Effect of fabrication parameters on morphology and drug loading of polymer particles for rosiglitazone delivery.

For the past several decades, drug-encapsulated polymer particles have been investigated as locally-delivered, long-acting therapies. The most common method of producing such particles is the oil in water solvent extraction technique. Using this technique, we produced poly(lactide-co-glycolide) (PLG) microparticles encapsulating rosiglitazone, a small molecule anti-diabetic drug. We investigated the impact of modulating fabrication parameters, including choice of organic solvent, concentration of polymer, and speed of homogenization and centrifugation on particle morphology and drug loading. Additionally, we studied the ratio of air-water-interface area to the extraction bath volume, a previously unstudied fabrication parameter, and its impact on rosiglitazone loading when using dichloromethane as the organic solvent. Under the conditions tested, drug loading can be increased 5-fold by increasing this ratio, which may be achieved by simply selecting a larger extraction vessel. By changing the organic solvent from dichloromethane to ethyl acetate, we produced particles with 60% higher rosiglitazone loading. Interestingly, the particles made with ethyl acetate appeared phase dark under light microscopy suggesting the presence of internal pores. By increasing the proportion of organic phase in the emulsion we eliminated the aberrant morphology but did not alter drug loading. As a final step in the development of the particles, we established that rosiglitazone remained stable throughout the encapsulation process and its subsequent release from particles by demonstrating that rosiglitazone loaded particles enhanced adipocyte lipid storage and adiponectin secretion. Taken together, for this system, air-water-interface area to volume ratio of the extraction bath and organic solvent both arose as key parameters in maximizing rosiglitazone loading in PLG microparticles. This study of how fabrication parameters impact drug loading and particle morphology may be useful in other investigations to encapsulate small molecules in polymer particles for controlled release applications.

On-chip phenotypic analysis of inflammatory monocytes in atherogenesis and myocardial infarction.

Monocyte recruitment to inflamed arterial endothelium initiates plaque formation and drives progression of atherosclerosis. Three distinct monocyte subsets are detected in circulation (CD14(++)CD16(-), CD14(++)CD16(+), and CD14(+)CD16(++)), and each may play distinct roles during atherogenesis and myocardial infarction. We studied a range of subjects that included otherwise healthy patients with elevated serum triglyceride levels to patients presenting with acute myocardial infarction. Our objective was to correlate an individual's risk with the activation state of each monocyte subset as a function of changes in adhesion receptor expression using flow cytometric quantitation of integrins and l-selectin membrane expression. A microfluidic-based laboratory-on-a-chip was developed to quantify the adhesion efficiency of monocytes sheared in whole blood on vascular cell adhesion molecule-1, while characterizing adhesion receptor expression and topography on captured monocytes. CD14(++)CD16(+) monocytes adhered with sevenfold higher efficiency than other subsets, and in patients with myocardial infarction the capture efficiency of this subset was double that for healthy subjects. In patients with hypertriglyceridemia, this increase in monocyte adhesion was attributable to CD14(++)CD16(+) uptake of triglyceride-rich lipoproteins and subsequent signaling via a Phospholipase C-dependent mechanism to increase CD11c expression, very late antigen-4 function, and integrin coclustering within focal adhesive sites on vascular cell adhesion molecule-1. In summary, we introduce a unique laboratory-on-a-chip method for quantifying the activation state of monocyte subsets. These experiments reveal that CD11c/CD18 is an inducible integrin whose expression correlates with a monocyte inflammatory state in subjects at risk for atherogenesis and in patients with myocardial infarction.

CD11c/CD18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to vascular cell adhesion molecule-1.

