Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype.

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox'd Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. METHODS: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. RESULTS: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. CONCLUSIONS: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

An Inducible and Vascular Smooth Muscle Cell-Specific Pink1 Knockout Induces Mitochondrial Energetic Dysfunction during Atherogenesis.

DNA damage and mitochondrial dysfunction are defining characteristics of aged vascular smooth muscle cells (VSMCs) found in atherosclerosis. Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. Here, we generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro. A smooth muscle cell-specific and MHC-Cre-inducible flox'd Pink1f/f kinase knockout was made on a ROSA26+/0 and ApoE-/- C57Blk6/J background. Mice were high fat fed for 10 weeks and vasculature assessed for physiological and pathogical changes. Mitochondrial respiratory activity was then assessed in wild-type and knockout animals vessels and isolated cells for their reliance on oxidative and glycolytic metabolism. During atherogenesis, we find that Pink1 knockout affects development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin. Pink1 protein is important in the wild-type VSMC response to metabolic stress and induced a compensatory increase in hexokinase II, which catalyses the first irreversible step in glycolysis. Pink1 appears to play an important role in VSMC energetics during atherogenesis but may also provide insight into the understanding of mitochondrial energetics in other diseases where the regulation of energetic switching between oxidative and glycolytic metabolism is found to be important.

Future of Smart Cardiovascular Implants.

Cardiovascular disease remains the leading cause of death in Western society. Recent technological advances have opened the opportunity of developing new and innovative smart stent devices that have advanced electrical properties that can improve diagnosis and even treatment of previously intractable conditions, such as central line access failure, atherosclerosis and reporting on vascular grafts for renal dialysis. Here we review the latest advances in the field of cardiovascular medical implants, providing a broad overview of the application of their use in the context of cardiovascular disease rather than an in-depth analysis of the current state of the art. We cover their powering, communication and the challenges faced in their fabrication. We focus specifically on those devices required to maintain vascular access such as ones used to treat arterial disease, a major source of heart attacks and strokes. We look forward to advances in these technologies in the future and their implementation to improve the human condition.

Mitochondrial DNA damage can promote atherosclerosis independently of reactive oxygen species through effects on smooth muscle cells and monocytes and correlates with higher-risk plaques in humans.

BACKGROUND: Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. METHODS AND RESULTS: mtDNA damage occurred early in the vessel wall in apolipoprotein E-null (ApoE(-/-)) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE(-/-) mice deficient for mitochondrial polymerase-γ proofreading activity (polG(-/-)/ApoE(-/-)). polG(-/-)/ApoE(-/-) mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG(-/-)/ApoE(-/-) mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG(-/-)/ApoE(-/-) bone marrow increased the features of plaque vulnerability, and polG(-/-)/ApoE(-/-) monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. CONCLUSIONS: We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

Detection of Blood Clots Using a Whole Stent as an Active Implantable Biosensor.

Many cardiovascular problems stem from blockages that form within the vasculature and often treatment includes fitting a stent through percutaneous coronary intervention. This offers a minimally invasive therapy but re-occlusion through restenosis or thrombosis formation often occurs post-deployment. Research is ongoing into the creation of smart stents that can detect the occurrence of further problems. In this study, it is shown that selectively metalizing a non-conductive stent can create a set of electrodes that are capable of detecting a build-up of material around the stent. The associated increase in electrical impedance across the electrodes is measured, testing the stent with blood clot to mimic thrombosis. It is shown that the device is capable of sensing different amounts of occlusion. The stent can reproducibly sense the presence of clot showing a 16% +/-3% increase in impedance which is sufficient to reliably detect the clot when surrounded by explanted aorta (one sample t-test, p = 0.009, n = 9). It is demonstrated that this approach can be extended beyond the 3D printed prototypes by showing that it can be applied to a commercially available stent and it is believed that it can be further utilized by other types of medical implants.

Realtime monitoring of thrombus formation in vivo using a self-reporting vascular access graft.

