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In a diabetic milieu high levels of reactive oxygen species (ROS) are induced. This contributes to the vascular complications of diabetes. Recent studies have shown that ROS formation is exacerbated in diabetic monocytes and macrophages due to a glycolytic metabolic shift. Macrophages are important players in the progression of diabetes and promote inflammation through the release of pro-inflammatory cytokines and proteases. Because ROS is an important mediator for the activation of pro-inflammatory signaling pathways, obesity and hyperglycemia-induced ROS production may favor induction of M1-like pro-inflammatory macrophages during diabetes onset and progression. ROS induces MAPK, STAT1, STAT6 and NFκB signaling, and interferes with macrophage differentiation via epigenetic (re)programming. Therefore, a comprehensive understanding of the impact of ROS on macrophage phenotype and function is needed in order to improve treatment of diabetes and its vascular complications. In the current comprehensive review, we dissect the role of ROS in macrophage polarization, and analyze how ROS production links metabolism and inflammation in diabetes and its complications. Finally, we discuss the contribution of ROS to the crosstalk between macrophages and endothelial cells in diabetic complications.

Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to functionally replace retinal pericytes and normalize retinal microvasculature during disease progression. We hypothesized that Notch signaling in ASC underlies regulation and stabilization of dysfunctional retinal microvascular networks such as in DR. ASC prominently and constitutively expressed NOTCH2. Genetic knockdown of NOTCH2 in ASC (SH-NOTCH2) disturbed the formation of vascular networks of human umbilical cord vein endothelial cells both on monolayers of ASC and in organotypical three-dimensional cocultures with ASC. On ASC SH-NOTCH2, cell surface platelet-derived growth factor receptor beta was downregulated which disrupted their migration toward the chemoattractant platelet-derived growth factor beta subunits (PDGF-BB) as well as to conditioned media from EC and bovine retinal EC. This chemoattractant is secreted by pro-angiogenic EC in newly formed microvascular networks to attract pericytes. Intravitreal injected ASC SH-NOTCH2 in oxygen-induced retinopathy mouse eyes did not engraft in the preexisting retinal microvasculature. However, the in vivo pro-angiogenic capacity of ASC SH-NOTCH2 did not differ from controls. In this respect, multifocal electroretinography displayed similar b-wave amplitudes in the avascular zones when either wild type ASC or SH-NOTCH2 ASC were injected. In conclusion, our results indicate that NOTCH2 is essential to support in vitro vasculogenesis via juxtacrine interactions. In contrast, ongoing in vivo angiogenesis is influenced by paracrine signaling of ASC, irrespective of Notch signaling.

The number of diabetic patients in Europe and world-wide is growing. Diabetes confers a 2-fold higher risk for vascular disease. Lack of insulin production (Type 1 diabetes, T1D) or lack of insulin responsiveness (Type 2 diabetes, T2D) causes systemic metabolic changes such as hyperglycemia (HG) which contribute to the pathology of diabetes. Monocytes and macrophages are key innate immune cells that control inflammatory reactions associated with diabetic vascular complications. Inflammatory programming of macrophages is regulated and maintained by epigenetic mechanisms, in particular histone modifications. The aim of our study was to identify the epigenetic mechanisms involved in the hyperglycemia-mediated macrophage activation. Using Affymetrix microarray profiling and RT-qPCR we identified that hyperglycemia increased the expression of S100A9 and S100A12 in primary human macrophages. Expression of S100A12 was sustained after glucose levels were normalized. Glucose augmented the response of macrophages to Toll-like receptor (TLR)-ligands Palmatic acid (PA) and Lipopolysaccharide (LPS) i.e., pro-inflammatory stimulation. The abundance of activating histone Histone 3 Lysine 4 methylation marks (H3K4me1, H3K4me3) and general acetylation on histone 3 (AceH3) with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycemia increased acetylation of histones bound to the promoters of S100A9 and S100A12 in M1 macrophages. In contrast, hyperglycemia caused a reduction in total H3 which correlated with the increased expression of both S100 genes. The inhibition of histone methyltransferases SET domain-containing protein (SET)7/9 and SET and MYND domain-containing protein (SMYD)3 showed that these specifically regulated S100A12 expression. We conclude that hyperglycemia upregulates expression of S100A9, S100A12 via epigenetic regulation and induces an activating histone code on the respective gene promoters in M1 macrophages. Mechanistically, this regulation relies on action of histone methyltransferases SMYD3 and SET7/9. The results define an important role for epigenetic regulation in macrophage mediated inflammation in diabetic conditions.