OBJECTIVE: Atherosclerosis is associated with monocyte adhesion to the arterial wall that involves integrin activation and emigration across inflamed endothelium. Involvement of ß(2)-integrin CD11c/CD18 in atherogenesis was recently shown in dyslipidemic mice, which motivates our study of its inflammatory function during hypertriglyceridemia in humans. METHODS AND RESULTS: Flow cytometry of blood from healthy subjects fed a standardized high-fat meal revealed that at 3.5 hours postprandial, monocyte CD11c surface expression was elevated, and the extent of upregulation correlated with blood triglycerides. Monocytes from postprandial blood exhibited an increased light scatter profile, which correlated with elevated CD11c expression and uptake of lipid particles. Purified monocytes internalized triglyceride-rich lipoproteins isolated from postprandial blood through low-density lipoprotein-receptor-related protein-1, and this also elicited CD11c upregulation. Laboratory-on-a-chip analysis of whole blood showed that monocyte arrest on a vascular cell adhesion molecule-1 (VCAM-1) substrate under shear flow was elevated at 3.5 hours and correlated with blood triglyceride and CD11c expression. At 7 hours postprandial, blood triglycerides decreased and monocyte CD11c expression and arrest on VCAM-1 returned to fasting levels. CONCLUSIONS: During hypertriglyceridemia, monocytes internalize lipids, upregulate CD11c, and increase adhesion to VCAM-1. These data suggest that analysis of monocyte inflammation may provide an additional framework for evaluating individual susceptibility to cardiovascular disease.

Surface Functionalization of Polymer Particles for Cell Targeting by Modifying Emulsifier Chemistry.

The oil in water emulsion/solvent extraction method is used to fabricate many FDA approved, polymer particle formulations for drug delivery. However, these formulations do not benefit from surface functionalization that can be achieved through tuning particle surface chemistry. Poly(vinyl alcohol) (PVA) is the emulsifier used for many FDA approved formulations and remains associated with the particle surface after fabrication. We hypothesized that the hydroxyl groups in PVA could be conjugated with biomolecules using isothiocyanate chemistry and that these modifications would endow the particle surface with additional functionality. We demonstrate that fluorescein isothiocyanate and an isothiocyanate derivatized mannose molecule can be covalently attached to PVA in a one-step reaction. The modified PVA polymers perform as well as unmodified PVA in acting as an emulsifier for fabrication of poly(lactide-co-glycolide) particles. Particles made with the fluorescein modified PVA exhibit fluorescence confined to the particle surface, while particles made with mannose modified PVA bind concanavalin A. In addition, mannose modified PVA increases particle association with primary macrophages by three-fold. Taken together, we present a facile method for modifying the surface reactivity of polymer particles widely used for drug delivery in basic research and clinical practice. Given that methods are established for conjugating the isothiocyanate functional group to a wide range of biomolecules, our approach may enable PVA based biomaterials to engage a multitude of biological systems.

Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed microvasculature.

Mac-1-dependent crawling is a new step in the leukocyte recruitment cascade that follows LFA-1-dependent adhesion and precedes emigration. Neutrophil adhesion via LFA-1 has been shown to induce cytoskeletal reorganization through Vav1-dependent signaling, and the current study investigates the role of Vav1 in the leukocyte recruitment process in vivo with particular attention to the events immediately downstream of LFA-1-dependent adhesion. Intravital and spinning-disk-confocal microscopy was used to investigate intravascular crawling in relation to endothelial junctions in vivo in wild-type and Vav1(-/-) mice. Adherent wild-type neutrophils almost immediately began crawling perpendicular to blood flow via Mac-1 until they reached an endothelial junction where they often changed direction. This pattern of perpendicular, mechanotactic crawling was recapitulated in vitro when shear was applied. In sharp contrast, the movement of Vav1(-/-) neutrophils was always in the direction of flow and appeared more passive as if the cells were dragged in the direction of flow in vivo and in vitro. More than 80% of Vav1(-/-) neutrophils moved independent of Mac-1 and could be detached with LFA-1 Abs. An inability to release the uropod was frequently noted for Vav1(-/-) neutrophils, leading to greatly elongated tails. The Vav1(-/-) neutrophils failed to stop or follow junctions and ultimately detached, leading to fewer emigrated neutrophils. The Vav1(-/-) phenotype resulted in fewer neutrophils recruited in a relevant model of infectious peritonitis. Clearly, Vav1 is critical for the complex interplay between LFA-1 and Mac-1 that underlies the programmed intravascular crawling of neutrophils.

