Neuroprotective Effects of Endurance Exercise Against High-Fat Diet-Induced Hippocampal Neuroinflammation.

Obesity contributes to systemic inflammation, which is associated with the varied pathogenesis of neurodegenerative diseases. Growing evidence has demonstrated that endurance exercise (EE) mitigates obesity-induced brain inflammation. However, exercise-mediated anti-inflammatory mechanisms remain largely unknown. We investigated how treadmill exercise (TE) reverses obesity-induced brain inflammation, mainly focusing on toll-like receptor-4 (TLR-4)-dependent neuroinflammation in the obese rat brain after 20 weeks of a high-fat diet (HFD). TE in HFD-fed rats resulted in a significant lowering in the homeostasis model assessment of insulin resistance index, the area under the curve for glucose and abdominal visceral fat, and also improved working memory ability in a passive avoidance task relative to sedentary behaviour in HFD-fed rats, with the exception of body weight. More importantly, TE revoked the increase in HFD-induced proinflammatory cytokines (tumour necrosis factor α and interleukin-1ß) and cyclooxygenase-2, which is in parallel with a reduction in TLR-4 and its downstream proteins, myeloid differentiation 88 and tumour necrosis factor receptor associated factor 6, and phosphorylation of transforming growth factor ß-activated kinase 1, IkBα and nuclear factor-κB. Moreover, TE reduced an indicator of microglia activation, ionised calcium-binding adapter molecule-1, and also decreased glial fibrillary acidic protein, an indicator of gliosis formed by activated astrocytes in the cerebral cortex and the hippocampal dentate gyrus, compared to HFD-fed sedentary rats. Finally, EE up-regulated the expression of anti-apoptotic protein, Bcl-2, and suppressed the expression of pro-apoptotic protein, Bax, in the hippocampus compared to HFD-fed sedentary rats. Taken together, these data suggest that TE may exert neuroprotective effects as a result of mitigating the production of proinflammatory cytokines by inhibiting the TLR4 signalling pathways. The results of the present study suggest that the unique combination of the beneficial effects of TE on the restoration of the blood profile and the anti-inflammatory and anti-apoptotic effects on cognitive function should inspire further investigations into its therapeutic potential for metabolic disorders and neurodegenerative diseases.

Involvement of Ca2+ signalling in the vasoactive intestinal peptide and 8-Br-cAMP induction of c-fos mRNA expression.

Vasoactive intestinal peptide (VIP) is known to signal via Gs mediated pathways. VIP stimulated c-fos mRNA expression in a clonal GH3 pituitary tumour cell line, GH3Ca, whereas 8-Br-cAMP only moderately induced c-fos expression. The VIP-induced c-fos expression was inhibited in the presence of EGTA, or the L-type Ca2+ channel blockers verapamil and nifedipine. Measurement of intracellular Ca2+ concentration ([Ca2+]i) by Fura-2 indicates that VIP gradually elevates [Ca2+]i, with the maximum level attained at 4 min following hormone addition. No [Ca2+]i increase could be detected in Ca2+ free buffer or in buffer containing nifedipine or verapamil, which suggests that VIP induced Ca2+ entry from L-type Ca2+ channels. 8-Br-cAMP rapidly increased [Ca2+]i, with a maximum concentration attained within 1 min of its addition and the elevated level maintained for 15 min. In the absence of external Ca2+ or in the presence of verapamil or nifedipine, the sustained Ca2+ increase was abolished whereas the transient Ca2+ peak was unaffected. Depletion of the internal calcium pools by thapsigargin (1 microM, 30 min), on the other hand, blocked the rapid transient [Ca2+], rise, suggesting the biphasic [Ca2+]i elicited by 8-Br-cAMP was due to mobilization from internal Ca2+ pool followed by extracellular flow. Interestingly, pretreatment with thapsigargin greatly potentiated the 8-Br-cAMP-stimulated c-fos expression. Pretreatment of cells with cholera toxin (1 microg/ml, 9 h) to deplete Gs proteins abolished VIP stimulated-[Ca2+] elevation, while it had little effect on the 8-Br-cAMP induced [Ca2+]i rise. Our results show that VIP increased Ca2+ influx from L-type channel through a Gs-mediated mechanism and this Ca2+ entry across the plasma membrane plays a major role in the hormone induced c-fos mRNA expression.

Synergistic activation of cAMP and calcium on cAMP-response-element-mediated gene expression in GH3 pituitary tumor cells.

