Number Of Myofibroblasts Research Articles

Macrophage recruitment by fibrocystin-defective biliary epithelial cells promotes portal fibrosis in congenital hepatic fibrosis.

Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4)) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a β-catenin-dependent secretion of a range of chemokines, including chemokine (C-X-C motif) ligands 1, 10, and 12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvβ6 integrin, an activator of latent local transforming growth factor-β1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. Fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time.

Kinin receptors in skin wound healing

BackgroundWound healing is a complex and dynamic process that includes 3 different phases: inflammation, proliferation, and remodeling. Kinins are vasoactive peptides released after tissue injury, and are directly involved in the development and maintenance of inflammatory processes, and their actions are mediated by the activation of receptors called B1 and B2. ObjectiveWe aimed to evaluate the involvement of kinin receptors in the skin healing process. MethodsKnockout mice for kinin receptors (KOB1, KOB2 and KOB1B2) and wild type controls (WT) were subjected to a skin excision model, and tissue repair process was evaluated during different phases of wound healing. ResultsIn knockout animals for kinin receptors differences were observed in the resolution period of injury exceeding 17 days for the total closure of wounds. The absence of kinin receptors promotes a significant reduction in infiltration of polymorphonuclear cells on day 2 of the inflammatory phase. Already at the late stage of this phase (3 days) there was a negative influence on the infiltration of polymorphonuclear and mononuclear cells at the site of injury in comparison to WT. Collagen was significantly diminished in tissue of KOB1, KOB2 and KOB1B2 from day two to the end of the healing process. Moreover, wound tissue from KOB2 and KOB1B2, but not KOB1, presented impaired parameters of re-epitheliazation, reduced proliferation of cells (PCNA immunostaining), and a lower number of myofibroblasts (α-SMA immunostaining). ConclusionThese data reveal the involvement of kinin receptors in processes of skin repair. Both kinin receptors participate especially during the inflammatory phase, while B2 receptors seem to be more relevant in the quality of the wound scar. Thus, a better understanding of the contribution of kinins to skin wound healing may reveal novel options for therapy.

The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts

Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts.Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential.We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells.UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts in human fetal hearts. Hypoxia further increased the number of human fetal and rat neonatal myofibroblasts. However, chronically administered UDCA reduced the number of myofibroblasts and prevented hypoxia-induced depolarisation.In conclusion, our results show that the protective effect of UDCA involves both the reduction of fibroblast differentiation into myofibroblasts, and hyperpolarisation of myofibroblasts, most likely through the stimulation of potassium channels, i.e. KATP channels. This could be important in validating UDCA as an antifibrotic and antiarrhythmic drug for treatment of failing hearts and fetal arrhythmia.

Immunohistochemical evaluation of myofibroblasts in odontogenic cysts and tumors: A comparative study

Context:Myofibroblasts are fibroblasts with smooth muscle-like features characterized by the presence of a contractile apparatus and found in the connective tissue stroma of normal tissues such as blood vessels and lymph nodes. They are now thought to play a role in the synthesis and reorganization of extracellular matrix, which could contribute to the aggressive biologic behavior of the lesions.Aims:To compare the mean number of stromal myofibroblasts in dentigerous cysts (DCs), keratocystic odontogenic tumor (KCOT) and ameloblastoma; and to derive a correlation between the stromal myofibroblasts and the known biologic behavior of the lesions.Settings and Design:A cross-sectional immunohistochemical analysis of cases of DC, KCOT and ameloblastoma.Materials and Methods:Twenty paraffin-embedded tissue blocks each of DC, KCOT and multicystic ameloblastoma were selected for the study and diagnosis confirmed through hematoxylin and eosin staining. Tissue sections were analyzed for the number of myofibroblasts using alpha smooth muscle actin (α-SMA) immunostaining.Statistical Analysis:Differences in the mean number of α-SMA positive cells in each group were analyzed using one-way ANOVA test. Intergroup comparisons of mean values of α-SMA positive cells were performed using Mann-Whitney U-test.Results:Ameloblastoma showed the highest number of myofibroblasts, whereas DC showed the lowest. Among the groups, there were significant differences between the myofibroblast counts among DC and KCOT and between DC and ameloblastoma, whereas the difference in counts was not statistically significant between KCOT and ameloblastoma. A positive correlation was observed between the myofibroblast count and the known biologic behavior of the lesions.Conclusion:Myofibroblasts may act in close association with the epithelial cells to bring about changes in stromal microenvironment, favorable to the growth and progression of the lesion. They may be of great value in predicting the biologic behavior and growth potential of such lesions.

