Jeremy N. Skepper

Onset of Maternal Arterial Blood Flow and Placental Oxidative Stress : A Possible Factor in Human Early Pregnancy Failure

The aim was to measure changes in the oxygen tension within the human placenta associated with onset of the maternal arterial circulation at the end of the first trimester of pregnancy, and the impact on placental tissues. Using a multiparameter probe we established that the oxygen tension rises steeply from <20 mmHg at 8 weeks of gestation to >50 mmHg at 12 weeks. This rise coincides with morphological changes in the uterine arteries that allow free flow of maternal blood into the placenta, and is associated with increases in the mRNA concentrations and activities of the antioxidant enzymes catalase, glutathione peroxidase, and manganese and copper/zinc superoxide dismutase within placental tissues. Between 8 to 9 weeks there is a sharp peak of expression of the inducible form of heat shock protein 70, formation of nitrotyrosine residues, and derangement of the mitochondrial cristae within the syncytiotrophoblast. We conclude that a burst of oxidative stress occurs in the normal placenta as the maternal circulation is established. We speculate that this may serve a physiological role in stimulating normal placental differentiation, but may also be a factor in the pathogenesis of pre-eclampsia and early pregnancy failure if antioxidant defenses are depleted.

Human Vascular Smooth Muscle Cells Undergo Vesicle-Mediated Calcification in Response to Changes in Extracellular Calcium and Phosphate Concentrations: A Potential Mechanism for Accelerated Vascular Calcification in ESRD

Patients with ESRD have a high circulating calcium (Ca) x phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.

Apoptosis Regulates Human Vascular Calcification In Vitro: Evidence for Initiation of Vascular Calcification by Apoptotic Bodies

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after ≈28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by ≈40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with 45Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Direct imaging of single-walled carbon nanotubes in cells

The development of single-walled carbon nanotubes for various biomedical applications is an area of great promise. However, the contradictory data on the toxic effects of single-walled carbon nanotubes highlight the need for alternative ways to study their uptake and cytotoxic effects in cells. Single-walled carbon nanotubes have been shown to be acutely toxic in a number of types of cells, but the direct observation of cellular uptake of single-walled carbon nanotubes has not been demonstrated previously due to difficulties in discriminating carbon-based nanotubes from carbon-rich cell structures. Here we use transmission electron microscopy and confocal microscopy to image the translocation of single-walled carbon nanotubes into cells in both stained and unstained human cells. The nanotubes were seen to enter the cytoplasm and localize within the cell nucleus, causing cell mortality in a dose-dependent manner.

Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer

Objective Pancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA. Methods Using a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies. Results PEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility. Conclusions The authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics

The incorporation of silicate into hydroxyapatite (HA) has been shown to significantly increase the rate of bone apposition to HA bioceramic implants. However, uncertainty remains about the mechanism by which silicate increases the in vivo bioactivity of HA. In this study, high-resolution transmission electron microscopy was used to observe dissolution from HA, 0.8 wt% Si-HA and 1.5 wt% Si-HA implants after 6 and 12 weeks in vivo. Our observations confirmed that defects, in particular those involving grain boundaries, were the starting point of dissolution in vivo. Dissolution was observed to follow the order 1.5 wt% Si-HA>0.8 wt% Si-HA>pure HA and it was found to be particularly prevalent at grain boundaries and triple-junctions. These observations may help to explain the mechanism by which silicate ions increase the in vivo bioactivity of pure HA, and highlight the enhanced potential of these ceramics for biomedical applications.

Hypoxia-Reoxygenation A Potent Inducer of Apoptotic Changes in the Human Placenta and Possible Etiological Factor in Preeclampsia