Diabetic retinopathy (DR) is a hyperglycemia (HG)-mediated microvascular complication. In DR, the loss of pericytes and subsequently endothelial cells leads to pathologic angiogenesis in retina. Adipose-derived stromal cells (ASC) are a promising source of therapeutic cells to replace lost pericytes in DR. To date, knowledge of the influence of HG on the bioenergetics and pericytic function of ASC is negligible. Human ASC were cultured in normoglycemia medium (5mM d-glucose) or under HG (30mM D-glucose) and assessed. Our data showed that HG increased the level of apoptosis and reactive oxygen species production in ASC, yet their proliferation rate was not affected. HG induced alterations in mitochondrial function and morphology in ASC. HG also strongly affected the bioenergetic status of ASC in which both the maximum oxygen consumption rate and extracellular acidification rate were decreased. This was corroborated by a reduced uptake of glucose under HG. In spite of these observations, in vitro, ASC promoted the formation of vascular-like networks of human umbilical vein endothelial cells on monolayers of ASC under HG with minimally affected.

Stem cell therapy is a promising approach for repair, remodeling and even regenerate tissue of otherwise irreparable damage, such as after myocardial infarction (aMI). A severe limitation of cardiac stem cell therapy is the generally poor retention of administered cells in the target tissue. In tissue repair the main mode of action of adipose tissue-derived stem cells (ADSC) is the production of various growth factors, cytokines, anti-inflammatory and anti-apoptotic factors that together augment repair, remodeling, and regeneration. In this experiment, we used recombinant collagen peptide (RCP) with additional integrin-binding motives and different crosslinkers. Formulated as 50-100 μm microspheres with bound ADSC, we hypothesized that this would improve ADSC retention and function. Crosslinking was performed with chemical crosslinkers (EDC and HMDIC) at high and low concentrations or by thermal treatment (DHT). ADSC adhesion, proliferation, apoptosis/necrosis, and gene expressions in two-dimensional and three-dimensional were analyzed. In addition, the effect of ADSC conditioned medium (ADSC-CM) on proapoptotic/sprouting HUVEC was examined. Our results show that all materials support cell adhesion in short time point, however, EDC-High crosslinker induced ADSC apoptosis/necrosis. Gene expression results revealed lower expression of proinflammatory genes in chemical crosslinked materials, despite EDC-High the proinflammatory genes expressions were similar or higher than TCPS. In addition, cultured ADSC on DHT crosslinked RCP showed a proinflammatory phenotype compared to TCPS. Sprouting assay results confirmed the protective effect of ADSC-CM derived from TCPS and HMDIC-High crosslinked RCP proapoptotic HUVEC. We conclude that ADSC adhere to the materials and maintain their therapeutic profile.

Diabetic retinopathy (DR) is a multifactorial microvascular disease induced by hyperglycemia and subsequent metabolic abnormalities. The resulting cell stress causes a sequela of events that ultimately can lead to severe vision impairment and blindness. The early stages are characterized by activation of glia and loss of pericytes, endothelial cells (EC) and neuronal cells. The integrity of the retinal microvasculature becomes affected, and, as a possible late response, macular edema may develop as a common reason for vision loss in patients with non-proliferative DR. Moreover, the local ischemia can trigger vasoproliferation leading to vision-threating proliferative DR (PDR) in humans. Available treatment options include control of metabolic and hemodynamic factors. Timely intervention of advanced DR stages with laser photocoagulation, intraocular anti–vascular endothelial growth factor (VEGF) or glucocorticoid drugs can reduce vision loss. As the pathology involves cell loss of both the vascular and neuroglial compartments, cell replacement strategies by stem and progenitor cells have gained considerable interest in the past years. Compared to other disease entities, so far little is known about the efficacy and potential mode of action of cell therapy in treatment of DR. In preclinical models of DR different cell types have been applied ranging from embryonic or induced pluripotent stem cells, hematopoietic stem cells, and endothelial progenitor cells to mesenchymal stromal cells (MSC). The latter cell population can combine various modes of action (MoA), thus they are among the most intensely tested cell types in cell therapy. The aim of this review is to discuss the rationale for using MSC as potential cell therapy to treat DR. Accordingly, we will revise identified MoA of MSCs and speculate how these may support the repair of the damaged retina.