Modulation of adipocyte size and fat pad weight via resveratrol releasing scaffolds implanted into the epididymal adipose tissue.

Lipid overload of the adipose tissue, which can be caused by overnutrition, underlies metabolic disease. We hypothesized that increasing the energy demand of adipose tissue is a promising strategy to combat excessive lipid accumulation. Resveratrol, a natural polyphenol, activates lipid catabolism in fat tissue; however, its clinical success is hindered by poor bioavailability. Here, we implanted resveratrol releasing poly(lactide-co-glycolide) scaffolds into epididymal fat to overcome its poor bioavailability with the goal of enhancing local lipid catabolism. In lean mice, resveratrol scaffolds decreased adipocyte size relative to scaffolds with no drug, a response that correlated with AMP kinase activation. Immunohistochemistry indicated that macrophages and multinucleated giant cells within the scaffold expressed carnitine palmitoyltransferase 1 (CPT1) at higher levels than other cells in the adipose tissue. Furthermore, resveratrol increased CPT1 levels in cultured macrophages. Taken together, we propose that resveratrol scaffolds decrease adipocyte size because resveratrol increases lipid utilization in scaffold-infiltrating immune cells, possibly through elevating CPT1 levels or activity. In a follow-up study, mice that received resveratrol scaffolds 28-day prior to a high-fat diet exhibited decreased weight gain, adipose tissue expansion, and adipocyte hypertrophy compared to mice with control scaffolds. Notably, this scaffold-based strategy required a single resveratrol administration compared to the daily regiment generally needed for oral administration. These results indicate that localized delivery of metabolism modulating agents to the adipose tissue may overcome issues with bioavailability and that the role of biomaterials should be further investigated in this therapeutic strategy for metabolic disease.

Functional role of CD11c+ monocytes in atherogenesis associated with hypercholesterolemia.

BACKGROUND: Monocyte activation and migration into the arterial wall are key events in atherogenesis associated with hypercholesterolemia. CD11c/CD18, a beta2 integrin expressed on human monocytes and a subset of mouse monocytes, has been shown to play a distinct role in human monocyte adhesion on endothelial cells, but the regulation of CD11c in hypercholesterolemia and its role in atherogenesis are unknown. METHODS AND RESULTS: Mice genetically deficient in CD11c were generated and crossbred with apolipoprotein E (apoE)-/- mice to generate CD11c-/-/apoE-/- mice. Using flow cytometry, we examined CD11c on blood leukocytes in apoE-/- hypercholesterolemic mice and found that compared with wild-type and apoE-/- mice on a normal diet, apoE-/- mice on a Western high-fat diet had increased CD11c+ monocytes. Circulating CD11c+ monocytes from apoE-/- mice fed a high-fat diet exhibited cytoplasmic lipid vacuoles and expressed higher levels of CD11b and CD29. Deficiency of CD11c decreased firm arrest of mouse monocytes on vascular cell adhesion molecule-1 and E-selectin in a shear flow assay, reduced monocyte/macrophage accumulation in atherosclerotic lesions, and decreased atherosclerosis development in apoE-/- mice on a high-fat diet. CONCLUSIONS: CD11c, which increases on blood monocytes during hypercholesterolemia, plays an important role in monocyte recruitment and atherosclerosis development in an apoE-/- mouse model of hypercholesterolemia.

Fabrication of biodegradable particles with tunable morphologies by the addition of resveratrol to oil in water emulsions.

