Imaging the Proangiogenic Effects of Cardiovascular Drugs in a Diabetic Model of Limb Ischemia.

Purpose: Peripheral artery disease (PAD) causes narrowing of arteries in the limbs, leading to tissue ischemia, gangrene, and eventually limb amputation. The presence of diabetes greatly exacerbates the course of PAD, accounting for the majority of lower limb amputations. Therapeutic strategies focussing on macrovascular repair are less effective in diabetic patients where smaller vessels are affected, and proangiogenic therapies offer a viable adjunct to improve vascularisation in these at risk individuals. The purpose of the current study was to assess the proangiogenic effects of drugs routinely used to treat cardiovascular disease in a diabetic murine model of hind limb ischemia longitudinally using multimodal imaging. Procedures: Diabetic mice underwent surgical intervention to induce hind limb ischemia and were treated with simvastatin, metformin, or a combination orally for 28 days and compared to diabetic and nondiabetic mice. Neovascularisation was assessed using [18F]FtRGD PET imaging, and macrovascular volume was assessed by quantitative time of flight MRI. At each imaging time point, VEGF expression and capillary vessel density were quantified using immunohistochemical analysis, and functional recovery and disease progression were assessed. Results: Combined use of simvastatin and metformin significantly increased neovascularisation above levels measured with either treatment alone. Early angiogenic events were accurately assessed using PET [18F]FtRGD, showing maximal retention in the ischemic hind limb by day 8, which translated to a sustained increase in vascular volume at later time points. Immunohistochemical analysis shows that combined therapy significantly increased VEGF expression and capillary density (CD31+) in a similar time course and also slowed disease progression while simultaneously improving functional foot use. Conclusions: Combined treatment with simvastatin and metformin led to a significant improvement in limb angiogenesis, vascular volume, and sustained functional recovery in a diabetic murine model of HLI. PET imaging with [18F]FtRGD provides a robust method for early detection of these proangiogenic effects preclinically and may be useful for the assessment of proangiogenic therapies used clinically to treat diabetic PAD patients.

Characterisation of the nuclear receptors FXR, PXR and CAR in normal and cholestatic placenta.

Bile acids are the toxic end products of hepatic cholesterol metabolism. They are synthesised from early in gestation and excreted via the placenta. The mechanism for transplacental excretion of bile acids is not known. The gene and protein expression of the nuclear receptors responsible for hepatic bile acid metabolism and transport was studied in eight normal and fourteen cholestatic placentas, and in an ex vivo model. The expression of the nuclear receptor FXR and several of it's target genes and of PXR and CAR was found to be very low in both normal and cholestatic placenta.

Negative correlation between cerebral inorganic phosphate and the volumetric niacin response in male patients with schizophrenia who have seriously and dangerously violently offended: a (31)P magnetic resonance spectroscopy study.

The aim of the study was to examine the association of arachidonic acid-related signal transduction with cerebral metabolism in patients with schizophrenia who have violently and dangerously offended while psychotic. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 11 male patients with schizophrenia who had violently offended (homicide, attempted murder, or wounding with intent to cause grievous bodily harm) while psychotic. Spectra were obtained from 70 x 70 x 70 mm(3) voxels using an image-selected in vivo spectroscopy pulse sequence. Niacin flush testing results were quantified as the volumetric niacin response. There was a strong, and negative, correlation between the volumetric niacin response and the metabolite concentration of inorganic phosphate expressed as a ratio of the total 31-phosphorus signal (p<0.005). Our results suggest that patients with schizophrenia who have violently offended and have poor phospholipid-related signal transduction may have higher levels of cerebral energy metabolism.

Hierarchy of carbon source selection in Paracoccus pantotrophus: strict correlation between reduction state of the carbon substrate and aerobic expression of the nap operon.