The Influence of Bone Marrow-Secreted IL-10 in a Mouse Model of Cerulein-Induced Pancreatic Fibrosis.

This study aimed to understand the role of IL-10 secreted from bone marrow (BM) in a mouse model of pancreatic fibrosis. The severity of cerulein-induced inflammation, fibrosis, and the frequency of BM-derived myofibroblasts were evaluated in the pancreas of mice receiving either a wild-type (WT) BM or an IL-10 knockout (KO) BM transplantation. The area of collagen deposition increased significantly in the 3 weeks after cerulein cessation in mice with an IL-10 KO BM transplant (13.7 ± 0.6% and 18.4 ± 1.1%, p < 0.05), but no further increase was seen in WT BM recipients over this time. The percentage of BM-derived myofibroblasts also increased in the pancreas of the IL-10 KO BM recipients after cessation of cerulein (6.7 ± 1.1% and 11.9 ± 1.3%, p < 0.05), while this figure fell in WT BM recipients after cerulein withdrawal. Furthermore, macrophages were more numerous in the IL-10 KO BM recipients than the WT BM recipients after cerulein cessation (23.2 ± 2.3 versus 15.3 ± 1.7 per HPF, p < 0.05). In conclusion, the degree of fibrosis, inflammatory cell infiltration, and the number of BM-derived myofibroblasts were significantly different between IL-10 KO BM and WT BM transplanted mice, highlighting a likely role of IL-10 in pancreatitis.

Effectiveness of rosiglitazone in reducing flexion contracture in a rabbit model of arthrofibrosis with surgical capsular release: A biomechanical, histological, and genetic analysis.

AimsAnimal models have been developed that allow simulation of post-traumatic joint contracture. One such model involves contracture-forming surgery followed by surgical capsular release. This model allows testing of antifibrotic agents, such as rosiglitazone.MethodsA total of 20 rabbits underwent contracture-forming surgery. Eight weeks later, the animals underwent a surgical capsular release. Ten animals received rosiglitazone (intramuscular initially, then orally). The animals were sacrificed following 16 weeks of free cage mobilisation. The joints were tested biomechanically, and the posterior capsule was assessed histologically and via genetic microarray analysis.ResultsThere was no significant difference in post-traumatic contracture between the rosiglitazone and control groups (33° (standard deviation (sd) 11) vs 37° (sd14), respectively; p = 0.4). There was no difference in number or percentage of myofibroblasts. Importantly, there were ten genes and 17 pathways that were significantly modulated by rosiglitazone in the posterior capsule.DiscussionRosiglitazone significantly altered the genetic expression of the posterior capsular tissue in a rabbit model, with ten genes and 17 pathways demonstrating significant modulation. However, there was no significant effect on biomechanical or histological properties.Cite this article: M. P. Abdel. Effectiveness of rosiglitazone in reducing flexion contracture in a rabbit model of arthrofibrosis with surgical capsular release: A biomechanical, histological, and genetic analysis. Bone Joint Res 2016;5:11–17. DOI: 10.1302/2046-3758.51.2000593

Deletion of CD28 Co-stimulatory Signals Exacerbates Left Ventricular Remodeling and Increases Cardiac Rupture After Myocardial Infarction.

Inflammatory responses, especially by CD4(+)T cells activated by dendritic cells, are known to be important in the pathophysiology of cardiac repair after myocardial infarction (MI). Although co-stimulatory signals through B7 (CD80/86) and CD28 are necessary for CD4(+)T cell activation and survival, the roles of these signals in cardiac repair after MI are still unclear. C57BL/6 (Control) mice and CD28 knockout (CD28KO) mice were subjected to left coronary artery permanent ligation. The ratio of death by cardiac rupture within 5 days after MI was significantly higher in CD28KO mice compared with Control mice. Although there were no significant differences in the infarct size between the 2 groups, left ventricular end-diastolic and end-systolic diameters were significantly increased, and fractional shortening was significantly decreased in CD28KO mice compared with Control mice. Electron microscopic observation revealed that the extent of extracellular collagen fiber was significantly decreased in CD28KO mice compared with Control mice. The number of α-smooth muscle actin-positive myofibroblasts was significantly decreased, and matrix metalloproteinase-9 activity and the mRNA expression of interleukin-1β were significantly increased in CD28KO mice compared with Control mice. Deletion of CD28 co-stimulatory signals exacerbates left ventricular remodeling and increases cardiac rupture after MI through prolongation of the inflammatory period and reduction of collagen fiber in the infarct scars. (Circ J 2016; 80: 1971-1979).