BACKGROUND: Chronic kidney disease (CKD) affects 10% of the global population costing over a hundred billion dollars per annum and leading to increased risk of cardiovascular disease. Many patients with CKD require regular haemodialyses. Synthetic arteriovenous grafts (AVG) are increasingly used to provide rapid vascular connection for dialysis. Initially, they have excellent patency rates but are critically limited by neointimal hyperplasia at the venous anastomosis, which drives subsequent thrombosis, graft failure and death. METHODS: Here, we describe a system in which electrical impedance spectroscopy sensors are incorporated circumferentially into the wall of a synthetic arteriovenous graft. This is combined with an implantable radiotelemetry system for data transmission outside the patient. The system was tested using monolayers of endothelial and smooth muscle cells as well as swine blood and clots with explanted human carotid artery plaques. Sensor testing was then performed in vitro and the device was implanted in vivo in female swine. RESULTS: The device can wirelessly report the accumulation of biological material, both cells and blood. Differences are also detected when comparing controls with pathological atheroma. In swine differences between blockage formation in a graft were remotely obtained and wireless reported. CONCLUSIONS: Combining electrical impedance spectroscopy and an implantable radiotelemetry system enables graft surveillance. This has the potential to be used for early detection of venous stenosis and blood clot formation in real-time in vivo. In principle, the concept could apply to other cardiovascular diseases and vascular implantable devices.

Chronic kidney disease is common throughout the world and required treatments are expensive. People with chronic kidney disease require frequent blood dialysis treatment to filter their blood and remove waste products and toxic substances circulating in the blood. For some patients, implantable tubular structures called AV grafts are used for providing access to dialysis. These grafts frequently block sometimes without warning leading to patients not being able to undergo dialysis. Through a series of laboratory experiments looking at cells that block the graft, fatty deposits and blood clots, we evaluated whether sensors could detect blockages in an AV graft. We also tested the device in an animal model. From these results we were able to show that our device could detect blockages within a graft. In the future we hope that introduction to the clinic of an optimized version of our device will reduce costs to healthcare systems and improve patient outcomes.

The methyl xanthine caffeine inhibits DNA damage signaling and reactive species and reduces atherosclerosis in ApoE(-/-) mice.

OBJECTIVE: Caffeine remains one of the most widely consumed drugs in the world. Caffeine has multiple actions, including inhibition of the DNA damage response, and its metabolites, 1-methylxanthine and 1-methyluric acid, are potent antioxidants. Combined, these properties can exert direct effects on cell proliferation, cell death, inflammation, and DNA repair, all important processes that occur in atherosclerosis. METHODS AND RESULTS: We first examined the effects of caffeine on mouse vascular smooth muscle cells. Caffeine inhibited activation of the DNA damage response regulator ataxia telangiectasia mutated protein and its downstream targets. Caffeine delayed DNA repair, had a concentration-dependent effect on cell proliferation, and protected against apoptosis. In vitro caffeine reduced oxygen consumption and decreased generation of reactive oxygen species. In vivo caffeine reduced DDR activation in vascular and nonvascular tissues, reduced reactive nitrogen species and serum levels of the DNA adduct 8-oxo-guanine, and inhibited atherogenesis in fat-fed ApoE(-/-) mice. Reduction in atherosclerosis was independent of the effects on blood pressure and serum lipids but associated with reduced cell proliferation and ataxia telangiectasia mutated protein activation. CONCLUSIONS: The Methyl Xanthine caffeine inhibits the DNA damage response in vitro and in vivo, regulates both cell proliferation and apoptosis after DNA damage, inhibits reactive species, and reduces atherogenesis in ApoE(-/-) mice.

Poor maternal nutrition programmes a pro-atherosclerotic phenotype in ApoE-/- mice.

Numerous animal studies have consistently shown that early life exposure to LP (low-protein) diet programmes risk factors for CVD (cardiovascular disease) such as dyslipidaemia, high BP (blood pressure) and cardiac dysfunction in the offspring. However, studies on the effect of maternal under-nutrition on offspring development of atherosclerosis are scarce. Applying our LP model to the ApoE(-/-) atherosclerosis-prone mouse model, we investigated the development of atherosclerotic lesions in the aortic root of 6-month-old offspring. In addition, markers of plaque progression including SMA (smooth muscle actin) and Mac3 (macrophage marker 3) were studied. Pregnant dams were fed on a control (20% protein) or on an isocaloric LP diet (8% protein) throughout pregnancy and lactation. After weaning, male offspring were maintained on 20% normal laboratory chow. At 6 months of age, LP offspring showed a significantly greater plaque area (P<0.05) with increased cholesterol clefts and significantly higher indices of DNA damage compared with controls (P<0.05). The expression of HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) (P<0.05) and LDL (low-density lipoprotein) receptor in the liver of LP offspring were increased. Furthermore, LP offspring had higher LDL-cholesterol levels (P<0.05) and a trend towards elevated insulin. There were no differences in other lipid measurements and fasting glucose between groups. These observations suggest that early exposure to an LP diet accelerates the development and increases the progression of atherosclerotic lesions in young adult offspring. Future studies are needed to elucidate the specific mechanisms linking in utero exposure to a diet low in protein to the development of atherosclerosis.

