The myofibroblast at a glance.

In 1971, Gabbiani and co-workers discovered and characterized the "modification of fibroblasts into cells which are capable of an active spasm" (contraction) in rat wound granulation tissue and, accordingly, named these cells 'myofibroblasts'. Now, myofibroblasts are not only recognized for their physiological role in tissue repair but also as cells that are key in promoting the development of fibrosis in all organs. In this Cell Science at a Glance and the accompanying poster, we provide an overview of the current understanding of central aspects of myofibroblast biology, such as their definition, activation from different precursors, the involved signaling pathways and most widely used models to study their function. Myofibroblasts will be placed into context with their extracellular matrix and with other cell types communicating in the fibrotic environment. Furthermore, the challenges and strategies to target myofibroblasts in anti-fibrotic therapies are summarized to emphasize their crucial role in disease progression.

Correction: Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development.

[This corrects the article DOI: 10.1371/journal.pone.0017830.].

A Rodent Model of Hypertrophic Scarring: Splinting of Rat Wounds.

Human hypertrophic scars are the result of imperfect healing of skin, which is particularly evident from the scars developing after severe burns. In contrast, mouse and rat full-thickness skin wounds heal normally without forming visible scar tissue, which reduces the suitability of rodent models for the study of skin scarring. We here provide a simple procedure to splint the edges of full-thickness rodent skin with a sutured plastic frame that prevents wound closure by granulation tissue contraction. The resulting mechanical tension in the wound bed and the lack of neo-epithelium amplify myofibroblast formation and generate hypertrophic features, not unlike those of human skin. In addition to producing scar tissue, the splint provides a reservoir that can be used for the delivery of cellular and acellular wound treatment regimen. Despite being simple and almost historical, wound splinting is a robust and reliable model to study myofibroblast biology.

Myofibroblast Markers and Microscopy Detection Methods in Cell Culture and Histology.

The identification of myofibroblasts is essential for mechanistic in vitro studies, cell-based drug tests, and to assess the level of fibrosis in experimental animal or human fibrosis. The name myo-fibroblast was chosen in 1971 to express that the formation of contractile features-stress fibers is the essential criterion to define these cells. Additional neo-expression of α-smooth muscle actin (α-SMA) in stress fibers has become the most widely used molecular marker. Here, we briefly introduce the concept of different myofibroblast activation states, of which the highly contractile α-SMA-positive phenotype represents a most advanced functional stage. We provide targeted immunofluorescence protocols to assess this phenotype, and publicly accessible image analysis tools to quantify the level of myofibroblast activation in culture and in tissues.

Compromised dental cells viability following teeth-whitening exposure.

This study aimed to assess the viability of dental cells following time-dependent carbamide peroxide teeth-whitening treatments using an in-vitro dentin perfusion assay model. 30 teeth were exposed to 5% or 16% CP gel (4 h daily) for 2-weeks. The enamel organic content was measured with thermogravimetry. The time-dependent viability of human dental pulp stem cells (HDPSCs) and gingival fibroblast cells (HGFCs) following either indirect exposure to 3 commercially available concentrations of CP gel using an in-vitro dentin perfusion assay or direct exposure to 5% H2O2 were investigated by evaluating change in cell morphology and by hemocytometry. The 5% and 16% CP produced a significantly lower (p < 0.001) enamel protein content (by weight) when compared to the control. The organic content in enamel varied accordingly to the CP treatment: for the 16% and 5% CP treatment groups, a variation of 4.0% and 5.4%, respectively, was observed with no significant difference. The cell viability of HDPSCs decreased exponentially over time for all groups. Within the limitation of this in-vitro study, we conclude that even low concentrations of H2O2 and CP result in a deleterious change in enamel protein content and compromise the viability of HGFCs and HDPSCs. These effects should be observed in-vivo.

Combined use of GABA and sitagliptin promotes human ß-cell proliferation and reduces apoptosis.