Monocytes are actively recruited at sites of chronic inflammation. However, molecular factors involved in this process are not fully elucidated. Here, we show that cytokine IL-4 which is implicated in the development of chronic inflammatory disease atopic dermatitis (AD) induces expression of transcription factor FoxQ1 in human monocytes and macrophages. FoxQ1 mRNA levels were elevated in monocytes of AD patients compared to healthy donors. Overexpression of FoxQ1 in RAW 264.7 monocytic cells facilitated their migration towards MCP-1 and was associated with decreased expression of migration-regulating genes (claudin 11 and plexin C1). Furthermore, FoxQ1 overexpression in RAW cells accelerated TNFα secretion after LPS challenge. Overall, our results indicate that FoxQ1 stimulates monocyte motility, increases pro-inflammatory potential, and directs monocyte migration towards MCP-1 that is crucial for monocyte influx into inflammatory sites. This mechanism could contribute to the pathogenesis of chronic inflammatory disorders such as AD.

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytoldne profile that can support of diabetes-associated inflammation and development of vascular complications. (C) 2016 Elsevier GmbH. All rights reserved.

Adipose derived stromal cells (ADSC) are relevant therapeutic agents to treat myocardial infarction (MI) in clinical trials. Soluble factors secreted by ADSC, such as growth factors and cytokines, suppress inflammation and apoptosis while promoting angiogenesis and the proliferation of cardiomyocytes (CM). Moreover, ADSC synthesize extracellular matrix (ECM) components into the intercellular space which might contribute to their therapeutic capacity. Thus, ADSC might directly modulate the post-MI microenvironment through a combination of paracrine and juxtacrine signaling. In this study, the juxtacrine role of ADSC and ADSC-derived ECM on the organization and maturation of CM was investiagated. Human ADSC synthesized and deposited a heterogenous and complex mixture of ECM components such as collagen I, III, IV, fibronectin, elastin as well as the matricellular protein periostin. Cocultures of rodent CM with human ADSC or with human ADSC-derived ECM components enhanced the cardiomyocyte alignment, their intercellular connections and the maturation of their sarcomeres, while the proliferation rate of the CM was increased and their level of hypertrophy reduced. The use of human ADSC-derived ECM could serve both to augment in vitro tissue-engineered myocardial constructs and to improve myocardial remodeling after infarction. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1840-1848, 2015.

The pandemic of cardiovascular disease is continuously expanding as the result of changing life styles and diets throughout the Old and New World. Immediate intervention therapy saves the lives of many patients after acute myocardial infarction (MI). However, for many this comes at the price of adverse cardiac remodeling and heart failure. Currently, no conventional therapy can prevent the negative aftermath of MI and alternative treatments are warranted. Therefore, cardiac stem cell therapy has been put forward over the past decade, albeit with modest successes. Mesenchymal Stem Cells (MSC) are promising because these are genuine cellular factories of a host of secreted therapeutic factors. MSC are obtained from bone marrow or adipose tissue (ADSC). However, the heart itself also contains mesenchymal- like stem cells, though more difficult to acquire than ADSC. Interestingly, mesenchymal cells such as fibroblasts can be directly or indirectly reprogrammed to all myocardial cell types that require replacement after MI. To date, the paracrine and juxtacrine mechanisms of ADSC and other MSC on vessel formation are best understood. The preconditioning of, otherwise naive, stem cells is gaining more interest: previously presumed deleterious stimuli such as hypoxia and inflammation, i.e. causes of myocardial damage, have the opposite effect on mesenchymal stem cells. MSC gain a higher therapeutic capacity under hypoxia and inflammatory conditions. In this review, mesenchymal stem cells and their working mechanisms are put into the perspective of clinical cardiac stem cell therapy.

