Steroid-induced polycystic ovaries in rats: effect of electro-acupuncture on concentrations of endothelin-1 and nerve growth factor (NGF), and expression of NGF mRNA in the ovaries, the adrenal glands, and the central nervous system

Previous studies on the effect of repeated electro-acupuncture (EA) treatments in rats with steriod-induced polycystic ovaries (PCO), EA has been shown to modulate nerve growth factor (NGF) concentration in the ovaries as well as corticotropin releasing factor (CRF) in the median eminence (ME). In the present study we tested the hypothesis that repeated EA treatments modulates sympathetic nerve activity in rats with PCO. This was done by analysing endothelin-1 (ET-1), a potent vasoconstrictor involved in ovarian functions, as well as NGF and NGF mRNA expression involved in the pathophysiological process underlying steroid-induced PCO. The main result in the present study was that concentrations of ET-1 in the ovaries were significantly lower in the PCO group receiving EA compared with the healthy control group (p < 0.05). In the hypothalamus, however, ET-1 concentrations were found to be significantly higher in the PCO group receiving EA than in the healthy control group (p < 0.05). Concentrations of ovarian NGF protein were significantly higher in the PCO control group compared with the healthy control group (p < 0.001), and these concentrations decreased significantly after repeated EA treatments compared with those in the PCO control group (p < 0.05) and were found to be the same as those in the healthy control group. In conclusion, these results indicate that EA modulates the neuroendocrinological state of the ovaries, most likely by modulating the sympathetic nerve activity in the ovaries, which may be a factor in the maintenance of steroid-induced PCO

Transcriptional regulation of cardiac genes balance pro- and anti-hypertrophic mechanisms in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy. HCM is often hereditary, but our knowledge of the mechanisms leading from mutation to phenotype is incomplete. The transcriptional expression patterns in the myocar - dium of HCM patients may contribute to understanding the mechanisms that drive and stabilize the hypertrophy. Cardiac myectomies/biopsies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM) and 5 controls were studied with whole genome Illumina microarray gene expression (detecting 18 189 mRNA). When comparing HOCM myocardium to controls, there was significant transcriptional down-regulation of the MYH6, EGR1, APOB and FOS genes, and significant transcriptional up-regulation of the ACE2, JAK2, NPPA (ANP), APOA1 and HDAC5 genes. The transcriptional regulation revealed both pro- and anti-hypertrophic mechanisms. The pro-hypertrophic response was explained by the transcriptional down-regulation of MYH6, indicating that the switch to the fetal gene program is maintained, and the transcriptional up-regulation of JAK2 in the JAK-STAT pathway. The anti-hypertrophic response was seen as a transcriptional down-regulation of the immediate early genes (IEGs), FOS and EGR1, and a transcriptional up-regulation of ACE2 and HDAC5. This can be interpreted as a transcriptional endogenous protection system in the heart of the HOCM patients, neither growing nor suppressing the already hypertrophic myocardium

Confocal microscopy images of fibroblasts incubated with microvesicles/exosomes stained with acridine orange.

<p>Confocal microscopy picture of DNA-stained microvesicles/exosomes after dialysis, ultracentrifugation and resuspension in DMEM. After incubation with fibroblasts for 3 h at 37°C the DNA-staining localizes in fibroblasts to and inside the nuclear membrane. Additional light microscopy was used to add a layer in images to visualize cell borders. Arrows in A) and B) indicate acridine orange staining inside nuclei. B) also visualizes red wave length which detects acridine orange staining for RNA. Yellow staining shows colocalization of DNA and RNA.</p

Filtering of differentially expressed genes.

<p>Fibroblasts incubated for 48 h with Claycomb medium, previously incubated for 24 h with cardiomyocytes were compared to fibroblasts incubated with fresh Claycomb medium.</p><p>Fibroblasts incubated for 48 h with supernatant from ultracentrifuged Claycomb medium, previously incubated for 24 h with cardiomyocytes were compared to fibroblasts incubated with fresh Claycomb medium.</p><p>Fibroblasts incubated for 48 h with pellet from ultracentrifuged Claycomb medium, previously incubated for 48 h with cardiomyocytes. Pellet was dissolved in DMEM and compared to fibroblasts incubated with fresh DMEM.</p><p>False Discovery Rate (FDR) was used for corrections for multiple testing. Significant up-regulation was defined as a foldchange >1.5 and significant down-regulation was defined as foldchange <0.67. A minimum signal intensity value of 50 was utilized. Abbreviations: Cm, cardiomyocytes; Fb, fibroblasts; sup. supernatant after ultracentrifugation; Avg. sign., average signal; FDR, False Discovery Rate; ↑, up-regulated; ↓, down-regulated; DMEM, Dulbecco's modified Eagle's medium.</p

Flow cytometry of DNA-stained microvesicles/exosomes.

