Christopher Foote

Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light.

Cells of Dictyostelium discoideum are highly resistant to DNA damaging agents such as UV-light, gamma-radiation and chemicals. The genes encoding nucleotide excision repair (NER) and base excision repair (BER) enzymes are rapidly upregulated in response to UV-irradiation and DNA-damaging chemicals, suggesting that this is at least partially responsible for the resistance of this organism to these agents. Although Dictyostelium is also unusually resistant to high concentrations of H(2)O(2), little is known about the response of this organism to oxidative stress. To determine if transcriptional upregulation is a common mechanism for responding to DNA-damaging agents, we have studied the Dictyostelium catalase and Cu/Zn superoxide dismutase antioxidant enzymes. We show that there are two catalase genes and that each is differentially regulated both temporally and spatially during multicellular development. The catA gene is expressed throughout growth and development and its corresponding enzyme is maintained at a steady level. In contrast, the catB gene encodes a larger protein and is only expressed during the final stages of morphogenesis. Cell type fractionation showed that the CatB enzyme is exclusively localized to the prespore cells and the CatA enzyme is found exclusively in the prestalk cells. Each enzyme has a different subcellular localization. The unique developmental timing and cell type distribution suggest that the role for catB in cell differentiation is to protect the dormant spores from oxidative damage. We found that exposure to H(2)O(2) does not result in the induction of the catalase, superoxide dismutase, NER or BER mRNAs. A mutant with greatly reduced levels of catA mRNA and enzyme has greatly increased sensitivity to H(2)O(2) but normal sensitivity to UV. These results indicate that the natural resistance to oxidative stress is not due to an ability to rapidly raise the level of antioxidant or DNA repair enzymes and that the response to UV-light is independent from the response to reactive oxygen compounds.

The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast

Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F→A)–alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F→A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of ∼60 min. Delivery of A(F→A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F→A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F→A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN→EE→PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F→A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN.

Mechanisms of the inward remodeling process in resistance vessels: is the actin cytoskeleton involved?

The resistance arteries and arterioles are the vascular components of the circulatory system where the greatest drop in blood pressure takes place. Consequently, these vessels play a preponderant role in the regulation of blood flow and the modulation of blood pressure. For this reason, the inward remodeling process of the resistance vasculature, as it occurs in hypertension, has profound consequences on the incidence of life‐threatening cardiovascular events. In this manuscript, we review some of the most prominent characteristics of inwardly remodeled resistance arteries including their changes in vascular passive diameter, wall thickness, and elastic properties. Then, we explore the known contribution of the different components of the vascular wall to the characteristics of inwardly remodeled vessels, and pay particular attention to the role the vascular smooth muscle actin cytoskeleton may play on the initial stages of the remodeling process. We end by proposing potential ways by which many of the factors and mechanisms known to participate in the inward remodeling process may be associated with cytoskeletal modifications and participate in reducing the passive diameter of resistance vessels.

Mutations of DMYPT cause over constriction of contractile rings and ring canals during Drosophila germline cyst formation

Ring canals, also known as stable intercellular bridges, are derived from the contractile rings of incomplete cytokinesis (IC) in most organisms. Formation of ring canals is necessary to generate functional eggs and sperm in multiple organisms including insects, birds, mammals and various plants. How the constriction of a contractile ring is arrested and how an arrested contractile ring is transformed into a ring canal is unknown. We describe here the function of the Drosophila melanogaster myosin binding subunit of myosin phosphatase (DMYPT) in both processes. We have found that DMYPT is highly enriched in the cytoplasm of cells undergoing IC during oogenesis. DMYPT mutations in germ cells, but not in somatic follicle cells, resulted in over-constriction of contractile rings and ring canals. This leads to formation of small ring canals and mis-regulation of centriole migration during female germline cyst formation. Our results suggest that there may be two parallel mechanisms to prevent the contractile rings from being completely closed, physical resistance and inhibition of myosin II activity via DMYPT.

