Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis.

OBJECTIVE: Excessive extra-cellular-matrix production and uncontrolled proliferation of the fibroblasts are characteristics of many fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). The fibroblasts have enhanced glutaminolysis with up-regulated glutaminase, GLS1, which converts glutamine to glutamate. Here, we investigated the role of glutaminolysis and glutaminolysis-derived metabolite α-ketoglutarate (α-KG) on IPF fibroblast phenotype and gene expression. METHODS: Reduced glutamine conditions were carried out either using glutamine-free culture medium or silencing the expression of GLS1 with siRNA, with or without α-KG compensation. Cell phenotype has been characterized under these different conditions, and gene expression profile was examined by RNA-Seq. Specific profibrotic genes (Col3A1 and PLK1) expression were examined by real-time PCR and western blots. The levels of repressive histone H3K27me3, which demethylase activity is affected by glutaminolysis, were examined and H3K27me3 association with promoter region of Col3A1 and PLK1 were checked by ChIP assays. Effects of reduced glutaminolysis on fibrosis markers were checked in an animal model of lung fibrosis. RESULTS: The lack of glutamine in the culture medium alters the profibrotic phenotype of activated fibroblasts. The addition of exogenous and glutaminolysis-derived metabolite α-KG to glutamine-free media barely restores the pro-fibrotic phenotype of activated fibroblasts. Many genes are down-regulated in glutamine-free medium, α-KG supplementation only rescues a limited number of genes. As α-KG is a cofactor for histone demethylases of H3K27me3, the reduced glutaminolysis alters H3K27me3 levels, and enriches H3K27me3 association with Col3A1 and PLK1 promoter region. Adding α-KG in glutamine-free medium depleted H3K27me3 association with Col3A1 promoter region but not that of PLK1. In a murine model of lung fibrosis, mice with reduced glutaminolysis showed markedly reduced fibrotic markers. CONCLUSIONS: This study indicates that glutamine is critical for supporting pro-fibrotic fibroblast phenotype in lung fibrosis, partially through α-KG-dependent and -independent mechanisms, and supports targeting fibroblast metabolism as a therapeutic method for fibrotic diseases.

Using a Radial Diffusion Method to Investigate the Role of Plasmin Degradation of Fibrin in a Physical Model of an Early-phase Wound.

BACKGROUND: Fibrin clot formation, which acts to stabilize a wound following injury, is among the key early aspects of dermal wound healing. This preliminary matrix is eventually degraded via a process known as fibrinolysis and replaced with a collagen-rich matrix that continues to be remodeled to minimize scarring. Disruptions in these carefully coordinated events lead to certain undesirable conditions such as fibrosis and the formation of abnormal scars that are associated with excess amounts of collagen. The hypothesis proposed herein is that the presence of collagen (and potentially other molecules) in an early-phase model of healing alters fibrinolysis and that this effect can be attenuated with mediators of the process. MATERIALS AND METHODS: Laboratory in vitro experiments were conducted using agarose-fibrin gel systems with and without collagen to study fibrinolysis caused by plasmin (a serine protease that degrades fibrin) and the effects of aprotinin (a serine protease inhibitor) and bromelain (an extract from pineapple) on fibrin clot breakdown. The extent of fibrinolysis was monitored at various times (0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours) by measuring the size of rings of fibrinolysis following the diffusion of plasmin. The data obtained at 0.5, 12, and 24-hour time points were considered (because there was no difference found in the data collected for closer intermediates nor for the longer times beyond 24 hours) and were compared using the nonparametric Mann-Whitney U statistical significance test. RESULTS: The results obtained showed aprotinin significantly inhibited fibrinolysis in systems containing collagen, while bromelain improved fibrinolysis. In general, the presence of increasing amounts of collagen in the system decreased the extent of fibrinolysis. CONCLUSIONS: These findings support the notion that early-phase deposition of collagen contributes to disrupted fibrinolysis, which could lead to impaired healing as well as potentially facilitate control of fibrinolysis.

Mathematical models of wound healing and closure: a comprehensive review.

Wound healing is a complex process comprised of overlapping phases and events that work to construct a new, functioning tissue. Mathematical models describe these events and yield understanding about the overall process of wound healing. Generally, these models are focused on only one phase (or a few phases) to explain healing for a specific system. A review of the literature reveals insights as reported on herein regarding the variety of overlapping inputs and outputs for any given type of model. Specifically, these models have been characterized with respect to the phases of healing and their mathematical/physical basis in an effort to shed light on new opportunities for model development. Though all phases of wound healing have been modeled, previous work has focused mostly on the proliferation and related contraction phases of healing with fewer results presented regarding other phases. As an example, a gap in the literature has been identified regarding models to describe facilitated wound closure techniques (e.g., suturing and its effect on resultant scarring). Thus, an opportunity exists to create models that tie the transient processes of wound healing, such as cell migration, to resultant scarring when considering tension applied to skin with given suturing techniques.

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.

Pleural fluid exchange in rabbits.

BACKGROUND AND OBJECTIVES: The study was designed to better characterize pleural fluid absorption in rabbits with the following two objectives: to determine the relative absorption of saline versus high-protein solutions, and to identify the relative rates of absorption of dextran molecules of varying sizes. METHODS: Twenty New Zealand white rabbits received a 12-mL intrapleural injection of saline solution and a 10% protein solution on opposite sides, each solution containing dextran molecules with varying MWs. At sacrifice at 1, 4, 8, 18 and 24 h, the volume of pleural fluid and the concentrations of the dextran molecules were determined. RESULTS: Saline was absorbed faster than the high-protein fluid (P < 0.001). Dextran concentrations in the saline were significantly higher than those in the protein solution at all times after injection (P = 0.005; P < 0.001, respectively). The higher-MW dextrans were cleared more slowly than the lower-MW dextrans in a continuously graded manner. CONCLUSIONS: Saline was absorbed faster than a solution with a high protein content. There was a continuous spectrum in the rate of absorption of the dextran molecules, with the larger molecules being absorbed more slowly.