Signals responsible for expression of the vasoactive intestinal peptide (VIP)-stimulated prolactin gene in GH3 pituitary tumor cells were examined. Transfection with a deoxyribonucleic acid (DNA) construct containing the chloramphenicol acetyltransferase (CAT) gene fused to the 2.5-kb prolactin 5'-upstream regulatory sequence indicated that VIP stimulated CAT expression. However, this effect could not be mimicked by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and was inhibited by the L-type Ca(2+)-channel blocker verapamil. While KCl had little effect on CAT activity, combined treatment with KCl and 8-Br-cAMP synergistically activated CAT expression. Potentiation between KCl and 8-Br-c-AMP was also seen with c-fos messenger ribonucleic acid (mRNA) expression. In addition, KCl and 8-Br-cAMP synergistically activated cAMP response element (CRE)-mediated CAT expression, and the synergism was abolished by verapamil. In the presence of okadaic acid, cAMP had no significant activation on CRE-driven CAT expression, whereas KCl-stimulated CAT expression was greatly potentiated. These results indicate that cAMP and Ca2+ synergistically activated CRE-driven gene expression through non-overlapping phosphorylation events in GH3 cells.

Vitronectin deficiency is associated with increased wound fibrinolysis and decreased microvascular angiogenesis in mice.

BACKGROUND: Vitronectin has several putative functions including regulating hemostasis, cell adhesion, and cell migration. However, the targeted deletion of vitronectin in mice results in normal development and normal coagulation parameters. To determine whether vitronectin may be necessary for nondevelopmental processes, we examined the response to tissue injury in vitronectin-null mice. METHODS: We examined wound healing in control and vitronectin-null mice by healing rate, zymography, reverse zymography, and Western blots. RESULTS: We found that dermal wound healing was slightly delayed in mice lacking vitronectin. More importantly, we found extensive areas of delayed hemorrhage near the sprouting tips of microvessels between days 7 and 14, which temporally coincided with increased urokinase-type plasminogen activator and tissue-type plasminogen activator activity by zymography. Though Western blots confirmed the presence of plasminogen activator inhibitor-1 protein throughout wound repair and reverse zymograms showed decreased plasminogen activator inhibitor-1 activity between days 7 and 14. CONCLUSIONS: Loss of vitronectin in mice was associated with changes in the fibrinolytic balance, and this may have led to focal sites of delayed hemorrhage. The mechanism that resulted in decreased angiogenesis and the formation of larger blood vessels in response to tissue injury remains unknown. This study suggests that vitronectin may have several distinct functions that are not required for normal development but are manifested in response to tissue injury.

Temporal activity of plasminogen activators and matrix metalloproteinases during cutaneous wound repair.

BACKGROUND: Response to tissue injury begins with the deposition of a fibrin-rich clot or the provisional matrix. The provisional matrix consists of plasma-borne matrix molecules that serve as scaffolding for the ensuing migration of cells. During wound repair multiple cell types must migrate through the clot-matrix scaffolding. The migration of these cells through the matrix is dependent on the activity of the fibrinolytic and proteolytic systems, which include the plasminogen activator (PA) system and matrix metalloproteinases (MMP). The aim of this study was to better understand the temporal activity of these enzymes during normal wound repair. METHODS: We used the murine excisional wound model and extracted proteins under nonreducing conditions. With use of gelatin and casein zymography, we determined the activity of the MMPs during the course of wound repair. In addition, we quantified the activity of MMP-2 and MMP-9 by a standardized assay. Plasminogen zymograms were used to detect urokinase PA and tissue PA activity. Western blots were used to detect the natural inhibitor of PAs, plasminogen activator inhibitor type 1. RESULTS: Our results demonstrate the temporal activity of MMP-2, MMP-3, MMP-7, and MMP-9 during the course of normal dermal repair. The activity of urokinase PA and tissue PA were also determined; it preceded the activity of the MMPs. CONCLUSIONS: We demonstrate the temporal activity of the 2 protease families, MMPs and PAs, in the normal process of cutaneous wound healing.

The optimal management of pediatric steam burn from electric rice-cooker: STSG or FTSG?

The steam burn caused by an electric rice-cooker is a unique mode of burn injury in Asian countries, especially Korea and Japan. This type of burn injury is characterized by 1) occurring most frequently on the volar aspect of the hand in toddlers younger than 2 years of age (92.8%); 2) the depth of burns are normally deep second-degree to third-degree (98%) and usually need surgery at the time of injury; 3) flexion contractures of multiple finger joints and web space contracture are common sequelae. We hypothesized that primary full-thickness skin graft (FTSG) would give more reliable results and eliminate the late reconstructive procedures. Between January 1997 and September 1999, 36 patients underwent primary FTSG, and the results of this primary FTSG group were compared with 124 patients who were treated with split-thickness skin graft (STSG; 79/124; 63.7%) or by conservative management (45/124; 36.3%), and readmitted for the correction of hand deformities between September 1995 and September 1999. In the primary FTSG group, 11.1% (4/36) of mild web contractures and 5.5% (2/36) of finger joint contractures were documented, and these did not require the reconstructive procedure during a follow-up period of 8.8 +/- 4.8 months. In 124 patients of the primary STSG or conservative group, the mean time interval to reoperation was 8.9 +/- 4.0 months and all patients received FTSG for correction of late hand deformities. In a retrospective study of the primary STSG group, 42 of 53 patients (79.2%) received reconstructive procedure during a 5-year follow-up period. In this report, we introduce the nature of steam burn caused by electric rice-cooker and propose that primary FTSG may be a reliable method for the treatment of this more severe type of acute burn in pediatric patients.

Occurrence of organic pollutants in recovered soil fines from construction and demolition waste.

The objective of this study was to characterize recovered soil fines from construction and demolition (C&D) waste recycling facilities for trace organic pollutants. Over a period of 18 months, five sampling trips were made to 14 C&D waste recycling facilities in Florida. Screened soil fines were collected from older stockpiles and newly generated piles at the sites. The samples were analyzed for the total concentration (mg/kg) of a series of volatile organic compound (VOCs) and semi-volatile organic compounds (semi-VOCs). The synthetic precipitation leaching procedure (SPLP) test was also performed to evaluate the leachability of the trace organic chemicals. During the total analysis only a few volatile organic compounds were commonly found in the samples (trichlorofluoromethane, toluene, 4-isopropyltoluene, trimethylbenzene, xylenes, and methylene chloride). A total of nine VOCs were detected in the leaching test. Toluene showed the highest leachability among the compounds (61.3-92.0%), while trichlorofluoromethane, the most commonly detected compound from both the total and leaching tests, resulted in the lowest leachability (1.4-39.9%). For the semi-VOC analysis, three base-neutral semi-VOC compounds (bis(2-ethylhexyl)phthalate, butyl benzyl phthalate, and di-n-butyl phthalate) and several PAHs (acenaphthene, pyrene, fluoranthene, and phenanthrene) were commonly detected in C&D fines samples. These compounds also leached during the SPLP leaching test (0.1-25%). No acid extractable compounds, pesticides, or PCBs were detected. The results of this study were further investigated to assess risk from land applied recovered soil fines by comparing total and leaching concentrations of recovered soil fines samples to risk-based standards. The results of this indicate that the organic chemicals in recovered soil fines from C&D debris recycling facilities were not of a major concern in terms of human risk and leaching risk to groundwater under reuse and contact scenarios.

Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function.

Homeostatic and regenerative replacement of skeletal muscle fibers requires the activity of a dedicated pool of myogenic stem cells, called satellite cells, that are activated by muscle injury and act as a renewable source of muscle-forming cells throughout adult life. Satellite cell function is controlled by both intrinsic and extrinsic regulatory cues, whose integration determines the success of muscle regenerative responses. Pathological deregulation of satellite cell function through perturbation of these signaling pathways appears to play an important role in age-dependent deterioration of muscle function and in muscle dystrophic disease. The regenerative activity of skeletal muscle also appears to be tightly linked to metabolism, and alterations in metabolic state can directly influence the activity of these tissue-specific stem cells. Here, we review recent and emerging insights into the molecular and biochemical signals that control satellite cell function and discuss these in the context of muscle degenerative diseases such as dystrophy and sarcopenia. Novel discoveries from this ongoing work bring new opportunities to enhance or restore muscle repair and are likely to facilitate satellite cell transplantation in clinical applications.

Nerve distribution in hemangiomas depends on the proliferative state of the microvasculature.

Hemangiomas appear at birth and undergo gradual regression within several years. Recent published studies have documented increased nerve numbers in port-wine stains and intramuscular vascular tumors. The aim of this study was to establish a relationship between angiogenesis and nerve growth in lesions that undergo neovascular proliferation followed by vessel involution. Twenty-two hemangiomas and arteriovenous malformations were studied using indirect immunocytochemistry with antibodies against the nerve markers protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). Nerves and vessels were counted and compared. Our results indicate that PGP 9.5(+) and CGRP(+) nerves were most numerous in growing hemangiomas and numbers were reduced in involuting hemangiomas and vascular malformations. The percentage of CGRP(+) sensory nerves was markedly increased in growing hemangiomas (45.3%) compared with involuting hemangiomas (21.2). These data indicate that hemangiomas with increasing neovascularization have increased sensory nerve growth. Sensory nerve-derived neuropeptides are known to act as endothelial cell mitogens and may contribute to the angiogenesis in these vascular tumors. Conversely, angiogenic endothelial cells may secrete mediators that promote nerve fiber growth. These results suggest that endothelial cell proliferation and sensory nerve fiber growth may be closely related.

