A (Sub)field Guide to Quality Control in Hippocampal Subfield Segmentation on Highresolution T2-weighted MRI.

Inquiries into properties of brain structure and function have progressed due to developments in magnetic resonance imaging (MRI). To sustain progress in investigating and quantifying neuroanatomical details in vivo, the reliability and validity of brain measurements are paramount. Quality control (QC) is a set of procedures for mitigating errors and ensuring the validity and reliability of brain measurements. Despite its importance, there is little guidance on best QC practices and reporting procedures. The study of hippocampal subfields in vivo is a critical case for QC because of their small size, inter-dependent boundary definitions, and common artifacts in the MRI data used for subfield measurements. We addressed this gap by surveying the broader scientific community studying hippocampal subfields on their views and approaches to QC. We received responses from 37 investigators spanning 10 countries, covering different career stages, and studying both healthy and pathological development and aging. In this sample, 81% of researchers considered QC to be very important or important, and 19% viewed it as fairly important. Despite this, only 46% of researchers reported on their QC processes in prior publications. In many instances, lack of reporting appeared due to ambiguous guidance on relevant details and guidance for reporting, rather than absence of QC. Here, we provide recommendations for correcting errors to maximize reliability and minimize bias. We also summarize threats to segmentation accuracy, review common QC methods, and make recommendations for best practices and reporting in publications. Implementing the recommended QC practices will collectively improve inferences to the larger population, as well as have implications for clinical practice and public health.

Jugular Anomalies in Multiple Sclerosis Are Associated with Increased Collateral Venous Flow.

BACKGROUND AND PURPOSE: To date, research on extracranial venous collaterals has been focused on structure, with relatively little attention paid to hemodynamics. We addressed this limitation by quantitatively comparing collateral flow in patients with multiple sclerosis and healthy controls by using phase-contrast MR imaging. We hypothesize that patients with MS with structurally anomalous internal jugular veins will have elevated collateral venous flow compared with healthy controls. MATERIALS AND METHODS: The sample consisted of 276 patients with MS and 106 healthy controls. We used MRV to classify internal jugular veins as stenotic and nonstenotic based on an absolute cross-sectional area threshold in 276 patients with MS and 60 healthy controls; 46 healthy controls lacked this imaging. Individual and total vessel flows were quantified by using phase-contrast MR imaging on all patients. Veins were classified by extracranial drainage type: internal jugular veins (I), paraspinal (II), and superficial (III). Differences among healthy controls, patients with MS, nonstenotic patients, and stenotic subgroups in total venous flow by vessel type were evaluated in a general linear model for statistical analysis. RESULTS: In the MS group, 153 patients (55%) evidenced stenosis, whereas 12 (20%) healthy controls were classified as stenotic (P < .001). Compared with healthy controls, the MS group showed lower type I flow and increased type II flow. Stenosis was associated with reduced flow in the type I vessels [F(1272) = 68; P < .001]. The stenotic MS group had increased flow in the type II vessels compared with the nonstenotic MS group [F(1272) = 67; P < .001]. CONCLUSIONS: Compared with healthy controls, patients with MS exhibit reduced venous flow in the main extracerebral drainage vein (internal jugular vein). In contrast, flow in the paraspinal venous collaterals is elevated in patients with MS and exacerbated by venous stenosis. Collateral drainage may be a compensatory response to internal jugular vein flow reduction.

Regional brain shrinkage over two years: individual differences and effects of pro-inflammatory genetic polymorphisms.