Comparison of Myofibroblasts Between Solid/Multicystic Ameloblastoma and Unicystic Ameloblastoma: An Immunohistochemical Analysis.

Microenvironment is crucial for the maintenance of cellular functions and tissue integrity suggesting that cancer-induced changes in the stroma may contribute to cancer invasion and its biological behaviour. One of the major constituent of the tumour stroma is myofibroblasts. Myofibroblasts are differentiated host fibroblasts that express α-Sma as cytoplasmic microfilaments. They are considered as one of the modified stromal component which in recent years have been thought to have a role in the invasion and aggressive behaviour of odontogenic tumours too. To detect immunohistochemically the presence of myofibroblasts in solid/multicystic ameloblastoma and in unicystic ameloblastoma and to see if a relationship exists between the frequency and pattern of distribution of myofibroblasts and the behaviour of ameloblastomas. Ten cases each of solid/multicystic ameloblastoma and unicystic ameloblastoma were stained immunohistochemically for vimentin, α-SMA and desmin. The frequency and pattern of distribution of myofibroblasts in the two study groups were analysed and then compared with clinical and radiographic features of pain and cortical perforation respectively. Immunohistochemical reaction for α-SMA (alpha Smooth Muscle Actin) showed positive cells in the stroma of both solid/multicystic and unicystic ameloblastomas. The mean number of myofibroblasts was more in unicystic ameloblastoma (UA) compared to Solid/Multicystic Ameloblastoma (SMA). Myofibroblasts expression was dense and arranged in the form of fascicles with indistinct cell borders in one case of follicular ameloblastoma, two cases of plexiform ameloblastoma and in a focal area of one case of type 1UA. In all other cases where the expression was noted, the myofibroblasts were spindle in shape with distinct cell boundaries. The results of the study indicate that myofibroblasts alone may not play a role in the behaviour of ameloblastomas. This calls for determining the role of various other stromal components in the biological behaviour of ameloblastomas. Our study could not establish a correlation between pattern of distribution of myofibroblasts and the behaviour of ameloblastomas.

Mechanical Stress Changes the Complex Interplay Between HO-1, Inflammation and Fibrosis, During Excisional Wound Repair.

Mechanical stress following surgery or injury can promote pathological wound healing and fibrosis, and lead to functional loss and esthetic problems. Splinted excisional wounds can be used as a model for inducing mechanical stress. The cytoprotective enzyme heme oxygenase-1 (HO-1) is thought to orchestrate the defense against inflammatory and oxidative insults that drive fibrosis. Here, we investigated the activation of the HO-1 system in a splinted and non-splinted full-thickness excisional wound model using HO-1-luc transgenic mice. Effects of splinting on wound closure, HO-1 promoter activity, and markers of inflammation and fibrosis were assessed. After seven days, splinted wounds were more than three times larger than non-splinted wounds, demonstrating a delay in wound closure. HO-1 promoter activity rapidly decreased following removal of the (epi)dermis, but was induced in both splinted and non-splinted wounds during skin repair. Splinting induced more HO-1 gene expression in 7-day wounds; however, HO-1 protein expression remained lower in the epidermis, likely due to lower numbers of keratinocytes in the re-epithelialization tissue. Higher numbers of F4/80-positive macrophages, αSMA-positive myofibroblasts, and increased levels of the inflammatory genes IL-1β, TNF-α, and COX-2 were present in 7-day splinted wounds. Surprisingly, mRNA expression of newly formed collagen (type III) was lower in 7-day wounds after splinting, whereas, VEGF and MMP-9 were increased. In summary, these data demonstrate that splinting delays cutaneous wound closure and HO-1 protein induction. The pro-inflammatory environment following splinting may facilitate higher myofibroblast numbers and increase the risk of fibrosis and scar formation. Therefore, inducing HO-1 activity against mechanical stress-induced inflammation and fibrosis may be an interesting strategy to prevent negative effects of surgery on growth and function in patients with orofacial clefts or in patients with burns.

Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide.

The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in order to avoid possible unwanted side effects such as a delayed healing process.

The natural behavior of mononuclear phagocytes in HTS formation.

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.

Abstract 17796: Toll-like Receptor 9 Signaling Pathway Ameliorates Cardiac Rupture After Myocardial Infarction by Promoting the Proliferation of Myofibroblasts

Background: Toll-like receptor (TLR) 9 signaling pathway plays an important role in inflammatory responses in failing hearts in response to pressure overload and in the pathogenesis of heart failure. However, the role of the signaling pathway in left ventricular (LV) remodeling after myocardial infarction, where necrotic cardiomyocyte death is prominent, has not been elucidated. Methods and Results: Wild-type (WT) (n=9) and TLR9 knockout (KO) mice (n=11) were subjected to the left coronary artery ligation to induce myocardial infarction (MI). The survival ratio 7 days after MI was significantly reduced in KO mice (36.4%) than in WT mice (88.9%) (p&lt;0.05). All of dead mice in both groups exhibited cardiac rupture. The peak time occurring rupture was 4 days after MI. The ischemia area at risk, identified by a lack of Evans blue staining were not significantly different between the WT and KO hearts (29.7% and 28.9%). Cardiovascular MRI imaging perfomed at 3 days post MI showed no difference in infarct size, LV volume and ejection fraction between WT and KO mice. The mRNA expression levels of inflammatory cytokines including IL-6 and TNFα were not significantly different in KO mice compared with WT mice. Immunohistochemical analysis did not show the significant difference in the extent of neutrophilic infiltration (CD45 positive cells) and macrophage accumulation (CD68 positive cells) in the infarct region between WT and KO mice. The number of myofibroblasts (αsmooth muscle action-positive cells), one of the cellular components in the formation of granulation tissue, was significantly reduced in KO mice (346 cells /mm2) compared to WT mice (1547 cells /mm2) (p&lt;0.05). The number of myofibroblasts exhibiting Ki-67 immunoreactivity was also markedly lower in KO mice (113 cells /mm2) than in WT mice (789 cells /mm2) (p&lt;0.05), indicating that the proliferative activity of myofibroblasts was significantly decreased in the infarcted regions of KO hearts. Conclusions: TLR9 signaling pathway is not involved in inflammatory responses in the infarcted regions of MI hearts but ameliorates cardiac rupture after MI by promoting the proliferation of myofibroblasts.

Adenosine A2A receptor plays an important role in radiation-induced dermal injury.

Ionizing radiation is a common therapeutic modality and following irradiation dermal changes, including fibrosis and atrophy, may lead to permanent changes. We have previously demonstrated that occupancy of A2A receptor (A2AR) stimulates collagen production, so we determined whether blockade or deletion of A2AR could prevent radiation-induced fibrosis. After targeted irradiation (40 Gy) of the skin of wild-type (WT) or A2AR knockout (A2ARKO) mice, the A2AR antagonist ZM241385 was applied daily for 28 d. In irradiated WT mice treated with the A2AR antagonist, there was a marked reduction in collagen content and skin thickness, and ZM241385 treatment reduced the number of myofibroblasts and angiogenesis. After irradiation, there is an increase in loosely packed collagen fibrils, which is significantly diminished by ZM241385. Irradiation also induced an increase in epidermal thickness, prevented by ZM241385, by increasing the number of proliferating keratinocytes. Similarly, in A2ARKO mice, the changes in collagen alignment, skin thickness, myofibroblast content, angiogenesis, and epidermal hyperplasia were markedly reduced following irradiation. Radiation-induced changes in the dermis and epidermis were accompanied by an infiltrate of T cells, which was prevented in both ZM241385-treated and A2ARKO mice. Radiation therapy is administered to a significant number of patients with cancer, and radiation reactions may limit this therapeutic modality. Our findings suggest that topical application of an A2AR antagonist prevents radiation dermatitis and may be useful in the prevention or amelioration of radiation changes in the skin.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial‐to‐mesenchymal transition (EndoMT)