Changes in matrix composition during the growth and regression of human hemangiomas.

BACKGROUND: Hemangiomas offer an uncommon opportunity to study rapid vessel growth and spontaneous regression of a vascular human tumor. In contrast, venous malformations are another type of vascular tumor that grows slowly without spontaneous involution. Extracellular matrix (ECM) molecules modulate the responsiveness of endothelial cells to mitogenic stimuli such as basic fibroblast growth factor (bFGF), a well-recognized stimulant of angiogenesis. In this study we hypothesized that in hemangiomas, sites of angiogenesis may have a different ECM composition than sites of vascular regression. MATERIALS AND METHODS: Using immunohistochemistry, we analyzed proliferating hemangiomas, regressing hemangiomas, venous malformations, and normal skin for the basement membrane ECM molecules collagen IV and laminin and plasma-borne ECM molecules fibronectin and vitronectin. We used metabolic labeling to determine whether primary human dermal microvascular endothelial cells regulated FGFR-1 or FGFR-2 when grown on these different matrices. RESULTS: We found that proliferating hemangiomas showed extensive deposition of vitronectin in the subendothelial space. In contrast, regressing hemangiomas or venous malformations did not show vitronectin deposition. Venous malformations, which are composed of ectatic lakes of venous channels, also lacked laminin in their basement membranes. We also found that cultured microvascular endothelial cells grown on vitronectin increased synthesis of FGFR-1 and FGFR-2 protein. CONCLUSIONS: Changes in the ECM environment occur in conjunction with the angiogenic state of a vascular human tumor. Furthermore, changes in the ECM environment alone can directly regulate synthesis of angiogenic growth factor receptors.

Vitronectin decreases microvascular endothelial cell apoptosis.

Angiogenesis after tissue injury occurs in a matrix environment consisting of fibrin, fibronectin, and vitronectin as the major extracellular matrix (ECM) constituents. ECM-integrin interactions is critical for angiogenesis and failure to bind a ligand to certain integrin receptors (alpha[v]beta3 or alpha[v]beta5) inhibits angiogenesis. The ligand that binds to alpha(v)beta3 or alpha(v)beta5 integrin receptors during microvascular angiogenesis has not been identified. Our hypothesis is that provisional matrix molecules provide the environmental context cues to microvascular endothelial cells and promote angiogenesis by decreased programmed cell death. Using cultured human microvascular endothelial cells, we show that vitronectin, in comparison to growth on alternative provisional matrix molecules (fibronectin, fibrinogen plus thrombin), collagen I, and basement membrane molecules (collagen IV), significantly reduces microvascular endothelial cell death in vitro. This reduction was observed using morphologic criteria, TdT-mediated dUTP nick end labeling (TUNEL) assay, histone release into the cytoplasm, and thymidine release into the supernatant. Though our data confirm that vitronectin may bind to more than one integrin receptor to reduce MEC apoptosis, binding to the alpha(v) component appears to be the critical integrin subcomponent for reducing apoptosis.

Role of alpha(v) integrins and angiogenesis during wound repair.

Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, is thought to be critical for wound repair. Yet few studies have critically examined dermal wound repair in a system in which angiogenesis was impaired. Since alpha(v)-containing integrins are critical for angiogenesis, we administered either an alpha(v) integrin blocking antibody or cyclic Arg-Gly-Asp peptide into a murine excisional wound model to restrict wound angiogenesis. Although both methods markedly decreased wound angiogenesis, decreased angiogenesis had no significant effect on wound epithelization, contraction, or ultimate wound closure. These results suggest that if other cellular components of wound healing are intact, moderate impairment of angiogenesis alone does not necessarily retard normal wound healing.

Simulatory hypothyroidism pituitary function in vitro: dynamics of TSH release after TRH stimulation in perifused rat hemipituitaries.

The production of immunoreactive TSH by hemipituitaries (Hps) to the stimulation of TRH in perifusion with negligible influence of the feedback of secretogogues and hormones was analysed. The stimulations were of long term continuous (3 hrs) and short term (15 min), with the dose levels of 1, 10 and 100 ng per ml of medium (for 3 hrs) or in 2 ml (for 15 min). The largest amount in production and the fastest rate in release of TSH found in present report is at 10 ng level. Only total TSH, in tissue plus in medium, after continuous TRH stimulation were dose-related, but not in release alone. We present herein an analysis of pituitary TSH production, in a perifusion system under steady TRH stimulation. This arrangement is believed to be a condition simulating hypothyroidism in pituitary level and suitable for study of the functions of the pituitary with hyperactivated thyrotrophs.