We examined regional changes in brain volume in healthy adults (N=167, age 19-79years at baseline; N=90 at follow-up) over approximately two years. With latent change score models, we evaluated mean change and individual differences in rates of change in 10 anatomically-defined and manually-traced regions of interest (ROIs): lateral prefrontal cortex (LPFC), orbital frontal cortex (OF), prefrontal white matter (PFw), hippocampus (Hc), parahippocampal gyrus (PhG), caudate nucleus (Cd), putamen (Pt), insula (In), cerebellar hemispheres (CbH), and primary visual cortex (VC). Significant mean shrinkage was observed in the Hc, CbH, In, OF, and PhG, and individual differences in change were noted in all regions, except the OF. Pro-inflammatory genetic variants modified shrinkage in PhG and CbH. Carriers of two T alleles of interleukin-1ß (IL-1ß C-511T, rs16944) and a T allele of methylenetetrahydrofolate reductase (MTHFR C677T, rs1801133) polymorphisms showed increased PhG shrinkage. No effects of a pro-inflammatory polymorphism for C-reactive protein (CRP-286C>A>T, rs3091244) or apolipoprotein (APOE) ε4 allele were noted. These results replicate the pattern of brain shrinkage observed in previous studies, with a notable exception of the LPFC, thus casting doubt on the unique importance of prefrontal cortex in aging. Larger baseline volumes of CbH and In were associated with increased shrinkage, in conflict with the brain reserve hypothesis. Contrary to previous reports, we observed no significant linear effects of age and hypertension on regional brain shrinkage. Our findings warrant further investigation of the effects of neuroinflammation on structural brain change throughout the lifespan.

Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation.

BACKGROUND: Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. METHODS AND RESULTS: To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes. CONCLUSIONS: A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Paradoxical reduction of atherosclerosis in apoE-deficient mice with obesity-related type 2 diabetes.

OBJECTIVE: The effect of obesity and insulin resistance on the development of atherosclerosis was evaluated in apoE-deficient (ApoE(-/-)) mice. A previously described obesity model, in which the hypothalamic satiety center can be destroyed by a single gold thioglucose (GTG) injection, was used. To evaluate the effect of starvation on atherosclerosis ApoE(-/-) mice were food-restricted with 25% less chow than ad libitum-fed control mice. METHODS: Sixty-eight ApoE(-/-) mice were allocated into a control group (n=20), a GTG-injected group (n=28), and a food-restricted group (n=20). The control and food-restricted mice were injected with saline instead of GTG. The control and GTG-injected mice had free access to food, and all mice had free access to water during the study period. RESULTS: After 4 months, the GTG-injected mice were significantly overweight (mean body weight (g): 33 +/- 2.11 vs. 23 +/- 0.24 and 17 +/- 0.31 in control and food-restricted mice, respectively), obese, hypertriglyceridemic, insulin-resistant, hyperinsulinemic (mean plasma insulin (ng/ml): 2.45 and 0.43 in obese and control mice, respectively), and hyperglycemic (mean plasma glucose (mmol/l): 11.03 and 7.80 in obese and control mice, respectively). Unexpectedly, these obese and diabetic mice developed significantly less atherosclerosis compared with lean non-diabetic control mice. Food-restricted mice also developed less atherosclerosis compared to control mice. CONCLUSIONS: These findings may question the usefulness of mouse models in studying the relation of obesity-related type 2 diabetes to atherosclerosis and also the relevance of results obtained in apoE(-/-) mice with reduced weight gain during intervention.

Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.

1. We have recently demonstrated that chronic infusion of Angiotensin II into apoE-/- mice promotes the development of abdominal aortic aneurysms. To determine the involvement of specific Angiotensin II receptors in this response, we co-infused Angiotensin II (1000 ng kg(-1) min(-1) for 28 days) with losartan (30 mg kg(-1) day(-1)) or PD123319 (3 mg kg(-1) day(-1)) to antagonize AT1 and AT2 receptors, respectively. 2. Infusion of Angiotensin II promoted the development of abdominal aortic aneurysms in 70% of mature female apoE-/- mice. The formation of aortic aneurysms was totally inhibited by co-infusion of Angiotensin II with losartan (30 mg kg(-1) day(-1); P=0.003). In contrast, the co-infusion of Angiotensin II with PD123319 resulted in a marked increase in the incidence and severity of aortic aneurysms. 3. To determine whether AT2 antagonism also promoted Angiotensin II-induced atherosclerosis, Angiotensin II was infused into young female apoE-/- mice that had little spontaneous atherosclerosis. In these mice, co-infusion of PD123319 led to a dramatic increase in the extent of atherosclerosis. This increase was associated with no change in plasma lipid concentrations and only transient and modest increases in blood pressure during co-infusion with PD123319. 4. While antagonism of AT1 receptors totally prevented the formation of aneurysms, antagonism of AT2 receptors promoted a large increase in the severity of Angiotensin II-induced vascular pathology.

Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and specific immunization against TNFalpha confers no additional benefit.

TNFalpha participates in the pathogenesis of atherosclerosis. The effect of immunization against TNFalpha on development of advanced vascular lesions in atherosclerosis-susceptible apoE-deficient mice was investigated. At 5-7 weeks of age, animals received immunization with either Freunds adjuvant and a recombinant antigenic TNFalpha molecule (TNF106), Freunds adjuvant alone, or no immunization. All mice received a Western-type high-fat diet for 12 weeks. Aortic sinus lesion area was determined by microscopic morphometry, the total aortic arch cholesterol content was determined by gas chromatography, and antibodies against TNFalpha, malondialdehyde-modified low density lipoprotein, or heat shock protein 60, were assessed by ELISAs. Immunization with TNF106 induced high-titered circulating antibodies against TNFalpha (n=23), and these antibodies were not detected in mice immunized with Freunds adjuvant alone (n=22), or in non-immunized control animals (n=25). After 12 weeks, the atherosclerotic lesion size was significantly reduced in immunized animals, whether they had been immunized with TNF106 or Freunds adjuvant alone, and the total lesional cholesterol content was decreased in mice immunized with TNF106. There were no correlations between circulating antibody titers and plaque size, total aortic arch cholesterol content, or plasma lipid levels, respectively. Administration of Freunds adjuvant alone can thus reduce formation of mature atherosclerotic lesions in apoE-deficient mice and this response is not modified by specific immunization against TNFalpha.

Macrophage-specific expression of class A scavenger receptors enhances granuloma formation in the absence of increased lipid deposition.

Class A scavenger receptors (SR-A) have several proposed functions that could impact atherosclerosis and inflammatory processes. To define the function of SR-A in vivo, we created C57BL/6 transgenic mice that expressed bovine SR-A under the control of the restricted macrophage promoter, lysozyme (lyso-bSR-A). bSR-A mRNA was present in cultured peritoneal macrophages of transgenic mice and tissues that contain significant macrophages including spleen, lung, and ileum. Functional overexpression of SR-A was demonstrated in peritoneal macrophages both by augmented cholesterol ester deposition in response to AcLDL and enhanced adhesion in transgenic mice compared with nontransgenic littermates. To determine whether macrophage-specific expression of bSR-A regulated inflammatory responses, granulomas were generated by subcutaneous injection of carrageenan. Granuloma size was significantly increased in lyso-bSR-A transgenic mice compared with wild-type littermates [421 +/- 51 mg (n = 11) vs. 127 +/- 22 mg (n = 10), P < 0.001]. However, the larger granulomas in lyso-bSR-A transgenic mice were only associated with an increase in unesterified cholesterol, and not cholesterol esters. Furthermore, granulomas from transgenic mice had an increase in the number of macrophages within the tissue.Therefore, macrophage expression of bSR-A increased presence of this cell type in granulomas without enhancing the deposition of cholesterol esters, consistent with a role of the adhesive property of the protein.

Development of poly-(D,L-lactide--coglycolide) microsphere formulations containing recombinant human vascular endothelial growth factor to promote local angiogenesis.

Although preclinical animal studies have demonstrated the utility of recombinant human vascular endothelial growth factor (rhVEGF) in promoting neovascularization in regions of ischemia, rhVEGF systemic administration did not provide clinical benefit to patients in recent placebo-controlled Phase II clinical trials. The amount of rhVEGF localized in the ischemic region after systemic administration is minimal and does not persist for more than 1 day. A greater persistence of rhVEGF at the region of ischemia may provide an increased angiogenesis with the eventual formation of patent blood vessels to restore nourishment to the tissues. We sought to develop a formulation of rhVEGF in poly(D,L-lactide--co-glycolide) (PLG) microspheres that would provide a continuous local delivery of intact protein. A stable formulation of rhVEGF for encapsulation contained a small amount of a stabilizing sugar, trehalose. Addition of excess trehalose increased the rate of release from the PLG. In addition, PLG with free acid end groups appeared to retard the initial release of rhVEGF by associating with it through ionic interactions at the positively charged heparin binding domain. rhVEGF was released continuously for 21 days with a very low (less than 10%) initial burst. The released rhVEGF aggregated and hydrolyzed over time and lost heparin affinity but not receptor affinity. The compression molding of rhVEGF PLG microspheres into disks yielded formulations with a low initial release and a lag of 10 days followed by complete release. The PLG microsphere formulations were assessed in the corneal implant model of angiogenesis and generated a dose-dependent angiogenic response. These formulations were also administered intravitreally and subretinally, generating local neovascularization comparable to the human disease states, vitroretinopathy and age-related macular degeneration, respectively. The rhVEGF PLG formulations may increase local angiogenesis without systemic side effects and may also be useful in the development of ocular disease models.