Paracoccus pantotrophus can express a periplasmic nitrate reductase (Nap) during aerobic growth. A proposed role for this enzyme is the dissipation of excess redox energy during oxidative metabolism of reduced carbon substrates. To investigate the regulation of nap expression, a transcriptional fusion between the nap promoter region of P. pantotrophus and the lacZ gene was constructed. When this fusion was used, analyses showed that transcription from the nap promoter increases as the average reduction state of the carbon atoms increases. Thus, beta-galactosidase activities increase as the carbon source changes in the order succinate-acetate-butyrate. This result was obtained regardless of which of the three carbon sources was used for culture of the inoculum. If two carbon sources were presented together, the beta-galactosidase activity was always the same as it was when the least-reduced carbon source was added alone. This suggests that the regulation is dependent upon metabolism of the more-reduced carbon sources rather than just their presence in the medium. Analysis of culture medium by (1)H nuclear magnetic resonance showed that for aerobic growth P. pantotrophus strictly selected its carbon source in the order succinate-acetate-butyrate. This was reflected by diauxic growth kinetics on medium containing mixed carbon substrates. The regulatory mechanism underpinning such a selection is unknown but is likely to be related to the mechanism which controls the transcription of the nap operon.

Swelling-activated taurine and creatine effluxes from rat cortical astrocytes are pharmacologically distinct.

Primary cultures of rat cortical astrocytes undergo a swelling-activated loss of taurine and creatine. In this study, the pharmacological characteristics of the taurine and creatine efflux pathways were compared, and significant differences were shown to exist between the two. Both taurine and creatine effluxes were rapidly activated upon exposure of astrocytes to hypo-osmotic media, and rapidly inactivated upon their return to iso-osmotic media. The relative rates of taurine and creatine efflux depended upon the magnitude of the hypo-osmotic shock. Anion-transport inhibitors strongly inhibited taurine efflux, with the order of potency being NPPB > DIDS > niflumic acid. DIDS and NPPB had less of an inhibitory effect on creatine efflux, whereas tamoxifen and niflumic acid actually stimulated creatine efflux. These data are consistent with separate pathways for taurine and creatine loss during astrocyte swelling.

A comparison of cell and tissue extraction techniques using high-resolution 1H-NMR spectroscopy.

Analysis of brain metabolites by a wide range of analytical techniques is typically achieved using biochemical extraction methodologies that require either two separate samples or two separate extraction steps to prepare both aqueous and organic metabolite fractions. However there are a number of brain pathologies in which both aqueous metabolite and lipid changes occur so that a simultaneous extraction of both fractions would be valuable. The methanol-chloroform (M/C) technique enables extraction of both aqueous metabolites and lipids simultaneously. It is already well established for lipid extraction of cells and tissue but its efficiency and reproducibility for extraction of aqueous metabolites is unknown. Therefore, we compared the aqueous metabolite yield and the reproducibility of the M/C method to the commonly used perchloric acid (PCA) method, using 1H-NMR spectroscopy of adult rat brain and purified rat astrocyte culture extracts. The results indicate that M/C is a superior technique for aqueous metabolite extraction from both brain tissue and cells when compared to the PCA method. The M/C extraction technique enables the simultaneous extraction of both lipids and aqueous metabolites from a single sample using small solvent-volumes, making it well suited for NMR investigations of both tissues and cells.

Developmental and regional distribution of aspartoacylase in rat brain tissue.

The function of N-acetyl-aspartate (NAA), a predominant molecule in the brain, has not yet been determined. However, NAA is commonly used as a putative marker of viable neurones. To investigate the possible function of NAA, we determined the anatomical, developmental and cellular distribution of aspartoacylase, which catalyses the hydrolysis of NAA. Levels of aspartoacylase activity were measured during postnatal development in several brain regions. The differential distribution of aspartoacylase activity in purified populations of cells derived from the rat CNS was also investigated. The developmental and anatomical distribution of aspartoacylase correlated with the maturation of white matter tracts in the rat brain. Activity increased markedly after 7 days and coincided with the time course for the onset of myelination in the rat brain. Gray matter showed little activity or developmental trend. There was a 60-fold excess in optic nerve (a white matter tract) when compared with cortex at 21 days of development. In the adult brain there was a 18-fold difference in corpus callosum compared with cortex (stripped of corpus callosum). Cellular studies demonstrated that purified cortical neurons and cerebellar granular neurones have no activity. Primary O-2A progenitor cells had moderate activity, with three-fold higher activity in immature oligodendrocyte and 13-fold increase in mature oligodendrocytes (myelinating cells of the CNS). The highest activity was seen in type-2 astrocytes (20-fold difference compared with O-2A progenitors) derived from the same source. Aspartoacylase activity increased with time in freshly isolated astrocytes, with significantly higher activity after 15 days in culture. We conclude that aspartoacylase activity in the developing postnatal brain corresponds with maturation of myelination, and that the cellular distribution is limited to glial cells.

