Extra-viral DNA in adeno-associated viral vector preparations induces TLR9-dependent innate immune responses in human plasmacytoid dendritic cells.

Adeno-associated viral (AAV) vector suspensions produced in either human derived HEK cells or in Spodoptera frugiperda (Sf9) insect cells differ in terms of residual host cell components as well as species-specific post-translational modifications displayed on the AAV capsid proteins. Here we analysed the impact of these differences on the immunogenic properties of the vector. We stimulated human plasmacytoid dendritic cells with various lots of HEK cell-produced and Sf9 cell-produced AAV-CMV-eGFP vectors derived from different manufacturers. We found that AAV8-CMV-eGFP as well as AAV2-CMV-eGFP vectors induced lot-specific but not production platform-specific or manufacturer-specific inflammatory cytokine responses. These could be reduced or abolished by blocking toll-like receptor 9 signalling or by enzymatically reducing DNA in the vector lots using DNase. Successful HEK cell transduction by DNase-treated AAV lots and DNA analyses demonstrated that DNase did not affect the integrity of the vector but degraded extra-viral DNA. We conclude that both HEK- and Sf9-cell derived AAV preparations can contain immunogenic extra-viral DNA components which can trigger lot-specific inflammatory immune responses. This suggests that improved strategies to remove extra-viral DNA impurities may be instrumental in reducing the immunogenic properties of AAV vector preparations.

Synthetic Material Abdominal Swabs Reduce Activation of Platelets and Leukocytes Compared to Cotton Materials.

During surgical procedures, cotton abdominal swabs with their high absorptive capacity and malleability are used to retain organs and absorb blood or other body fluids. Such properties of the natural material cotton are advantageous for most operations, but in cardiopulmonary bypass (CPB) surgery, a high blood volume can accumulate in the thoracic cavity that is quickly retransfused via the heart-lung machine (HLM). This common practice is supposed to be safe due to the high anticoagulation. However, in vitro analyses showed that blood cells and plasma proteins were activated despite a high anticoagulation, which can propagate especially an inflammatory response in the patient. Thus, we investigated patients' blood during CPB surgery for inflammatory and coagulation-associated activation after contact to the HLM and either cotton or synthetic abdominal swabs. Contact with cotton significantly increased thrombocyte and neutrophil activation measured as ß-thromboglobulin and PMN-elastase secretion, respectively, compared to synthetic abdominal swabs. Both inflammatory cytokines, interleukin (IL) 1ß and IL6, were also significantly increased in the cotton over the synthetic patient group, while SDF-1α was significantly lower in the synthetic group. Our data show for the first time that cotton materials can activate platelets and leukocytes despite a high anticoagulation and that this activation is lower with synthetic materials. This additional activation due to the material on top of the activation exerted by the tissue contact that blood is exposed to during CPB surgery can propagate further reactions in patients after surgery, which poses a risk for this already vulnerable patient group.

Pancreatic Steatosis Associates With Impaired Insulin Secretion in Genetically Predisposed Individuals.

CONTEXT: Pancreatic steatosis leading to beta-cell failure might be involved in type 2 diabetes (T2D) pathogenesis. OBJECTIVE: We hypothesized that the genetic background modulates the effect of pancreatic fat on beta-cell function and investigated genotype × pancreatic fat interactions on insulin secretion. DESIGN: Two observational studies. SETTING: University hospital. PATIENTS OR PARTICIPANTS: A total of 360 nondiabetic individuals with elevated risk for T2D (Tuebingen Family Study [TUEF]), and 64 patients undergoing pancreatectomy (Pancreas Biobank [PB], HbA1c <9%, no insulin therapy). MAIN OUTCOME MEASURES: Insulin secretion calculated from 5-point oral glucose tolerance test (TUEF) and fasting blood collection before surgery (PB). A genome-wide polygenic score for T2D was computed from 484,788 genotyped variants. The interaction of magnetic resonance imaging-measured and histologically quantified pancreatic fat with the polygenic score was investigated. Partitioned risk scores using genome-wide significant variants were also computed to gain insight into potential mechanisms. RESULTS: Pancreatic steatosis interacted with genome-wide polygenic score on insulin secretion (P = 0.003), which was similar in the replication cohort with histological measurements (P = 0.03). There was a negative association between pancreatic fat and insulin secretion in participants with high genetic risk, whereas individuals with low genetic risk showed a positive correlation between pancreatic fat and insulin secretion. Consistent interactions were found with insulin resistance-specific and a liver/lipid-specific polygenic scores. CONCLUSIONS: The associations suggest that pancreatic steatosis only impairs beta-cell function in subjects at high genetic risk for diabetes. Genetically determined insulin resistance specifically renders pancreatic fat deleterious for insulin secretion.