Interleukin 4 induces transcription of the 15-lipoxygenase I gene in human endothelial cells.

The reticulocyte-type 15-lipoxygenase (15-LO-I) has been implicated in atherogenesis because of its capability of oxidizing low density lipoprotein. Therefore, we investigated the expression of the 15-LO-I gene in human umbilical vein endothelial cells (HUVEC). Nonactivated HUVEC did not exhibit detectable 15-LO-I mRNA. However, exposure of the cells to interleukin 4 (IL-4) induced the transcription of the 15-LO-I gene in a time- and concentration-dependent manner. Interestingly, this induction was not paralleled by a concomitant production of the functional 15-LO-I enzyme, as indicated by activity assays and immunoblotting. To gain more information about the mechanism of the induction process, we investigated IL-4-dependent activation of nuclear transcription factors for which binding sites were previously identified in the 5'-flanking region of the human 15-LO-I gene. Electrophoretic mobility shift assays revealed that IL-4 can activate signal transducer and activator of transcription 6, activator protein 2, GATA motif-binding transcription factor 1, nuclear factor 1, and SP-1 in HUVEC in a time- and concentration-dependent manner. Activation of these transcription factors was observed as early as 30 min after cytokine exposure. These data indicate that IL-4 upregulates the transcription of the 15-LO-I gene in human vascular endothelial cells, and this process may involve the activation of several nuclear transcription factors. The lack of active 15-LO-I protein in the presence of functional 15-LO-I mRNA suggests additional regulatory elements of 15-LO expression at posttranscriptional levels.

Sidestream cigarette smoke accelerates atherogenesis in apolipoprotein E-/- mice.

Epidemiological studies have strongly implicated active and passive smoking with increased risk of cardiovascular diseases. The present study was performed to determine if exposure to sidestream cigarette smoke (SSCS), a surrogate of environmental tobacco smoke, promotes atherogenesis in a mouse model of human atherosclerosis. Female ApoE-deficient mice, maintained on a Western diet, were exposed to SSCS in a whole-body exposure chamber for a total of 6 h each day, 5 days a week for 7, 10 and 14 weeks. Animals exposed to filtered ambient air served as controls. Elevated concentrations of blood carboxyhemoglobin and pulmonary CYP1A1 ascertained effective exposure of animals to SSCS. There were no consistent changes in serum concentrations of cholesterol between control and SSCS-exposed mice. Morphometric assessment of grossly discernible lesions covering the intimal area of aorta showed remarkable increases in SSCS-exposed mice at all three exposure durations studied. Increases in the lesion area defined by en face measurements were accompanied by parallel increases in the levels of esterified and unesterified cholesterol in the aortic tissues of SSCS mice. These results clearly demonstrate promotion of atherosclerotic lesion development by tobacco smoke in an atherosclerosis-susceptible mouse model.

Emerging protein delivery methods.

The efficient and safe delivery of therapeutic proteins is the key to commercial success and, in some cases, the demonstration of efficacy in current and future biotechnology products. Numerous delivery technologies and companies have evolved over the past year. To critically evaluate the available options, each method must be assessed in terms of how easily it can be manufactured, impact on protein quality, bioavailability, and toxicity. Recent advances in depot delivery systems have, for the most part, overcome all of these obstacles except for complex and costly manufacturing. On the other hand, pulmonary delivery usually involves efficient manufacturing, but low protein bioavailability resulting in higher doses compared with injections. Although recent advances in transdermal and oral delivery have been significant, both of these delivery routes require logarithmic increases in bioavailability to make them viable candidates for commercialization. In the next few years, protein delivery for commercial products will probably be limited to injection devices, depot systems and pulmonary administration.