Hypo-osmotic swelling-activated release of organic osmolytes in brain slices: implications for brain oedema in vivo.

A decrease in the intracellular levels of osmotically active species has invariably been seen after swelling of mammalian brain tissue preparations. The exact identity of the species, and the manner of their decrease, remain to be described. We investigated the swelling-activated decrease of organic osmolytes in rat cortical brain slices using (1)H- and (31)P-magnetic resonance spectroscopy. We found that acute hypo-osmotic shock causes decreases in the levels of a range of intracellular amino acids and amino acid derivatives, N-acetyl-aspartate, creatine, GABA, glutamate, hypotaurine, and also in the levels of the methylamines glycerol-phosphorylcholine, phosphorylcholine and choline. Incubation of cortical slices with the anion channel blockers niflumic acid and tamoxifen caused inhibition of organic osmolyte efflux, suggesting that such osmolyte efflux occurs through anion channels. Intracellular phosphocreatine was also seen to decrease during acute hypo-osmotic superfusion, although intracellular ATP remained constant. In addition, the acidification of an intracellular compartment was observed during hypo-osmotic superfusion. Our results suggest a link between brain energy reserve and brain osmoregulation.

Differential expression of carnosine, homocarnosine and N-acetyl-L-histidine hydrolytic activities in cultured rat macroglial cells.

N-acetyl-L-histidine (NAH) and N-acetyl-L-aspartate (NAA) are representatives of two series of substances that are synthesized by neurons and other cells in the vertebrate central nervous system (CNS). Histidine containing homologs of NAH are beta-alanyl-L-histidine or carnosine (Carn) and gamma-aminobutyrl-L-histidine or homocarnosine (Hcarn). A homolog of NAA is N-acetylaspartylglutamate (NAAG). These substances belong to a unique group of osmolytes in that they are synthesized in cells that may not to be able to hydrolyze them, and are released in a regulated fashion to a second compartment where they can be rapidly hydrolyzed. In this investigation, the catabolic activities for NAH, Carn, and Hcarn in cultured macroglial cells and neurons have been measured, and the second compartment for NAH and Hcarn has been identified only with astrocytes. In addition, oligodendrocytes can only hydrolyze Carn, although Carn can also be hydrolyzed by astrocytes. Thus, astrocytes express hydrolytic activity against all three substrates, but oligodendrocytes can only act on Carn. The cellular separation of these hydrolytic enzyme activities, and the possible nature of the enzymes involved are discussed.

In vitro expression of N-acetyl aspartate by oligodendrocytes: implications for proton magnetic resonance spectroscopy signal in vivo.

Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N-Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA-specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte-type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.

Metabolic changes underlying 31P MR spectral alterations in human hepatic tumours.

Magnetic resonance spectroscopy (MRS) remains the technique of choice for observing tumour metabolism non-invasively. Although initially 31P MR spectroscopy showed much promise as a non-invasive diagnostic tool, studies of a wide range of hepatic tumours have conclusively shown that this technique cannot be utilized to distinguish between different tumour types. This lack of specificity and sensitivity appears to be a consequence of the fact that hepatic tumours develop with a range of modalities and not as a single abnormal disease process, and also because of the limited availability of MR detectable metabolic markers. This has led, in recent years, to a re-evaluation of the role of 31P MR spectroscopy, re-emerging as a non-invasive tool to follow the efficacy of the treatment regime. Furthermore, since the principal changes observed in tumours by 31P MRS appear to be an elevation in the concentration of phosphorylcholine (PCho) and phosphoethanolamine (PEth), new research using a combination of MRS and tissue culture of cell lines which carry a combination of known inducible oncogenes, are helping to elucidate some of the metabolic pathways that give rise to these metabolic alterations.

Hormonal regulation of the mRNA encoding the ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in neonatal primary cultures of cortical astrocytes and meningeal fibroblasts.

We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3-hydroxy-3-methylglutaryl-CoA cycle, namely, mitochondrial acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum-free/hormone-free medium.

Molecular cloning of rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase and detection of the corresponding mRNA and of those encoding the remaining enzymes comprising the ketogenic 3-hydroxy-3-methylglutaryl-CoA cycle in central nervous system of suckling rat.