PurposeThe fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial‐to‐mesenchymal transition (EndoMT) and/or bone marrow‐derived circulating fibrocytes contribute to the myofibroblast population present in PDR membranes.MethodsEpiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor‐β1 (TGF‐ β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐a (TNF‐a).ResultsAll membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co‐expressed the fibroblast/myofibroblast markers fibroblast‐specific protein‐1 (FSP‐1) and a‐SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP‐1, a‐SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co‐localization of CD45 with FSP‐1 and a‐SMA and co‐localization of CD11b with a‐SMA and matrix metalloproteinase‐9, demonstrating the presence of infiltrating fibrocytes. Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial‐cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP‐1).ConclusionsThese results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial‐to‐mesenchymal transition (EndoMT)

PurposeThe fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial‐to‐mesenchymal transition (EndoMT) and/or bone marrow‐derived circulating fibrocytes contribute to the myofibroblast population present in PDR membranes.MethodsEpiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor‐β (TGF‐β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐a (TNF‐a).ResultsAll membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co‐expressed the fibroblast/myofibroblast markers fibroblast‐specific protein‐1 (FSP‐1) and a‐SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP‐1, a‐SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co‐localization of CD45 with FSP‐1 and a‐SMA and co‐localization of CD11b with a‐SMA and matrix metalloproteinase‐9, demonstrating the presence of infiltrating fibrocytes. Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial‐cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP‐1).ConclusionsThese results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Revisiting stroma in pancreatic cancer.

Revisiting stroma in pancreatic cancer.

Mast cell involvement in the progression of peritoneal fibrosis in rats with chronic renal failure.

Peritoneal fibrosis is a serious complication in patients with end stage renal disease (ESRD), especially those undergoing long-term peritoneal dialysis therapy. Since the peritoneum is a major site of mast cell accumulation, and since mast cells are known to facilitate the progression of organ fibrosis, they would also contribute to the pathogenesis of peritoneal fibrosis. The aim of this study was to reveal the involvement of mast cells in the progression of peritoneal fibrosis in chronic renal failure. Using a rat model with chronic renal failure (CRF) resulting from 5/6 nephrectomy, we examined the histopathological features of the rat peritoneum and compared them to those of age-matched sham-operated rat peritoneum. By treating the CRF rats with a potent mast cell stabilizer, tranilast, we also examined the involvement of mast cells in the progression of peritoneal fibrosis. The CRF rat peritoneum was characterized by the wide staining of collagen III and an increased number of myofibroblasts, indicating the progression of fibrosis. Compared to sham-operated rat peritoneum, the number of toluidine blue-stained mast cells was significantly higher in the fibrotic peritoneum of CRF rats. The mRNA expression of fibroblast-activating factors and stem cell factor was significantly higher in peritoneal mast cells obtained from CRF rats than in those obtained from sham-operated rats. Treatment with tranilast significantly suppressed the progression of peritoneal fibrosis in CRF rats. This study demonstrated for the first time that the number of mast cells was significantly increased in the fibrotic peritoneum of CRF rats. The proliferation of mast cells and their increased activity in the peritoneum were thought to be responsible for the progression of peritoneal fibrosis.