Exogenous interferon-gamma enhances atherosclerosis in apolipoprotein E-/- mice.

A role for interferon-gamma (IFN-gamma) has been implied in the atherogenic process. To determine whether exogenously administered IFN-gamma exerts an effect on the development of atherosclerosis, we intraperitoneally administered either recombinant IFN-gamma (100 U/g body weight) or phosphate buffered saline daily for 30 days to atherosclerosis-susceptible apolipoprotein E-/- mice (16-week-old male mice, n = 11 per group) fed a normal diet. Atherosclerotic lesion size was quantified in the ascending aorta. The number of T lymphocytes and major histocompatibility complex (MHC) class II-positive cells within lesions were also quantified in this region. IFN-gamma administration reduced serum cholesterol concentrations by 15% (P = 0.02). For both groups, the majority of cholesterol was present in very low density lipoproteins, which were modestly reduced in mice receiving IFN-gamma. Despite the decrease in serum cholesterol concentrations, IFN-gamma injections significantly increased lesion size twofold compared to controls (119,980 +/- 18, 536 vs. 59,396 +/- 20,017 micrometer(2); P = 0.038). IFN-gamma also significantly increased the mean number of T lymphocytes (19 +/- 4 vs. 7 +/- 1 cells; P = 0.03) and MHC class II-positive cells (10 +/- 3 vs. 3 +/- 1 cells; P = 0.04) within lesions. These data lend further support to a pro-atherogenic role of IFN-gamma.

Polymorphism of class A scavenger receptors in C57BL/6 mice.

Scavenger receptors class A (SR-A) have been hypothesized to regulate the development of atherosclerotic lesions through recognition of modified low density lipoprotein (LDL) and macrophage adhesion to substrata. Supporting data have been collected from studies using the monoclonal antibody 2F8, an antibody developed from the BALB/c strain-derived macrophage cell line, RAW.264. Although 2F8 immunostained both cultured peritoneal macrophages (MPM) and thymic macrophages from Swiss, BALB/c, and DBA/2 mice, no immunostaining was detected in cells and tissues from C57BL/6 mice, one of the most commonly used atherosclerosis-susceptible mouse strains. Similarly, 2F8 detected SR-A protein in MPM by Western blotting in all strains except C57BL/6. However, a guinea pig antiserum developed to a fusion protein of the extracellular SR-A domain detected appropriately sized bands in all strains. Incubation with 2F8 antagonized acetylated low-density lipoprotein (AcLDL)-induced cholesterol esterification in MPM from BALB/c, Swiss, and DBA/2 strains but had no effect on MPM from C57BL/6 mice. Sequencing of SR-A cDNA from C57BL/6 mice demonstrated complete identity with published sequence in the collagen-like domain. However, four single-residue substitutions were noted in the alpha-helical coiled-coil domain. Site-directed mutagenesis demonstrated that a single substitution (L168S) in this domain accounted for the loss of 2F8 immunoreactivity. Differing reactivities toward a commonly used monoclonal antibody were used to identify polymorphism of SR-A in C57BL/6 mice.

Macrophage colony-stimulating factor rapidly enhances beta-migrating very low density lipoprotein metabolism in macrophages through activation of a Gi/o protein signaling pathway.