We have investigated, by RNase protection assays in rat brain regions and primary cortical astrocyte cultures, the presence of the mRNA species encoding the three mitochondrially located enzymes acetoacetyl-CoA thiolase, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mt. HMG-CoA synthase) and HMG-CoA lyase (HMG-CoA lyase) that together constitute the ketogenic HMG-CoA cycle. As a prerequisite we obtained a full-length cDNA encoding rat HMG-CoA lyase by degenerate oligonucleotide-primed PCR coupled to a modification of PCR-rapid amplification of cDNA ends (PCR-RACE). We report here: (1) the nucleotide sequence of rat mt. HMG-CoA lyase, (2) detection of the mRNA species encoding all three HMG-CoA cycle enzymes in all regions of rat brain during suckling, (3) approximately twice the abundance of mt. HMG-CoA synthase mRNA in cerebellum than in cortex in 11-day-old suckling rat pups, (4) significantly lower abundances of mt. HMG-CoA synthase mRNA in brain regions derived from rats weaned to a high-carbohydrate/low-fat diet compared with the corresponding regions derived from the suckling rat, and (5) the presence of mt. HMG-CoA synthase mRNA in primary cultures of neonatal cortical astrocytes at an abundance similar to that found in liver of weaned animals. These results provide preliminary evidence that certain neural cell types possess ketogenic potential and might thus have a direct role in the provision of fatty acid-derived ketone bodies during the suckling period.

The application of NMR spectroscopy to the study of apoptosis.

Programmed cell death is a physiological process whereby multicellular organisms eliminate unwanted cells. The unravelling of this process has thus far remained in the hands of molecular and cell biologists. These studies have therefore led to a detailed understanding of the genetic nature and some control mechanisms regulating this complex process. However, the biochemical consequences of apoptosis within the cell still remain elusive. NMR spectroscopy, which allows the detection, identification and quantitation of a large number of metabolites and macromolecules, is being utilized to define some of these metabolic processes. The initial application of NMR spectroscopy, to solving the mysteries of apoptosis were confined to elucidating the macromolecular structure of the proteins involved in initiating programmed cell death. The ability of NMR to monitor energy metabolites non-invasively was so put to use in correlating the temporal changes in the rate of apoptosis to these metabolites. More recent studies have shown changes in both a range of small molecules and membrane components. The power of NMR spectroscopy is being used in more laboratories and is likely to become the method of choice for the possible detection of apoptosis in vivo once the NMR signals associated with the apoptotic process have been established in vitro.

Immortalization and transformation are associated with specific alterations in choline metabolism.

Analysis of transformed, immortalized, and primary rat Schwann cells by high-resolution proton nuclear magnetic resonance spectroscopy reveals that immortalization of Schwann cells (by SV40 large T antigen) induced a decrease in sn-glycero-3-phosphocholine (GPCho), whereas H-ras alone, which is known to cause growth arrest in these cells, induced a marked increase in GPCho and a decrease in phosphocholine (PCho). An increase of PCho was found only in cells fully transformed by both oncogenes together. Moreover, we examined 11 human tumor cell lines, all of which expressed a PCho:GPCho ratio similar to that of fully transformed rat Schwann cells. Importantly, neither the absolute levels of PCho nor the ratio of PCho:GPCho were correlated with the rate of cell division across a range of normal (primary cultures) and transformed cells. Thus, raised PCho:GPCho ratios may serve as an indicator of multiple oncogenic lesions and malignancy in noninvasive tumor investigations.

Regional and developmental variations in metabolite concentration in the rat brain and eye: a study using 1H NMR spectroscopy and high performance liquid chromatography.

Regional and developmental changes in metabolite concentrations were measured by 1H NMR spectroscopy and HPLC of perchloric acid extracts from rat brain and eye. The highest concentrations of N-acetylaspartate were found in grey matter as opposed to white matter with concentration increasing with age from neonate to adult, while the related compound N-acetylaspartylglutamate was highest in adult optic nerve. Creatine and choline-containing compounds were present in all regions throughout development, with higher levels of creatine found in grey matter compared to other regions. Choline-containing compounds were present at the highest concentrations in the eye at all ages examined, and tended to decrease in concentration to minimum values in adulthood in all regions. The presence of hypotaurine in corpus callosum and optic nerve was consistent with the metabolic profiles of O-2A progenitor cells and oligodendrocytes, which are cells composing these tissues. The neurotransmitters glutamate and GABA reached their highest concentrations in the olfactory bulb (higher than in adult cortex).