Metabolic crosstalk between fatty pancreas and fatty liver: effects on local inflammation and insulin secretion.

AIMS/HYPOTHESIS: Obesity-linked ectopic fat accumulation is associated with the development of type 2 diabetes. Whether pancreatic and liver steatosis impairs insulin secretion is controversial. We examined the crosstalk of human pancreatic fat cells with islets and the role of diabetogenic factors, i.e. palmitate and fetuin-A, a hepatokine released from fatty liver. METHODS: Human pancreatic resections were immunohistochemically stained for insulin, glucagon, somatostatin and the macrophage/monocyte marker CD68. Pancreatic adipocytes were identified by Oil Red O and adiponectin staining. Primary pancreatic pre-adipocytes and differentiated adipocytes were co-cultured with human islets isolated from organ donors and the metabolic crosstalk between fatty liver and fatty pancreas was mimicked by the addition of palmitate and fetuin-A. Insulin secretion was evaluated by ELISA and RIA. Cytokine expression and secretion were assessed by RT-PCR and multiplex assay, respectively. Subcellular distribution of proteins was examined by confocal microscopy and protein phosphorylation by western blotting. RESULTS: In human pancreatic parenchyma, highly differentiated adipocytes were detected in the proximity of islets with normal architecture and hormone distribution. Infiltration of adipocytes was associated with an increased number of CD68-positive cells within islets. In isolated primary pancreatic pre-adipocytes and differentiated adipocytes, palmitate and fetuin-A induced IL6, CXCL8 and CCL2 mRNA expression. Cytokine production was toll-like receptor 4 (TLR4)-dependent and further accentuated in pre-adipocytes when co-cultured with islets. In islets, IL6 and CXCL8 mRNA levels were also increased by fetuin-A and palmitate. Only in macrophages within the isolated islets, palmitate and fetuin-A stimulated the production of the cytotoxic cytokine IL-1ß. Palmitate, but not fetuin-A, exerted pro-apoptotic effects in islet cells. Instead, fetuin-A impaired glucose-induced insulin secretion in a TLR4-independent, but c-Jun N-terminal kinase- and Ca2+-dependent, manner. CONCLUSIONS/INTERPRETATION: These results provide the first evidence that fetuin-A-mediated metabolic crosstalk of fatty liver with islets may contribute to obesity-linked glucose blindness of beta cells, while fatty pancreas may exacerbate local inflammation.

Fetuin-A influences vascular cell growth and production of proinflammatory and angiogenic proteins by human perivascular fat cells.