Cell therapy strategies that use adult peripheral blood-derived CD34+ progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34+ cells to treat ischaemic diseases is under debate. Interaction between CD34+ cells and CD14+ cells results in superior endothelial differentiation of CD14+ cells in vitro, indicating that cell therapy approaches utilizing both CD34+ and CD14+ cells may be advantageous in therapeutic neovascularization. Here, human CD34+ and CD14+ cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34+ and CD14+ cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34+ and CD14+ cells produced and secreted a pentad of pro-angiogenic mediators, such as HGF, MCP-1 and IL-8, bFGF and VEGFa in monoculture. The production and secretion of pro-angiogenic mediators by CD14+ cells was highly amplified upon incubation with conditioned medium from CD34+ cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34+ or CD14+ cells, but depended on the paracrine effects of IL-8, MCP-1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell-induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases. Copyright (c) 2011 John Wiley & Sons, Ltd.

Progenitor cell based therapies have emerged for the treatment of ischemic cardiovascular diseases where there is insufficient endogenous repair. However, clinical success has been limited, which challenges the original premise that transplanted progenitor cells would orchestrate repair. In this review, we discuss the basics of endothelial progenitor cell therapy and describe how microenvironmental changes (i.e., trophic and mechano-structural factors) in the damaged myocardium influence progenitor cell plasticity and hamper beneficial therapeutic outcome. Further understanding of these microenvironmental clues will enable optimization of cell therapy at all levels. We discuss current concepts and provide future perspectives for the enhancement of progenitor cell therapy, and merge these advances into a combined approach for ischemic tissue repair.

Backgrounds/Aims: Pericyte loss, vasoregression and neuroglial activation are characteristic changes in incipient diabetic retinopathy. In this study, the effect of the antioxidant and antiglycating dipeptide carnosine was studied on the development of experimental diabetic retinopathy. Materials/Methods: STZ-induced diabetic Wistar rats were orally treated with carnosine (1g/kg body weight/day). Retinal vascular damage was assessed by quantitative morphometry. Retinal protein extracts were analyzed for markers of oxidative stress, AGE-formation, activation of the hexosamine pathway and changes in the expression of Ang-2, VEGF and heat shock proteins Hsp27 and HO-1. Glial cell activation was analyzed using Western blot analysis and immunofluorescence of GFAP expression and retinal neuronal damage was histologically examined. Results: Oral carnosine treatment prevented retinal vascular damage after 6 months of experimental hyperglycemia. The protection was not caused by ROS-or AGE-inhibition, but associated with a significant induction of Hsp27 in activated glial cells and normalization of increased Ang-2 levels in diabetic retinas. A significant reduction of photoreceptors in retinas of carnosine treated animals was noted. Conclusion: Oral carnosine treatment protects retinal capillary cells in experimental diabetic retinopathy, independent of its biochemical function. The vasoprotective effect of carnosine might be mediated by the induction of protective Hsp27 in activated glial cells and normalization of hyperglycemia-induced Ang-2. Copyright (C) 2011 S. Karger AG, Basel

In order to get more insight into the role of neutrophils on the micro-environment and consequently on macrophages in the foreign body reaction in mice, we investigated the fate of the two differently crosslinked dermal sheep collagen disks (glutaraldehyde = GDSC, hexamethylenediisocyanate = HDSC) in mice implanted in one anatomical location, namely subcutaneously. In GDSC massive infiltration of neutrophils is seen at day 2 and day 21, whereas in HDSC only minor infiltration is seen at day 2. The presence of neutrophils coincided with high levels of IFN-gamma, a cytokine that activates macrophages. Major differences were seen in degradation rate of the two disks: GDSC was almost completely degraded after 28 days, whereas HDSC remained intact. Degradation of GDSC occurred through collagenolytic activity and phagocytosis by macrophages. Phagocytosis was observed at day 2 and day 21. IL-13 was only observed in HDSC, and this resulted in the presence of giant cells in HDSC. These giant cells produced IL-10, that promoted TIMP-1 expression and that inhibits collagenolytic and phagocytic activity. We conclude that the function of macrophages in mice is largely influenced by differences in micro-environment induced by GDSC and HDSC and that the presence/absence of neutrophils play a major role in the shaping of this micro-environment. (C) 2010 Elsevier Ltd. All rights reserved.