Proton nuclear magnetic resonance spectroscopy of primary cells derived from nervous tissue.

Cell culture techniques, high-resolution in vitro 1H nuclear magnetic resonance (NMR) spectroscopy, and chromatographic analyses were used to compare the properties of purified cell populations derived from the PNS and cortical neurones. Cell cultures were immunocytochemically characterised with specific antibodies to ensure purity of the individual cultures. Spectra of perchoric acid extracts of cultured Schwann cells, perineural fibroblasts, dorsal root ganglion neurones, and cortical neurones displayed several common features. However, statistically significant differences were found by 1H NMR spectroscopy in most metabolites among the cell types studied. In addition, cells could be distinguished by the presence or absence of certain amino acids. For example, N-acetylaspartate was present in dorsal root ganglion neurones and cortical neurones, gamma-aminobutyric acid was present in large amounts in cortical neurones, and Schwann cell spectra displayed a large signal from glycine. These results extend our earlier findings that different cell types of the CNS exhibit highly characteristic metabolite profiles to now include the major cell types of the PNS. These latter cell types also exhibit characteristic metabolite compositions, such that even Schwann cells and oligodendrocyte type 2 astrocyte (O-2A) progenitor cells-precursors of the myelinating cells of the CNS and PNS, respectively-can be readily distinguished from each other.

Characteristic metabolic profiles revealed by 1H NMR spectroscopy for three types of human brain and nervous system tumours.

Cell culture techniques, high-resolution in vitro 1H NMR spectroscopy, and chromatographic analyses were used to compare the properties of three types of human brain and nervous system tumours. Cell lines were immunocytochemically characterized at all stages in culture with specific antibodies. Intracellular metabolites present in cell extracts were analysed by 1H NMR spectroscopy and by high performance liquid chromatography (HPLC). The spectra from meningiomas, neuroblastomas, and glioblastomas displayed, in addition to similarities-including the presence of signals from leucine, isoleucine, valine, threonine, lactate, acetate, glutamate, choline-containing compounds and glycine-certain distinguishing metabolic features. Spectra from meningiomas featured relatively high signals from alanine. Intense signals from creatine were present in neuroblastoma spectra, while in spectra from glioblastoma they were not detectable. We found statistically significant differences by 1H NMR spectroscopy in the amounts of alanine, glutamate, creatine, phosphorylcholine and threonine among the types of tumours examined. HPLC determinations confirmed that there were also other metabolites specific to a type of tumour, such as taurine, gamma-aminobutyric acid, and serine. We suggest that these findings have potential relevance for the development of non-invasive diagnosis of tumour lineage by 1H NMR spectroscopy in vivo.

Cell type-specific fingerprinting of meningioma and meningeal cells by proton nuclear magnetic resonance spectroscopy.

We compared the properties of six human meningiomas with normal rat meningeal cells using cell culture techniques, high resolution in vitro 1H-NMR (nuclear magnetic resonance) spectroscopy, and chromatographic analysis. Cell cultures were immunocytochemically characterized at all stages with specific antibodies. Quantitative and qualitative metabolite assessments in cell extracts were obtained from 1H-NMR spectra and chromatographic analysis. Human meningioma cells expressed a characteristic spectrum of metabolites including free amino acids, compounds related to membrane phospholipid metabolism, energy metabolites, and other intermediary products. These spectral characteristics, although different in some respects, were strikingly similar to the ones of rat meningeal cells. Particularly, several metabolites that allow discrimination between meningeal cells and other cell types of the central nervous system were preserved in meningiomas. These similarities suggest that the regulation of intracellular levels of such metabolites is so intrinsic to the identity of cell type as to be conserved across species and through transformation. Additionally, human meningioma cultures expressed some spectroscopic characteristics that enabled them to be clearly distinguished from primary rat meningeal cultures. Thus, human meningiomas may be both specifically recognizable by 1H-NMR spectroscopy and also distinguishable from normal rat meningeal tissue. Our results raise the eventual possibility of using NMR in the noninvasive diagnosis of brain tumors in vivo.