Hormetic modulation of angiogenic factors by exercise-induced mechanical and metabolic stress in human skeletal muscle.

This study used an integrative experimental model in humans to investigate whether muscle angiogenic factors are differentially modulated by exercise stimuli eliciting different degrees of mechanical and metabolic stress. In a randomized crossover design, 12 men performed two low-volume high-intensity exercise regimens, including short sprint intervals (SSI) or long sprint intervals (LSI) inducing pronounced mechanical/metabolic stress, and a high-volume moderate-intensity continuous exercise protocol (MIC) inducing mild but prolonged mechanical/metabolic stress. Gene and protein expression of angiogenic factors was determined in vastus lateralis muscle samples obtained before and after exercise. Exercise upregulated muscle VEGF mRNA to a greater extent in LSI and MIC compared with SSI. Analysis of angiogenic factors sensitive to shear stress revealed more marked exercise-induced VEGF receptor 2 (VEGF-R2) mRNA responses in MIC than SSI, as well as greater platelet endothelial cell adhesion molecule (PECAM-1) and endothelial nitric oxide synthase (eNOS) mRNA responses in LSI than SSI. No apparent exercise-induced phosphorylation of shear stress-sensory proteins VEGF-R2Tyr1175, PECAM-1Tyr713, and eNOSSer1177 was observed despite robust elevations in femoral artery shear stress. Exercise evoked greater mRNA responses of the mechanical stretch sensor matrix metalloproteinase-9 (MMP9) in SSI than MIC. Exercise-induced mRNA responses of the metabolic stress sensor hypoxia-inducible factor-1α (HIF-1α) were more profound in LSI than SSI. These results suggest that low-volume high-intensity exercise transcriptionally activates angiogenic factors in a mechanical/metabolic stress-dependent manner. Furthermore, the angiogenic potency of low-volume high-intensity exercise appears similar to that of high-volume moderate-intensity exercise, but only on condition of eliciting severe mechanical/metabolic stress. We conclude that the angiogenic stimulus produced by exercise depends on both magnitude and protraction of myocellular homeostatic perturbations.NEW & NOTEWORTHY Skeletal muscle capillary growth is orchestrated by angiogenic factors sensitive to mechanical and metabolic signals. In this study, we employed an integrative exercise model to synergistically target, yet to different extents and for different durations, the mechanical and metabolic components of muscle activity that promote angiogenesis. Our results suggest that the magnitude of the myocellular perturbations incurred during exercise determines the amplitude of the angiogenic molecular signals, implying hormetic modulation of skeletal muscle angiogenesis by exercise-induced mechanical and metabolic stress.

Metabolic stress-dependent regulation of the mitochondrial biogenic molecular response to high-intensity exercise in human skeletal muscle.

KEY POINTS: Low-volume high-intensity exercise training promotes muscle mitochondrial adaptations that resemble those associated with high-volume moderate-intensity exercise training. These training-induced mitochondrial adaptations stem from the cumulative effects of transient transcriptional responses to each acute exercise bout. However, whether metabolic stress is a key mediator of the acute molecular responses to high-intensity exercise is still incompletely understood. Here we show that, by comparing different work-matched low-volume high-intensity exercise protocols, more marked metabolic perturbations were associated with enhanced mitochondrial biogenesis-related muscle mRNA responses. Furthermore, when compared with high-volume moderate-intensity exercise, only the low-volume high-intensity exercise eliciting severe metabolic stress compensated for reduced exercise volume in the induction of mitochondrial biogenic mRNA responses. The present results, besides improving our understanding of the mechanisms mediating exercise-induced mitochondrial biogenesis, may have implications for applied and clinical research that adopts exercise as a means to increase muscle mitochondrial content and function in healthy or diseased individuals. ABSTRACT: The aim of the present study was to examine the impact of exercise-induced metabolic stress on regulation of the molecular responses promoting skeletal muscle mitochondrial biogenesis. Twelve endurance-trained men performed three cycling exercise protocols characterized by different metabolic profiles in a randomized, counter-balanced order. Specifically, two work-matched low-volume supramaximal-intensity intermittent regimes, consisting of repeated-sprint (RS) and speed endurance (SE) exercise, were employed and compared with a high-volume continuous moderate-intensity exercise (CM) protocol. Vastus lateralis muscle samples were obtained before, immediately after, and 3 h after exercise. SE produced the most marked metabolic perturbations as evidenced by the greatest changes in muscle lactate and pH, concomitantly with higher post-exercise plasma adrenaline levels in comparison with RS and CM. Exercise-induced phosphorylation of CaMKII and p38 MAPK was greater in SE than in RS and CM. The exercise-induced PGC-1α mRNA response was higher in SE and CM than in RS, with no difference between SE and CM. Muscle NRF-2, TFAM, MFN2, DRP1 and SOD2 mRNA content was elevated to the same extent by SE and CM, while RS had no effect on these mRNAs. The exercise-induced HSP72 mRNA response was larger in SE than in RS and CM. Thus, the present results suggest that, for a given exercise volume, the initial events associated with mitochondrial biogenesis are modulated by metabolic stress. In addition, high-intensity exercise seems to compensate for reduced exercise volume in the induction of mitochondrial biogenic molecular responses only when the intense exercise elicits marked metabolic perturbations.