γ-Aminobutyric acid (GABA) and glucagon-like peptide-1 receptor agonist (GLP-1RA) improve rodent ß-cell survival and function. In human ß-cells, GABA exerts stimulatory effects on proliferation and anti-apoptotic effects, whereas GLP-1RA drugs have only limited effects on proliferation. We previously demonstrated that GABA and sitagliptin (Sita), a dipeptidyl peptidase-4 inhibitor which increases endogenous GLP-1 levels, mediated a synergistic ß-cell protective effect in mice islets. However, it remains unclear whether this combination has similar effects on human ß-cell. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-gamma mice with streptozotocin-induced diabetes, and then treated them with GABA, Sita, or both. The oral administration of either GABA or Sita ameliorated blood glucose levels, increased transplanted human ß-cell counts and plasma human insulin levels. Importantly, the combined administration of the drugs generated significantly superior results in all these responses, as compared to the monotherapy with either one of them. The proliferation and/or regeneration, improved by the combination, were demonstrated by increased Ki67+, PDX-1+, or Nkx6.1+ ß-cell numbers. Protection against apoptosis was also significantly improved by the drug combination. The expression level of α-Klotho, a protein with protective and stimulatory effects on ß cells, was also augmented. Our study indicates that combined use of GABA and Sita produced greater therapeutic benefits, which are likely due to an enhancement of ß-cell proliferation and a decrease in apoptosis.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-ß1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

Combined effect of GABA and glucagon-like peptide-1 receptor agonist on cytokine-induced apoptosis in pancreatic ß-cell line and isolated human islets.

BACKGROUND: Treatment with GABA or glucagon-like peptide-1 (GLP-1) can preserve pancreatic ß-cell mass and prevent diabetes. Recently, we reported that the combination of GABA and sitagliptin (a dipeptidyl peptidase-4 inhibitor that increases endogenous GLP-1) was more effective than either agent alone in reducing drug-induced ß-cell damage and promoting ß-cell regeneration in mice. However, in human islets, it remains unclear whether GABA and GLP-1 exert similar effects. METHODS: To investigate GABA and GLP-1 interactions, human islets or INS-1 cells were treated with GABA and/or exendin-4, a GLP-1 receptor agonist (GLP-1RA) in clinical use, and incubated with a cytokine mixture for 24 hours. Cleaved caspase-3 and annexin V binding were measured by western blot and flow cytometry analysis, respectively, to investigate effects on cytokine-induced apoptosis. RESULTS: Cytokine-induced apoptosis was reduced by either GABA or exendin-4 alone. This was markedly improved by combining GABA and exendin-4, resulting in a reversal of apoptosis. The combination notably increased Akt pathway signaling. Furthermore, sirtuin-1 (SIRT1) and α-Klotho, both reported to have protective effects on ß-cells, were increased. Importantly, the combination ameliorated insulin secretion by human ß-cells. CONCLUSIONS: The combination of GABA and a GLP-1RA exerted additive effects on ß-cell survival and function, suggesting that this combination may be superior to either drug alone in the treatment of diabetes.

Inhibitory effect of carnosine on interleukin-8 production in intestinal epithelial cells through translational regulation.

The enhanced intestinal production of pro-inflammatory cytokines leads to inflammation and carcinogenesis, and therefore its down-regulation by nutrients could represent a promising therapeutic approach. We found for the first time that the secretion of interleukin-8 (IL-8) in intestinal epithelial cells stimulated by hydrogen peroxide or TNF-alpha was suppressed in the presence of carnosine (beta-Ala-His), a dietary dipeptide. Interestingly, carnosine had no influence on the stimulus-induced IL-8 mRNA expression, although the intracellular production and secretion of IL-8 were significantly inhibited by carnosine. The inhibitory effect of carnosine on the IL-8 secretion differed from that of other histidine-containing dipeptides like Gly-His, Ala-His, and anserine (beta-Ala-1-methyl-His), which inhibited both the hydrogen peroxide-induced secretion and mRNA expression of IL-8. These observations indicate that carnosine inhibited IL-8 secretion along a unique pathway, in which IL-8 production was suppressed at a post-transcriptional level, for instance, translation. The hypothesis that carnosine inhibited the translation of IL-8 mRNA is supported by the finding that the phosphorylation of eIF4E, an initiation factor, in stimulated Caco-2 cells was inhibited by carnosine. These results suggest that carnosine is a novel type of anti-inflammatory agent that down-regulates the inflammatory response in intestinal epithelial cells by a unique mechanism.