AIMS/HYPOTHESIS: Fetuin-A (alpha2-Heremans-Schmid glycoprotein), a liver-derived circulating glycoprotein, contributes to lipid disorders, diabetes and cardiovascular diseases. In a previous study we found that perivascular fat cells (PVFCs) have a higher angiogenic potential than other fat cell types. The aim was to examine whether fetuin-A influences PVFC and vascular cell growth and the expression and secretion of proinflammatory and angiogenic proteins, and whether TLR4-independent pathways are involved. METHODS: Mono- and co-cultures of human PVFCs and endothelial cells were treated with fetuin-A and/or palmitate for 6-72 h. Proteins were quantified by ELISA and Luminex, mRNA expression by real-time PCR, and cell growth by BrDU-ELISA. Some PVFCs were preincubated with a nuclear factor κB NFκBp65 inhibitor, or the toll-like receptor 4 (TLR4) inhibitor CLI-095, or phosphoinositide 3-kinase (PI3K)/Akt inhibitors and/or stimulated with insulin. Intracellular forkhead box protein O1 (FoxO1), NFκBp65 and inhibitor of κB kinase ß (IKKß) localisation was visualised by immunostaining. RESULTS: PVFCs expressed and secreted IL-6, IL-8, plasminogen activator inhibitor 1 (PAI-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), placental growth factor (PLGF) and hepatocyte growth factor (HGF). Fetuin-A upregulated IL-6 and IL-8, and this was potentiated by palmitate and blocked by CLI-095. Immunostaining and electrophoretic mobility shift assay (EMSA) showed partial NFκBp65 activation. MCP-1 was upregulated and blocked by CLI-095, but not by palmitate. However, HGF was downregulated, which was slightly potentiated by palmitate. This effect persisted after TLR4 pathway blockade. Stimulation of insulin-PI3K-Akt signalling by insulin resulted in nuclear FoxO1 extrusion and HGF upregulation. Fetuin-A counteracted these insulin effects. CONCLUSIONS/INTERPRETATION: Fetuin-A and/or palmitate influence the expression of proinflammatory and angiogenic proteins only partially via TLR4 signalling. HGF downregulation seems to be mediated by interference with the insulin-dependent receptor tyrosine kinase pathway. Fetuin-A may also influence angiogenic and proinflammatory proteins involved in atherosclerosis.

Inhibition of foam cell formation using a soluble CD68-Fc fusion protein.

The appearance of lipid-rich foam cells is a major feature of vulnerable atherosclerotic plaque formation. The transformation of macrophages into foam cells results from excessive uptake of cholesterol-rich particles by scavenger receptors such as CD68. We cloned a CD68-Fc immunoadhesin, a fusion protein consisting of the extracellular domain of the human CD68 and a human Fc domain, and investigated the function in vitro. Specific binding of CD68-Fc to OxLDL with an affinity of 10 nmol/L was determined by surface plasmon resonance and increased binding to lipid-rich human and ApoE(-/-) mice plaque tissue. This was confirmed both by immunohistochemical staining of CD68-Fc-treated paraffin sections from human plaques and by ELISA-based quantification of CD68-Fc binding to human atherosclerotic plaque extracts. In an in vitro model of macrophage/foam cell formation, CD68-Fc reduced foam cell formation significantly. This was caused both by interference of CD68-Fc with OxLDL uptake into macrophages and platelets and by the inhibition of platelet/OxLDL phagocytosis. Finally, expression of metalloproteinases by macrophages/foam cells was inhibited by CD68-Fc. In conclusion, CD68-Fc seems to be a promising new tool for preventing macrophage/foam cell formation. Thus, CD68-Fc might offer a novel therapeutic strategy for patients with acute coronary syndrome by modulating the generation of vulnerable plaques.

Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro.

Patients with myocardial infarction reveal an altered number of circulating mesenchymal stem cells (MSCs). Recently, it was shown that MSCs are able to regenerate myocardial tissue and to differentiate into endothelial cells. The homing mechanisms of MSCs from the circulation into the target tissue, however, are not understood so far. In this study, we evaluated the impact of platelets on MSC recruitment, proliferation, migration and integration into the endothelium. MSCs expressing alpha(v)beta(3) integrin were recruited to human arterial endothelial cells exposed to isolated platelets or IL-1 beta under high shear conditions. Furthermore, induction of vascular injury in vivo resulted in increased recruitment of injected MSCs as assessed by intravital microscopy and depletion of platelets significantly reduced this adhesion. The interaction of platelets and MSCs was inhibited by pre-incubation with the mAb 7E3 or an RGD protein both blocking beta(3) integrin mediated adhesion. Platelets had a chemotactic effect on MSCs, promoted a migratory MSC phenotype and dose- and activation-dependently enhanced migration of MSCs, a process, which was mediated by basic fibroblast growth factor (bFGF). Similarly, platelet derived bFGF increased proliferation of MSCs. Coincubation of MSCs with platelets facilitated integration into an endothelial monolayer, which was significantly reduced by pre-incubation with a blocking mAb to bFGF. We conclude that platelets may play a critical part in the recruitment of MSCs to the endothelium, influence MSC function and promote integration of MSCs into the endothelium.

