-
HOXA4 protein levels and localization in the aorta and in human abdominal aortic aneurysms
This report presents evidence for the specificities of select commercially available HOXA4 antibodies in regards to concerns about the specificity of the HOXA4 antibody used by Lillvis et al. (Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms. BMC Physiol 2011, 11:9). Using an antibody characterized extensively by us, Lillvis et al. report detecting HOXA4 at a size of 33 kDa despite our previous reports that HOXA4 is detected at ~37-39 kDa and that the ~30-33 kDa band is non-specific. Using small interfering RNA targeting HOXA4, forced expression of full-length HOXA4 and HOXA4-positive and -negative ovarian cancer cell lines, we confirm our previous findings that the ~30-33 kDa band is non-specific and that HOXA4 is detected at ~37-39 kDa. Moreover, we demonstrate that HOXA4 small interfering RNA reduces the ~37-39 kDa HOXA4 band, but not the ~30-33 kDa non-specific band, in a human acute monocytic leukemia cell line used by Lillvis et al. Western blot analysis performed with two additional commercially available HOXA4 antibodies also detected HOXA4 at ~37-39 kDa. Lastly, immunofluorescent staining of a HOXA4-negative ovarian cancer cell line with the antibody used by Lillvis et al. yields strong perinuclear staining, similar to that observed by Lillvis et al., which cannot be attributed to HOXA4. Our results highlight and briefly discuss the importance of careful antibody validation and selection for use in various applications.
-
Effect of prolonged standardized bed rest on cystatin C and other markers of cardiovascular risk
Background:
Sedentary lifestyle is associated with coronary artery disease but even shorter periods of physical inactivity may increase cardiovascular risk. Cystatin C is independently associated with cardiovascular disease and our objective was to investigate the relation between this novel biomarker and standardized bed rest. Research of immobilization physiology in humans is challenging because good biological models are in short supply. From the Women International Space simulation for Exploration study (WISE) we studied markers of atherosclerosis and kidney function, including cystatin C, in a standardized bed rest study on healthy volunteers. Fifteen healthy female volunteers participated in a 20-day ambulatory control period followed by 60 days of bed rest in head-down tilt position (-6degrees) 24 h a day, finalized by 20 days of recovery. The subjects were randomized into two groups during bed rest: a control group (n=8) that remained physically inactive and an exercise group (n=7) that participated in both supine resistance and aerobic exercise training.
Results:
Compared to baseline values there was a statistically significant increase in cystatin C in both groups after bed rest (P<0.001). Glomerular filtration rate (GFR), calculated by both cystatin C and Cockcroft-Gault equation, decreased after bed rest while there were no differences in creatinine or creatine kinase levels. CRP did not change during bed rest in the exercise group, but there was an increase of CRP in the control group during recovery compared to both the baseline and the bed rest periods. The apo-B/apo-Ai ratio increased during bed rest and decreased again in the recovery period. Subjects experienced a small but statistically significant reduction in weight during bed rest and compared to baseline weights remained lower at day 8 of recovery.
Conclusion:
During and following prolonged standardized bed rest the concentrations of several clinically relevant cardiovascular risk markers change.
-
VEGF signaling mediates bladder neuroplasticity and inflammation in response to BCG
Background:
This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity.
Methods:
Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder.
Results:
The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density.
Conclusions:
For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.
-
Calcium sparks in the intact gerbil spiral modiolar artery
Background:
Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery.
Results:
Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times.
Conclusions:
This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.
-
Metabolomic phenotyping of a cloned pig model
Background:
Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs.
Results:
The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established.
Conclusions:
From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.
-
Monitoring the wild black bear's reaction to human and environmental stressors
Background:
Bears are among the most physiologically remarkable mammals. They spend half their life in an active state and the other half in a state of dormancy without food or water, and without urinating, defecating, or physical activity, yet can rouse and defend themselves when disturbed. Although important data have been obtained in both captive and wild bears, long-term physiological monitoring of bears has not been possible until the recent advancement of implantable devices.
Results:
Insertable cardiac monitors that were developed for use in human heart patients (Reveal® XT, Medtronic, Inc) were implanted in 15 hibernating bears. Data were recovered from 8, including 2 that were legally shot by hunters. Devices recorded low heart rates (pauses of over 14 seconds) and low respiration rates (1.5 breaths/min) during hibernation, dramatic respiratory sinus arrhythmias in the fall and winter months, and elevated heart rates in summer (up to 214 beats/min (bpm)) and during interactions with hunters (exceeding 250 bpm). The devices documented the first and last day of denning, a period of quiescence in two parturient females after birthing, and extraordinary variation in the amount of activity/day, ranging from 0 (winter) to 1084 minutes (summer). Data showed a transition toward greater nocturnal activity in the fall, preceding hibernation. The data-loggers also provided evidence of the physiological and behavioral responses of bears to our den visits to retrieve the data.
Conclusions:
Annual variations in heart rate and activity have been documented for the first time in wild black bears. This technique has broad applications to wildlife management and physiological research, enabling the impact of environmental stressors from humans, changing seasons, climate change, social interactions and predation to be directly monitored over multiple years.
-
Receptors and effects of gut hormones in three osteoblastic cell lines
Background:
In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells.
Methods:
mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production.
Results:
The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2.
Conclusions:
These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.
-
Regulation of Caenorhabditis elegans vitellogenesis by DAF-2/IIS through separable transcriptional and posttranscriptional mechanisms
Background:
Evolutionary theories of aging propose that longevity evolves as a competition between reproduction and somatic maintenance for a finite pool of resources. Reproduction is thought to shorten lifespan by depleting resources from processes promoting somatic maintenance. Maternal yolk production, vitellogenesis, represents a significant maternal cost for reproduction and is suppressed under genetic and environmental conditions that extend lifespan. However, little is known about the pathways regulating vitellogenesis in response to prolongevity cues.
Results:
In order to identify mechanisms that suppress vitellogenesis under prolongevity conditions, we studied factors regulating vitellogenesis in C. elegans nematodes. In C. elegans, vitellogenesis is depressed in the absence of insulin-like signaling (IIS). We found that the C. elegans daf-2/IIS pathway regulates vitellogenesis through two mechanisms. vit-2 transcript levels in daf-2 mutants were indirectly regulated through a germline-dependent signal, and could be rescued by introduction of daf-2(+) sperm. However, yolk protein (YP) levels in daf-2 mutants were also regulated by germline-independent posttranscriptional mechanisms.
Conclusions:
C. elegans vitellogenesis is regulated transcriptionally and posttranscriptionally in response to environmental and reproductive cues. The daf-2 pathway suppressed vitellogenesis through transcriptional mechanisms reflecting reproductive phenotypes, as well as distinct posttranscriptional mechanisms. This study reveals that pleiotropic effects of IIS pathway mutations can converge on a common downstream target, vitellogenesis, as a mechanism to modulate longevity.
-
The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes
Background:
The branched-chain amino acid (BCAA) leucine has been identified to be a key regulator of skeletal muscle anabolism. Activation of anabolic signalling occurs via the mammalian target of rapamycin (mTOR) through an undefined mechanism. System A and L solute carriers transport essential amino acids across plasma membranes; however it remains unknown whether an exogenous supply of leucine regulates their gene expression. The aim of the present study was to investigate the effects of acute and chronic leucine stimulation of anabolic signalling and specific amino acid transporters, using cultured primary human skeletal muscle cells.
Results:
Human myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34) and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine.
Conclusions:
mTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.
-
Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysm
Background:
The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.
Results:
We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007).
Conclusions:
Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.
|