Combined Oral Administration of GABA and DPP-4 Inhibitor Prevents Beta Cell Damage and Promotes Beta Cell Regeneration in Mice.

γ-aminobutyric acid (GABA) or glucagon-like peptide-1 based drugs, such as sitagliptin (a dipeptidyl peptidase-4 inhibitor), were shown to induce beta cell regenerative effects in various diabetic mouse models. We propose that their combined administration can bring forth an additive therapeutic effect. We tested this hypothesis in a multiple low-dose streptozotocin (STZ)-induced beta cell injury mouse model (MDSD). Male C57BL/6J mice were assigned randomly into four groups: non-treatment diabetic control, GABA, sitagliptin, or GABA plus sitagliptin. Oral drug administration was initiated 1 week before STZ injection and maintained for 6 weeks. GABA or sitagliptin administration decreased ambient blood glucose levels and improved the glucose excursion rate. This was associated with elevated plasma insulin and reduced plasma glucagon levels. Importantly, combined use of GABA and sitagliptin significantly enhanced these effects as compared with each of the monotherapies. An additive effect on reducing water consumption was also observed. Immunohistochemical analyses revealed that combined GABA and sitagliptin therapy was superior in increasing beta cell mass, associated with increased small-size islet numbers, Ki67+ and PDX-1+ beta cell counts; and reduced Tunel+ beta cell counts. Thus, beta cell proliferation was increased, whereas apoptosis was reduced. We also noticed a suppressive effect of GABA or sitagliptin on alpha cell mass, which was not significantly altered by combining the two agents. Although either GABA or sitagliptin administration delays the onset of MDSD, our study indicates that combined use of them produces superior therapeutic outcomes. This is likely due to an amelioration of beta cell proliferation and a decrease of beta cell apoptosis.

Histidine inhibits oxidative stress- and TNF-alpha-induced interleukin-8 secretion in intestinal epithelial cells.

We investigated the effect of several amino acids on the secretion of such inflammatory cytokines as interleukin-8 (IL-8) induced by hydrogen peroxide or tumor necrosis factor-alpha (TNF-alpha) in intestinal epithelial-like Caco-2 and HT-29 cells. We found that histidine, one of the conditionally essential amino acids, significantly inhibited both hydrogen peroxide- and TNF-alpha-induced IL-8 secretion and mRNA expression in Caco-2 cells and HT-29 cells. These inhibitions were dose dependent and the inhibition rate of hydrogen peroxide-induced IL-8 secretion reached more than 50% at a concentration of 25mM, with over 95% inhibition at a concentration of 50mM. TNF-alpha increased the transcriptional activity of the IL-8 promoter which was significantly inhibited by treating Caco-2 cells with histidine. Histidine also abolished the NF-kappaB-dependent activation of the IL-8 promoter induced by TNF-alpha. These results indicate that histidine inhibited the hydrogen peroxide- and TNF-alpha-induced IL-8 secretion at the transcriptional level in intestinal epithelial cells, suggesting that histidine has the potential to attenuate intestinal inflammation.

Characterization of carnosine uptake and its physiological function in human intestinal epithelial Caco-2 cells.

Carnosine (beta-Ala-L-His) is known to have the physiological functions of an antioxidant. Although dietary carnosine is thought to be absorbed across intestinal epithelial cells, the mechanism for this absorption is not yet well understood and its function in the intestinal tract is also obscure. The intestinal transport of carnosine was characterized in the present study by using human intestinal Caco-2 cells, and its physiological function in these cells was further examined. The carnosine uptake was proton-dependent, being activated by lowering the apical pH value. Its uptake was significantly inhibited by other dipeptides, whereas it was not inhibited by other amino acids. These characteristics of the carnosine uptake strongly suggest its transport into the cells via peptide transporter 1 (PepT1). Since carnosine has antioxidative activity, we studied its effect on the H2O2-induced secretion of inflammatory cytokines in Caco-2 cells. The H2O2 induced increase in IL-8 secretion was inhibited by a pretreatment with carnosine for 3 h, this inhibition being presented in a dose-dependent manner. These results suggest that carnosine had a protective effect against oxidative stress in intestinal epithelial cells.

