Periostin differentially induces proliferation, contraction and apoptosis of primary Dupuytren's disease and adjacent palmar fascia cells

Dupuytren's disease, (DD), is a fibroproliferative condition of the palmar fascia in the hand, typically resulting in permanent contracture of one or more fingers. This fibromatosis is similar to scarring and other fibroses in displaying excess collagen secretion and contractile myofibroblast differentiation. In this report we expand on previous data demonstrating that POSTN mRNA, which encodes the extra-cellular matrix protein periostin, is up-regulated in Dupuytren's disease cord tissue relative to phenotypically normal palmar fascia. We demonstrate that the protein product of POSTN, periostin, is abundant in Dupuytren's disease cord tissue while little or no periostin immunoreactivity is evident in patient-matched control tissues. The relevance of periostin up-regulation in DD was assessed in primary cultures of cells derived from diseased and phenotypically unaffected palmar fascia from the same patients. These cells were grown in type-1 collagen-enriched culture conditions with or without periostin addition to more closely replicate the in vivo environment. Periostin was found to differentially regulate the apoptosis, proliferation, α smooth muscle actin expression and stressed Fibroblast Populated Collagen Lattice contraction of these cell types. We hypothesize that periostin, secreted by disease cord myofibroblasts into the extra-cellular matrix, promotes the transition of resident fibroblasts in the palmar fascia toward a myofibroblast phenotype, thereby promoting disease progression.     

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.     

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.     

In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's disease fibroblasts displays unique drug related properties

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex?--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex? against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex? exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex? did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex? significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex? enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex? on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.     

In Vitro Study of Novel Collagenase (XIAFLEX?) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

Dupuytren''s disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH) - Xiaflex® - is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.     

Genome-Wide Analysis Using Exon Arrays Demonstrates an Important Role for Expression of Extra-Cellular Matrix,Fibrotic Control and Tissue Remodelling Genes in Dupuytren's Disease

Dupuytren''s disease (DD) is a classic example of pathological fibrosis which results in a debilitating disorder affecting a large sector of the human population. It is characterized by excessive local proliferation of fibroblasts and over-production of collagen and other components of extracellular matrix (ECM) in the palmar fascia. The fibrosis progressively results in contracture of elements between the palmar fascia and skin causing flexion deformity or clawing of the fingers and a severe reduction in hand function. While much is known about the pathogenesis and surgical treatment of DD, little is known about the factors that cause its onset and progression, despite many years of research. Gene expression patterns in DD patients now offers the potential to identify genes that direct the pathogenesis of DD. In this study we used primary cultures of fibroblasts derived from excisional biopsies of fibrotic tissue from DD patients to compare the gene expression profiles on a genome-wide basis with normal control fibroblasts. Our investigations have identified genes that may be involved with DD pathogenesis including some which are directly relevant to fibrosis. In particular, these include significantly reduced expression levels of three matrix metallopeptidases (MMP1, MMP3, MMP16), follistatin, and STAT1, and significantly increased expression levels of fibroblast growth factors (FGF9, FGF11), a number of collagen genes and other ECM genes in DD patient samples. Many of these gene products are known to be involved in fibrosis, tumour formation and in the normal processes of tissue remodelling. In addition, alternative splicing was identified in some DD associated genes. These highly sensitive genomic investigations provide new insight into the molecular mechanisms that may underpin the development and progression of DD.     

Post‐transcriptional regulation of α‐smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells

The objective was to study Dupuytren's myofibroblast cells in constrained collagen matrices in order to more closely emulate their in vivo environment and, to correlate their contractility with α‐smooth muscle actin (α‐SMA) expression and determine if dermal fibroblasts regulate Dupuytren's myofibroblast phenotype. Isotonic and isometric force contraction by cells isolated from Dupuytren's nodules, palmar and non‐palmar skin fibroblasts was measured in collagen matrices. The effect of co‐culturing nodule cells with dermal fibroblasts on isometric contraction was examined. Isometric contraction was correlated with levels of α‐SMA mRNA by pcr and protein by Western blotting, and α‐SMA distribution assessed by immunofluorescence. Dupuytren's nodule cells exhibited similar levels of isotonic contraction to both palmar and non‐palmar dermal fibroblasts. However, nodule cells generated high levels of isometric force (mean: 3.5 dynes/h), which continued to increase over 24 h to a maximum of 173 dynes. In contrast, dermal fibroblasts initially exhibited low levels of contraction (mean: 0.5 dynes/h) and reached tensional homeostasis on average after 15 h (range: 4–20 h), with a maximum force of 52 dynes. Although all three cell types had similar α‐SMA mRNA levels, increased levels of α‐SMA protein were observed in nodule cells compared to dermal fibroblasts. α‐SMA localised to stress fibres in 35% (range: 26–50%) of nodule cells compared to only 3% (range:0–6%) of dermal fibroblasts. Co‐cultures of Dupuytren's cells and dermal fibroblasts showed no contractile differences. The contractile phenotype of Dupuytren's myofibroblasts is determined by increased α‐SMA protein distributed in stress fibres, not by cellular mRNA levels. Dupuytren's cell contractility is not influenced by dermal fibroblasts. J. Cell. Physiol. 224: 681–690, 2010. © 2010 Wiley‐Liss, Inc.     

Proliferation characteristics and polyploidization of cultured myofibroblasts from a patient with fibroblastic rheumatism

Fibroblast-like cells were obtained from a nodule of a patient with fibroblastic rheumatism, and grown in culture for different times (from passage 3 to 21). These cells as well as the fibroblasts taken from an unaffected skin area (controls) of the same patient, have been investigated by fluorescence microscopy, cytochemical methods and cytometry, to evaluate their cytodifferentiation features and cytokinetic characteristics. In addition, in low-passage cultures, the secretion of collagen and of non-collagenic proteins was evaluated using electrophoretic techniques. The immunolabeling with antibodies against sm-specific a-actin (which was taken as a marker of myofibroblasts) showed that, already in low-passage cultures, the percentage of myofibroblasts was higher in the nodule-derived cell populations, and progressively increased with increasing passages. This suggests that myofibroblasts have higher proliferation potential than control fibroblasts. Myofibroblasts were also found to undergo polyploidization and hypertrophy, especially in high-passage cultures. Based on these results, it may be hypothesized that in fibroblastic rheumatism the development of the typical nodules could depend on the intrinsic capability of myofibroblats of proliferating faster than normal fibroblasts and of becoming polyploid and hypertrophic. Nodule-derived cells in culture synthesized slightly less collagen and non-collagen proteins than did the control fibroblasts; this suggests that the increased fibrosis observed in nodules in situ could be likely dependent on a reduced degradation of the extracellular matrix components.     

Increased CCT-eta expression is a marker of latent and active disease and a modulator of fibroblast contractility in Dupuytren’s contracture

Dupuytren’s contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts.     

Abnormal behavior of cultured fibroblasts from nodule and nonaffected aponeurosis of Dupuytren's disease

Vimentin-positive, desmin-negative cells were established in culture from the nodule and from apparently normal palmar aponeurosis of a patient with Dupuytren's disease and compared with normal human embryonic and adult fibroblasts or sarcomatous cells. Cells from the nodule display in vitro biological properties that are intermediate between those expressed by normal fibroblasts and sarcoma cells or cells from the nodule transformed with SV40 virus. Thus, they represent an interesting in vitro model of partially transformed human cells. This behavior is not evolutive and justifies the classification of Dupuytren's disease among the benign mesenchymal tumors. The production of high level of plasminogen activator probably explains the local reactive pathology, and could act as a mitogenic stimulus for the proliferation of the nodule itself. Cultures derived from the apparently normal palmar aponeurosis show some but not all the abnormal growth properties of cells from nodules; this may help to explain the onset of local recurrences. Our results suggest that Dupuytren's disease is not strictly local and limited to the nodules, but affects, at least partially, the whole aponeurosis. Dupuytren's nodules could be considered as a model of tumor progression in a benign situation.     

Induced Pluripotent Stem Cells Show Metabolomic Differences to Embryonic Stem Cells in Polyunsaturated Phosphatidylcholines and Primary Metabolism

Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.     