Response to cooling of pony stallion semen selected by glass wool filtration.

The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Retained semen was considered the portion of cells that did not transpose glass wool barrier. Total motility from the control, filtered and retained groups after 24 h of cooling was 35.5%, 43.3% and 10% (p < .0001) respectively. Sperm membrane integrity percentage at the CFDA/PI test was 37.9%, 44.8% and 14.8% (p < .0001), on the control, filtered and retained groups respectively. The results confirmed that the passage of spermatozoa through glass wool increased the selection of spermatozoa from pony stallions with higher motility, mitochondrial viability and membrane integrity for cooling in milk extender up to 24 h. Moreover, it was not obtained higher sperm parameters to control after cooling 48 h under the conditions that the study was conducted.

Altered localization and functionality of TAR DNA Binding Protein 43 (TDP-43) in niemann- pick disease type C.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by the occurrence of visceral and neurological symptoms. At present, the molecular mechanisms causing neurodegeneration in this disease are unknown. Here we report the altered expression and/or mislocalization of the TAR-DNA binding protein 43 (TDP-43) in both NPC mouse and in a human neuronal model of the disease. We also report the neuropathologic study of a NPC patient's brain, showing that while TDP-43 is below immunohistochemical detection in nuclei of cerebellar Purkinje cells, it has a predominant localization in the cytoplasm of these cells. From a functional point of view, the TDP-43 mislocalization, that occurs in a human experimental neuronal model system, is associated with specific alterations in TDP-43 controlled genes. Most interestingly, treatment with N-Acetyl-cysteine (NAC) or beta-cyclodextrin (CD) can partially restore TDP-43 nuclear localization. Taken together, the results of these studies extend the role of TDP-43 beyond the Amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD)/Alzheimer disease (AD) spectrum. These findings may open novel research/therapeutic avenues for a better understanding of both NPC disease and the TDP-43 proteinopathy disease mechanism.

TCL1 transgenic mouse model as a tool for the study of therapeutic targets and microenvironment in human B-cell chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy with a mature phenotype. In spite of its relatively indolent nature, no radical cure is as yet available. CLL is not associated with either a unique cytogenetic or a molecular defect, which might have been a potential therapeutic target. Instead, several factors are involved in disease development, such as environmental signals which interact with genetic abnormalities to promote survival, proliferation and an immune surveillance escape. Among these, PI3-Kinase signal pathway alterations are nowadays considered to be clearly important. The TCL1 gene, an AKT co-activator, is the cause of a mature T-cell leukemia, as well as being highly expressed in all B-CLL. A TCL1 transgenic mouse which reproduces leukemia with a distinct immunophenotype and similar to the course of the human B-CLL was developed several years ago and is widely used by many groups. This is a review of the CLL biology arising from work of many independent investigators who have used TCL1 transgenic mouse model focusing on pathogenetic, microenviroment and therapeutic targets.

Accuracy of GPS devices for measuring high-intensity running in field-based team sports.