GABA promotes human ß-cell proliferation and modulates glucose homeostasis.

γ-Aminobutyric acid (GABA) exerts protective and regenerative effects on mouse islet ß-cells. However, in humans it is unknown whether it can increase ß-cell mass and improve glucose homeostasis. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-γ mice with streptozotocin-induced diabetes. GABA treatment increased grafted ß-cell proliferation, while decreasing apoptosis, leading to enhanced ß-cell mass. This was associated with increased circulating human insulin and reduced glucagon levels. Importantly, GABA administration lowered blood glucose levels and improved glucose excursion rates. We investigated GABA receptor expression and signaling mechanisms. In human islets, GABA activated a calcium-dependent signaling pathway through both GABA A receptor and GABA B receptor. This activated the phosphatidylinositol 3-kinase-Akt and CREB-IRS-2 signaling pathways that convey GABA signals responsible for ß-cell proliferation and survival. Our findings suggest that GABA regulates human ß-cell mass and may be beneficial for the treatment of diabetes or improvement of islet transplantation.

Histone demethylase JMJD2B functions as a co-factor of estrogen receptor in breast cancer proliferation and mammary gland development.

Estrogen is a key regulator of normal function of female reproductive system and plays a pivotal role in the development and progression of breast cancer. Here, we demonstrate that JMJD2B (also known as KDM4B) constitutes a key component of the estrogen signaling pathway. JMJD2B is expressed in a high proportion of human breast tumors, and that expression levels significantly correlate with estrogen receptor (ER) positivity. In addition, 17-beta-estradiol (E2) induces JMJD2B expression in an ERα dependent manner. JMJD2B interacts with ERα and components of the SWI/SNF-B chromatin remodeling complex. JMJD2B is recruited to ERα target sites, demethylates H3K9me3 and facilitates transcription of ER responsive genes including MYB, MYC and CCND1. As a consequence, knockdown of JMJD2B severely impairs estrogen-induced cell proliferation and the tumor formation capacity of breast cancer cells. Furthermore, Jmjd2b-deletion in mammary epithelial cells exhibits delayed mammary gland development in female mice. Taken together, these findings suggest an essential role for JMJD2B in the estrogen signaling, and identify JMJD2B as a potential therapeutic target in breast cancer.

Food-derived peptides and intestinal functions.

The intestines are an important organ responsible for nutrient absorption, metabolism and recognition of food signals. The organ also acts as a physical and biological barrier against harmful substances including food pathogens and environmental chemicals. Food-derived peptides with a variety of physiological functions have been discovered in the past several decades. Although dietary peptides would mostly be hydrolyzed by digestive enzymes in the intestinal tract, possibly losing their biological functions during this step, some could be absorbed intact and act in their target organs. The intestines are also one of the targets for functional peptides. The intestine-modulatory peptides can be classified into two categories: (1) peptides that express their functions in the intestinal tract and (2) peptides that modulate intestinal epithelial cell functions. The 1(st) group includes peptides that regulate the intestinal absorption of nutrients. Enhancing mineral absorption by casein phosphopeptides, and suppressing dietary cholesterol absorption by soybean peptides are typical examples. The 2(nd) group includes such glutamine-containing peptides as Ala-Gln that show interesting properties in preventing and/or repairing damage caused by oxidative stress and inflammatory reactions. We have found that carinosine (beta-Ala-His) suppressed the secretion of such inflammatory cytokines as IL-8 in human intestinal epithelial cells, suggesting its anti-inflammatory function in the intestines. Peptides that modulate such intestinal immune functions as secretory IgA production and cytokine secretion, and opioid peptides regulating intestinal motility are also included in this group. These intestine-modulatory peptides would be useful as ingredients of future functional foods to prevent lifestyle-related diseases and promote gut health.