Ischemic cardiovascular events are a major cause of death globally. Endothelial progenitor cell (EPC)-based approaches can result in improvement of vascular perfusion and might offer clinical benefit. However, although functional improvement is observed, the lack of long-term engraftment of EPCs into neovessels has raised controversy regarding their mechanism of action. We and others have hypothesized that after ischemic injury, EPCs induce neovascularization through the secretion of cytokines and growth factors, which act in a paracrine fashion and induce sprouting angiogenesis by the surrounding endothelium. In this concise review, we discuss the (patho)physiology of EPC-induced neovascularization and focus on the paracrine signals secreted by EPCs and the effects they elicit. In future therapies, clinical administration of these paracrine modulators using slow-release depots might induce neovascularization and might therefore hold promise for vascular regenerative medicine.

Background: Ischemia/reperfusion injury (IRI) is a risk factor for the development of interstitial fibrosis. Previously we had shown that after renal IRI, bone marrow-derived cells (BMDC) can differentiate to interstitial myofibroblasts. Here we hypothesized that the immunosuppressant ciclosporin A (CsA), known for its profibrotic side effect, promotes myofibroblast differentiation of BMDC in the postischemic kidney. Methods: Using a model of unilateral renal IRI in rats reconstituted with R26-human placental alkaline phosphatase transgenic bone marrow, CsA was administered in a previously defined critical window for differentiation of BMDC to myofibroblasts. We evaluated fibrotic changes in the kidney and myofibroblast differentiation of BMDC on day 14 after CsA treatment. Results: CsA treatment for 14 days led to increased transforming growth factor-β transcript levels and collagen III deposition in the postischemic kidney. However, neither the total number of α-smooth-muscle-actin-positive interstitial myofibroblasts, nor the bone marrow-derived fraction thereof was affected by CsA administration, irrespective of dosage and duration of treatment. Conclusions: In the critical postischemic window of BMDC differentiation to myofibroblasts, CsA did not promote BMDC differentiation to myofibroblasts, suggesting that, in the clinical setting, CsA is not involved in myofibroblastic differentiation of BMDC.

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable Source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-beta and PDGF-BB). Expression of smooth muscle protein 22 alpha and alpha-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies. (C) 2008 Elsevier Ltd. All rights reserved.

The human pancarcinoma-associated epithelial cell adhesion molecule (EpCAM) (EGP-2, CO17-1A) is a well-known target for carcinoma-directed immunotherapy. Mouse-derived mAbs directed to EpCAM have been used to treat colon carcinoma patients showing well-tolerable toxic side effects but limited antitumor effects. Humanized or fully human anti-EpCAM mAbs may induce stronger antitumor activity, but proved to produce severe pancreatitis upon use in patients. To evaluate treatment-associated effects before a clinical trial, we have generated a transgenic mouse tumor model that expresses human EpCAM similar to carcinoma patients. In this study, we use this model to study the in vivo behavior of two humanized and one mouse-derived anti-EpCAM mAb, i.e., MOC31-hFc, UBS54, and MOC31. The pharmacokinetics and tissue distribution of the fully human mAb UBS54 and the mouse-derived MOC31 were largely the same after injection in tumor-bearing transgenic mice, whereas the molecularly engineered, humanized MOC31-hFc behaved differently. Injection of UBS54 and MOC31 resulted in significant, dose-dependent uptake of mAb in EpCAM-expressing normal and tumor tissues, accompanied by a drop in serum level, whereas injection of MOC31-hFc resulted in uptake in tumor tissue, limited uptake by normal tissues, and slow blood clearance. It is concluded that the EpCAM-transgenic mouse model provides valuable insights into the potential behavior of humanized anti-EpCAM mAbs in patients. mAbs sharing the same epitope and isotype but constructed differently were shown to behave differently in the model, indicating that the design of mAbs is important for eventual success in in vivo application. Copyright © 2007 by The American Association of Immunologists, Inc.

