scholarly journals Predictive Value of Precision-Cut Kidney Slices as an Ex Vivo Screening Platform for Therapeutics in Human Renal Fibrosis

Pharmaceutics ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 459 ◽  
Author(s):  
Emilia Bigaeva ◽  
Nataly Puerta Cavanzo ◽  
Elisabeth G. D. Stribos ◽  
Amos J. de Jong ◽  
Carin Biel ◽  
...  
Keyword(s):  
Animal Models ◽  
Renal Fibrosis ◽  
Ex Vivo ◽  
Collagen Type I ◽  
Type I ◽  
Preclinical Research ◽  
Predictive Tool ◽  
Predictive Capacity ◽  
Kidney Slices

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 TGFβ 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 TGFβ 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.

scholarly journals Oncofetal Protein CRIPTO Is Involved in Wound Healing and Fibrogenesis in the Regenerating Liver and Is Associated with the Initial Stages of Cardiac Fibrosis

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3325
Author(s):  
Sofia Karkampouna ◽  
Danny van der Helm ◽  
Mario Scarpa ◽  
Bart van Hoek ◽  
Hein W. Verspaget ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Mouse Models ◽  
Cardiac Fibrosis ◽  
Ex Vivo ◽  
Pressure Overload ◽  
Collagen Type I ◽  
Type I ◽  
Liver Fibrogenesis ◽  
End Stage

Oncofetal protein, CRIPTO, is silenced during homeostatic postnatal life and often re-expressed in different neoplastic processes, such as hepatocellular carcinoma. Given the reactivation of CRIPTO in pathological conditions reported in various adult tissues, the aim of this study was to explore whether CRIPTO is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of CRIPTO expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesis. Circulating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and 1 year after LT. The expression of CRIPTO and fibrotic markers (αSMA, collagen type I) was determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure, and in mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4), were evaluated for CRIPTO expression and fibrotic markers. CRIPTO was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for CRIPTO and fibrotic markers in three models of heart injury following myocardial infarction, pressure overload, and ex vivo induced fibrosis. Patients with end-stage liver cirrhosis showed elevated CRIPTO levels in plasma, which decreased 1 year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of CRIPTO in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. CRIPTO expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, CRIPTO-positive cells were observed in damaged liver areas around the central vein, which preceded the expression of αSMA-positive stellate cells, i.e., mediators of fibrosis. In the chronic mouse models, the fibrosis and CRIPTO expression were still present after 11 weeks, whereas in the acute model the liver regenerated and the fibrosis and CRIPTO expression resolved. In vivo overexpression of CRIPTO in this model led to an increase in fibrotic markers, while blockage of CRIPTO secreted function inhibited the extent of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. CRIPTO expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction CRIPTO is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, CRIPTO expression decreased concomitantly with the αSMA expression. Temporal expression of CRIPTO in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, CRIPTO expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis. Our results are indicative for a functional role of CRIPTO in the induction of fibrogenesis as well as a potential target in the antifibrotic treatments and stimulation of tissue regeneration.

scholarly journals Ex vivo observation of granulocyte activity during thrombus formation

2020 ◽  
Author(s):  
Daria S. Morozova ◽  
Alexey A. Martyanov ◽  
Sergei I. Obydennyi ◽  
Julia-Jessica D. Korobkin ◽  
Alexey V. Sokolov ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Average Rate ◽  
Fluorescent Microscopy ◽  
Collagen Type I ◽  
Type I ◽  
Average Cell ◽  
Cell Velocity

AbstractInfiltration of growing thrombi by leukocytes, being the key part of the thromboinflammation, is well established in vivo. The study was aimed at the development of an ex vivo simulation of this phenomenon. Thrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips.Moving CD66b-positive cells (granulocytes) were observed in hirudinated or recalcified blood under low wall shear rate conditions (<200 s−1). These cells crawled around thrombi in a step-wise manner with an average rate of 70 nm/s. Pre-incubation of blood with leukocyte priming agents lead to a significant increase in average cell velocity. On the contrary, leukocytes from Wiskott-Aldrich syndrome patients demonstrated a 1.5-fold lower average velocity, in line with their impaired actin polymerization.Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. We conclude that the proposed ex vivo experimental setting allows us to observe granulocytes activity in near-physiological conditions.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Properties of a bovine collagen type I membrane for guided bone regeneration applications

e-Polymers ◽  
2021 ◽  
Vol 21 (1) ◽  
pp. 210-221
Author(s):  
Igor S. Brum ◽  
Carlos N. Elias ◽  
Jorge J. de Carvalho ◽  
Jorge L. S. Pires ◽  
Mario J. S. Pereira ◽  
...  
Keyword(s):  
Bone Regeneration ◽  
Collagen Type ◽  
Mechanical Stability ◽  
Collagen Type I ◽  
Guided Bone Regeneration ◽  
Bone Volume ◽  
Male Rats ◽  
Toxicity Tests ◽  
Type I

Abstract Dental implant treatment requires an available bone volume in the implantation site to ensure the implant’s mechanical stability. When the bone volume is insufficient, one must resort to surgical means such as guided bone regeneration (GBR). In GBR surgery, bone grafts and membranes are used. The objective of this work is to manufacture and characterize the in vitro and in vivo properties of resorbable collagen type I membranes (Green Membrane®) for GBR. Membrane surface morphology was characterized by SEM and roughness was measured using an interferometric noncontact 3D system. In vivo skin sensitization and toxicity tests have been performed on Wistar rats. Bone defects were prepared in 24 adult male rats, filled with biomaterials (Blue Bone® and Bio Oss®) and covered with collagen membranes to maintain the mechanical stability of the site for bone regeneration. The incisions were closed with simple stitches; and 60 days after the surgery, the animals were euthanized. Results showed that the analyzed membrane was homogeneous, with collagen fiber webs and open pores. It had no sign of cytotoxicity and the cells at the insertion site showed no bone morphological changes. There was no tissue reaction and no statistical difference between Blue Bone® and Bio Oss® groups. The proposed membrane has no cytotoxicity and displays a biocompatibility profile that makes it suitable for GBR.