Previous studies have examined lipoprotein metabolism by macrophages following prolonged exposure (>24 h) to macrophage colony-stimulating factor (M-CSF). Because M-CSF activates several signaling pathways that could rapidly affect lipoprotein metabolism, we examined whether acute exposure of macrophages to M-CSF alters the metabolism of either native or modified lipoproteins. Acute incubation of cultured J774 macrophages and resident mouse peritoneal macrophages with M-CSF markedly enhanced low density lipoproteins (LDL) and beta-migrating very low density lipoproteins (beta-VLDL) stimulated cholesteryl [(3)H]oleate deposition. In parallel, M-CSF treatment increased the association and degradation of (125)I-labeled LDL or beta-VLDL without altering the amount of lipoprotein bound to the cell surface. The increase in LDL and beta-VLDL metabolism did not reflect a generalized effect on lipoprotein endocytosis and metabolism because M-CSF did not alter cholesterol deposition during incubation with acetylated LDL. Moreover, M-CSF did not augment beta-VLDL cholesterol deposition in macrophages from LDL receptor (-/-) mice, indicating that the effect of M-CSF was mediated by the LDL receptor. Incubation of macrophages with pertussis toxin, a specific inhibitor of G(i/o) protein signaling, had no effect on cholesterol deposition during incubation with beta-VLDL alone, but completely blocked the augmented response promoted by M-CSF. In addition, incubation of macrophages with the direct G(i/o) protein activator, mastoparan, mimicked the effect of M-CSF by enhancing cholesterol deposition in cells incubated with beta-VLDL, but not acetylated LDL. In summary, M-CSF rapidly enhances LDL receptor-mediated metabolism of native lipoproteins by macrophages through activation of a G(i/o) protein signaling pathway. Together, these findings describe a novel pathway for regulating lipoprotein metabolism.

Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.

Increased plasma concentrations of angiotension II (Ang II) have been implicated in atherogenesis. To examine this relationship directly, we infused Ang II or vehicle for 1 month via osmotic minipumps into mature apoE(-/-) mice. These doses of Ang II did not alter arterial blood pressure, body weight, serum cholesterol concentrations, or distribution of lipoprotein cholesterol. However, Ang II infusions promoted an increased severity of aortic atherosclerotic lesions. These Ang II-induced lesions were predominantly lipid-laden macrophages and lymphocytes; moreover, Ang II promoted a marked increase in the number of macrophages present in the adventitial tissue underlying lesions. Unexpectedly, pronounced abdominal aortic aneurysms were present in apoE(-/-) mice infused with Ang II. Sequential sectioning of aneurysmal abdominal aorta revealed two major characteristics: an intact artery that is surrounded by a large remodeled adventitia, and a medial break with pronounced dilation and more modestly remodeled adventitial tissue. Although no atherosclerotic lesions were visible at the medial break point, the presence of hyperlipidemia was required because infusions of Ang II into apoE(+/+) mice failed to generate aneurysms. These results demonstrate that increased plasma concentrations of Ang II have profound and rapid effects on vascular pathology when combined with hyperlipidemia, in the absence of hemodynamic influences.

Sustained release of recombinant human insulin-like growth factor-I for treatment of diabetes.

Recombinant human insulin-like growth factor-I (rhIGF-I) was found to improve glycemic control and enhance insulin sensitivity in patients with a syndrome of severe insulin resistance. Therefore, the protein may be considered as an alternative therapy in the treatment of diabetes when the patients become insensitive to insulin treatment. Because the protein was administered twice per day in the clinical trials, a sustained release polylactic-co-glycolic acid (PLGA) formulation for rhIGF-I with low initial burst (<20%), maximum possible protein loading (15-20%) and a continuous release of 1-2 weeks may provide greater patient convenience and compliance. The protein was encapsulated in PLGA for sustained release using a spray freeze-drying technique. Formulation parameters such as protein loading, polymer end group, and the presence of zinc carbonate were studied for their effects on in vitro release of rhIGF-I from PLGA microspheres. As the protein loading was increased, the initial burst increased. Due to the hydrophilic properties of the polymers, rhIGF-I encapsulated in unblocked PLGA (free acid end groups) gave a lower initial burst and a more steady-state release profile than the blocked PLGA (hydrocarbon end groups) with the same protein loading and PLGA molecular weight. At 15% w/w protein loading, the addition of 6% w/w zinc carbonate as a protein release modifier to the unblocked PLGA (12 kDa) decreased the initial burst of rhIGF-I. Therefore, a formulation consisting of 15% rhIGF-I and 6% zinc carbonate in 12 kDa, unblocked 50:50 PLGA can provide the required release characteristics in vitro. Rat studies revealed that rhIGF-I in this formulation was released in vivo at a rate which was comparable to that observed in vitro. These studies demonstrate the potential for a sustained release, 14-day formulation for rhIGF-I.

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins.

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment. These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.