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Contains fulltext : 218131.pdf (Publisher’s version ) (Open Access) Cleft lip with or without cleft palate is a congenital deformity that occurs in about 1 of 700 newborns, affecting the dentition, bone, skin, muscles and mucosa in the orofacial region. A cleft can give rise to problems with maxillofacial growth, dental development, speech, and eating, and can also cause hearing impairment. Surgical repair of the lip may lead to impaired regeneration of muscle and skin, fibrosis, and scar formation. This may result in hampered facial growth and dental development affecting oral function and lip and nose esthetics. Therefore, secondary surgery to correct the scar is often indicated. We will discuss the molecular and cellular pathways involved in facial and lip myogenesis, muscle anatomy in the normal and cleft lip, and complications following surgery. The aim of this review is to outline a novel molecular and cellular strategy to improve musculature and skin regeneration and to reduce scar formation following cleft repair. Orofacial clefting can be diagnosed in the fetus through prenatal ultrasound screening and allows planning for the harvesting of umbilical cord blood stem cells upon birth. Tissue engineering techniques using these cord blood stem cells and molecular targeting of inflammation and fibrosis during surgery may promote tissue regeneration. We expect that this novel strategy improves both muscle and skin regeneration, resulting in better function and esthetics after cleft repair.

Chronic kidney disease (CKD) is a major global health concern and renal fibrosis is an integral part of the pathophysiological mechanism underlying disease progression. In CKD patients, the majority of metabolic pathways are in disarray and perturbations in enzyme activity most likely contribute to the wide variety of comorbidities observed in these patients. To illustrate, catabolism of tryptophan by indoleamine 2,3-dioxygenase (IDO) gives rise to numerous biologically active metabolites implicated in CKD progression. Here, we evaluated the effect of antagonizing IDO on renal fibrogenesis. To this end, we antagonized IDO using 1-methyl-D-tryptophan (1-MT) and BMS-98620 in TGF-β-treated murine precision-cut kidney slices (mPCKS) and in mice subjected to unilateral ureteral obstruction (UUO). The fibrotic response was evaluated on both the gene and protein level using qPCR and western blotting. Our results demonstrated that treatment with 1-MT or BMS-985205 markedly reduced TGF-β-mediated fibrosis in mPCKS, as seen by a decreased expression of collagen type 1, fibronectin, and α-smooth muscle actin. Moreover, IDO protein expression clearly increased following UUO, however, treatment of UUO mice with either 1-MT or BMS-986205 did not significantly affect the gene and protein expression of the tested fibrosis markers. However, both inhibitors significantly reduced the renal deposition of collagen in UUO mice as shown by Sirius red and trichrome staining. In conclusion, this study demonstrates that IDO antagonism effectively mitigates fibrogenesis in mPCKS and reduces renal collagen accumulation in UUO mice. These findings warrant further research into the clinical application of IDO inhibitors for the treatment of renal fibrosis.

Background and Purpose Renal fibrosis plays an important role in the development and progression of chronic kidney disease (CKD). Clinical studies have shown that CKD progresses differently in males and females, which may be related to circulating levels of sex hormones. In this study, we investigated the effect of tamoxifen (TAM), a selective estrogen receptor modulator (SERM), on renal fibrosis in male and female rats subjected to unilateral ureteral obstruction (UUO) and human precision-cut kidney slices (PCKS). Experimental Approach Female, ovariectomized female (OVX), and male rats were subjected to 7 days of UUO and treated with TAM by oral gavage. Moreover, we studied individual responses to TAM treatment in PCKS prepared from female and male patients. In all models, the expression of fibrosis markers was examined by western blot, qPCR, and immunohistochemistry. Key Results TAM decreased the expression of fibronectin, α-smooth muscle actin, and collagen-1 and -3 in female, OVX, and male rats. In addition, TAM mitigated TGF‐β-induced fibrosis in human PCKS, irrespective of sex, yet interindividual differences in treatment response were observed. Conclusion and Implications TAM ameliorates renal fibrosis in males and females, although we did observe sex differences in drug response. These findings warrant further research into the clinical applicability of TAM, or other SERMs, for the personalized treatment of renal disease.