scholarly journals Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

2021 ◽  
Author(s):  
Michel Haagdorens ◽  
Elle Edin ◽  
Per Fagerholm ◽  
Marc Groleau ◽  
Zvi Shtein ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Fibrils ◽  
Type I ◽  
Safe Alternative ◽  
Human Donor ◽  
Human Collagen ◽  
Donor Corneas

Abstract Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

RUNX2 promotes malignant progression in gastric cancer by regulating COL1A1

2021 ◽  
pp. 1-12
Author(s):  
Yanlei Li ◽  
Ran Sun ◽  
Xiulan Zhao ◽  
Baocun Sun
Keyword(s):  
Gastric Cancer ◽  
Human Cancer ◽  
Clinical Stage ◽  
Collagen Type I ◽  
Migration And Invasion ◽  
Type I ◽  
Aberrant Expression ◽  
Invasion And Migration ◽  
Protein Levels

BACKGROUND: Runt-related transcription factor 2 (RUNX2) is an important gene that has been implicated in the progression of human cancer. Aberrant expression of RUNX2 predicts gastric cancer (GC) metastasis. However, the molecular mechanism of RUNX2 remains unknown. OBJECTIVE: We hypothesize that RUNX2 promotes GC metastasis by regulating the extracellular matrix component collagen type I alpha 1 (COL1A1). METHODS: The GEPIA database and immunohistochemical staining of 60 GC tissues were used to analyse the correlations between RUNX2 or COL1A1 expression and clinicopathological features, and the Kaplan-Meier method was used to evaluate survival. RT-PCR, western blotting and immunofluorescence were used to detect RUNX2 and COL1A1 expression in GC cells. Migration and invasion assays were performed to assess the influence of RUNX2 and COL1A1 on metastasis. RESULTS: RUNX2 and COL1A1 were highly expressed at both the gene and protein levels in GC, and patients who were positive for RUNX2 and COL1A1 had shorter survival. RUNX2 and COL1A1 expression linearly correlated with each other (r= 0.15, p< 0.01) and with clinical stage and lymph node metastasis (p< 0.05). Overexpressing RUNX2in vitro enhanced COL1A1 expression and promoted GC cell invasion and migration, whereas COL1A1 knockdown inhibited the increase in cell metastatic capacity promoted by RUNX2. In vivo, GC cells overexpressing RUNX2 promoted lung metastasis, and the downregulation of COL1A1 reduced the metastasis promoted by RUNX2. CONCLUSIONS: RUNX2 may promote GC metastasis by regulating COL1A1. RUNX2/COL1A1 can be employed as a novel target for therapy in GC.

A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

2022 ◽  
Author(s):  
Michael Valente ◽  
Nils Collinet ◽  
Thien-Phong Vu Manh ◽  
Karima Naciri ◽  
Gilles Bessou ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Steady State ◽  
Mouse Model ◽  
Single Cell ◽  
Ex Vivo ◽  
High Specificity ◽  
Type I ◽  
Specific Expression ◽  
Physiological Functions

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

scholarly journals Precision-Cut Kidney Slices as a Tool to Understand the Dynamics of Extracellular Matrix Remodeling in Renal Fibrosis

2016 ◽  
Vol 11 ◽  
pp. BMI.S38439 ◽  
Author(s):  
Federica Genovese ◽  
Zsolt S. Kàrpàti ◽  
Signe H. Nielsen ◽  
Morten A. Karsdal
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Interstitial Fibrosis ◽  
Ex Vivo ◽  
Collagen Type I ◽  
Extracellular Matrix Remodeling ◽  
Type I ◽  
Matrix Remodeling ◽  
Renal Interstitial Fibrosis ◽  
Ex Vivo Model

The aim of this study was to set up an ex vivo model for renal interstitial fibrosis in order to investigate the extracellular matrix (ECM) turnover profile in the fibrotic kidney. We induced kidney fibrosis in fourteen 12-week-old male Sprague Dawley rats by unilateral ureteral obstruction (UUO) surgery of the right ureter. The left kidney (contralateral) was used as internal control. Six rats were sham operated and used as the control group. Rats were terminated two weeks after the surgery; the kidneys were excised and precision-cut kidney slices (PCKSs) were cultured for five days in serum-free medium. Markers of collagen type I formation (P1NP), collagen type I and III degradation (C1M and C3M), and α-smooth muscle actin (αSMA) were measured in the PCKS supernatants by enzyme-linked immunosorbent assay. P1NP, C1M, C3M, and α-SMA were increased up to 2- to 13-fold in supernatants of tissue slices from the UUO-ligated kidneys compared with the contralateral kidneys ( P < 0.001) and with the kidneys of sham-operated animals ( P < 0.0001). The markers could also reflect the level of fibrosis in different animals. The UUO PCKS ex vivo model provides a valuable translational tool for investigating the extracellular matrix remodeling associated with renal interstitial fibrosis.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

scholarly journals Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0254557
Author(s):  
Christian Freise ◽  
Hyunho Lee ◽  
Christopher Chronowski ◽  
Doug Chan ◽  
Jessica Cziomer ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Collagen Type ◽  
Stellate Cell ◽  
Smooth Muscle Actin ◽  
Stellate Cells ◽  
Collagen Type I ◽  
Serum Starvation ◽  
Type I ◽  
Linear Peptide

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-β, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)—derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

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