DNA damage links mitochondrial dysfunction to atherosclerosis and the metabolic syndrome.

RATIONALE: DNA damage is present in both genomic and mitochondrial DNA in atherosclerosis. However, whether DNA damage itself promotes atherosclerosis, or is simply a byproduct of the risk factors that promote atherosclerosis, is unknown. OBJECTIVE: To examine the effect of DNA damage on atherosclerosis, we studied apolipoprotein (Apo)E(-/-) mice that were haploinsufficient for the protein kinase ATM (ataxia telangiectasia mutated), which coordinates DNA repair. METHODS AND RESULTS: ATM(+/-)/ApoE(-/-) mice developed accelerated atherosclerosis and multiple features of the metabolic syndrome, including hypertension, hypercholesterolemia, obesity, steatohepatitis, and glucose intolerance. Transplantation with ATM(+/+) bone marrow attenuated atherosclerosis but not the metabolic syndrome. ATM(+/-) smooth muscle cells and macrophages showed increased nuclear DNA damage and defective DNA repair signaling, growth arrest, and apoptosis. Metabolomic screening of ATM(+/-)/ApoE(-/-) mouse tissues identified metabolic changes compatible with mitochondrial defects, with increased ß-hydroxybutyrate but reduced lactate, reduced glucose, and alterations in multiple lipid species. ATM(+/-)/ApoE(-/-) mouse tissues showed an increased frequency of a mouse mitochondrial "common" deletion equivalent and reduced mitochondrial oxidative phosphorylation. CONCLUSIONS: We propose that failure of DNA repair generates defects in cell proliferation, apoptosis, and mitochondrial dysfunction. This in turn leads to ketosis, hyperlipidemia, and increased fat storage, promoting atherosclerosis and the metabolic syndrome. Prevention of mitochondrial dysfunction may represent a novel target in cardiovascular disease.

Radiation Dosimetry of Theragnostic Pairs for Isotopes of Iodine in IAZA.

Theragnostic pairs of isotopes are used to infer radiation dosimetry for a therapeutic radiopharmaceutical from a diagnostic imaging study with the same tracer molecule labelled with an isotope better suited for the imaging task. We describe the transfer of radiation dosimetry from the diagnostic radioiodine isotope 123I, labelled for the hypoxia tracer molecule iodoazomycin arabinoside ([123I]IAZA), to isotopes 131I (therapeutic) and 124I (PET imaging). Uncertainties introduced by the dissimilar isotope half-lives are discussed in detail. Radioisotope dosimetries for [123I]IAZA were obtained previously. These data are used here to calculate residence times for 131I and 124I and their uncertainties. We distinguish two cases when extrapolating to infinity: purely physical decay (case A) and physical decay plus biological washout (case B). Organ doses were calculated using the MIRD schema with the OLIDNA/EXM code. Significant increases in some organ doses (in mSv per injected activity) were found for 131I and 124I. The most affected organs were the intestinal walls, thyroid, and urinary bladder wall. Uncertainty remained similar to 123I for case A but considerably greater for case B, especially for long biological half-lives (GI tract). Normal tissue dosimetries for IAZA must be considered carefully when substituting isotope species. A long biological half-life can significantly increase dosimetric uncertainties. These findings are relevant when considering PET imaging studies with [124I]IAZA or therapeutic administration of [131I]IAZA.

An Impedance Sensor for Pathologically Relevant Detection of In-Stent Restenosis In Vitro.

Cardiovascular disease (CVD) is the biggest cause of death globally. CVD is caused by atherosclerosis which is the accumulation of fatty deposits, often within the fine arteries of the heart or brain. These blockages reduce blood flow and lead to oxygen starvation (ischemia) which can lead to heart attacks and strokes. To treat blocked arteries an implantable device called a stent re-opens the artery to reinstate blood flow to the organ. The stent itself can become blocked over time by cell growth (intimal hyperplasia) which is characterised by excessive smooth muscle cell proliferation. Sensors based on electrical impedance spectroscopy (EIS) embedded in a stent could detect this re-blocking to allow for early intervention. Using platinum interdigitated electrodes on silicon sensor wafers we were able to co-culture different ratios of mouse smooth muscle cells and mouse endothelial cells on these sensors. This mimics the complex, multicellular environment which a stent is found in vivo when undergoing neo-intimal hyperplasia. Trends in the cell impedances were then characterised using the detection frequency and the gradient of change between populations over time which we termed 'Peak Cumulative Gradients (PCG). PCGs were calculated to successfully discriminate each cell type. This work moves towards a sensor that may help guide clinician's decision-making in a disease that is historically silent and difficult to detect. Clinical Relevance-This moves towards an early warning system for the detection of neo intimal hyperplasia ultimately leading to a reduction in stent complications.

