Novel Ex Vivo Culture Method for the Study of Dupuytren's Disease: Effects of TGFβ Type 1 Receptor Modulation by Antisense Oligonucleotides

Abstract

Dupuytren's disease (DD) is a benign fibroproliferative disease of the hand. It is characterized by the excessive production of extracellular matrix (ECM) proteins, which form a strong fibrous tissue between the handpalm and fingers, permanently disrupting the fine movement ability. The major contractile element in DD is the myofibroblast (MFB). This cell has both fibroblast and smooth muscle cell-type characteristics and causes pathological collagen deposition. MFBs generate contractile forces that are transmitted to the surrounding collagen matrix. Μajor profibrotic factors are members of the transforming growth factor-β (TGFβ) pathway which directly regulate the expression levels of several fibrous proteins such as collagen type 1, type 3, and α-smooth muscle actin. Molecular modulation of this signaling pathway could serve as a therapeutic approach. We, therefore, have developed an ex vivo “clinical trial” system to study the properties of intact, patient-derived resection specimens. In these culture conditions, Dupuytren's tissue retains its three-dimensional (3D) structure and viability. As a novel antifibrotic therapeutic approach, we targeted TGFβ type 1 receptor (also termed activin receptor-like kinase 5) expression in cultured Dupuytren's specimens by antisense oligonucleotide-mediated exon skipping. Antisense oligonucleotides targeting activin receptor-like kinase 5 showed specific reduction of ECM and potential for clinical application.

CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer

“…Zebrafish embryos (including nonimplanted controls) were maintained at 33 °C, to compromise between the optimal temperature requirements for fish and mammalian cells. 43 , 44 Data are representative of at least two independent and blind experiments with ⩾30 embryos per group. Experiments with survival rate of control group lower than 80% were discarded.…”

Developing an animal model of Dupuytren’s disease by orthotopic transplantation of human fibroblasts into athymic rat

“…Recently, Karkampouna et al (2014) [ 30 ] have reported an ex vivo “clinical trial” system by culturing DD specimens after surgical removal, observing that the tissue so cultured preserved the pathological status of the disease by maintaining the complex organization of the ECM and its three-dimensional (3D) structure. The authors also demonstrated that targeting TGFβ 1 receptor expression in cultured DD specimens by antisense oligonucleotide-mediated exon skipping showed a specific reduction in fibrotic proteins and caused a reversal of the fibrotic phenotype.…”

Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

“…This system has helped to overcome the limiting approach of 2D fibroblast cultures and to define a role for the partial down regulation, achieved by exon skipping, of TGFβ pathway activation in mediating fibrosis 5 .…”

“…We have developed a method to culture ex vivo human resection specimens from DD patients to study the interaction between myofibroblasts and the surrounding ECM 5,6 . The study of DD connective tissue fibrosis as well as other fibrotic diseases relies on histopathological analysis of the excised surgical specimens, isolation of fibroblasts from the tissue and establishment of primary cultures or cell sorting procedures.…”

“…The cultures are maintained for a maximum of ten days after which the tissue is fixed in 4% paraformaldehyde (PFA), processed through 30% sucrose solution, embedded in O.C.T. compound and stored at -80 °C, as previously described 5 .…”