Limits...
Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture.

Satish L, LaFramboise WA, Johnson S, Vi L, Njarlangattil A, Raykha C, Krill-Burger JM, Gallo PH, O'Gorman DB, Gan BS, Baratz ME, Ehrlich GD, Kathju S - BMC Med Genomics (2012)

Bottom Line: Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses.These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells.In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Division of Plastic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA. latsat@hotmail.com.

ABSTRACT

Background: Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC.

Methods: To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram.

Results: We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder.

Conclusions: These data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

Show MeSH

Related in: MedlinePlus

Real time RT-PCR Quantitation of ANGPTL7, LAMA5 and SHROOM2 in CT-, PF-, and DC-derived cells. Differential expression of (a) ANGPTL7, (b) LAMA5 and (c) SHROOM2 was directly confirmed by qRT-PCR in CT-, PF- and DC-derived fibroblasts. Values are mean ± SEM of two independent experiments performed in triplicate. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ Ct values between CT-, PF- and DC- derived fibroblasts. p < 0.05 was considered significant
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3375203&req=5

Figure 6: Real time RT-PCR Quantitation of ANGPTL7, LAMA5 and SHROOM2 in CT-, PF-, and DC-derived cells. Differential expression of (a) ANGPTL7, (b) LAMA5 and (c) SHROOM2 was directly confirmed by qRT-PCR in CT-, PF- and DC-derived fibroblasts. Values are mean ± SEM of two independent experiments performed in triplicate. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ Ct values between CT-, PF- and DC- derived fibroblasts. p < 0.05 was considered significant

Mentions: Quantitative real time RT-PCR was performed on the same RNA extracted for the microarray analysis on select genes to validate the microarray results (shown in Figure 5). The genes selected for such confirmation have not previously been reported to be differentially expressed in CT-, PF- or DC-derived fibroblasts. Angiopoietin-like7 mRNA (ANGPTL7) was significantly decreased in DC-derived fibroblasts compared to both PF- and CT- derived cells, amongst which no significant difference was seen (Figure 6a). LAMA5 mRNA was similarly least in DC-derived cells, but was also significantly less in PF- compared to DC-derived cells (Figure 6b). In contrast, Shroom2 message was dramatically increased in DC-derived fibroblasts compared to both CT- and PF-derived fibroblasts (Figure 6c). As can be seen by comparing the gene expression patterns in Figures 5 and 6, in each case quantitative RT-PCR findings closely matched the results from microarray analysis.


Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture.

Satish L, LaFramboise WA, Johnson S, Vi L, Njarlangattil A, Raykha C, Krill-Burger JM, Gallo PH, O'Gorman DB, Gan BS, Baratz ME, Ehrlich GD, Kathju S - BMC Med Genomics (2012)

Real time RT-PCR Quantitation of ANGPTL7, LAMA5 and SHROOM2 in CT-, PF-, and DC-derived cells. Differential expression of (a) ANGPTL7, (b) LAMA5 and (c) SHROOM2 was directly confirmed by qRT-PCR in CT-, PF- and DC-derived fibroblasts. Values are mean ± SEM of two independent experiments performed in triplicate. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ Ct values between CT-, PF- and DC- derived fibroblasts. p < 0.05 was considered significant
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375203&req=5

Figure 6: Real time RT-PCR Quantitation of ANGPTL7, LAMA5 and SHROOM2 in CT-, PF-, and DC-derived cells. Differential expression of (a) ANGPTL7, (b) LAMA5 and (c) SHROOM2 was directly confirmed by qRT-PCR in CT-, PF- and DC-derived fibroblasts. Values are mean ± SEM of two independent experiments performed in triplicate. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ Ct values between CT-, PF- and DC- derived fibroblasts. p < 0.05 was considered significant
Mentions: Quantitative real time RT-PCR was performed on the same RNA extracted for the microarray analysis on select genes to validate the microarray results (shown in Figure 5). The genes selected for such confirmation have not previously been reported to be differentially expressed in CT-, PF- or DC-derived fibroblasts. Angiopoietin-like7 mRNA (ANGPTL7) was significantly decreased in DC-derived fibroblasts compared to both PF- and CT- derived cells, amongst which no significant difference was seen (Figure 6a). LAMA5 mRNA was similarly least in DC-derived cells, but was also significantly less in PF- compared to DC-derived cells (Figure 6b). In contrast, Shroom2 message was dramatically increased in DC-derived fibroblasts compared to both CT- and PF-derived fibroblasts (Figure 6c). As can be seen by comparing the gene expression patterns in Figures 5 and 6, in each case quantitative RT-PCR findings closely matched the results from microarray analysis.

Bottom Line: Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses.These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells.In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Division of Plastic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA. latsat@hotmail.com.

ABSTRACT

Background: Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC.

Methods: To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram.

Results: We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder.

Conclusions: These data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

Show MeSH
Related in: MedlinePlus