Particles for biomedical applications can be produced by emulsifying biocompatible polymers dissolved in an organic solvent in water. The emulsion is then transferred to an extraction bath that removes the solvent from the dispersed droplets, which leads to polymer precipitation and particle formation. Typically, the particles are smooth and spherical, likely because the droplets remain fluid throughout the solvent extraction process allowing minimization of surface area as the volume decreases. Few modifications to this technique exist that alter the spherical geometry, even though particle performance, from drug delivery to engaging cells of the body, can be tuned with morphology. Here we demonstrate that incorporation of resveratrol, with the aid of ethanol, into the oil phase of an emulsion of poly(lactide-co-glycolide) and dichloromethane in aqueous poly(vinyl alcohol) leads to a crumpled particle morphology. Video microscopy of particle formation revealed that during solvent extraction the droplet crumples in on itself, which does not occur when only ethanol is added to the emulsion. It is unclear why this occurs with resveratrol, but its hydroxyl groups appear to be optimally positioned because removal of the 4' hydroxyl or addition of a 3' hydroxyl resulted in a loss of crumpled particle morphology. We demonstrate that particle morphology can be tuned from that of a crumpled sheet of paper to a deflated sphere by switching out ethanol for a different cosolvent. We quantify the degree of particle deformation with surface area calculated from krypton adsorption isotherms and BET theory and find surface area correlates with resveratrol loading in the particle. Furthermore, spherical particles are achieved when ethyl acetate is used in lieu of dichloromethane and a cosolvent. We propose that during solvent extraction, resveratrol accumulates at the droplet surface where it inhibits polymer chain motion necessary to maintain a spherical geometry and the role of cosolvent is to redistribute resveratrol from the droplet bulk to its surface. This method of producing nonspherical particles extends to polycaprolactone and poly(L-lactic acid) and is compatible with the encapsulation of a hydrophobic fluorescent dye, suggesting hydrophobic bioactive agents could be encapsulated. Taken together, we demonstrate an ability to control morphology of biocompatible polymer particles produced by the widely practiced oil-in-water/solvent extraction protocol via the addition of resveratrol and a cosolvent to the oil phase. The methodology reported is straight forward, and scalable, and expected to be of utility in applications in which a deviation from the default smooth, spherical morphology is desired.

Scaffold Implant Into the Epididymal Adipose Tissue Protects Mice From High Fat Diet Induced Ectopic Lipid Accumulation and Hyperinsulinemia.

Ectopic lipid accumulation, the deposition of lipids in lean tissue, is linked to type 2 diabetes through an association with insulin resistance. It occurs when adipose tissue fails to meet lipid storage needs and there is lipid spillover into tissues not equipped to store them. Ectopic lipid contributes to organ dysfunction because lipids can interfere with insulin signaling and other signaling pathways. Clinical studies indicate that decreasing ectopic lipids through diet and exercise is effective in treating type 2 diabetes; however, its prevalence continues to rise. We propose that strategies to improve lipid handling in the adipose tissue would be adjunctive to healthy lifestyle modification and may address difficulties in treating type 2 diabetes and other syndromes spurred by ectopic lipid. Herein, we investigate biomaterial implants as a means to increase lipid utilization in adipose tissue through the recruitment of highly metabolic cells. Poly(lactide-co-glycolide) scaffolds were implanted into the epididymal fat of mice fed a high fat diet that overwhelms the adipose tissue and promotes ectopic lipid accumulation. Over 5 weeks, mice with scaffolds gained less weight compared to mice without scaffolds and were protected from hyperinsulinemia. These effects correlated with a 53% decrease in triglyceride in the gastrocnemius and a 25% decrease in the liver. Scaffolds increased CPT1A protein levels in the epididymal fat and histology revealed high expression of CTP1A in the cells infiltrating the scaffold relative to the rest of the fat pad. In addition, lacing the scaffold with resveratrol increased CPT1A expression in the epididymal fat over scaffolds with no drug; however, this did not result in further decreases in weight gain or ectopic lipid. Mechanistically, we propose that the cellular activity caused by scaffold implant mitigates the lipid load imposed by the high fat diet and leads to a substantial decrease in lipid accumulation in the muscle and liver. In conclusion, this study establishes that a tissue engineering approach to modulate lipid utilization in the epididymal fat tissue can mitigate ectopic lipid accumulation in mice fed a high fat diet with positive effects on weight gain and whole-body insulin resistance.

Nicotinamide Augments the Anti-Inflammatory Properties of Resveratrol through PARP1 Activation.