Aim: Renal fibrosis plays a pivotal role in the development and progression of chronic kidney disease, which affects 10% of the adult population. Previously, it has been demonstrated that the cyclooxygenase-2 (COX-2)/prostaglandin (PG) system influences the progression of renal injury. Here, we evaluated the impact of butaprost, a selective EP2 receptor agonist, on renal fibrosis in several models of kidney injury, including human tissue slices. Methods: We studied the anti-fibrotic efficacy of butaprost using Madin-Darby Canine Kidney (MDCK) cells, mice that underwent unilateral ureteral obstruction and human precision-cut kidney slices. Fibrogenesis was evaluated on a gene and protein level by qPCR and Western blotting. Results: Butaprost (50 μM) reduced TGF-β-induced fibronectin (FN) expression, Smad2 phosphorylation and epithelial-mesenchymal transition in MDCK cells. In addition, treatment with 4 mg/kg/day butaprost attenuated the development of fibrosis in mice that underwent unilateral ureteral obstruction surgery, as illustrated by a reduction in the gene and protein expression of α-smooth muscle actin, FN and collagen 1A1. More importantly, a similar anti-fibrotic effect of butaprost was observed in human precision-cut kidney slices exposed to TGF-β. The mechanism of action of butaprost appeared to be a direct effect on TGF-β/Smad signalling, which was independent of the cAMP/PKA pathway. Conclusion: In conclusion, this study demonstrates that stimulation of the EP2 receptor effectively mitigates renal fibrogenesis in various fibrosis models. These findings warrant further research into the clinical application of butaprost, or other EP2 agonists, for the inhibition of renal fibrosis.

Chronic kidney disease (CKD) is associated with renal fibrosis, a pathological process that is characterized by excessive accumulation of extracellular matrix proteins resulting in loss of organ architecture and function. Currently, renal transplantation and dialysis are the sole treatment options for advanced CKD, yet these therapies have limited impact on fibrogenesis. Even though antifibrotic therapies are being developed, the search for effective antifibrotic drugs is being hampered by the lack of appropriate cell and animal models to study renal fibrosis. In vitro models lack cellular heterogeneity whereas in vivo models do not fully reflect human pathology. Precision-cut tissue slices, prepared from human or rodent tissue, provide a unique ex vivo model system that captures the complexity of organs, and they are widely used for ADME/Tox drug testing. Moreover, precision-cut kidney slices (PCKS) have been recently established as a useful model to study renal fibrosis. This review summarizes the currently available models for renal fibrosis, describes the wide array of possibilities with PCKS and shows its role in the search for antifibrotic drugs. (C) 2016 Elsevier B.V. All rights reserved.

Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and studied the efficacy of multiple putative antifibrotic compounds.Our results demonstrated that human PCLS remained viable for 48 h and the early onset of fibrosis was observed during culture, as demonstrated by an increased gene expression of Heat Shock Protein 47 (HSP47) and Pro-Collagen 1A1 (PCOL1A1) as well as increased collagen 1 protein levels. SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK) showed a marked decrease in HSP47 and PCOL1A1 gene expression, whereas specific inhibitors of Smad 3 and Rac-1 showed no or only minor effects. Regarding the studied antifibrotics, gene levels of HSP47 and PCOL1A1 could be down-regulated with sunitinib and valproic acid, while PCOL1A1 expression was reduced following treatment with rosmarinic acid, tetrandrine and pirfenidone. These results are in contrast with prior data obtained in rat PCLS, indicating that antifibrotic drug efficacy is clearly species-specific.Thus, human PCLS is a promising model for liver fibrosis. Moreover, MAPK signaling plays an important role in the onset of fibrosis in this model and transforming growth factor beta pathway inhibitors appear to be more effective than platelet-derived growth factor pathway inhibitors in halting fibrogenesis in PCLS. (C) 2016 Elsevier Ltd. All rights reserved.

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGF&beta, or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation, (2) expression of markers of TGF&beta, and PDGF signaling, and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1β, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regards to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis.

Renal fibrosis is characterized by progressive accumulation of extracellular matrix (ECM) proteins, resulting in loss of organ function and eventually requiring renal replacement therapy. Unfortunately, no efficacious treatment options are available to halt renal fibrosis and translational models to test pharmacological agents are not always representative. Here, we evaluated murine precision-cut kidney slices (mPCKS) as a promising ex vivo model of renal fibrosis in which pathophysiology as well as therapeutics can be studied. Unique to this model is the use of rodent as well as human renal tissue, further closing the gap between animal models and clinical trials. Kidneys from C57BL/6 mice were used to prepare mPCKS and slices were incubated up to 96h. Viability, morphology, gene expression of fibrosis markers (Col1a1, Acta2, Serpinh1, Fn1, and Pai-1), inflammatory markers (Il1b, Il6, Cxcl1), and protein expression (collagen type 1, α-smooth muscle actin, HSP47) were determined. Furthermore, to understand the role of the transforming-growth factor β (TGF-β) pathway in mPCKS, slices were incubated with a TGF-β receptor inhibitor (LY2109761) for 48 h. Firstly, viability and morphology revealed an optimal incubation period of 48 h. Secondly, we demonstrated an early inflammatory response in mPCKS, which was accompanied by subsequent spontaneous fibrogenesis. Finally, LY2109761 showed great antifibrotic capacity in mPCKS by decreasing fibrosis markers on mRNA level as well as by reducing HSP47 protein expression. To conclude, we here present an ex vivo model of renal fibrosis, which can be used to further unravel the mechanisms of renal fibrogenesis and to screen antifibrotic therapy efficacy.

