Activation of PPARs α, β/δ, and γ Impairs TGF-β1-Induced Collagens' Production and Modulates the TIMP-1/MMPs Balance in Three-Dimensional Cultured Chondrocytes

Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs) α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1-) induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP-) 1/Matrix Metalloproteinase (MMP) balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9) or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes

Modulation of Hepatocarcinoma Cell Morphology and Activity by Parylene-C Coating on PDMS

BACKGROUND: The ability to understand and locally control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology as well as tissue engineering research. We present parylene-C (ParC) deposited on polydimethylsiloxane (PDMS) as a new substratum for in vitro advanced cell culture in the case of Human Hepatocarcinoma (HepG2) cells. PRINCIPAL FINDINGS: Our findings establish that the intrinsic properties of ParC-coated PDMS (ParC/PDMS) influence and modulate initial extracellular matrix (ECM; here, type-I collagen) surface architecture, as compared to non-coated PDMS substratum. Morphological changes induced by the presence of ParC on PDMS were shown to directly affect liver cell metabolic activity and the expression of transmembrane receptors implicated in cell adhesion and cell-cell interaction. These changes were characterized by atomic force microscopy (AFM), which elucidated differences in HepG2 cell adhesion, spreading, and reorganization into two- or three-dimensional structures by neosynthesis of ECM components. Local modulation of cell aggregation was successfully performed using ParC/PDMS micropatterns constructed by simple microfabrication. CONCLUSION/SIGNIFICANCE: We demonstrated for the first time the modulation of HepG2 cells' behavior in relation to the intrinsic physical properties of PDMS and ParC, enabling the local modulation of cell spreading in a 2D or 3D manner by simple microfabrication techniques. This work will provide promising insights into the development of cell-based platforms that have many applications in the field of in vitro liver tissue engineering, pharmacology and therapeutics

Etude des potentialités des agonistes des récepteurs activés par les proliférateurs de péroxysomes (PPAR) sur les réponses chondrocytaires induites par le transforming growth factor beta (TGF-b)

[Résumé en français] Considérant les effets bi-potentiels du TGF-b1 et les effets anti-fibrotiques des agonistes PPAR dans divers tissus d'origine non articulaire, le but de cette thèse est d'étudier la capacité de certains agonistes des trois isotypes PPAR (a, b/ et g) à contrecarrer les réponses métaboliques induites par le TGF-b1 dans les chondrocytes de rat. Dans ce rapport, nous démontrons que l'alginate constitue un système de culture optimal. De plus, au moyen d'un panel de gènes cibles spécifiques des PPAR, nous démontrons que le Wy14643, le GW501516, et la rosiglitazone induisent préférentiellement des réponses dépendantes de PPARa, b/ et g respectivement, alors que la pioglitazone induit des réponses dépendantes de PPAR" g>a". En présence de TGF-b1, nous montrons que les tous agonistes réduisent la biosynthèse des protéoglycanes ou des collagènes via une inhibition précoce et/ou tardive des voies dépendantes de Smad2/3, ERK1/2 et PGE2. Nous démontrons aussi que les agonistes régulent le turnover de la matrice extracellulaire et inhibent les activités cruciales pour la biosynthèse des composants matriciels en restaurant l'activité des MMPs. Ces régulations sont dépendantes du profil de sélectivité de chaque agoniste. Ceci suggère que la part de chondroprotection, fournie par les agonistes via une diminution de la production des cytokines pro-inflammatoires, semble être diminuée par une réduction de la sensibilité des chondrocytes à certains facteurs de croissance. Nos résultats sous-tendent une complexité des mécanismes mis en jeu qui nécessitent davantage d'investigations vis-à-vis du rôle du TGF-b1 et des PPAR au sein même de l'articulation.[Résumé en anglais] When considering the bipotential effects of TGF-b1 and the antifibrotic effect associated with PPAR activation in many organs, we investigated the ability of synthetic PPAR agonists of the three isotypes (a, b/ and g) to affect TGF-b1-induced metabolism responses in rat chondrocytes. ln our report, we choose alginate as the optimal culture system. ln addition, using a panel of PPAR target genes, we demonstrate that Wy14643, GW501516 and rosiglitazone induced preferentially a-, b/ - and g-dependent responses respectively, while pioglitazone induced "g >a responses. Moreover, the stimulating effect of TGF-b1 on proteoglycans or collagens synthesis is reduced dramatically by all PPAR agonists via early and/or tardive inhibitions of Smads, ERK1/2 and/or PGE2 pathways. This could suggest that PPAR agonists inhibit crucial activities and regulate extracellular matrix (ECM) turnover biosynthesis by restoring MMPs activities. These regulations were found to be dependant of the selectivity profile of each agonist in rat chondrocytes. Taken all together, our data demonstrate that PPAR agonists reduce TGF-b1-induced responses in rat chondrocytes and may favour an altered repair capacity of cartilage in response to some articular joint growth factors, like TGF-b. Due to the complexity of the involved mechanisms related to the physiopathological role of TGF-b1 and to the lack of chondroprotection provided by PPAR agonists in cartilage, further studies are required.NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells.

International audienceIn response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities

Investigation of ifosfamide nephrotoxicity induced in a liver-kidney co-culture biochip.

International audienceIn this article, we present a liver-kidney co-culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver-kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG-MDCK) when compared to untreated co-cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3-dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG-MDCK co-cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG-MDCK co-culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3-dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG-MDCK co-culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver-kidney micro fluidic co-culture model using HepaRG-MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a-MDCK model. This study demonstrates the interest in the development of systemic organ-organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods