Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux.

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

Macrophage SR-BI modulates autophagy via VPS34 complex and PPARα transcription of Tfeb in atherosclerosis.

Autophagy modulates lipid turnover, cell survival, inflammation, and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-b1-/- macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPARα activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34-Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescued the defective autophagy in Sr-b1-/- macrophages. Taken together, our results show that macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34-Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and potentially novel targets for the treatment of atherosclerosis.

2-Hydroxypropyl-gamma-cyclodextrin overcomes NPC1 deficiency by enhancing lysosome-ER association and autophagy.

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 and NPC2 genes that result in an accumulation of cholesterol in lysosomes. The majority of children with NPC die in adolescence. Currently, no FDA-approved therapies exist for NPC and the mechanisms of NPC disease are not fully understood. Our recent study and the reports from other laboratories showed that 2-hydroxypropyl-γ-cyclodextrin (HPγCD) alleviates cholesterol accumulation in NPC1-deficient cells in spite of its low binding affinity for cholesterol. In this study, we explored the cellular changes that are induced upon HPγCD treatment in NPC1 patient-derived fibroblasts. We show that HPγCD treatment increases lysosome-ER association and enhances autophagic activity. Our study indicates that HPγCD induces an activation of the transcription factor EB (TFEB), a master regulator of lysosomal functions and autophagy. Lysosome-ER association could potentially function as conduits for cholesterol transport from lysosomes to the ER. Accumulating evidence suggests a role for autophagy in rescuing the cholesterol accumulation in NPC and other degenerative diseases. Collectively, our findings suggest that HPγCD restores cellular homeostasis in NPC1-deficient cells via enhancing lysosomal dynamics and functions. Understanding the mechanisms of HPγCD-induced cellular pathways could contribute to effective NPC therapies.

7-Ketocholesterol in disease and aging.

7-Ketocholesterol (7KC) is a toxic oxysterol that is associated with many diseases and disabilities of aging, as well as several orphan diseases. 7KC is the most common product of a reaction between cholesterol and oxygen radicals and is the most concentrated oxysterol found in the blood and arterial plaques of coronary artery disease patients as well as various other disease tissues and cell types. Unlike cholesterol, 7KC consistently shows cytotoxicity to cells and its physiological function in humans or other complex organisms is unknown. Oxysterols, particularly 7KC, have also been shown to diffuse through membranes where they affect receptor and enzymatic function. Here, we will explore the known and proposed mechanisms of pathologies that are associated with 7KC, as well speculate about the future of 7KC as a diagnostic and therapeutic target in medicine.

Bid maintains mitochondrial cristae structure and function and protects against cardiac disease in an integrative genomics study.

Bcl-2 family proteins reorganize mitochondrial membranes during apoptosis, to form pores and rearrange cristae. In vitro and in vivo analysis integrated with human genetics reveals a novel homeostatic mitochondrial function for Bcl-2 family protein Bid. Loss of full-length Bid results in apoptosis-independent, irregular cristae with decreased respiration. Bid-/- mice display stress-induced myocardial dysfunction and damage. A gene-based approach applied to a biobank, validated in two independent GWAS studies, reveals that decreased genetically determined BID expression associates with myocardial infarction (MI) susceptibility. Patients in the bottom 5% of the expression distribution exhibit >4 fold increased MI risk. Carrier status with nonsynonymous variation in Bid's membrane binding domain, BidM148T, associates with MI predisposition. Furthermore, Bid but not BidM148T associates with Mcl-1Matrix, previously implicated in cristae stability; decreased MCL-1 expression associates with MI. Our results identify a role for Bid in homeostatic mitochondrial cristae reorganization, that we link to human cardiac disease.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL.

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Modification by isolevuglandins, highly reactive γ-ketoaldehydes, deleteriously alters high-density lipoprotein structure and function.

Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia (n = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls (n = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [3H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1ß expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.

Microsomal triglyceride transfer protein contributes to lipid droplet maturation in adipocytes.

Previous studies in our laboratory have established the presence of MTP in both white and brown adipose tissue in mice as well as in 3T3-L1 cells. Additional studies demonstrated an increase in MTP levels as 3T3-L1 cells differentiate into adipocytes concurrent with the movement of MTP from the juxtanuclear region of the cell to the surface of lipid droplets. This suggested a role for MTP in lipid droplet biogenesis and/or maturation. To probe the role of MTP in adipocytes, we used a Cre-Lox approach with aP2-Cre and Adipoq-Cre recombinase transgenic mice to knock down MTP expression in brown and white fat of mice. MTP expression was reduced approximately 55% in white fat and 65-80% in brown fat. Reducing MTP expression in adipose tissue had no effect on weight gain or body composition, whether the mice were fed a regular rodent or high fat diet. In addition, serum lipids and unesterified fatty acid levels were not altered in the knockdown mice. Importantly, decreased MTP expression in adipose tissue was associated with smaller lipid droplets in brown fat and smaller adipocytes in white fat. These results combined with our previous studies showing MTP lipid transfer activity is not necessary for lipid droplet initiation or growth in the early stages of differentiation, suggest that a structural feature of the MTP protein is important in lipid droplet maturation. We conclude that MTP protein plays a critical role in lipid droplet maturation, but does not regulate total body fat accumulation.

Aerosol Delivery of Curcumin Reduced Amyloid-ß Deposition and Improved Cognitive Performance in a Transgenic Model of Alzheimer's Disease.

We report a novel approach for the delivery of curcumin to the brain via inhalation of the aerosol for the potential treatment of Alzheimer's disease. The percentage of plaque fraction in the subiculum and hippocampus reduced significantly when young 5XFAD mice were treated with inhalable curcumin over an extended period of time compared to age-matched nontreated counterparts. Further, treated animals demonstrated remarkably improved overall cognitive function, no registered systemic or pulmonary toxicity associated with inhalable curcumin observed during the course of this work.

Mechanisms of Lipid Accumulation in the Bone Morphogenetic Protein Receptor Type 2 Mutant Right Ventricle.

RATIONALE: In heritable pulmonary arterial hypertension with germline mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene, right ventricle (RV) dysfunction is associated with RV lipotoxicity; however, the underlying mechanism for lipid accumulation is not known. OBJECTIVES: We hypothesized that lipid accumulation in cardiomyocytes with BMPR2 mutation occurs owing to alterations in lipid transport and impaired fatty acid oxidation (FAO), which is exacerbated by a high-lipid (Western) diet (WD). METHODS: We used a transgenic mouse model of pulmonary arterial hypertension with mutant BMPR2 and generated a cardiomyocyte cell line with BMPR2 mutation. Electron microscopy and metabolomic analysis were performed on mouse RVs. MEASUREMENTS AND MAIN RESULTS: By metabolomics analysis, we found an increase in long-chain fatty acids in BMPR2 mutant mouse RVs compared with controls, which correlated with cardiac index. BMPR2-mutant cardiomyocytes had increased lipid compared with controls. Direct measurement of FAO in the WD-fed BMPR2-mutant RV showed impaired palmitate-linked oxygen consumption, and metabolomics analysis showed reduced indices of FAO. Using both mutant BMPR2 mouse RVs and cardiomyocytes, we found an increase in the uptake of (14)C-palmitate and fatty acid transporter CD36 that was further exacerbated by WD. CONCLUSIONS: Taken together, our data suggest that impaired FAO and increased expression of the lipid transporter CD36 are key mechanisms underlying lipid deposition in the BMPR2-mutant RV, which are exacerbated in the presence of dietary lipids. These findings suggest important features leading to RV lipotoxicity in pulmonary arterial hypertension and may point to novel areas of therapeutic intervention.

Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes.

Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology.

Quantification of acute vocal fold epithelial surface damage with increasing time and magnitude doses of vibration exposure.