Mining for treatment-specific and general changes in target compounds and metabolic fingerprints in response to herbivory and phytohormones in Plantago lanceolata

Induction studies focusing on target metabolites may not reveal metabolic changes occurring in plants after various challenges. By contrast, metabolic fingerprinting can be a powerful tool to find patterns that are either treatment-specific or general and was therefore used to depict plant responses after various challenges. Plants of Plantago lanceolata were challenged by mechanical damage, specialist herbivores (aphids or sawfly larvae), generalist herbivores (Lepidopteran caterpillars) or phytohormones (jasmonic or salicylic acid). After 3 d of treatment, local and systemic leaves were analyzed for characteristic target metabolites (iridoid glucosides and verbascoside) by gas chromatography coupled with mass spectrometry (GC-MS) and for metabolic fingerprints by liquid chromatography coupled with time of flight mass spectrometry (LC-TOF-MS). Whereas only marginal changes in target metabolite concentrations were found, metabolic fingerprints were substantially affected especially by generalist and phytohormone treatments. By contrast, mechanical damage and specialist herbivory caused fewer changes. Responses to generalists partly overlapped with the changes caused by jasmonic acid, but many additional peaks were up-regulated. Furthermore, many peaks were co-induced by jasmonic and salicylic acid. The surprisingly high co-induction of peaks by both phytohormones suggests that the signaling pathways regulate a set of common targets. Furthermore, only metabolic fingerprinting could reveal that herbivores induce additional species-specific pathways beyond these phytohormone responses.     

Free radicals and Dupuytren's contracture.

The concentration of substrate expressed as hypoxanthine capable of reacting with xanthine oxidase to release superoxide free radicals (O2-) was measured in control and Dupuytren''s contracture palmar fascia. In Dupuytren''s contracture palmar fascia the concentration of hypoxanthine was six times that of control and was greatest in "nodular" areas. Xanthine oxidase activity was also detected in Dupuytren''s contracture palmar fascia. These results suggest a greater potential for hypoxanthine-xanthine oxidase generated oxygen free radical formation in Dupuytren''s contracture than in control palmar fascia. Production of free radicals may be an important factor in the pathogenesis of Dupuytren''s contracture. The benefit of allopurinol in the management of Dupuytren''s contracture and other fibrotic conditions may thus be explained, as allopurinol binds to xanthine oxidase and prevents release of free radicals.     

N-acetyl-L-cysteine abrogates fibrogenic properties of fibroblasts isolated from Dupuytren's disease by blunting TGF-beta signalling

Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-beta1 (TGF-beta1) and its downstream Smad signalling system is well established as a key player during fibrogenesis. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies. One such promising treatment involves the substance N-acetyl-L-cysteine (NAC), shown to have antifibrotic properties in hepatic stellate cells and rat fibroblasts. In order to investigate antifibrotic effects of N-acetyl-L-cysteine (NAC), fibroblasts were isolated from surgically resected fibrotic palmar tissues (Dupuytren fibroblasts, DF) and exposed to different concentrations of NAC and recombinant TGF-beta1. Fibroblasts isolated from tendon pulleys served as controls (control fibroblasts, CF). Smad signalling was investigated by a Smad binding element driven reporter gene analysis. Both cell types express TGF-beta1, indicating autocrine signalling in DF and CF. This was confirmed by comparing reporter gene activity from LacZ and Smad7 adenovirus infected cells. NAC treatment resulted in abrogation of Smad mediated signalling comparable to ectopically overexpressed Smad7, even when the cells were stimulated with recombinant TGF-beta1 or ectopically expressed a constitutively active TGF-beta receptor type I. Additionally, NAC dose-dependently decreased expression of three major indicators of impaired fibrotic matrix turnover, namely alpha-smooth muscle actin (alpha-SMA), alpha 1 type I procollagen (Col1A1), and plasminogen activator inhibitor-type I (PAI-1). Our results suggest that TGF-beta signalling and subsequent expression of fibrogenesis related proteins in Dupuytren's disease is abrogated by NAC thus providing a basis for a therapeutic strategy in Dupuytren's disease and other fibroproliferative disorders.     

Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts

Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.     

Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts

Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.     

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.