelial cells.

Conclusions: Our results do not support a role for AQP5 in KC etiopathogeny or as a disease marker. Genetic background differences or a distinct pathogenetic KC cascade specific to the analyzed population could account for the dissimilarities observed in KC-related AQP5 expression.

Show MeSH
Related in: MedlinePlus