The Role of Reactive Oxygen Species in Microvascular Remodeling

The microcirculation is a portion of the vascular circulatory system that consists of resistance arteries, arterioles, capillaries and venules. It is the place where gases and nutrients are exchanged between blood and tissues. In addition the microcirculation is the major contributor to blood flow resistance and consequently to regulation of blood pressure. Therefore, structural remodeling of this section of the vascular tree has profound implications on cardiovascular pathophysiology. This review is focused on the role that reactive oxygen species (ROS) play on changing the structural characteristics of vessels within the microcirculation. Particular attention is given to the resistance arteries and the functional pathways that are affected by ROS in these vessels and subsequently induce vascular remodeling. The primary sources of ROS in the microcirculation are identified and the effects of ROS on other microcirculatory remodeling phenomena such as rarefaction and collateralization are briefly reviewed.

The obligatory role of the actin cytoskeleton on inward remodeling induced by dithiothreitol activation of endogenous transglutaminase in isolated arterioles

Inward remodeling is the most prevalent structural change found in the resistance arteries and arterioles of hypertensive individuals. Separate studies have shown that the inward remodeling process requires transglutaminase activation and the polymerization of actin. Therefore, we hypothesize that inward remodeling induced via endogenous transglutaminase activation requires and depends on actin cytoskeletal structures. To test this hypothesis, isolated and cannulated rat cremaster arterioles were exposed to dithiothreitol (DTT) to activate endogenous transglutaminases. DTT induced concentration-dependent vasoconstriction that was suppressed by coincubation with cystamine or cytochalasin-D to inhibit tranglutaminase activity or actin polymerization, respectively. Prolonged (4 h) exposure to DTT caused arteriolar inward remodeling that was also blocked by the presence of cystamine or cytochalasin-D. DTT inwardly remodeled arterioles had reduced passive diameters, augmented wall thickness-to-lumen ratios and altered elastic characteristics that were reverted upon disruption of the actin cytoskeleton with mycalolide-B. In freshly isolated arterioles, exposure to mycalolide-B caused no changes in their passive diameters or their elastic characteristics. These results suggest that, in arterioles, the early stages of the inward remodeling process induced by prolonged endogenous transglutaminase activation require actin dynamics and depend on changes in actin cytoskeletal structures.

Maternal Hyperleptinemia Is Associated with Male Offspring’s Altered Vascular Function and Structure in Mice

Children of mothers with gestational diabetes have greater risk of developing hypertension but little is known about the mechanisms by which this occurs. The objective of this study was to test the hypothesis that high maternal concentrations of leptin during pregnancy, which are present in mothers with gestational diabetes and/or obesity, alter blood pressure, vascular structure and vascular function in offspring. Wildtype (WT) offspring of hyperleptinemic, normoglycemic, Leprdb/+ dams were compared to genotype matched offspring of WT-control dams. Vascular function was assessed in male offspring at 6, and at 31 weeks of age after half the offspring had been fed a high fat, high sucrose diet (HFD) for 6 weeks. Blood pressure was increased by HFD but not affected by maternal hyperleptinemia. On a standard diet, offspring of hyperleptinemic dams had outwardly remodeled mesenteric arteries and an enhanced vasodilatory response to insulin. In offspring of WT but not Leprdb/+ dams, HFD induced vessel hypertrophy and enhanced vasodilatory responses to acetylcholine, while HFD reduced insulin responsiveness in offspring of hyperleptinemic dams. Offspring of hyperleptinemic dams had stiffer arteries regardless of diet. Therefore, while maternal hyperleptinemia was largely beneficial to offspring vascular health under a standard diet, it had detrimental effects in offspring fed HFD. These results suggest that circulating maternal leptin concentrations may interact with other factors in the pre- and post -natal environments to contribute to altered vascular function in offspring of diabetic pregnancies.

Arterial Stiffening in Western Diet-Fed Mice Is Associated with Increased Vascular Elastin, Transforming Growth Factor-β, and Plasma Neuraminidase

