Probes for TGF-β

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.

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.