We compared the accuracy of 2 GPS systems with different sampling rates for the determination of distances covered at high-speed and metabolic power derived from a combination of running speed and acceleration. 8 participants performed 56 bouts of shuttle intermittent running wearing 2 portable GPS devices (SPI-Pro, GPS-5 Hz and MinimaxX, GPS-10 Hz). The GPS systems were compared with a radar system as a criterion measure. The variables investigated were: total distance (TD), high-speed distance (HSR>4.17 m·s(-1)), very high-speed distance (VHSR>5.56 m·s(-1)), mean power (Pmean), high metabolic power (HMP>20 W·kg(-1)) and very high metabolic power (VHMP>25 W·kg(-1)). GPS-5 Hz had low error for TD (2.8%) and Pmean (4.5%), while the errors for the other variables ranged from moderate to high (7.5-23.2%). GPS-10 Hz demonstrated a low error for TD (1.9%), HSR (4.7%), Pmean (2.4%) and HMP (4.5%), whereas the errors for VHSR (10.5%) and VHMP (6.2%) were moderate. In general, GPS accuracy increased with a higher sampling rate, but decreased with increasing speed of movement. Both systems could be used for calculating TD and Pmean, but they cannot be used interchangeably. Only GPS-10 Hz demonstrated a sufficient level of accuracy for quantifying distance covered at higher speeds or time spent at very high power.

A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-ß-cyclodextrin.

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1(-/-) mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1(-/-) mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1(-/-) mice. A single injection of hydroxypropyl-ß-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.

Cholesterol metabolism-associated molecules in late onset Alzheimer's disease.

Alzheimer’s disease (AD) is the most common cause of dementia and, with an aging population, poses a huge public health problem. Although a small per cent is caused by single gene changes, most AD is sporadic and unexplained. Of many modifying factors, changes in brain cholesterol homeostasis are the best studied. We present a review of the role of altered cholesterol metabolism and hypercholesterolemia in APP processing and Abeta generation. We also provide an overview of the potential pharmacological modulation of cholesterol homeostasis in the brain by cholesterol-lowering agents and beta-cyclodextrins.

Are all cases of low-grade mosaic trisomy 13 in amniotic fluid with no fetal malformation in fact confined placental mosaicism? A case report.

We report on a case of true prenatal mosaic trisomy 13 on amniotic fluid associated with a normal phenotype at the age of 6 years. The amniocentesis was performed because of advanced maternal age and was controlled by a second sample. Morphological and cardiac ultrasonography did not reveal any fetal malformations. No trisomic cells were found in the fetal blood and a nuclear magnetic resonance imaging (IRM) of the brain was performed during the third trimester found no abnormality of the brain. Finally, at birth cytogenetic analysis was performed on two placental samples for chromosomal analysis: one in an area where the placenta seemed normal, and the other one in an area with infarcted and hemorrhagic aspect. We found a high rate of trisomic cells in the sample with abnormal aspect. Furthermore, no trisomic cell was observed by fluorescent in situ hybridation (FISH) on the buccal smears of the baby. We concluded to a confined placental mosaicism. The good outcome of the child aged 6 years confirms this diagnosis. So in the aim to predict a good development for the child in case of low rate mosaic trisomy 13 in amniotic fluid, we propose at birth: i) to take several samples from the placenta to confirm placental mosaicism; ii) to label by FISH buccal smears with a LSI 13 probe to prove that the baby is not a carrier of the trisomy.

Impact of SLCO1B1 (OATP1B1) and ABCG2 (BCRP) genetic polymorphisms and inhibition on LDL-C lowering and myopathy of statins.

Statins are the preferred class of drugs for treating patients with atherosclerosis and related coronary heart disease. Treatment with statins leads to significant low-density lipoprotein cholesterol (LDL-C) lowering, resulting in reductions in major coronary and vascular events. Statins are generally well tolerated and safe; however, their use is complicated by infrequent, but often serious, muscular adverse events. For many statins, both efficacy and risk of adverse muscle events can be influenced by membrane transporters, which are important determinants of statin disposition. Genetic polymorphisms and drug-drug interactions (DDIs) involving organic anion-transporting polypeptide 1B1 and breast cancer resistance protein have shown the capacity to reduce the activity of these transporters, resulting in changes in LDL-C lowering by statins, as well as changes in the frequency of adverse muscle events associated with their use. This review presents evidence for how reduced transporter activity impacts the safety and pharmacology of statins. It expands on the scope of other recent statin reviews by providing recommendations on in vitro evaluation of statin interaction potential, discussing how reduced transporter activity impacts statin management during drug development, and proposing ideas on how to evaluate the impact of DDI on statin efficacy during clinical trials. Furthermore, the potential clinical consequences of perturbing statin efficacy via DDI are discussed.