Kidney allograft failure due to chronic injury/rejection remains the main cause of graft loss in renal transplant recipients (RTR). Here, we investigated whether specific biomarkers of extracellular matrix (ECM) turnover are associated with allograft function and chronic kidney disease (CKD) stage in RTR. Seventy-eight patients who attended the University Medical Center Groningen for a routine check-up after kidney transplantation were enrolled in the study. Plasma and/or 24h-urine samples were collected and specific matrix-metalloproteinase- generated neo-epitope fragments of collagens were measured by enzyme-linked immunosorbent assay. Our results demonstrated that urinary levels of C3M, a marker for collagen type III degradation, correlated with estimated glomerular filtration rate (eGFR; r = 0.58, p

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Chronic kidney disease (CKD) is a major global health concern, and the uremic state is highly associated with fibrogenesis in several organs and tissues. Fibrosis is characterized by excessive production and deposition of extracellular matrix proteins with a detrimental impact on organ function. Another key feature of CKD is the retention and subsequent accumulation of solutes that are normally cleared by the healthy kidney. Several of these uremic retention solutes, including indoxyl sulfate and p-cresyl sulfate, have been suggested to be CKD-specific triggers for the development and perpetuation of fibrosis. The purpose of this brief review is to gather and discuss the current body of evidence linking uremic retention solutes to the fibrotic response during CKD, with a special emphasis on the pathophysiological mechanisms in the kidney.

Fibrosis is a common endpoint of a multiplicity of chronic diseases resulting in scarring and loss of organ function. To improve and accelerate antifibrotic drug discovery, there is an urgent need for reliable and reproducible human in vitro methods that also include the cellular diversity that epitomize specific organs. The objective of our study was to investigate fibrogenesis in precision-cut tissue slices (PCTS) from human liver and kidney. Moreover, we strove to elucidate the effect of imatinib - a promising antifibrotic compound in rat - in the early stages of liver fibrosis. PCTS were successfully prepared from human livers and kidneys, and cultured up to 72 hours. Viability of PCTS was assessed by the ATP content of the slices. Furthermore, gene expression of several fibrosis markers was determined. The antifibrotic effect of imatinib was studied at the maximal non-toxic concentrations. Both liver and renal PCTS were viable up to 72 hours. Furthermore, after 48 hours of incubation, the early onset of fibrosis was observed in liver PCTS, as demonstrated by an increased expression of Pro-Collagen 1A1 and Heat Shock Protein 47. Moreover, treatment with imatinib for 48 hours did neither impact the gene expression of fibrosis markers nor the protein expression of collagen I in human liver PCTS. Which is in stark disparity to the antifibrotic effects observed in animal models. The gene expression profile of renal PCTS during incubation is currently being evaluated. Thus, human PCTS can be used to study the onset of fibrosis, are promising models to test the efficacy of antifibrotic compounds and can reveal species differences in fibrosis.

Background: Recently, we developed a novel model for drug screening by culturing ex-vivo precision-cut tissue slices (PCTS). The tissue slice consists of multiple cell types still in their normal matrix environment and structure provides numerous advantages compare to other models. Our objective was to use this model to investigate the effect of various potential compounds. In this study precision-cut intestinal slices (PCIS) were used to evaluate some transforming growth factor (TGF- β) and platelet-derived growth factor (PDGF) pathway inhibitors. TGF-β and PDGF are key cytokines in fibrotic and cancer diseases and are the main targets for treatment. Methods: Murine PCIS were cultured for 48 h in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using a variety of markers including (pro)collagen 1a1 (Col1a1), heat shock protein 47 (Hsp47), fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1). The effects of potential drugs mainly inhibiting the TGFb pathway i.e. valproic acid, tetrandrine, pirfenidone, SB203580 and LY2109761 as well as compounds mainly acting on the PDGF pathway i.e. imatinib, sorafenib and sunitinib were assessed in the model at maximum non-toxic concentrations. Results: Murine PCIS remained viable for 48 h and the onset of fibrosis was observed during culture, as demonstrated by an increased expression of, amongst others, Hsp47, Fn2 and Pai-1. Furthermore, TGFb1 stimulated fibrogenesis while PDGF had no effect. Regarding the tested antifibrotics, pirfenidone, LY2109761 and sunitinib had the most pronounced impact on fibrogenesis, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1a1, Hsp47, Fn2 and Pai-1 following treatment. Moreover, LY2109761 significantly reduced fibronectin protein expression in the presence of TGFb1. Conclusion: PCIS can successfully be used to test drug efficacy. Using the model we demonstrated that tetrandrine, pirfenidone, LY2109761 and sunitinib showed potential antifibrotic effects on a gene level, warranting further evaluation of these compounds for the treatment of fibrosis disease. By using tissue extracted from patient, PCIS could also be a promising model to screen drug for personalized treatment in fibrotic and cancer disease.

Contains fulltext : 139044.pdf (Publisher’s version ) (Closed access)

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