Objective: To investigate the feasibility of intracoronary application of endothelial progenitor cells and the subsequent distribution within the heart. Methods: Endothelial progenitors cells (EPCs) cultured from rat bone marrow were identified by double-positive staining with Dil-Ac-LDL and BS1-lectin. Twenty-four hours before cell transplantation, EPCs were labeled with 5-bromo-2'-deoxyuridine (BrdU). Cells (5x10(5) in 250-mu l medium) were injected into healthy rats, either as intracoronary application (n=11) or as intramyocardial injection (n=6). At 15 min or 3 days posuransplantation, hearts as well as other organs (lung, liver, kidney, and spleen) were collected and processed for subsequent BrdU immunohistochemistry. The number of BrdU-positive cells per tissue area was counted. Results: Compared to intramyocardial injection, intracoronary administration resulted in more than twice as much positive cells in the heart (P

Macrophages have been suggested to be beneficial for myocardial wound healing. We investigated the role of macrophages in myocardial wound healing by inhibition of macrophage infiltration after myocardial injury. We used a murine cryoinjury model to induce left ventricular damage. Infiltrating macrophages were depleted during the 1st week after cryoinjury by serial intravenous injections of clodronate-containing liposomes. After injury, the presence of macrophages, which secreted high levels of transforming growth factor-beta and vascular endothelial growth factor-A, led to rapid removal of cell debris and replacement by granulation tissue containing inflammatory cells and blood vessels, followed by myofibroblast infiltration and collagen deposition. In macrophage-depleted hearts, nonresorbed cell debris was still observed 4 weeks after injury. Secretion of transforming growth factor-beta and vascular endothelial growth factor-A as well as neovascularization, myofibroblast infiltration, and collagen deposition decreased. Moreover, macrophage depletion resulted in a high mortality rate accompanied by increased left ventricular dilatation and wall thinning. In conclusion, infiltrating macrophage depletion markedly impairs wound healing and increases remodeling and mortality after myocardial injury, identifying the macrophage as a key player in myocardial wound healing. Based on these findings, we propose that increasing macrophage numbers early after myocardial infarction could be a clinically relevant option to promote myocardial wound healing and subsequently to reduce remodeling and heart failure.

Vascular tissue engineering aims at creating self-renewing, anti-thrombogenic, vascular grafts, which can be based on endothelial progenitor cells (EPC). EPC harbor essential features such as plasticity and longevity. Unfortunately, the archetype CD34+ EPC is rare in peripheral blood. Monocytes, i.e. CD14+ cells also have the ability to differentiate into endothelial-like cells and are by far more abundant in peripheral blood than are CD34+ EPC. Therefore, CD14+ cells would seem appropriate candidates for tissue engineering of small-diameter blood vessels. In this study, we investigated the differentiation of CD14+ cells on three biodegradable biomaterials under angiogenic conditions. Morphological analyses, gene transcript analyses, endothelial marker (i.e. VE-Cadherin and eNOS) and macrophage marker (i.e. CD68 and CD163) expression analyses, revealed that a small fraction (15-25%) of cultured CD14+ cells differentiated into macrophages after 21 days of culture. The majority of CD14+ cells (>75%) differentiated into endothelial-like cells (ELC) on all biomaterials used. The expression of endothelial markers was similar to their expression on HUVEC. Since CD14+ cells are present in high numbers in adult peripheral blood, easy to isolate and because they easily differentiate into ELC on biomaterials, we conclude that CD14+ cells are a suitable cell source for progenitor-based vascular tissue engineering.

