Inorganic Phosphate (Pi) Signaling in Endothelial Cells: A Molecular Basis for Generation of Endothelial Microvesicles in Uraemic Cardiovascular Disease.

Hyperphosphataemia increases cardiovascular mortality in patients with kidney disease. Direct effects of high inorganic phosphate (Pi) concentrations have previously been demonstrated on endothelial cells (ECs), including generation of procoagulant endothelial microvesicles (MVs). However, no mechanism directly sensing elevated intracellular Pi has ever been described in mammalian cells. Here, we investigated the hypothesis that direct inhibition by Pi of the phosphoprotein phosphatase PP2A fulfils this sensing role in ECs, culminating in cytoskeleton disruption and MV generation. ECs were treated with control (1 mM [Pi]) vs. high (2.5 mM [Pi]), a condition that drives actin stress fibre depletion and MV generation demonstrated by confocal microscopy of F-actin and NanoSight Nanoparticle tracking, respectively. Immuno-blotting demonstrated that high Pi increased p-Src, p-PP2A-C and p-DAPK-1 and decreased p-TPM-3. Pi at 100 µM directly inhibited PP2A catalytic activity. Inhibition of PP2A enhanced inhibitory phosphorylation of DAPK-1, leading to hypophosphorylation of Tropomyosin-3 at S284 and MV generation. p-Src is known to perform inhibitory phosphorylation on DAPK-1 but also on PP2A-C. However, PP2A-C can itself dephosphorylate (and therefore inhibit) p-Src. The direct inhibition of PP2A-C by Pi is, therefore, amplified by the feedback loop between PP2A-C and p-Src, resulting in further PP2A-C inhibition. These data demonstrated that PP2A/Src acts as a potent sensor and amplifier of Pi signals which can further signal through DAPK-1/Tropomyosin-3 to generate cytoskeleton disruption and generation of potentially pathological MVs.

Vesicles bearing gifts: the functional importance of micro-RNA transfer in extracellular vesicles in chronic kidney disease.

Extracellular vesicles (EVs), including microparticles (MPs) and exosomes (EXOs), are derived from a wide range of mammalian cells including blood platelets, endothelial cells, and kidney cells and can be detected in body fluids including blood and urine. While EVs are well established as diagnostic markers under pathophysiological and stress conditions, there is also mounting evidence of their functional significance as vehicles for communication between cells mediated by the presence of nucleic acids, especially microRNAs (miRs), encapsulated in the EVs. miRs regulate gene expression, are transported both in MPs and EXOs, and exert profound effects in the kidney. Here we review current understanding of the links between EVs and miRs, discuss the importance of miRs in kidney disease, and shed light on the role of EVs in transferring miRs through the circulation among the renal, vascular, and inflammatory cell populations that are functionally important in patients with chronic kidney disease.

Regulation of low-density lipoprotein cholesterol by intestinal inflammation and the acute phase response.

Systemic inflammation, induced by disease or experimental intervention, is well established to result in elevated levels of circulating triglycerides, and reduced levels of high-density lipoprotein-cholesterol (HDL-C), in most mammalian species. However, the relationship between inflammation and low-density lipoprotein-cholesterol (LDL-C) concentrations is less clear. Most reports indicate that systemic inflammation, as observed during sepsis or following high dose experimental endotoxaemia, lowers total, and LDL-C in man. However, isolated reports have suggested that certain inflammatory conditions are associated with increased LDL-C. In this review, we summarize the emerging evidence that low-grade inflammation specifically of intestinal origin may be associated with increased serum LDL-C levels. Preliminary insights into potential mechanisms that may mediate these effects, including those connecting inflammation to trans-intestinal cholesterol efflux (TICE), are considered. We conclude that this evidence supports the potential downregulation of major mediators of TICE by inflammatory mediators in vitro and during intestinal inflammation in vivo. The TICE-inflammation axis therefore merits further study in terms of its potential to regulate serum LDL-C, and as a readily druggable target for hypercholesterolaemia.

Regulation of vascular function and blood pressure by circadian variation in redox signalling.

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock.

Integrated metabolic models for xenobiotic induced mitochondrial toxicity in skeletal muscle.

There is a need for robust in vitro models to sensitively capture skeletal muscle adverse toxicities early in the research and development of novel xenobiotics. To this end, an in vitro rat skeletal muscle model (L6) was used to study the translation of transcriptomics data generated from an in vivo rat model. Novel sulfonyl isoxazoline herbicides were associated with skeletal muscle toxicity in an in vivo rat model. Gene expression pathway analysis on skeletal muscle tissues taken from in vivo repeat dose studies identified enriched pathways associated with mitochondrial dysfunction, oxidative stress, energy metabolism, protein regulation and cell cycle. Mitochondrial dysfunction and oxidative stress were further explored using in vitro L6 metabolic models. These models demonstrated that the sulfonyl isoxazoline compounds induced mitochondrial dysfunction, mitochondrial superoxide production and apoptosis. These in vitro findings accurately concurred with the in vivo transcriptomics data, thereby confirming the ability of the L6 skeletal muscle models to identify relevant in vivo mechanisms of xenobiotic-induced toxicity. Moreover, these results highlight the sensitivity of the L6 galactose media model to study mitochondrial perturbation associated with skeletal muscle toxicity; this model may be utilised to rank the potency of novel xenobiotics upon further validation.

Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells.

Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-µm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.

Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity.

Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR) was significantly increased whereas extracellular acidification rate (ECAR), a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2 (•-) level compared to cells in the glucose model. An antimycin A (AA) dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a galactose media model and are consequently more susceptible to mitochondrial toxicants.

A role for mitochondrial oxidants in stress-induced premature senescence of human vascular smooth muscle cells.

Mitochondria are a major source of cellular oxidants and have been implicated in aging and associated pathologies, notably cardiovascular diseases. Vascular cell senescence is observed in experimental and human cardiovascular pathologies. Our previous data highlighted a role for angiotensin II in the induction of telomere-dependent and -independent premature senescence of human vascular smooth muscle cells and suggested this was due to production of superoxide by NADPH oxidase. However, since a role for mitochondrial oxidants was not ruled out we hypothesise that angiotensin II mediates senescence by mitochondrial superoxide generation and suggest that inhibition of superoxide may prevent vascular smooth muscle cell aging in vitro. Cellular senescence was induced using a stress-induced premature senescence protocol consisting of three successive once-daily exposure of cells to 1×10(-8) mol/L angiotensin II and was dependent upon the type-1 angiotensin II receptor. Angiotensin stimulated NADPH-dependent superoxide production as estimated using lucigenin chemiluminescence in cell lysates and this was attenuated by the mitochondrial electron transport chain inhibitor, rotenone. Angiotensin also resulted in an increase in mitoSOX fluorescence indicating stimulation of mitochondrial superoxide. Significantly, the induction of senescence by angiotensin II was abrogated by rotenone and by the mitochondria-targeted superoxide dismutase mimetic, mitoTEMPO. These data suggest that mitochondrial superoxide is necessary for the induction of stress-induced premature senescence by angiotensin II and taken together with other data suggest that mitochondrial cross-talk with NADPH oxidases, via as yet unidentified signalling pathways, is likely to play a key role.

Oxidative and non-oxidative DNA damage and cardiovascular disease.

Evidence for the association of DNA damage with cardiovascular disease has been obtained from in vitro cell culture models, experimental cardiovascular disease and analysis of samples obtained from humans with disease. There is general acceptance that several factors associated with the risk of developing cardiovascular disease cause oxidative damage to DNA in cell culture models with both nuclear and mitochondrial DNA as targets. Moreover, evidence obtained over the past 10 years points to a possible mechanistic role for DNA damage in experimental atherosclerosis culminating in recent studies challenging the assumption that DNA damage is merely a biomarker of the disease process. This kind of mechanistic insight provides a renewed impetus for further studies in this area.

Blood leucocyte telomere DNA content predicts vascular telomere DNA content in humans with and without vascular disease.

AIMS: Previous studies have suggested that reduced telomere length in circulating leucocytes in humans is associated with premature vascular disease and by implication, accelerated vascular ageing. Importantly, a link between telomere length in circulating leucocytes and the blood vessel wall has never been established. We, thus, investigated the relationship between vascular wall and circulating leucocyte telomere length in humans with and without overt vascular disease. METHODS AND RESULTS: Aortic biopsies and paired blood leucocytes were obtained from 20 patients with asymptomatic abdominal aortic aneurysms (AAAs), undergoing elective open repair, and 12 morphologically normal aortas from a group of cadaveric organ donors of similar mean age. Telomere content was compared by quantitative PCR and expressed as telomere:genomic DNA ratio. The telomere:genomic DNA content was significantly reduced in wall biopsies of AAA vs. normal aorta, and this difference remained after adjusting for age and gender. There were strong correlations between leucocyte and vascular telomere content when the AAA and control groups were analysed either separately or grouped irrespective of the presence of vascular disease (r = 0.62, P < 0.001). CONCLUSION: The findings demonstrate that leucocyte DNA content is predictive of vascular telomere content and is an accurate surrogate for human vascular age.

Angiotensin II-mediated oxidative DNA damage accelerates cellular senescence in cultured human vascular smooth muscle cells via telomere-dependent and independent pathways.

