Impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cutaneous wound healing.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a representative of a large group of polyhalogenated aromatic hydrocarbons that are widespread environmental contaminants. Administration of TCDD to laboratory animals or cultured cells results in a number of adverse effects that are well documented. For example, the effects of TCDD observed in developing organisms indicate that exposure to this class of environmental contaminants significantly alters embryo morphogenesis. However, it is not clear whether tissue regeneration in adult animals may be similarly affected. With this in mind, we examined the impact of TCDD exposure on wound healing using a murine cutaneous wound healing model. Our results indicate that TCDD exposure did not significantly alter the time needed for wound closure. However, in the TCDD-treated mice, a significant decrease in tensile strength in the healed wounds was observed which is indicative of an aberrantly healed wound. Immunostaining revealed that exposure to TCDD increased the population of macrophages detected within the wounded tissue at the latter stages of wound healing. Our findings support the idea that exposure to environmental contaminants such as TCDD is proinflammatory in the wounded tissue, disrupts normal healing and ultimately produces in a poorly healed wound.

Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.

Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin.

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

Nucleoside reverse transcriptase inhibitors prevent HIV protease inhibitor-induced atherosclerosis by ubiquitination and degradation of protein kinase C.

HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Calpha (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.

Caveolae, lipid rafts, and vascular disease.

Caveolae and lipid rafts are discrete regions within the plasma membrane that coordinate and regulate a variety of signaling processes. The exact relationship between caveolae and lipid rafts is unclear. However, caveolae contain a protein called caveolin that serves as a biochemical marker for caveolae. In addition, caveolin plays a role in maintaining the lipid composition of caveolae, the morphology of caveolae, and the signals that emanate from caveolae. The physiologic importance of caveolae is evidenced by recent studies using caveolin knockout mice that show dramatic abnormalities in the cardiovascular system, such as pulmonary hypertension and cardiac hypertrophy. In this review, we will focus on the role of caveolae in the cardiovascular system.

Lipoprotein aggregation protects human monocyte-derived macrophages from OxLDL-induced cytotoxicity.

Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large variations in human foam cell formation in individuals. A fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis. 148: 243-253). Here, we tested the hypothesis that aggregation of OxLDL enhances its clearance by human macrophages and thus may protect macrophages from OxLDL-induced cytotoxicity. We found that increased aggregation of OxLDL correlated with decreased macrophage injury. Using 3H-labeled and Alexa546-labeled OxLDL, we found that aggregation enhanced OxLDL uptake and increased cholesteryl ester accumulation but did not alter free cholesterol levels in macrophages. Acetylated LDL was a potent competitor of aggregated oxidized LDL (AggOxLDL) uptake, suggesting that scavenger receptor A plays an important role in the clearance of AggOxLDL. Inhibitors of actin polymerization, cytochalasin B, cytochalasin D, and latrunculin A, also prevented AggOxLDL uptake and restored OxLDL-induced cytotoxicity. This suggests that OxLDL-induced macrophage injury does not require OxLDL uptake and may occur on the cell surface. Our data demonstrate that aggregation of cytotoxic OxLDL enhances its clearance by macrophages without damage to the cells, thus allowing macrophages to avoid OxLDL-induced cell injury.

Estrogen in cardiovascular disease.

PURPOSE OF REVIEW: The controversy surrounding hormone replacement therapy has induced fear in patients and left many researchers with the impression that estrogen produces negative effects on cardiovascular function. The aim of this review is to summarize recent findings illustrating that estrogen also has positive effects even if estrogen replacement therapy is not a cure-all. RECENT FINDINGS: Studies have unveiled new aspects of estrogen action in the cardiovascular system; however, clinical trials have not demonstrated a protective effect of the most widely used modalities of hormone replacement therapy against cardiovascular disease. New information has emerged showing that estrogen has both beneficial and detrimental effects. Further mechanistic studies and use of well defined forms of estrogens and selective estrogen receptor modulators will continue to provide novel mechanistic information that will likely lead to the development of new avenues for therapeutic interventions. SUMMARY: Estrogens, like other steroid hormones, are potent actors in the cardiovascular system. Since half the population have high levels of estrogen most of their lives it is plain that estrogen has a variety of beneficial physiologic functions. Clinical studies, however, have demonstrated that a specific formulation of a combination of potent estrogens and metabolites is not a magic bullet, but induces both positive and negative impacts on different organ systems. More research into the mechanistic actions of estrogens in specific pathways in individual cell types is necessary to determine appropriate therapeutic interventions to replace the loss of positive effects of estrogens while minimizing the negative effects in postmenopausal women.

Autofluorescence of Toxoplasma gondii and related coccidian oocysts.

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

Cholesteryl ester is transported from caveolae to internal membranes as part of a caveolin-annexin II lipid-protein complex.

We previously demonstrated that in Chinese hamster ovary cells scavenger receptor, class B, type I-dependent selective cholesteryl ester uptake occurs in caveolae. In the present study we hypothesized that cholesteryl ester is transported from caveolae through the cytosol to an internal membrane by a caveolin chaperone complex similar to the one we originally described for the transport of newly synthesized cholesterol. To test this hypothesis we incubated Chinese hamster ovary cells expressing scavenger receptor, class B, type I with [(3)H]cholesteryl ester-labeled high density lipoprotein, subfractionated the cells and looked for a cytosolic pool of [(3)H]cholesteryl ester. The radiolabeled sterol initially appeared in the caveolae fraction, then in the cytosol, and finally in the internal membrane fraction. Caveolin IgG precipitated all of the [(3)H]cholesteryl ester associated with the cytosol. Co-immunoprecipitation studies demonstrated that in the presence of high density lipoprotein, but not low density lipoprotein or lipoprotein-deficient serum, caveolin IgG precipitated four proteins: annexin II, cyclophilin 40, caveolin, and cyclophilin A. Caveolin acylation-deficient mutants were used to demonstrate that acylation of cysteine 133 but not cysteine 143 or 156 is required for annexin II association with caveolin and the rapid transport of cholesteryl esters out of caveolae. We conclude that a caveolin-annexin II lipid-protein complex facilitates the rapid internalization of cholesteryl esters from caveolae.