Because the vocal folds undergo repeated trauma during continuous cycles of vibration, the epithelium is routinely susceptible to damage during phonation. Excessive and prolonged vibration exposure is considered a significant predisposing factor in the development of vocal fold pathology. The purpose of the present study was to quantify the extent of epithelial surface damage following increased time and magnitude doses of vibration exposure using an in vivo rabbit phonation model. Forty-five New Zealand white breeder rabbits were randomized to nine groups and received varying phonation time-doses (30, 60, or 120 minutes) and magnitude-doses (control, modal intensity phonation, or raised intensity phonation) of vibration exposure. Scanning electron microscopy and transmission electron microscopy was used to quantify the degree of epithelial surface damage. Results revealed a significant reduction in microprojection density, microprojection height, and depth of the epithelial surface with increasing time and phonation magnitudes doses, signifying increased epithelial surface damage risk with excessive and prolonged vibration exposure. Destruction to the epithelial cell surface may provide significant insight into the disruption of cell function following prolonged vibration exposure. One important goal achieved in the present study was the quantification of epithelial surface damage using objective imaging criteria. These data provide an important foundation for future studies of long-term tissue recovery from excessive and prolonged vibration exposure.

Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography.

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.

Dysfunctional high-density lipoprotein in patients on chronic hemodialysis.

OBJECTIVES: This study examined the functionality of high-density lipoprotein (HDL) in individuals with end-stage renal disease on dialysis (ESRD-HD). BACKGROUND: The high rate of cardiovascular disease (CVD) in chronic kidney disease is not explained by standard risk factors, especially in patients with ESRD-HD who appear resistant to benefits of statin therapy. HDL is antiatherogenic because it extracts tissue cholesterol and reduces inflammation. METHODS: Cellular cholesterol efflux and inflammatory response were assessed in macrophages exposed to HDL of patients with ESRD-HD or controls. RESULTS: HDL from patients with ESRD-HD was dramatically less effective than normal HDL in accepting cholesterol from macrophages (median 6.9%; interquartile range [IQR]: 1.4% to 10.2%) versus control (median 14.9%; IQR: 9.8% to 17.8%; p < 0.001). The profound efflux impairment was also seen in patients with ESRD-HD and diabetes compared with patients with diabetes without renal disease (median 8.1%; IQR: 3.3% to 12.9%) versus control (median 13.6%; IQR: 11.0% to 15.9%; p = 0.009). In vitro activation of cellular cholesterol transporters increased cholesterol efflux to both normal and uremic HDL. HDL of patients with ESRD-HD had reduced antichemotactic ability and increased macrophage cytokine response (tumor necrosis factor-alpha, interleukin-6, and interleukin-1-beta). HDL of patients with ESRD-HD on statin therapy had reduced inflammatory response while maintaining impaired cholesterol acceptor function. Interestingly, impaired HDL-mediated efflux did not correlate with circulating C-reactive protein levels or cellular inflammatory response. CONCLUSIONS: These findings suggest that abnormal HDL capacity to mediate cholesterol efflux is a key driver of excess CVD in patients on chronic hemodialysis and may explain why statins have limited effect to decrease CV events. The findings also suggest cellular cholesterol transporters as potential therapeutic targets to decrease CV risk in this population.

Regulation of late endosomal/lysosomal maturation and trafficking by cortactin affects Golgi morphology.

