miR-338-3p blocks TGFß-induced myofibroblast differentiation through the induction of PTEN.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease. The pathogenesis of IPF is not completely understood. However, numerous genes are associated with the development and progression of pulmonary fibrosis, indicating there is a significant genetic component to the pathogenesis of IPF. Epigenetic influences on the development of human disease, including pulmonary fibrosis, remain to be fully elucidated. In this paper, we identify miR-338-3p as a microRNA severely downregulated in the lungs of patients with pulmonary fibrosis and in experimental models of pulmonary fibrosis. Treatment of primary human lung fibroblasts with miR-338-3p inhibits myofibroblast differentiation and matrix protein production. Published and proposed targets of miR-338-3p such as TGFß receptor 1, MEK/ERK 1/2, Cdk4, and Cyclin D are also not responsible for the regulation of pulmonary fibroblast behavior by miR-338-3p. miR-338-3p inhibits myofibroblast differentiation by preventing TGFß-mediated downregulation of phosphatase and tensin homolog (PTEN), a known antifibrotic mediator.

Development of low-cost cardiac and skeletal muscle laboratory activities to teach physiology concepts and the scientific method.

Anatomy and Physiology courses taught at community colleges tend to focus laboratory hours primarily on anatomy as opposed to physiology. However, research demonstrates that, when instructors utilize active learning approaches (such as in laboratory settings) where students participate in their own learning, students have improved outcomes, such as higher test scores and better retention of material. To provide community college students with opportunities for active learning in physiology, we developed two laboratory exercises to engage students in cardiac and skeletal muscle physiology. We utilized low-cost SpikerBox devices to measure electrical activity during cardiac (electrocardiogram) and skeletal muscle (electromyogram) contraction. Laboratory activities were employed in Anatomy and Physiology courses at two community colleges in southeast Michigan. A 2-h laboratory period was structured with a 20-min slide presentation covering background material on the subject and experiments to examine the effects of environmental variables on nervous system control of cardiac and skeletal muscle contraction. Students were asked to provide hypotheses and proposed mechanisms, complete a results section, and provide conclusions for the experiments based on their results. Our laboratory exercises improved student learning in physiology and knowledge of the scientific method and were well-received by community college students enrolled in Anatomy and Physiology. Our results demonstrate that the use of a SpikerBox for cardiac and skeletal muscle physiology concepts is a low-cost and effective approach to integrate physiology activities into an Anatomy and Physiology course.

Denervation and senescence markers data from old rats with intrinsic differences in responsiveness to aerobic training.

The data described below is related to the manuscript "Late life maintenance and enhancement of functional exercise capacity in low and high responding rats after low intensity treadmill training" [1]. Rodents exhibit age-related declines in skeletal muscle function that is associated with muscle denervation and cellular senescence. Exercise training is a proven method to delay or even reverse some aging phenotypes, thus improving healthspan in the elderly. The beneficial effects of exercise to preserve muscle may be reliant on an individual's innate ability to adapt to aerobic training. To examine this question, we assessed aged rats that were selectively bred to be either minimally or highly responsive to aerobic exercise training. We specifically asked whether mild treadmill training initiated late in life would be beneficial to preserve muscle function in high response and low response trainer rats. We examined gene expression data on markers of denervation and senescence. We also evaluated measures of aerobic training and neuromuscular muscle function through work capacity, contractile properties, and endplate fragmentation for further analysis of the aging phenotype in older rodents.

Prevention and treatment of bleomycin-induced pulmonary fibrosis with the lactate dehydrogenase inhibitor gossypol.

Pulmonary fibrosis is a chronic and irreversible scarring disease in the lung with poor prognosis. Few therapies are available; therefore it is critical to identify new therapeutic targets. Our lab has previously identified the enzyme lactate dehydrogenase-A (LDHA) as a potential therapeutic target in pulmonary fibrosis. We found increases in LDHA protein and its metabolic product, lactate, in patients with idiopathic pulmonary fibrosis (IPF). Importantly, we described lactate as a novel pro-fibrotic mediator by acidifying the extracellular space, and activating latent transforming growth factor beta (TGF-ß1) in a pH-dependent manner. We propose a pro-fibrotic feed-forward loop by which LDHA produces lactate, lactate decreases pH in the extracellular space and activates TGF-ß1 which can further perpetuate fibrotic signaling. Our previous work also demonstrates that the LDHA inhibitor gossypol inhibits TGF-ß1-induced myofibroblast differentiation and collagen production in vitro. Here, we employed a mouse model of bleomycin-induced pulmonary fibrosis to test whether gossypol inhibits pulmonary fibrosis in vivo. We found that gossypol dose-dependently inhibits bleomycin-induced collagen accumulation and TGF-ß1 activation in mouse lungs when treatment is started on the same day as bleomycin administration. Importantly, gossypol was also effective at treating collagen accumulation when delayed 7 days following bleomycin. Our results demonstrate that inhibition of LDHA with the inhibitor gossypol is effective at both preventing and treating bleomycin-induced pulmonary fibrosis, and suggests that LDHA may be a potential therapeutic target for pulmonary fibrosis.

The Lactate Dehydrogenase Inhibitor Gossypol Inhibits Radiation-Induced Pulmonary Fibrosis.