Challenges to the Development of the Next Generation of Self-Reporting Cardiovascular Implantable Medical Devices.

Cardiovascular disease (CVD) is a group of heart and vasculature conditions which are the leading form of mortality worldwide. Blood vessels can become narrowed, restricting blood flow, and drive the majority of hearts attacks and strokes. Reactive surgical interventions are frequently required; including percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). Despite successful opening of vessels and restoration of blood flow, often in-stent restenosis (ISR) and graft failure can still occur, resulting in subsequent patient morbidity and mortality. A new generation of cardiovascular implants that have sensors and real-time monitoring capabilities are being developed to combat ISR and graft failure. Self-reporting stent/graft technology could enable precision medicine-based and predictive healthcare by detecting the earliest features of disease, even before symptoms occur. Bringing an implantable medical device with wireless electronic sensing capabilities to market is complex and often obstructive undertaking. This critical review analyses the obstacles that need to be overcome for self-reporting stents/grafts to be developed and provide a precision-medicine based healthcare for cardiovascular patients. Here we assess the latest research and technological advancement in the field, the current devices; including smart cardiovascular implantable biosensors and associated wireless data and power transfer solutions. We include an evaluation of devices that have reached clinical trials and the market potential for their end-user implementation.

Predicting Cardiovascular Stent Complications Using Self-Reporting Biosensors for Noninvasive Detection of Disease.

Self-reporting implantable medical devices are the future of cardiovascular healthcare. Cardiovascular complications such as blocked arteries that lead to the majority of heart attacks and strokes are frequently treated with inert metal stents that reopen affected vessels. Stents frequently re-block after deployment due to a wound response called in-stent restenosis (ISR). Herein, an implantable miniaturized sensor and telemetry system are developed that can detect this process, discern the different cell types associated with ISR, distinguish sub plaque components as demonstrated with ex vivo samples, and differentiate blood from blood clot, all on a silicon substrate making it suitable for integration onto a vascular stent. This work shows that microfabricated sensors can provide clinically relevant information in settings closer to physiological conditions than previous work with cultured cells.

Impedimetric Detection and Electromediated Apoptosis of Vascular Smooth Muscle Using Microfabricated Biosensors for Diagnosis and Therapeutic Intervention in Cardiovascular Diseases.

Cardiovascular diseases remain a significant global burden with 1-in-3 of all deaths attributable to the consequences of the disease. The main cause is blocked arteries which often remain undetected. Implantable medical devices (IMDs) such as stents and grafts are often used to reopen vessels but over time these too will re-block. A vascular biosensor is developed that can report on cellularity and is amenable to being mounted on a stent or graft for remote reporting. Moreover, the device is designed to also receive currents that can induce a controlled form of cell death, apoptosis. A combined diagnostic and therapeutic biosensor would be transformational for the treatment of vascular diseases such as atherosclerosis and central line access. In this work, a cell sensing and cell apoptosing system based on the same interdigitated electrodes (IDEs) is developed. It is shown that the device is scalable and that by miniaturizing the IDEs, the detection sensitivity is increased. Apoptosis of vascular smooth muscle cells is monitored using continuous impedance measurements at a frequency of 10 kHz and rates of cell death are tracked using fluorescent dyes and live cell imaging.

Target specificity of 188Re-labeled B27.1 monoclonal antibodies to ovarian cancer cells in vivo.

BACKGROUND: Currently we are exploring a new multistep pretargeting approach involving administration of a bispecific antibody (B27.1 x P54) which has an anti-CA-125 (B27.1) and antibiotin (P54) paratope. It is followed by the administration of radiolabeled biotinylated liposomes to target the 188Re to the ovarian cancer cells. As a preliminary step to realize this goal, we determined the target specificity of the monoclonal antibodies (B27.1) to the ovarian cancer cells in vivo. MATERIALS AND METHODS: B27.1 monoclonal antibodies were photoreduced using UV light and incubated with reduced 188Re for 30 min at 25degreesC. 188Re-labeled B27.1 antibodies were purified using size exclusion chromatography. A comparative biodistribution of Re-B27.1 and 188Re was performed in nude mice xenografted with NIH:OVCAR-3 cells. RESULTS: While free rhenium distributed preferentially into thyroid and stomach with insignificant accumulation in the cancer cells, about 20% of the injected dose of 188Re-B27.1 was recovered in ascites cells with insignificant localization in other organs four hours after administration. CONCLUSION: The study validates the affinity of the B27.1 antibodies to the ovarian cancer cells in vivo.

