Dual inhibition of TGFßR and ROCK reverses the epithelial to mesenchymal transition in collectively migrating zebrafish keratocytes.

There is a growing controversy about the role of the epithelial to mesenchymal transition (EMT) in the fibrosis associated with chronic disease. Recent studies suggest that it is not the EMT transcriptional program but differentiation of progenitor cells, response to chronic inflammation, or some combination of both which cause the appearance of fibroblasts and the production of the extracellular matrix. To address this issue, we study the EMT process in the zebrafish keratocytes which migrate from primary explants of epithelial tissue as these cells are both terminally differentiated and able to divide. To firmly place this EMT process in the context of other systems, we first demonstrate that the zebrafish keratocyte EMT process involves nuclear accumulation of twist and snail/slug transcription factors as part of a TGFßR-mediated EMT process. As assessed by the expression and localization of EMT transcription factors, the zebrafish keratocyte EMT process is reversed by the addition of Rho-activated kinase (ROCK) in combination with TGFßR inhibitors. The complete cycle of EMT to MET observed in this system links these in vitro results more closely to the process of wound healing in vivo. However, the absence of observable activation of EMT transcription factors when keratocytes are cultured on compliant substrata in a TGFß1-containing medium suggests that ROCK signaling, initiated by tension within the sheet, is an essential contributor to the EMT process. Most importantly, the requirement for ROCK activation by culturing on noncompliant substrata suggests that EMT in these terminally differentiated cells would not occur in vivo.

Mild aerobic exercise blocks elastin fiber fragmentation and aortic dilatation in a mouse model of Marfan syndrome associated aortic aneurysm.

Regular low-impact physical activity is generally allowed in patients with Marfan syndrome, a connective tissue disorder caused by heterozygous mutations in the fibrillin-1 gene. However, being above average in height encourages young adults with this syndrome to engage in high-intensity contact sports, which unfortunately increases the risk for aortic aneurysm and rupture, the leading cause of death in Marfan syndrome. In this study, we investigated the effects of voluntary (cage-wheel) or forced (treadmill) aerobic exercise at different intensities on aortic function and structure in a mouse model of Marfan syndrome. Four-week-old Marfan and wild-type mice were subjected to voluntary and forced exercise regimens or sedentary lifestyle for 5 mo. Thoracic aortic tissue was isolated and subjected to structural and functional studies. Our data showed that exercise improved aortic wall structure and function in Marfan mice and that the beneficial effect was biphasic, with an optimum at low intensity exercise (55-65% V̇o2max) and tapering off at a higher intensity of exercise (85% V̇o2max). The mechanism underlying the reduced elastin fragmentation in Marfan mice involved reduction of the expression of matrix metalloproteinases 2 and 9 within the aortic wall. These findings present the first evidence of potential beneficial effects of mild exercise on the structural integrity of the aortic wall in Marfan syndrome associated aneurysm. Our finding that moderate, but not strenuous, exercise protects aortic structure and function in a mouse model of Marfan syndrome could have important implications for the medical care of young Marfan patients.NEW & NOTEWORTHY The present study provides conclusive scientific evidence that daily exercise can improve aortic health in a mouse model of Marfan syndrome associated aortic aneurysm, and it establishes the threshold for the exercise intensity beyond which exercise may not be as protective. These findings establish a platform for a new focus on promoting regular exercise in Marfan patients at an optimum intensity and create a paradigm shift in clinical care of Marfan patients suffering from aortic aneurysm complications.

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition.

BACKGROUND: The BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes. METHODS: SW13 subtype morphology, chemotaxis, growth rates, and gene expression were assessed by standard immunofluorescence, transwell, growth, and qPCR assays. Metastatic potential was measured by anchorage-independent growth and MMP activity. The efficacy of HDAC inhibitors in inducing subtype switching was determined by immunofluorescence and qPCR. Histone modifications were assessed by western blot. RESULTS: Treatment of SW13- cells with HDAC1 inhibitors most effectively promotes re-expression of BRM and VIM, characteristic of the SW13+ phenotype. During treatment, hyperacetylation of histone residues and hypertrimethylation of H3K4 is pronounced. Furthermore, histone modification enzymes, including HDACs and KDM5C, are differentially expressed during treatment but several features of this differential expression pattern differs from that seen in the SW13- and SW13+ subtypes. As the SW13- subtype is more proliferative while the SW13+ subtype is more metastatic, treatment with HDACi increases the metastatic potential of SW13 cells while restoring expression of the BRM tumor suppressor. CONCLUSIONS: When compared to the SW13- subtype, SW13+ cells have restored BRM expression, increased metastatic capacity, and significantly different expression of a variety of chromatin remodeling factors including those involved with histone acetylation and methylation. These data are consistent with a multistep mechanism of SW13- to SW13+ conversion and subtype stabilization: histone hypermodification results in the altered expression of chromatin remodeling factors and chromatin epigenetic enzymes and the re-expression of BRM which results in restoration of SWI/SNF complex function and leads to changes in chromatin structure and gene expression that stabilize the SW13+ phenotype.

Zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Prevalence of podiatric conditions seen in Special Olympics athletes: a comparison of USA data to an international population.

BACKGROUND: Persons with intellectual disabilities frequently have podiatric conditions. Limited information exists on their prevalence in international cohorts of Special Olympics (SO) athletes. Findings from multiple United States (US) venues are compared to those from athletes screened at the 2011 Special Olympics World Summer Games in Athens, Greece (ATHENS). METHODS: Data from Fit Feet screenings from 2096 ATHENS participants was compared to 7192 US participants. RESULTS: Frequently noted in the ATHENS population were motion restriction in both the ankle and the first metatarsal phalangeal joint (1st MTPJ), pes planus, metatarsus adductus, brachymetatarsia, hallux abducto valgus (HAV), onychomycosis, onychocryptosis, and tinea pedis. ATHENS differed from the US cohort as HAV and restricted ankle joint and 1st MTPJ joint motion was less frequent. Significantly more tinea pedis, xerosis, and hyperhidrosis were present in the ATHENS population. DISCUSSION/CONCLUSION: SO athletes have a higher prevalence of podiatric structural conditions compared to the general population, and some vary between ATHENS versus US. Less prevalent in ATHENS was HAV, and restricted motion in both the ankle and 1st MTPJ. This may reflect differences due to varied clinical observers. The higher rates of several dermatological conditions in ATHENS may reflect venue seasonal climate, or social factors.

Collective cell migration of primary zebrafish keratocytes.

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 µm/h but over the course of 24h the rate of leading edge advance decreases to ~23 µm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell-cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell-cell interactions determine the role of keratocytes within the migrating sheet.

Zebrafish keratocyte explant cultures as a wound healing model system: differential gene expression & morphological changes support epithelial-mesenchymal transition.

The control of collective cell migration of zebrafish keratocyte sheets in explant culture is of interest for cell migration and epithelial wound healing and depends on the gene expression profile. In a zebrafish genome array, ∼17.5% of the probe sets were differentially expressed greater than two-fold (p≤0.003) between 1 and 7 days of explant culture. Among the differentially expressed genes were a variety of wound healing-related genes and many of the biomarkers for epithelial-mesenchymal transition (EMT), including a switch from keratin and E-cadherin to vimentin and N-cadherin expression and several EMT-related transcription factors were found to be differentially expressed. Supporting evidence for EMT is seen in both morphological change and rearrangement of the actin cytoskeleton and in expression of cadherins during explant culture with a visible disassembly of the cell sheet. TGFß1 and TNFα expression were analyzed by qPCR at various time points and peak differential expression of both cytokines occurred at 3 days, indicating that the EMT process is ongoing under conditions routinely used in the study of fish keratocyte motility. These data establish that an EMT process is occurring during zebrafish keratocyte explant culture and support the use of this system as a wound healing model.

Foot-to-shoe mismatch and rates of referral in Special Olympics athletes.

BACKGROUND: Improperly fitted shoes are frequently seen in athletes participating in Special Olympics competitions. This foot-to-shoe mismatch may result in deformities as well as discomfort and reduced performance or injuries in competitions. A primary purpose for providing medical screenings is to identify conditions unknown and to promptly refer to an appropriate provider for evaluation and care. This study attempts to determine the prevalence of improperly fitted shoes and the rate of referral for Special Olympics athletes screened at Fit Feet venues. METHODS: To evaluate the foot-to-shoe mismatch and rate of referral, 4,094 Fit Feet screenings of Special Olympics athletes participating in US competitions in 2005 to 2009 were analyzed. The participants were 58.5% male and 41.5% female, with a median age of 25.6 years. A power analysis and the χ(2) test were used. The athletes voluntarily underwent a foot screening that followed the standardized Special Olympics Fit Feet protocol. The Brannock Device for measuring feet was used to assess proper fit. RESULTS: A proper fit was found in 58.56% of the athletes, with 28.60% wearing shoes too big and 12.84% wearing shoes too small. Unrelated to shoe fit, 20% of the athletes required referrals for professional follow-up based on abnormal clinical findings. CONCLUSIONS: There is a significant (41.44%) mismatch of foot to shoe in Special Olympics athletes. The most common mismatch is a shoe too big, with a much smaller number of athletes having shoes too small. Awareness of this foot-to-shoe incompatibility may be useful for the development of shoes better designed for athletes with a foot structure not consistent with conventional shoes. Because 20% of the athletes required a referral for professional follow-up, Fit Feet examinations are important for identifying athletes with conditions that can be more readily evaluated and treated, thus improving the athletes' comfort and performance. Beyond knowing the rate of referral, future studies can determine the conditions or findings that necessitate a referral and the ultimate outcome of that referral.

TGFß (transforming growth factor ß) and keratocyte motility in 24 h zebrafish explant cultures.