Exposure of the lung to ionizing radiation that occurs in radiotherapy, as well as after accidental or intentional mass casualty incident can result in pulmonary fibrosis, which has few treatment options. Pulmonary fibrosis is characterized by an accumulation of extracellular matrix proteins that create scar tissue. Although the mechanisms leading to radiation-induced pulmonary fibrosis remain poorly understood, one frequent observation is the activation of the profibrotic cytokine transforming growth factor-beta (TGF-ß). Our laboratory has shown that the metabolite lactate activates latent TGF-ß by a reduction in extracellular pH. We recently demonstrated that lactate dehydrogenase-A (LDHA), the enzyme that produces lactate, is upregulated in patients with radiation-induced pulmonary fibrosis. Furthermore, genetic silencing of LDHA or pharmacologic inhibition using the LDHA inhibitor gossypol prevented radiation-induced extracellular matrix secretion in vitro through inhibition of TGF-ß activation. In the current study, we hypothesized that LDHA inhibition in vivo prevents radiation-induced pulmonary fibrosis. To test this hypothesis, C57BL/6 mice received 5 Gy total-body irradiation plus 10 Gy thoracic irradiation from a 137Cs source to induce pulmonary fibrosis. Starting at 4 weeks postirradiation, mice were treated with 5 mg/kg of the LDHA inhibitor gossypol or vehicle daily until sacrifice at 26 weeks postirradiation. Exposure to radiation resulted in pulmonary fibrosis, characterized by an increase in collagen content, fibrosis area, extracellular matrix gene expression and TGF-ß activation. Irradiated mice treated with gossypol had significantly reduced fibrosis outcomes, including reduced collagen content in the lungs, reduced expression of active TGF-ß, LDHA and the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). These findings suggest that inhibition of LDHA protects against radiation-induced pulmonary fibrosis, and may be a novel therapeutic strategy for radiation-induced pulmonary fibrosis.

Pharmacologic inhibition of lactate production prevents myofibroblast differentiation.

Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-ß. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-ß-induced myofibroblast differentiation. Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-ß and or gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Gossypol inhibited TGF-ß-induced expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in a dose-dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3, and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose-dependent manner. Furthermore, gossypol inhibited TGF-ß bioactivity in a dose-dependent manner. Concurrent treatment with an LDH siRNA increased the ability of gossypol to inhibit TGF-ß-induced myofibroblast differentiation. Gossypol inhibits TGF-ß-induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid, and inhibition of TGF-ß bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis.

Interleukin-12 preserves the cutaneous physical and immunological barrier after radiation exposure.

The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation and dispersal of radiological material are legitimate threats. Such attacks could have devastating consequences to large populations, in the form of radiation injury to various human organ systems. One of these at risk organs is the cutaneous system, which forms both a physical and immunological barrier to the surrounding environment and is particularly sensitive to ionizing radiation. Therefore, increased efforts to develop medical countermeasures for treatment of the deleterious effects of cutaneous radiation exposure are essential. Interleukin-12 (IL-12) was shown to elicit protective effects against radiation injury on radiosensitive systems such as the bone marrow and gastrointestinal tract. In this article, we examined if IL-12 could protect the cutaneous system from a combined radiation injury in the form of sublethal total body irradiation and beta-radiation burn (ß-burn) directly to the skin. Combined radiation injury resulted in a breakdown in skin integrity as measured by transepidermal water loss, size of ß-burn lesion and an exacerbated loss of surveillant cutaneous dendritic cells. Interestingly, intradermal administration of IL-12 48 h postirradiation reduced transepidermal water loss and burn size, as well as retention of cutaneous dendritic cells. Our data identify IL-12 as a potential mitigator of radiation-induced skin injury and argue for the further development of this cytokine as a radiation countermeasure.

Exposure to ionizing radiation induces the migration of cutaneous dendritic cells by a CCR7-dependent mechanism.

In the event of a deliberate or accidental radiological emergency, the skin would likely receive substantial ionizing radiation (IR) poisoning, which could negatively impact cellular proliferation, communication, and immune regulation within the cutaneous microenvironment. Indeed, as we have previously shown, local IR exposure to the murine ear causes a reduction of two types of cutaneous dendritic cells (cDC), including interstitial dendritic cells of the dermis and Langerhans cells of the epidermis, in a dose- and time-dependent manner. These APCs are critical regulators of skin homeostasis, immunosurveillance, and the induction of T and B cell-mediated immunity, as previously demonstrated using conditional cDC knockout mice. To mimic a radiological emergency, we developed a murine model of sublethal total body irradiation (TBI). Our data would suggest that TBI results in the reduction of cDC from the murine ear that was not due to a systemic response to IR, as a loss was not observed in shielded ears. We further determined that this reduction was due, in part, to the upregulation of the chemoattractant CCL21 on lymphatic vessels as well as CCR7 expressed on cDC. Migration as a potential mechanism was confirmed using CCR7(-/-) mice in which cDC were not depleted following TBI. Finally, we demonstrated that the loss of cDC following TBI results in an impaired contact hypersensitivity response to hapten by using a modified contact hypersensitivity protocol. Taken together, these data suggest that IR exposure may result in diminished immunosurveillance in the skin, which could render the host more susceptible to pathogens.