Cortactin is a branched actin regulator and tumor-overexpressed protein that promotes vesicular trafficking at a variety of cellular sites, including endosomes and the trans-Golgi network. To better understand its role in secretory trafficking, we investigated its function in Golgi homeostasis. Here, we report that knockdown (KD) of cortactin leads to a dramatic change in Golgi morphology by light microscopy, dependent on binding the Arp2/3 actin-nucleating complex. Surprisingly, there was little effect of cortactin-KD on anterograde trafficking of the constitutive cargo vesicular stomatitis virus glycoprotein (VSVG), Golgi assembly from endoplasmic reticulum membranes upon Brefeldin A washout, or Golgi ultrastructure. Instead, electron microscopy studies revealed that cortactin-KD cells contained a large number of immature-appearing late endosomal/lysosomal (LE/Lys) hybrid organelles, similar to those found in lysosomal storage diseases. Consistent with a defect in LE/Lys trafficking, cortactin-KD cells also exhibited accumulation of free cholesterol and retention of the retrograde Golgi cargo mannose-6-phosphate receptor in LE. Inhibition of LE maturation by treatment of control cells with Rab7 siRNA or chloroquine led to a compact Golgi morphology similar to that observed in cortactin-KD cells. Furthermore, the Golgi morphology defects of cortactin-KD cells could be rescued by removal of cholesterol-containing lipids from the media, suggesting that buildup of cholesterol-rich membranes in immature LE/Lys induced disturbances in retrograde trafficking. Taken together, these data reveal that LE/Lys maturation and trafficking are highly sensitive to cortactin-regulated branched actin assembly and suggests that cytoskeletal-induced Golgi morphology changes can be a consequence of altered trafficking at late endosomes.

Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers.

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼ 12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ∼ 12 nm and 10 nm diameter, contained ∼ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ∼ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.

Reovirus replication protein µ2 influences cell tropism by promoting particle assembly within viral inclusions.

The double-stranded RNA virus mammalian reovirus displays broad cell, tissue, and host tropism. A critical checkpoint in the reovirus replication cycle resides within viral cytoplasmic inclusions, which are biosynthetic centers of genome multiplication and new-particle assembly. Replication of strain type 3 Dearing (T3) is arrested in Madin-Darby canine kidney (MDCK) cells at a step subsequent to inclusion development and prior to formation of genomic double-stranded RNA. This phenotype is primarily regulated by viral replication protein µ2. To understand how reovirus inclusions differ in productively and abortively infected MDCK cells, we used confocal immunofluorescence and thin-section transmission electron microscopy (TEM) to probe inclusion organization and particle morphogenesis. Although no abnormalities in inclusion morphology or viral protein localization were observed in T3-infected MDCK cells using confocal microscopy, TEM revealed markedly diminished production of mature progeny virions. T3 inclusions were less frequent and smaller than those formed by T3-T1M1, a productively replicating reovirus strain, and contained decreased numbers of complete particles. T3 replication was enhanced when cells were cultivated at 31°C, and inclusion ultrastructure at low-temperature infection more closely resembled that of a productive infection. These results indicate that particle assembly in T3-infected MDCK cells is defective, possibly due to a temperature-sensitive structural or functional property of µ2. Thus, reovirus cell tropism can be governed by interactions between viral replication proteins and the unique cell environment that modulate efficiency of particle assembly.

Enhanced expression of VEGF-A in ß cells increases endothelial cell number but impairs islet morphogenesis and ß cell proliferation.

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the ß cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and ß cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and ß cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in ß cell proliferation and ß cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in ß cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in ß cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs ß cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.

The structure of dimeric apolipoprotein A-IV and its mechanism of self-association.

Apolipoproteins are key structural elements of lipoproteins and critical mediators of lipid metabolism. Their detergent-like properties allow them to emulsify lipid or exist in a soluble lipid-free form in various states of self-association. Unfortunately, these traits have hampered high-resolution structural studies needed to understand the biogenesis of cardioprotective high-density lipoproteins (HDLs). We derived a crystal structure of the core domain of human apolipoprotein (apo)A-IV, an HDL component and important mediator of lipid absorption. The structure at 2.4 Å depicts two linearly connected 4-helix bundles participating in a helix swapping arrangement that offers a clear explanation for how the protein self-associates as well as clues to the structure of its monomeric form. This also provides a logical basis for antiparallel arrangements recently described for lipid-containing particles. Furthermore, we propose a "swinging door" model for apoA-IV lipid association.