Preeclampsia is a severe disorder of human pregnancy characterized by generalized activation of maternal endothelial cells. Oxidative stress of the placenta is considered a key intermediary step, precipitating deportation of apoptotic fragments into the maternal circulation, but the cause remains unknown. We hypothesize that intermittent placental perfusion, secondary to deficient trophoblast invasion of the endometrial arteries, leads to an ischemia-reperfusion–type insult. We therefore tested whether hypoxia-reoxygenation (H/R) in vitro stimulates apoptosis in human placental tissues compared with controls kept hypoxic or normoxic throughout. After H/R, release of cytochrome c from mitochondria was significantly increased and was associated with intense immunolabeling for active caspase 3 in the syncytiotrophoblast and fetal endothelial cells. There was also increased labeling of syncytiotrophoblastic nuclei for cleaved poly (ADP-ribose) polymerase (PARP), and higher cytosolic concentrations of cleaved PARP fragment were detected by Western blot. Syncytiotrophoblastic nuclei displayed increased chromatin condensation, and a significantly greater percentage was TUNEL positive. These changes were accompanied by increased lactate dehydrogenase release into the medium. Preadministration of the free radical scavenger, desferrioxamine, reduced cytochrome c release and the TUNEL-positive index, suggesting generation of hydroxyl radicals mediates these processes. By contrast, hypoxia alone caused a smaller increase in the TUNEL-positive index, and the majority of syncytiotrophoblastic nuclei displayed karyolysis, whereas normoxic controls remained euchromatic. We conclude that H/R stimulates apoptotic changes within the syncytiotrophoblast, whereas hypoxia principally induces necrosis. The quality of placental perfusion may therefore be a more important factor in the pathophysiology of preeclampsia than the absolute quantity.

Dialysis Accelerates Medial Vascular Calcification in Part by Triggering Smooth Muscle Cell Apoptosis

Background— Vascular calcification is associated with increased morbidity and mortality in stage V chronic kidney disease, yet its early pathogenesis and initiating mechanisms in vivo remain poorly understood. To address this, we quantified the calcium (Ca) load in arteries from children (10 predialysis, 24 dialysis) and correlated it with clinical, biochemical, and vascular measures. Methods and Results— Vessel Ca load was significantly elevated in both predialysis and dialysis and was correlated with the patients’ mean serum Ca×phosphate product. However, only dialysis patients showed increased carotid intima-media thickness and increased aortic stiffness, and calcification on computed tomography was present in only the 2 patients with the highest Ca loads. Importantly, predialysis vessels appeared histologically intact, whereas dialysis vessels exhibited evidence of extensive vascular smooth muscle cell (VSMC) loss owing to apoptosis. Dialysis vessels also showed increased alkaline phosphatase activity and Runx2 and osterix expression, indicative of VSMC osteogenic transformation. Deposition of the vesicle membrane marker annexin VI and vesicle component mineralization inhibitors fetuin-A and matrix Gla-protein increased in dialysis vessels and preceded von Kossa positive overt calcification. Electron microscopy showed hydroxyapatite nanocrystals within vesicles released from damaged/dead VSMCs, indicative of their role in initiating calcification. Conclusions— Taken together, this study shows that Ca accumulation begins predialysis, but it is the induction of VSMC apoptosis in dialysis that is the key event in disabling VSMC defense mechanisms and leading to overt calcification, eventually with clinically detectable vascular damage. Thus the identification of factors that lead to VSMC death in dialysis will be of prime importance in preventing vascular calcification.

In Vivo Detection of Macrophages in Human Carotid Atheroma: Temporal Dependence of Ultrasmall Superparamagnetic Particles of Iron Oxide-Enhanced MRI

Background— It has been suggested that inflammatory cells within vulnerable plaques may be visualized by superpara-magnetic iron oxide particle–enhanced MRI. The purpose of this study was to determine the time course for macrophage visualization with in vivo contrast–enhanced MRI using an ultrasmall superparamagnetic iron oxide (USPIO) agent in symptomatic human carotid disease. Methods— Eight patients scheduled for carotid endarterectomy underwent multisequence MRI of the carotid bifurcation before and 24, 36, 48, and 72 hours after Sinerem (2.6 mg/kg) infusion. Results— USPIO particles accumulated in macrophages in 7 of 8 patients given Sinerem. Areas of signal intensity reduction, corresponding to USPIO/macrophage–positive histological sections, were visualized in all 7 of these patients, optimally between 24 and 36 hours, decreasing after 48 hours, but still evident up to 96 hours after infusion. Conclusions— USPIO-enhanced MRI of carotid atheroma can be used to identify macrophages in vivo. The temporal change in the resultant signal intensity reduction on MRI suggests an optimal time window for the detection of macrophages on postinfusion imaging.