Angiotensin II (Ang II) induces reactive oxygen species (ROS) production by human vascular smooth muscle cells (hVSMCs). ROS have been implicated in the development of both acute stress-induced premature senescence (SIPS) and chronic replicative senescence. Global oxidative DNA damage triggers SIPS and telomere DNA damage accelerates replicative senescence, both mediated via p53. This study tests the hypothesis that DNA is an important target for Ang II-induced ROS leading to senescence via telomere-dependent and independent pathways. DNA damage was quantified using the Comet assay, telomere DNA length by Southern blotting and hVSMC senescence by senescence-associated beta-galactosidase staining. Exposure to Ang II increased DNA damage in hVSMCs within 4 hours. Inhibition by an AT1 receptor antagonist (losartan metabolite: E3174) or catalase, confirmed that Ang II-induced DNA damage was AT1 receptor-mediated, via the induction of ROS. Acute exposure to Ang II resulted in SIPS within 24 hours that was prevented by coincubation with E3174 or catalase. SIPS was associated with increased p53 expression but was not dependent on telomere attrition because overexpression of human telomerase did not prevent Ang II-induced SIPS. Exposure to Ang II over several population doublings accelerated the rate of telomere attrition (by >2-fold) and induced premature replicative senescence of hVSMCs--an effect that was also attenuated by E3174 or catalase. These data demonstrate that Ang II-induced ROS-mediated DNA damage results in accelerated biological aging of hVSMCs via 2 mechanisms: (1) Acute SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with accelerated telomere attrition.

Rapid telomere attrition in cardiac tissue of the ageing Wistar rat.

Many studies show an association between ageing and mean telomere length in DNA isolated from peripheral blood mononuclear cells, few studies have examined less accessible tissues. This study has two objectives: (i) to define the best method to prepare rodent DNA for telomere length measurement by Southern blotting and (ii) to determine whether there are differential rates of telomere attrition in different rodent tissues. We found that the use of agarose plugs for DNA isolation was essential for the accurate measurement of rodent telomere length. Tissue was collected from neonatal (3 days) or aged (18-24 months) male Wistar rats and terminal restriction fragment (TRF) length was measured by Southern blotting. Cardiac tissue from aged rats showed a 38% loss of TRF length compared with newborn animals (p<0.001, n=13), this contrasts with much smaller reductions in brain (1.6%), liver (14.2%), kidney (8.9%) and lung (9.7%). This study demonstrates that the methods of DNA preparation are critical for accurate measurement of telomeres in rodent tissues. Moreover, we show differential rates of telomere attrition in rat tissues, the heart being most susceptible to telomere loss. These observations could have important implications for the study of age-specific changes in tissue function.

Immunochemical detection of UV-induced DNA damage and repair.

The application of an antiserum to ultraviolet radiation (UVR)-damaged DNA is presented. A novel experimental system was employed to ascertain the limits of detection for this antiserum. Using a DNA standard containing a known amount of dimer, the limits of detection were found to be 0.9 fmol of dimer. This was compared to a limit of 20-50 fmol dimer using gas chromatography-mass spectrometry (GC-MS). Induction of thymine dimers in DNA following UVR exposure, as assessed using this antiserum in an enzyme-linked immunosorbent assay (ELISA), was compared with GC-MS measurements. The ELISA method successfully demonstrated the induction of lesions in DNA irradiated either with UVC or UVB, although despite high sensitivity, no discernible binding was seen to UVA-irradiated DNA. The antiserum was also shown to be applicable to immunocytochemistry, localising damage in the nuclei of UVR exposed keratinocytes in culture. The ability of the antiserum to detect DNA damage in skin biopsies of individuals exposed to sub-erythemal doses of UVR was also demonstrated. Moreover, the subsequent removal of this damage, as evidenced by a reduction in antiserum staining, was noted in sections of biopsies taken in the hours following irradiation.

Modulation of hOGG1 DNA repair enzyme in human cultured cells in response to pro-oxidant and antioxidant challenge.

The putative modulation of the base excision repair enzyme, human 8-oxoguanine glycosylase (hOGG1), important in the removal of the potentially mutagenic lesion 8-oxo-2'-deoxyguanosine (8-oxodG), was investigated in human cell culture models. The expression of specific mRNA and protein was measured following pro-oxidant and antioxidant treatments in one human lymphoblastoid and one keratinocyte line. The measurement of intracellular reactive oxygen species generation was monitored by a fluorogenic assay and potential genotoxic effects confirmed by the dose-dependent increase in formamidopyrimidine-DNA glycosylase (Fpg) sensitive sites by alkaline unwinding following sub-lethal doses of hydrogen peroxide. The generation of a potentially antioxidant environment was assessed by the intracellular increase and extracellular depletion in ascorbic acid, confirmed by capillary electrophoresis. Despite these pro-oxidant and antioxidant treatments no significant change in mRNA of hOGG1 was observed in either cell line. Western analysis revealed that relatively high, yet noncytotoxic, doses of hydrogen peroxide caused a consistent approximate 50% decrease in hOGG1 protein in lymphoblastoid cells. The lack of upregulation of hOGG1 suggests the gene is constitutively expressed, which is further supported by studies examining the sequence of its promoter region. However, hOGG1 protein turnover may be sensitive to intracellular redox changes.

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo.

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.