Strain-specific proportion of the two isoforms of the dopamine D2 receptor in the mouse striatum: associated neural and behavioral phenotypes.

Genetic variability in the proportion of the two alternative dopamine D2 receptor (D2R) mRNA splice variants, D2R-long (D2L) and D2R-short (D2S), influence corticostriatal functioning and could be implicated in liability to psychopathology. This study compared mesostriatal D2L/D2S ratios and associated neural and behavioral phenotypes in mice of the DBA/2J and C57BL/6J-inbred strains, which differ for schizophrenia- and addiction-like phenotypes. Results showed that DBA/2J mice lack the striatal predominance of D2L that has been reported in the rat and in C57BL/6J mice and confirmed in the latter strain by this study. Only C57BL/6J mice showed enhanced striatal c-Fos expression under D1R and D2/3R co-stimulation, indicating synergistic interaction between the subtypes of DA receptors. Instead, DBA/2J mice were characterized by opposing effects of D2/3R and D1R stimulation on striatal c-Fos expression, in line with a more pronounced influence of D2S isoform, and did not express stereotyped climbing under D1R and D2/3R co-stimulation, as reported for D2L-/- mice. Finally, strain-specific modulation of c-Fos expression by D1R and D2/3R co-stimulation was selectively observed in striatal compartments receiving inputs from the prefrontal cortex and involved in the control of motivated behaviors. These results show differences in tissue-specific D2R splicing in mice with intact genotypes and support a role for this phenotype in individual variability of corticostriatal functioning and in liability to psychopathology.

Familial 18 centromere variant resulting in difficulties in interpreting prenatal interphase FISH.

We report here on a familial case of centromeric heteromorphism of chromosome 18 detected by prenatal interphase fluorescence in situ hybridization (FISH) analysis transmitted by the mother to her fetus, and resulting in complete loss of one 18 signal. The prenatal diagnosis was performed by interphase FISH (AneuVysion probe set, and LSI DiGeorge 22q11.2 kit) because of the presence of an isolated fetal cardiac abnormality, and was first difficult to interpret: only one centromeric 18 signal was detectable on prenatal interphase nuclei, along with one signal for the Y and one for the X chromosome. The LSI DiGeorge 22q11.2 kit also showed the absence of one TUPLE 1 signal on all examined nuclei. In fact, the FISH performed on maternal buccal smear displayed the same absence of one chromosome 18 centromeric signal, combined with the presence of two TUPLE1 signals. All these results led to the diagnosis of an isolated 22q11.2 fetal microdeletion that was confirmed on metaphases spreads. This case illustrates once again that the locus specific (LSI) probes are more effective than the alpha centromeric probes for interphase analysis. The development of high-quality LSI probes for chromosomes 18, X and Y could avoid the misinterpretation of prenatal interphase FISH leading to numerous additional and expensive investigations.

Lymphatic mapping to tailor selective lymphadenectomy in cN0 tongue carcinoma: beyond the sentinel node concept.

PURPOSE: Cervical lymph node status is the most important pathological determinant of prognosis and decision making in head and neck squamous cell carcinoma (SCC). The aim of this study was to demonstrate that lymphoscintigraphy (LS) can supply a complete map of the lymphatic drainage before surgery, allowing planning of the type of intervention and serving to guide lymphadenectomy. METHODS: The study population comprised 14 patients with T2-4 SCCs of the tongue and clinically negative lymph nodes in the neck (cN0) who were scheduled to undergo tumour resection and selective level I-IV neck dissection extended to level V. LS was performed in all patients following the injection of (99m)Tc-colloidal sulphide in three aliquots around the primary lesion. Dynamic, static and tomographic images of the head and neck were acquired. The operative specimens were subjected to lymphoscintigraphic evaluation. Preoperative and postoperative imaging results were compared with the pathological findings. All nodes were examined using haematoxylin-eosin staining. RESULTS: Preoperative LS was successful in all patients. Preferential pathways of lymphatic drainage were identified: level II of the neck was the most common lymphatic drainage pattern, followed by levels IV and III. Contralateral drainage occurred in 11 patients and in two of them metastatic nodes were found on the contralateral side. Metastases were observed only in radioactive lymph nodes. CONCLUSION: LS is able to supply a complete map of the lymphatic drainage before surgery, making it possible to tailor selective neck dissection to each individual patient based on the results of preoperative mapping, thereby sparing healthy lymphatic tissue and reducing surgery-related morbidity.

Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: a pilot study.

In a previous study we applied a three-step avidin-biotin pretargeting approach to target 90Y-biotin to the tumour in patients with recurrent high grade glioma. The encouraging results obtained in this phase I-II study prompted us to apply the same approach in an adjuvant setting, to evaluate (i) time to relapse and (ii) overall survival. We enrolled 37 high grade glioma patients, 17 with grade III glioma and 20 with glioblastoma, in a controlled open non-randomized study. All patients received surgery and radiotherapy and were disease-free by neuroradiological examinations. Nineteen patients (treated) received adjuvant treatment with radioimmunotherapy. In the treated glioblastoma patients, median disease-free interval was 28 months (range=9-59); median survival was 33.5 months and one patient is still without evidence of disease. All 12 control glioblastoma patients died after a median survival from diagnosis of 8 months. In the treated grade III glioma patients median disease-free interval was 56 months (range=15-60) and survival cannot be calculated as only two, within this group, died. Three-step radioimmunotherapy promises to have an important role as adjuvant treatment in high grade gliomas, particularly in glioblastoma where it impedes progression, prolonging time to relapse and overall survival. A further randomized trial is justified.

90Y-DOTA-D-Phe1-Try3-octreotide in therapy of neuroendocrine malignancies.

Somatostatin receptors type 2 (sst(2)) are expressed in high concentration on numerous neudoendocrine tumors. The successful use of radiolabeled somatostatin analogs in imaging promoted further studies in utilizing them in radiopeptide therapy. The somatostatin analog [(90)Y-DOTA-D-Phe(1)-Try3]octreotide (DOTATOC) (DOTA: 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) possesses favorable characteristic for its therapeutic use; shows high affinity for sst(2), moderately high affinity for sst(5), and intermediate affinity for sst(3); high hydrophilicity; stable and facile labeling with (111) In and (90) Y. In this article we report our experience with (90)Y-DOTATOC in neuroendocrine tumors. Eighty-seven patients with neuroendocrine tumors were treated with a cumulated activity ranging from 7.4 to 20.2 GBq. Most patients responded with stabilization of disease (48%); however, objective responses were observed in 28% of patients (5% complete response). No major acute reactions were observed up to the activity of 5.55 GBq per cycle. The dose limiting was bone marrow toxicity and the maximal tolerated dose was defined as 5.18 GBq.

Expression screening for Lhx3 downstream genes identifies Thg-1pit as a novel mouse gene involved in pituitary development.

Thg-1pit, a novel mouse gene, was detected in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3 null mutant embryos. The predicted translation product of the Thg-1pit gene contains a C-terminal TSC-box adjacent to a leucine zipper motif. These features are characteristic for the TSC-22/DIP/bun family of proteins. The onset of prominent Thg-1pit expression coincides with Lhx3 activation at early stages of pituitary development. Expression is further enhanced as cells begin to differentiate within the developing pituitary gland. No expression is observed in the pituitary rudiment of mutants that lack Lhx3 function. A possible role is thus suggested for Lhx3 activities in the regulation of Thg-1pit function during early steps of pituitary organogenesis.

Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining.

We have investigated the ability of growing dictyate oocytes and early preimplantation embryos of the mouse to process extrachromosomal DNA molecules with free ends by intranuclearly microinjecting DNA fragments containing a region of homology of various extent at either the 5' or 3' terminus. Homologous recombination of these fragments by single-strand annealing (SSA), but not other DNA recombination/joining mechanisms, resulted in the formation of a full-length hsp-lacZ-pA fusion gene that was transcriptionally activated by heat shock in growing oocytes and spontaneously at the early two-cell stage in the embryos, making it possible to quantitatively evaluate SSA activities of these cells by the beta-galactosidase produced. SSA activities of oocytes and embryos were similar in their general properties and in the activity levels observed with saturating amounts of DNA. However, embryo SSA was almost one order of magnitude less effective than that of oocytes. Oocyte and embryo 5' --> 3' exonuclease (a key function of the SSA pathway) and DNA nonhomologous end joining (NHEJ) activities were also investigated using an asymmetric PCR assay. Results showed that NHEJ is lacking in oocytes and is very prominent in the embryos, where it competes with SSA for the injected DNA.