Resveratrol (RSV) and nicotinamide (NAM) have garnered considerable attention due to their anti-inflammatory and anti-aging properties. NAM is a transient inhibitor of class III histone deacetylase SIRTs (silent mating type information regulation 2 homologs) and SIRT1 is an inhibitor of poly-ADP-ribose polymerase-1 (PARP1). The debate on the relationship between RSV and SIRT1 has precluded the use of RSV as a therapeutic drug. Recent work demonstrated that RSV facilitates tyrosyl-tRNA synthetase (TyrRS)-dependent activation of PARP1. Moreover, treatment with NAM is sufficient to facilitate the nuclear localization of TyrRS that activates PARP1. RSV and NAM have emerged as potent agonists of PARP1 through inhibition of SIRT1. In this study, we evaluated the effects of RSV and NAM on pro-inflammatory macrophages. Our results demonstrate that treatment with either RSV or NAM attenuates the expression of pro-inflammatory markers. Strikingly, the combination of RSV with NAM, exerts additive effects on PARP1 activation. Consistently, treatment with PARP1 inhibitor antagonized the anti-inflammatory effect of both RSV and NAM. For the first time, we report the ability of NAM to augment PARP1 activation, induced by RSV, and its associated anti-inflammatory effects mediated through the induction of BCL6 with the concomitant down regulation of COX-2.

Development of microparticles for controlled release of resveratrol to adipose tissue and the impact of drug loading on particle morphology and drug release.

Resveratrol is a small molecule produced by various plants with a remarkable range of beneficial functions in animals. One of these is stimulating signaling pathways in adipose tissue that protect against obesity. Unfortunately, resveratrol suffers from poor bioavailability that inhibits its accumulation in target tissues, including fat, thus hindering the realization of its therapeutic potential. To address this, we are developing biodegradable microparticles as drug depots for controlled release of resveratrol within fat. In this study, resveratrol was encapsulated into poly(lactide-co-glycolide) microparticles using an oil-in-water emulsion/solvent evaporation technique. The oil phase consisted of resveratrol and poly(lactide-co-glycolide) dissolved in a mixture of dichloromethane and ethanol; meanwhile, the aqueous phase contained poly(vinyl alcohol) as the emulsifier. Increasing ethanol's volume ratio increased resveratrol's solubility in the oil phase and particle drug loading. The maximal loading achieved was 65 µg/mg (6.5%) and occurred when the ethanol to dichloromethane ratio was 1:3. Under these conditions, particles exhibited ruffled surfaces, which resulted in variable drug release over the first three days of a six-week release assay. By decreasing resveratrol and ethanol in the oil phase and increasing poly(vinyl alcohol) in the aqueous phase, smooth particles were achieved, but they suffered a 15-25-fold decrease in drug loading depending on size. Small particles exhibited higher drug loading and burst drug release compared to larger particles because of their higher specific surface area. Utilizing mild chemistry, we functionalized poly(vinyl alcohol) with fluorescein isothiocyanate and demonstrated that encapsulation of resveratrol in the particle decreases the amount of fluorescent polymer on the particle surface, suggesting resveratrol displaces the emulsifier during particle formation. Taken together, resveratrol can be encapsulated into poly(lactide-co-glycolide) microparticles, but it accumulates at the particle surface impacting drug loading, surface roughness, and drug release.

The host response to poly(lactide-co-glycolide) scaffolds protects mice from diet induced obesity and glucose intolerance.

Underlying metabolic disease is poor adipose tissue function characterized by impaired glucose tolerance and low expression of health promoting adipokines. Currently, no treatments specifically target the adipose tissue and we are investigating polymer scaffolds for localized drug delivery as a therapeutic platform. In this work we implanted porous poly(lactide-co-glycolide) scaffolds into the epididymal fat of mice. Surprisingly, "empty" scaffolds decreased blood glucose levels in healthy mice as well as epididymal fat pad size. By injecting a fluorescent glucose tracer into mice, we determined that glucose uptake increases by 60% in epididymal fat pads with scaffolds; in contrast, glucose uptake was not elevated in other major metabolic organs, suggesting the enhanced glucose uptake at the scaffold implant site was responsible for decreased blood glucose levels. Histology indicated increased cellularity and tissue remodeling around the scaffold and we found increased expression of glucose transporter 1 and insulin-like growth factor 1, which are proteins involved in wound healing that can also modulate blood glucose levels through their promotion of glucose uptake. Regarding clinical translation, "empty" scaffolds decreased obesity and improved glucose tolerance in mice fed a high fat diet. These findings demonstrate increased cellular activity in the adipose tissue, such as that associated with the host response to biomaterial implant, is beneficial in mice suffering from metabolic complications of over nutrition, possibly because it mitigates the positive energy balance that leads to the obese, diabetic state. More broadly, this work reaffirms that in addition to the local host response typically investigated, biomaterial implant has systemic physiological effects and suggests that there may be implications for therapy.

