Probes for TGF-β

Abstract

BACKGROUND:

Low back pain is one of the most common ailments in western countries afflicting more than 80% of the population and the main cause is considered to be degeneration of intervertebral discs (IVDs). IL-1β is a vital inflammatory cytokine found in abundance in degenerated disc environment whereas BMP-3 is believed to promote chondrogenesis through TGF-β pathway.

AIM:

The aim was to study the effects of BMP-3, IL-1β and combination (pre-treatment with IL-1β) on hMSCs encapsulated in PuraMatrix™ hydrogel (Phg) especially in the absence of TGF-β in order to investigate the proliferation, and differentiation ability of hMSCs over 28 days period.

METHOD:

100µL of hMSCs cell suspension was encapsulated between two layers of 100 µL hydrogels forming a sandwich-like structure. The encapsulated hMSCs were cultured in two sets of media, chondrogenic (C) and non-chondrogenic (nC) media along with addition of BMP-3 (10ng/mL) and IL-1β (10ng/mL). To study the combined effects of BMP-3 and IL-1β, the encapsulated hMSCs were first pre-treated with relevant media containing IL-1β for 24 hours, and then the media was replaced by media containing BMP-3 for the remaining experimental time period. IL-1β pre-treatment was carried out in both C and nC media. The samples were collected at day 7, 14, and 28.

RESULTS:

Proliferation and differentiation of hMSCs into chondrocyte-like cells was observed in all samples. Proteoglycans accumulation was observed in pre-treatment samples in C media. The protein and gene expression of Sox-9 and COL2A1 respectively, showed the occurrence of chondrogenesis in all samples.

CONCLUSION:

High cell viability, proliferation and differentiation was achieved in this in vitro model confirming that BMP-3 alone in the absence of TGF-β could drive hMSCs into chondrogenic lineage. Pre-treatment with IL-1β followed by BMP-3 stimulation resulted in high proteoglycans accumulation compared to stimulation with growth factors or cytokine alone. This suggests that pre-treatment with a pro-inflammatory cytokine before driving them into a chondrogeneic lineage might be of importance also in vivo.

Abstract

Purpose/Aim: Many genes have been associated with primary open-angle glaucoma (POAG). Knowing exactly where they are expressed in the eye helps to unravel POAG pathology and to select optimal targets for intervention. We investigated whether RNA in-situ hybridization (RNA-ISH) is a convenient technique to obtain detailed pan-ocular expression data of these genes. We tested this for four diverse candidate POAG genes, selected because of unclear ocular distribution (F5 and Dusp1) and relevance for potential new therapies (Tnf, Tgfβr3). Optn, a POAG gene with well-known ocular expression pattern served as control.

METHODS:

We made a list of candidate glaucoma genes reported in genetic studies. A table of their ocular expression at the tissue level was compiled using publicly available microarray data (the ocular tissue database). To add cellular detail we performed RNA-ISH for Optn, Tnf, Tgfβr3, F5, and Dusp1 on eyes of healthy, 2-month-old, pigmented and albino mice.

RESULTS:

Expression of the Optn control matched with published immunohistochemistry data. Ocular expression of Tnf was generally low, with patches of higher Tnf expression, superficially in the corneal epithelium. F5 had a restricted expression pattern with high expression in the non-pigmented ciliary body epithelium and moderate expression in the peripapillary region. Tgfβr3 and Dusp1 showed ubiquitous expression.

CONCLUSIONS:

RNA-ISH is a suitable technique to determine the ocular expression pattern of POAG genes, adding meaningful cellular detail to existing microarray expression data. For instance, the high expression of F5 in the non-pigmented ciliary body epithelium suggests a role of this gene in aqueous humor dynamics and intraocular pressure. In addition, the ubiquitous expression of Tgfβr3 has implications for designing TGF-β related glaucoma therapies, with respect to side effects. Creating pan-ocular expression maps of POAG genes with RNA-ISH will help to identify POAG pathways in specific cell types and to select targets for drug development.