Impaired mitochondrial respiration in human carotid plaque atherosclerosis: A potential role for Pink1 in vascular smooth muscle cell energetics.

BACKGROUND AND AIMS: DNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. METHODS: Human plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. RESULTS: Human VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMP kinase (AMPK) and hexokinase 2 (Hex2). CONCLUSIONS: We have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPK and Hex2.

Molecular imaging agents for clinical positron emission tomography in oncology other than fluorodeoxyglucose (FDG): applications, limitations and potential.

Recent efforts in radiopharmaceutical design for positron emission tomography (PET) imaging in clinical oncology have provided a library of tracers that have the potential to contribute to individualizing cancer patient management. These tracers can provide PET images that reveal aspects of the fundamental underlying biochemistry in the tumor before and during treatment. For a number of these PET tracers the cellular processing has been well described and they are now generally referred to as molecular imaging agents. Despite their recognized value in clinical oncology these tracers have not yet obtained widespread acceptance and are not generally available at centers performing PET scans. There are a number of barriers and challenges to the widespread use of these PET tracers that include; a limited clinical demand, challenges presented by the chemistry and formulation for clinical acceptability, the short physical half life of the PET radionuclides, regulatory issues and the overall costs associated with PET radiopharmaceutical production. In addition the interpretation of the PET images requires a clear understanding of the biochemical processes involved at the cellular level. A concerted effort is required among stakeholders including clinicians, scientists, industry and governmental agencies if the potential of these agents in clinical oncology is to be realized.

A 99mTc-labeled gemcitabine bisphosphonate drug conjugate as a probe to assess the potential for targeted chemotherapy of metastatic bone cancer.

INTRODUCTION: A novel compound with the potential for "targeted" therapy for cancer patients was prepared using a conjugate between the potent anticancer drug Gemzar (gemcitabine) and a bisphosphonate. This conjugate would be expected to accumulate at sites of bone metastatic cancer by virtue of an affinity of the bisphosphonate for bone undergoing osteoclastic and osteoblastic remodeling. Release of the anticancer drug at the site of the tumor would provide high local concentrations of the drug but avoid systemic toxicity. METHODS: The conjugate was tested for bone binding by labeling with technetium-99m and using an in vitro test procedure with either purified hydroxyapatite (HA) or powdered bovine bone. Biodistribution and pharmacokinetic studies in mice were used to determine the excretion and bone-binding characteristics of the test compound. RESULTS AND CONCLUSIONS: The conjugate binds readily to powdered bone and HA using the in vitro test systems. In animal studies, the conjugate is found predominantly in bone with low soft tissue uptake after intravenous dosing. Unbound compound undergoes renal excretion. The gemcitabine bisphosphonate complex is a promising lead compound for investigation in metastatic bone cancer that may provide a therapeutic effect without undue toxicity.

Lin28A induces energetic switching to glycolytic metabolism in human embryonic kidney cells.

BACKGROUND: Loss of a cell's capacity to generate sufficient energy for cellular functions is a key hallmark of the ageing process and ultimately leads to a variety of important age-related pathologies such as cancer, Parkinson's disease and atherosclerosis. Regenerative medicine has sought to reverse these pathologies by reprogramming somatic cells to a more juvenile energetic state using a variety of stem cell factors. One of these factors, Lin28, is considered a candidate for modification in the reprogramming of cellular energetics to ameliorate the ageing process while retaining cell phenotype. RESULTS: Over-expression of Lin28A resulted in key changes to cellular metabolism not observed in wild-type controls. Extracellular pH flux analysis indicated that Lin28A over expression significantly increased the rate of glycolysis, whilst high resolution oxygen respirometry demonstrated a reduced oxygen consumption. Western blot and real-time PCR analysis identified Hexokinase II as one of the key modulators of glycolysis in these cells which was further confirmed by increased glucose transport. A metabolic switching effect was further emphasised by Western blot analysis where the oxygen consuming mitochondrial complex IV was significantly reduced after Lin28A over expression. CONCLUSIONS: Results from this study confirm that Lin28A expression promotes metabolic switching to a phenotype that relies predominantly on glycolysis as an energy source, while compromising oxidative phosphorylation. Mechanisms to augment regulated Lin28A in age related pathologies that are characterised by mitochondria dysfunction or in differentiated and aged post-mitotic cells is the future goal of this work.