Resveratrol Delivery from Porous Poly(lactide- co-glycolide) Scaffolds Promotes an Anti-Inflammatory Environment within Visceral Adipose Tissue.

As biomaterial therapies emerge to address adipose tissue dysfunction that underlies metabolic disease, the immune response to these systems must be established. As a potential therapy, we are investigating resveratrol delivery from porous poly(lactide- co-glycolide) scaffolds designed to integrate with adipose tissue. Resveratrol was selected for its ability to protect mice and primates from high fat diet and broad anti-inflammatory properties. Herein, we report fabrication of scaffolds with high resveratrol loading that are stable and active for up to one year. In vitro release profiles indicate that drug release is biphasic with a burst release over 3 days followed by a plateau. Surprisingly, we find that PLG scaffolds implanted into adipose tissue of mice promote an anti-inflammatory environment characterized by high arginase-1 and low TNF-α and IL-6 compared to naïve unmanipulated fat. Resveratrol delivery from the scaffold augments this anti-inflammatory environment by decreasing monocyte and lymphocyte numbers at the implant site and increasing expression of IL-10 and IL-13, cytokines that promote healthy adipose tissue. In terms of therapeutic applications, implant of scaffolds designed to release resveratrol into the visceral fat decreases MCP-1 expression in mice fed a high fat diet, a molecule that drives both local and systemic inflammation during obesity. Taken together, resveratrol delivery to adipose tissue using poly(lactide- co-glycolide) scaffolds is a promising therapeutic strategy for the treatment of adipose tissue inflammation that drives metabolic disease.

Transforming growth factor-beta 1 delivery from microporous scaffolds decreases inflammation post-implant and enhances function of transplanted islets.

Biomaterial scaffolds are central to many regenerative strategies as they create a space for infiltration of host tissue and provide a platform to deliver growth factors and progenitor cells. However, biomaterial implantation results in an unavoidable inflammatory response, which can impair tissue regeneration and promote loss or dysfunction of transplanted cells. We investigated localized TGF-ß1 delivery to modulate this immunological environment around scaffolds and transplanted cells. TGF-ß1 was delivered from layered scaffolds, with protein entrapped within an inner layer and outer layers designed for cell seeding and host tissue integration. Scaffolds were implanted into the epididymal fat pad, a site frequently used for cell transplantation. Expression of cytokines TNF-α, IL-12, and MCP-1 were decreased by at least 40% for scaffolds releasing TGF-ß1 relative to control scaffolds. This decrease in inflammatory cytokine production corresponded to a 60% decrease in leukocyte infiltration. Transplantation of islets into diabetic mice on TGF-ß1 scaffolds significantly improved the ability of syngeneic islets to control blood glucose levels within the first week of transplant and delayed rejection of allogeneic islets. Together, these studies emphasize the ability of localized TGF-ß1 delivery to modulate the immune response to biomaterial implants and enhance cell function in cell-based therapies.

Modulation of leukocyte infiltration and phenotype in microporous tissue engineering scaffolds via vector induced IL-10 expression.