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFß (transforming growth factor ß) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFß in fish keratocyte migration. Studying the involvement of TGFß1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFß levels increase 6.2-fold in SFM (serum-free medium) and 2.4-fold in SFM+2% FBS (fetal bovine serum), while TGFß1 and TGFßRII (TGFß receptor II) mRNA levels increase ∼3- and ∼5-fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFß1 and culture media. The addition of 100 ng/ml exogenous TGFß1 in SFM increases both measures [3.3-fold (P = 4.5×10-5) and 26% (P = 2.1×10-2) respectively]. In contrast, 100 ng/ml of exogenous TGFß1 in medium containing 2% FBS decreases migration distance by 2.1-fold (P = 1.7×10-7), but does not affect sheet area. TGFß1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8-fold increase (P = 1.5×10-2) with 2% FBS. The variable response to TGFß1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFß1 and TGFßRII. Together, these results suggest that expression of TGFß and its receptor are up-regulated during zebrafish keratocyte explant culture and that TGFß promotes fish keratocyte migration.

Reducing the pain of local 1% lidocaine infiltration with a preceding bacteriostatic saline injection: a double-blind prospective trial.

BACKGROUND: Lidocaine injection for local anesthesia is a common podiatric medical procedure. We tested the hypothesis that injection of bacteriostatic saline solution containing 0.9% benzyl alcohol before the lidocaine infiltration can reduce the burning caused by lidocaine injection. METHODS: This double-blind prospective trial involved 45 participants who each received four injections in two areas of the dorsum of the foot and rated the perceived pain on a visual analog scale. The order of the injections was designed to disguise the control and intervention arms of the study. RESULTS: The sensation of the lidocaine injection after the injection of saline was reduced significantly (P = .028). The percentage of lidocaine injections with visual analog scale scores of 0 increased by 36% after preinjection with bacteriostatic saline solution containing 0.9% benzyl alcohol. CONCLUSIONS: The fact that 40% of the intervention visual analog scale pain scores for lidocaine injections were 0 suggests that a near painless lidocaine injection technique is an achievable goal and that the present technique is a simple and inexpensive method of reducing the pain of lidocaine injections.

Prevalence of podiatric conditions seen in Special Olympics athletes: Structural, biomechanical and dermatological findings.

BACKGROUND: Podiatric conditions are frequently seen in persons with intellectual disabilities. Unfortunately, many are not detected or addressed. This study attempts to determine the prevalence of a variety of selected podiatric conditions in Special Olympics athletes and compare the findings to studies of a similarly matched general population. METHODS: Screenings from 1580 Special Olympics athletes participating in a sampling of United States competitions in 2004-2006 were used for the analysis. The athletes voluntarily underwent a foot screening which followed the standardized Special Olympics Fit Feet protocol. RESULTS: The most commonly identified conditions included overpronated gait, abducted gait, restriction in both the ankle and the first metatarsal phalangeal joint, pes planus, metatarsus adductus, brachymetatarsia, hallux abducto valgus, onychomycosis, onychocryptosis and tinea pedis. DISCUSSION/CONCLUSION: The prevalence of all the structural conditions was notably higher than a similarly matched general population and both conditions and biomechanical/gait findings associated with ligamentous laxity like pes planus and hallux abducto valgus had especially high prevalence rates. Restricted motion in the ankle and first metatarsal phalangeal joint was surprising and may indicate some athletes have intellectual disabilities with contractures. The higher rates of dermatophyte related conditions may reflect increased exposure from the athletic competition environment, immunocompromise, issues with hygiene or both. Higher rates of onychocryptosis in the athletes may be due to ill fitting shoes and/or hygiene issues.

Is there a characteristic lipid profile in Alzheimer's disease?

OBJECTIVE: To characterize the lipid profile in Alzheimer's Disease (AD) and to determine whether it differs from the cardiac risk profile. BACKGROUND: Links between hypercholesterolemia and AD development continue to grow. Presently, limited information exists about the lipid profile characteristics in AD. METHODS: We examined the lipid profiles (total cholesterol (TC), high-density lipoprotein (HDL), lower-density lipoprotein (LDL), TC/HDL ratio, and triglyceride (TG) levels) of 153 subjects with probable/possible AD (mean age 77.2 +/- 8.6 years, mean MMSE 19.9 +/- 5.6) and 25 non-demented subjects with atherosclerotic heart disease (ASHD) (mean age 73.8 +/- 7.2 years); neither on lipid lowering therapy. RESULTS: Subjects with TC > 200 mg/dl composed 69% of AD and 72% of ASHD groups. Mean TC was 218.9 +/- 38.9 mg/dl and 218.5 +/- 9.2 mg/dl for AD and ASHD subjects respectively. AD subjects exhibited significantly higher HDL and lower TG and TC/HDL ratios. MMSE did not correlate with any lipid parameters in AD. DISCUSSION: Elevated TC, LDL and TG with normal HDL and TC/HDL ratio characterize the lipid profile in AD, which somewhat overlaps with but may be distinct from the cardiac risk profile. MMSE does not correlate with lipid parameters suggesting no interaction between cholesterol and cognition in AD.