The evil in atherosclerosis: adherent platelets induce foam cell formation.

Platelet interaction with circulating progenitor cells plays an important role for repair mechanisms at sites of vascular lesions. Foam cell formation represents a key process in atherosclerotic plaque formation. We revealed that platelets regulate recruitment and differentiation of CD34 (+) progenitor cells into foam cells and endothelial cells. Adhesion studies showed that platelets recruit CD34 (+) progenitor cells via specific adhesion receptors, including P-selection/P-selectin glycoprotein ligand 1, and beta (1) and beta (2) integrins. CD34 (+) progenitor cells were coincubated with human platelets for 1 week. We demonstrated that a substantial number of CD34 (+) cells differentiated into foam cells. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and agonists of peroxisome proliferator-activated receptor-alpha and -gamma (PPAR-alpha and -gamma agonists) reduced this foam cell generation via inhibition of matrix metalloproteinase 9 secretions. Foam cell formation is also induced by low-density lipoproteins (LDLs). It was revealed that platelets take up modified LDL (fluorochrome-conjugated acetylated LDL) that is stored in the dense granules and internalized rapidly into the foam cells. These findings emphasize that the balance between endothelial cell regeneration and platelet-mediated foam cell generation derived from CD34 (+) progenitor cells may play a critical role in atherogenesis and atheroprogression.

Effects of local all-trans-retinoic acid delivery on experimental atherosclerosis in the rabbit carotid artery.

BACKGROUND: Retinoids regulate a variety of biological processes and play an important role in cell differentiation and proliferation. All-trans retinoid acid (atRA) is known to inhibit smooth muscle cell growth and thus is supposed to have favorable effects on the incidence of restenosis after percutaneous coronary interventions. The broad biological spectrum, however, leads to numerous severe side effects which limit the clinical use of a systemic application of atRA. In order to avoid systemic side effects, local delivery of atRA is preferable. The aim of this study was to evaluate the effects of atRA on the response to injury in a second-injury model of experimental balloon angioplasty. METHODS: After induction of a fibromuscular plaque in the right carotid artery of 40 New Zealand rabbits, 35 animals underwent balloon angioplasty of the preformed plaque formation. Subsequent local atRA delivery (10 ml, 10 microM) with the double-balloon catheter was performed in 15 animals. Five animals received vehicle only as sham controls, and five animals were solely electrostimulated, 15 animals served as control group with balloon angioplasty only. Vessels were excised 7 days (n=15) and 28 days (n=30) after intervention. Immunocytochemistry with antibodies against smooth muscle alpha-actin and myosin, bromodeoxyuridine, macrophages, collagen I and III and von Willebrand factor was performed. Quantitative analysis was done by computerized morphometry. RESULTS: After local atRA delivery in vivo, the extent of stenosis was markedly reduced with 21.7+/-8.3% (mean+/-S.D.) 4 weeks after intervention compared to 31.8+/-13.4% in balloon-dilated animals (P=0.0937). Both a reduced early neointimal proliferation (P=0.0002) and an increase in overall vessel diameter (4 weeks after intervention, P=0.0264) contributed to a limitation of restenosis in atRA-treated animals. Immunocytochemistry revealed a more intense alpha-actin staining pattern after local atRA therapy indicating redifferentiating effects of atRA on vascular smooth muscle cells. CONCLUSIONS: Local delivery of atRA led to limitation of restenosis formation in this animal model. The concept of a local atRA therapy might be a promising way to exploit the potential of atRA for vascular indications while minimizing the severe side effects of systemic retinoid therapy.