Biomaterial scaffolds are central to many tissue engineering strategies as they create a space for tissue growth and provide a support for cell adhesion and migration. However, biomaterial implantation results in unavoidable injury resulting in an inflammatory response, which can impair integration with the host and tissue regeneration. Toward the goal of reducing inflammation, we investigated the hypothesis that a lentiviral gene therapy-based approach to localized and sustained IL-10 expression at a scaffold could modulate the number, relative proportions, and cytokine production of infiltrating leukocyte populations. Flow cytometry was used to quantify infiltration of six leukocyte populations for 21 days following implantation of PLG scaffolds into intraperitoneal fat. Leukocytes with innate immune functions (i.e., macrophages, dendritic cells, neutrophils) were most prevalent at early time points, while T lymphocytes became prevalent by day 14. Reporter gene delivery indicated that transgene expression persisted at the scaffold for up to 28 days and macrophages were the most common leukocyte transduced, while transduced dendritic cells expressed the greatest levels of transgene. IL-10 delivery decreased leukocyte infiltration by 50% relative to controls, increased macrophage IL-10 expression, and decreased macrophage, dendritic cell, and CD4 T cell IFN-γ expression. Thus, IL-10 gene delivery significantly decreased inflammation following scaffold implant into the intraperitoneal fat, in part by modulating cytokine expression of infiltrating leukocytes.

PLG scaffold delivered antigen-specific regulatory T cells induce systemic tolerance in autoimmune diabetes.

Islet transplantation is a promising treatment for human type 1 diabetes mellitus. Transplantation requires systemic immunosuppression, which has numerous deleterious side effects. Islet antigen-specific regulatory T cells (Tregs) have been shown to protect islet grafts from autoimmune destruction in the nonobese diabetic (NOD) model when co-localized in the kidney capsule. An extra-hepatic transplant site was established by transplanting islet-loaded microporous poly (lactide-co-glycolide) (PLG) scaffolds into abdominal fat. This study examined an autoimmune transplantation model and determined whether co-localized Tregs could protect islet grafts in an extra-hepatic and extra-renal transplant site. Normoglycemia was restored, and co-transplanted Tregs extended graft survival, including several instances of indefinite protection. Transplanted Tregs were replaced by recipient-derived Tregs over time, indicating that islet antigen-specific Tregs induce tolerance to islet grafts through host-derived Tregs. Thus, Tregs provided protection against a diverse repertoire of autoreactive T-cell-receptor specificities mediating diabetes in the NOD model, possibly through a phenomenon previously described as infectious tolerance. Interestingly, the infiltration by Tregs protected a second islet transplant, indicating systemic tolerance to islet antigens. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that allows for the co-localization of immunomodulatory cells within islet grafts and induces long-term graft survival in an autoimmune diabetes model. This method of co-localizing immunomodulatory cells with islets in a clinically translatable transplant site to affect the immune system on a local and systemic level has potential therapeutic implications for human islet transplantation.

Spatial regulation of inflammation by human aortic endothelial cells in a linear gradient of shear stress.

OBJECTIVE: Atherosclerosis is a focal disease that develops at sites of low and oscillatory shear stress in arteries. This study aimed to understand how endothelial cells sense a gradient of fluid shear stress and transduce signals that regulate membrane expression of cell adhesion molecules and monocyte recruitment. METHODS: Human aortic endothelial cells were stimulated with TNF-alpha and simultaneously exposed to a linear gradient of shear stress that increased from 0 to 16 dyne/cm2. Cell adhesion molecule expression and activation of NFkappa B were quantified by immunofluorescence microscopy with resolution at the level of a single endothelial cell. Monocyte recruitment was imaged using custom microfluidic flow chambers. RESULTS: VCAM-1 and E-selectin upregulation was greatest between 2-4 dyne/cm2 (6 and 4-fold, respectively) and above 8 dyne/cm2 expression was suppressed below that of untreated endothelial cells. In contrast, ICAM-1 expression and NFkappa B nuclear translocation increased with shear stress up to a maximum at 9 dyne/cm2. Monocyte recruitment was most efficient in regions where E-selectin and VCAM-1 expression was greatest. CONCLUSIONS: We found that the endothelium can sense a change in shear stress on the order of 0.25 dyne/cm2 over a length of approximately 10 cells, regulating the level of protein transcription, cellular adhesion molecule expression, and leukocyte recruitment during inflammation.