<p>A) Enhanced fluorescence at the 530±15 nm channel of membrane permeable acridine orange-stained microvesicles/exosomes (below) in comparison with unstained microvesicles/exosomes (above). B) Weak or no fluorescence at 670 nm/LP channel of membrane impermeable propidium iodide-stained microvesicles/exosomes (below) not differing from unstained microvesicles/exosomes (above).</p

Detection of proteins on microvesicle/exosome surface with flow cytometry.

<p>Microvesicles/exosomes prepared from Claycomb culture medium was incubated with antibodies conjugated with phycoerythrin (PE). A) Mouse anti-caveolin-3, was detected on approximately 30% of the microvesicles/exosomes. B) Mouse anti-flotillin-1, was detected on approximately 80% of the microvesicles/exosomes. C) Mouse anti-annexin-2, was not detected on the microvesicles/exosomes. D) Mouse anti-clathrin heavy chain, was not detected on the microvesicles/exosomes. The distribution of exosomes presenting caveolin-3 and flotillin-1 indicates that the sample contains more than one population of microvesicles/exosomes.</p

Transmission electron microscopy of purified microvesicles/exosomes.

<p>A) Microvesicles/exosomes displaying an electron dense appearance, and B) electron lucent appearance. Bar represents 100 nm.</p

Myocardial ischemic preconditioning in a porcine model leads to rapid changes in cardiac extracellular vesicle messenger RNA content

Extracellular vesicles (EVs) are thought to exert protective effects after ischemic and remote ischemic preconditioning. It is not well understood which EV content factors are most relevant for protective effects. We hypothesize that ischemic preconditioning leads to qualitative changes in EV mRNA content and quantitative changes in EV size and number. Using an in vivo porcine ischemic preconditioning model, EVs were collected from coronary venous blood, and isolated by differential ultracentrifugations. The presence and purity of EV were verified by electron microscopy and Western blot, and EV number was assessed by nanoparticle tracking analysis. The mRNA EV was identified by microarray. Gene ontology analysis showed enrichment of EV mRNA coding for proteins associated with regulation of transcription, translation, extracellular matrix, morphogenic development and feeding behavior. There were 11, 678 different mRNA transcripts detected in EV, where a total of 1103 was significantly increased or decreased after preconditioning, of which 638 mRNA sequences were up-regulated and/or emerged due to preconditioning. Several of them have known association with ischemic preconditioning. There was no significant difference in EV quantity or size before and after preconditioning. These findings demonstrate in an in vivo model that myocardial ischemic preconditioning influences the composition of mRNA in EV, including gene transcripts for proteins associated with the protective effect of ischemic preconditioning. The finding that preconditioned parental cells release EV containing mRNA that is qualitatively different from those released by non-preconditioned cells shows the importance of the external milieu on parental cell EV production

Myocardial ischemic preconditioning in a porcine model leads to rapid changes in cardiac extracellular vesicle messenger RNA content

Background: Extracellular vesicles (EVs) are thought to exert protective effects after ischemic and remote ischemic preconditioning. It is not well understood which EV content factors are most relevant for protective effects. We hypothesize that ischemic preconditioning leads to qualitative changes in EV mRNA content and quantitative changes in EV size and number. Methods: Using an in vivo porcine ischemic preconditioning model, EVs were collected from coronary venous blood, and isolated by differential ultracentrifugations. The presence and purity of EV were verified by electron microscopy and Western blot, and EV number was assessed by nanoparticle tracking analysis. The mRNA EV was identified by microarray. Results: Gene ontology analysis showed enrichment of EV mRNA coding for proteins associated with regulation of transcription, translation, extracellular matrix, morphogenic development and feeding behavior. There were 11,678 different mRNA transcripts detected in EV, where a total of 1103 was significantly increased or decreased after preconditioning, of which 638 mRNA sequences were up-regulated and/or emerged due to preconditioning. Several of them have known association with ischemic preconditioning. There was no significant difference in EV quantity or size before and after preconditioning. Conclusions: These findings demonstrate in an in vivo model that myocardial ischemic preconditioning influences the composition of mRNA in EV, including gene transcripts for proteins associated with the protective effect of ischemic preconditioning. The finding that preconditioned parental cells release EV containing mRNA that is qualitatively different from those released by non-preconditioned cells shows the importance of the external milieu on parental cell EV production