Abstract 372: Regulation of Ski and Scleraxis in the Infarcted Rat Heart

Introduction: Coronary heart disease is causal to myocardial infarction (MI) and cardiac fibrosis. Upon ischemic myocardial injury, resident cardiac fibroblasts phenoconvert to myofibroblasts and synthesize large amounts of fibrillar collagens to produce scar tissue. Although the myofibroblast numbers are reduced in the infarct scar following the completion of wound healing, a sub-population of cells persist in the wounded area, leading to maladaptive chronic remodeling of the scar area and eventually the non-infarcted myocardium. Ski has been identified as a repressor of the TGF-β1 signaling pathway, attenuating the myofibroblast phenotype and its functional properties. Scleraxis has been implicated in canonical TGF-β1 signaling to promote collagen1α2 expression. We investigated how Ski and Scleraxis contribute to physiological and pathological wound healing in vivo. Methods: The study was carried out using 64 male Sprague-Dawley rats. The left anterior descending (LAD) coronary artery was ligated to induce a myocardial infarction. Control (sham) operated animals underwent surgery without ligation of the LAD artery. Animals were sacrificed at 2, 4, and 8 weeks post-MI and tissue collected for Western blot and qPCR studies. Results: Scleraxis mRNA expression remained at baseline at 2 and 8 weeks post-MI, but was significantly increased 4 weeks post-MI. Scleraxis protein expression was down-regulated within the scar area of infarcted hearts when compared to control samples 2 and 4 weeks post-MI. Ski mRNA expression was up-regulated within the scar area of infarcted hearts 2, 4 and 8 weeks after infarction. Conclusions: Scleraxis protein is down-regulated in myofibroblasts of the infarct scar in the chronic stages of myocardial infarction, corresponding to the maturation of the scar. At these stages of wound healing, we have previously published that Ski is up-regulated in the cytosol of these same cells. We suggest reciprocal feedback in the expression of these two proteins exists in myofibroblasts in the infarct scar. We hope to learn more about the Ski/Scleraxis feedback loop in pathological wound healing to identify novel therapeutic targets.

Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease

Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem cell associated factor, controls liver growth in healthy adult mice. Increasing nuclear localization of YAP triggers accumulation of reactive-appearing ductular cells (YAP+RDC) with liver progenitor capabilities. The significance of YAP activation, and mechanisms involved, are unknown in diseased livers. We evaluated the hypothesis that YAP is more activated in injured livers that are scarring than in those that are regenerating effectively. Immunohistochemistry and qRT-PCR analysis were used to localize and quantify changes in YAP and RDC in 52 patients with non-alcoholic fatty liver disease (NAFLD) and two mouse models of diet-induced non-alcoholic steatohepatitis (NASH). Results were correlated with liver disease severity, metabolic risk factors, and factors proven to control NAFLD progression. YAP increased in NAFLD where it mainly localized in nuclei of RDC that expressed progenitor markers. Accumulation of YAP+RDC paralleled the severity of hepatocyte injury and accumulation of Sonic hedgehog, but not steatosis or metabolic risk factors. YAP+RDC expressed osteopontin, a Shh-regulated fibrogenic factor. Myofibroblast accumulation, fibrosis, and numbers of YAP+RDC strongly correlated. In murine NASH models, atrophic fibrotic livers contained significantly more YAP+RDC than livers with less severe NASH. YAP+RDC promote scarring, rather than effective regeneration, during NASH.

Combined effect of genetic background and gender in a mouse model of bleomycin-induced skin fibrosis.

IntroductionSystemic sclerosis (SSc) is a connective tissue disorder characterised by the development of skin fibrosis. Our current understanding of the disease pathogenesis is incomplete and the study of SSc is hindered, at least partially, by a lack of animal models that fully replicate the complex state of human disease. Murine model of bleomycin-induced dermal fibrosis encapsulates important events that take place early in the disease course.MethodsTo characterise the optimum in vivo parameters required for the successful induction of dermal fibrosis we subjected three commonly used mouse strains to repeated subcutaneous bleomycin injections. We aimed to identify the effects of genetic background and gender on the severity of skin fibrosis. We used male and female Balb/C, C57BL/6, and DBA/2 strains and assessed their susceptibility to bleomycin-induced fibrosis by measuring dermal thickness, hydroxyproline/collagen content and number of resident myofibroblasts, all of which are important indicators of the severity of skin fibrosis. All data are expressed as mean values ± SEM. The Mann–Whitney U test was used for statistical analysis with GraphPad Prism 6.04 software.ResultsDermal fibrosis was most severe in Balb/C mice compared to C57BL/6 and DBA/2 suggesting that Balb/C mice are more susceptible to bleomycin-induced fibrosis. Analysis of the effect of gender on the severity of fibrosis showed that male Balb/C, C57BL/6, DBA/2 mice had a tendency to develop more pronounced fibrosis phenotype than female mice. Of potential importance, male Balb/C mice developed the most severe fibrosis phenotype compared to male C57BL/6 and male DBA/2 as indicated by significantly increased number of dermal myofibroblasts.ConclusionOur study highlights the importance of genetic background and gender in the induction of murine dermal fibrosis. Robust and reproducible animal models of fibrosis are important research tools used in pharmacological studies which may lead to better understanding of the pathogenesis of fibrotic diseases and assist in identification of new drugs.