Consumption of excess fat and carbohydrate (Western diet, WD) is associated with alterations in the structural characteristics of blood vessels. This vascular remodeling contributes to the development of cardiovascular disease, particularly as it affects conduit and resistance arteries. Vascular remodeling is often associated with changes in the elastin-rich internal elastic lamina (IEL) and the activation of transforming growth factor (TGF)-β. In addition, obesity and type II diabetes have been associated with increased serum neuraminidase, an enzyme known to increase TGF-β cellular output. Therefore, we hypothesized that WD-feeding would induce structural modifications to the IEL of mesenteric resistance arteries in mice, and that these changes would be associated with increased levels of circulating neuraminidase and the up-regulation of elastin and TGF-β in the arterial wall. To test this hypothesis, a WD, high in fat and sugar, was used to induce obesity in mice, and the effect of this diet on the structure of mesenteric resistance arteries was investigated. 4-week old, Post-weaning mice were fed either a normal diet (ND) or WD for 16 weeks. Mechanically, arteries from WD-fed mice were stiffer and less distensible, with marginally increased wall stress for a given strain, and a significantly increased Youngs modulus of elasticity. Structurally, the wall cross-sectional area and the number of fenestrae found in the internal elastic lamina (IEL) of mesenteric arteries from mice fed a WD were significantly smaller than those of arteries from the ND-fed mice. There was also a significant increase in the volume of elastin, but not collagen in arteries from the WD cohort. Plasma levels of neuraminidase and the amount of TGF-β in mesenteric arteries were elevated in mice fed a WD, while ex vivo, cultured vascular smooth muscle cells exposed to neuraminidase secreted greater amounts of tropoelastin and TGF-β than those exposed to vehicle. These data suggest that consumption of a diet high in fat and sugar causes stiffening of the vascular wall in resistance arteries through a process that may involve increased neuraminidase and TGF-β activity, elevated production of elastin, and a reduction in the size and number of fenestrae in the arterial IEL.

Effects of the Use of Assisted Reproductive Technologies and an Obesogenic Environment on Resistance Artery Function and Diabetes Biomarkers in Mice Offspring

Maternal obesity affects the incidence of cardiovascular disease and diabetes in offspring. Also the use of assisted reproductive technologies (ART) has been associated with cardiovascular deficiencies in offspring. Obese women often suffer from infertility and use ART to achieve a pregnancy, but the combined effects of maternal obesity and ART on cardiovascular health and incidence of diabetes in the offspring is not known. Here, we report the effects of the use of ART within an obesogenic environment, consisting of feeding a western diet (WD) to dams and offspring, on resistance artery function and presence of diabetes biomarkers in juvenile mice offspring. Our results indicate that WD and ART interacted to induce endothelial dysfunction in mesenteric resistance arteries isolated from 7-week-old mice offspring. This was determined by presence of a reduced acetylcholine-induced dilation compared to controls. The arteries from these WD-ART mice also had greater wall cross-sectional areas and wall to lumen ratios indicative of vascular hypertrophic remodeling. Of the diabetes biomarkers measured, only resistin was affected by a WD×ART interaction. Serum resistin was significantly greater in WD-ART offspring compared to controls. Diet and sex effects were observed in other diabetes biomarkers. Our conclusion is that in mice the use of ART within an obesogenic environment interacts to favor the development of endothelial dysfunction in the resistance arteries of juvenile offspring, while having marginal effects on diabetes biomarkers.

Effects of the use of assisted reproduction and high-caloric diet consumption on body weight and cardiovascular health of juvenile mouse offspring

Maternal obesity and the use of assisted reproductive technologies (ART) are two suboptimal developmental environments that can lead to offspring obesity and cardiovascular disease. We hypothesized that these environments independently and synergistically adversely affect the offsprings weight and cardiovascular performance at ~7 weeks of age. Mice were fed either 24% fat and 17.5% high-fructose (HF) corn syrup or maintenance chow (5% fat; low-fat, no-fructose (LF)). Dams were subdivided into no ART and ART groups. ART embryos were cultured in Whittens medium and transferred into pseudopregnant recipients consuming the same diet as the donor. Offspring were fed the same diet as the mother. Body weights (BW) were measured weekly and mean arterial pressure (MAP) was collected through carotid artery catheterization at killing (55±0.5 days old). Expression of genes involved in cardiovascular remodeling was measured in thoracic aorta using qRT-PCR, and levels of reactive oxygen species (ROS) were measured intracellularly and extracellularly in mesenteric resistance arteries. ART resulted in increased BW at weaning. This effect decreased over time and diet was the predominant determinant of BW by killing. Males had greater MAP than females (P=0.002) and HF consumption was associated with greater MAP regardless of sex (P<0.05). Gene expression was affected by sex (P<0.05) and diet (P<0.1). Lastly, the use of ART resulted in offspring with increased intracellular ROS (P=0.05). In summary, exposure to an obesogenic diet pre- and/or post-natally affects weight, MAP, and gene expression while ART increases oxidative stress in mesenteric resistance arteries of juvenile offspring, no synergistic effects were observed.