N-octanoyl dopamine (NOD) treatment improves renal function when applied to brain dead donors and in the setting of warm ischaemia-induced acute kidney injury (AKI). Because it also activates transient receptor potential vanilloid type 1 (TRPV1) channels, we first assessed if NOD conveys its renoprotective properties in warm ischaemia-induced AKI via TRPV1 and secondly, if renal transplant recipients also benefit from NOD treatment.We induced warm renal ischaemia in Lewis, wild-type (WT) and TRPV1(-/-) Sprague-Dawley (sd) rats by clamping the left renal artery for 45 min. Transplantations were performed in allogeneic and syngeneic donor-recipient combinations (Fisher to Lewis and Lewis to Lewis) with a cold ischaemia time of 20 h. Treatment was instituted directly after restoration of organ perfusion. Renal function, histology and perfusion were assessed by serum creatinine, microscopy and magnetic resonance imaging (MRI) using arterial spin labelling (ASL).NOD treatment significantly improved renal function in Lewis rats after warm ischaemia-induced AKI. It was, however, not effective after prolonged cold ischaemia. The renoprotective properties of NOD were only observed in Lewis or WT, but not in TRPV1(-/-) sd rats. Renal inflammation was significantly abrogated by NOD. MRI-ASL showed a significantly lower cortical perfusion in ischaemic when compared with non-ischaemic kidneys. No overall differences were observed in renal perfusion between NOD- and NaCl-treated rats.NOD treatment reduces renal injury in warm ischaemia, but is not effective in renal transplant in our experimental animal models. The salutary effect of NOD appears to be TPRV1-dependent, not involving large changes in renal perfusion.

The foreign body reaction (FBR) differs between subcutaneously and supra-epicardially implanted materials. We hypothesize that this is a result of differences in cytokine, chemokine and matrix metalloproteinase (MNIP) dynamics. Therefore we applied collagen disks subcutaneously and on the epicardium in mice and analyzed the FBR from day 1 to 21. Both the influx of leukocytes and implant degradation were higher in supra-epicardially implanted collagen than in subcutaneously implanted material. This correlated with a higher gene expression of pro-inflammatory cytokines such as IL-1 and IL-6, and a lower expression of the anti-inflammatory cytokine IL-10. Furthermore, the higher supra-epicardial expression of PMN attractants CXCL1/KC and CXCL2/MIP2 correlated with a higher and prolonged PMN influx. The gene expression levels of collagen degrading MMPs, i.e. MMP8, MMP13 and MMP14 were similar in subcutaneous and supra-epicardial disks. However, the activity of these enzymes was markedly higher supra-epicardially. In addition, the MMP9 expression was higher supra-epicardially, suggesting a role for this enzyme in the degradation process. In conclusion, a strong pro-inflammatory milieu is generated after supra-epicardial implantation that enables prolonged PMN presence and activation. This, together with the high supra-epicardial MMP9 level, could explain the observed difference in Col-I degradation between locations. (c) 2006 Elsevier Ltd. All rights reserved.

Intracorporally implanted materials, such as medical devices, will provoke the body to initiate an inflammatory reaction. This inflammatory reaction to implanted materials is known as the foreign body reaction (FBR) and is characterized by 3 distinct phases: onset, progression, and resolution. The FBR proceeds in the creation of a dynamic microenvironment that is spatially well organized. The progression of the FBR is regulated by soluble mediators, such as cytokines, chemokines, and matrix metalloproteinases (MMPs), which are produced locally by tissue cells and infiltrated inflammatory cells. These soluble mediators orchestrate the cascade of cellular processes in the microenvironment that accompanies the FBR, consisting of cellular activation, angiogenesis, extravasation, migration, phagocytosis, and, finally, fibrosis. The nature of the FBR requires that the soluble mediators act in a spatial and temporally regulated manner as well. This regulation is well known for several inflammatory processes, but scarce knowledge exists about the intricate relationship between the FBR and the expression of soluble mediators. This review discusses the key processes during the initiation, progression, and resolution phase, with emphasis on the role of soluble mediators. Besides other sites of implantation, we focus on the subcutaneous implantation model.

Within the phenotypically and functionally heterogeneous group of circulating progenitor cells (CPC), a subclass of cells with vascular repair potential have been identified. These CPC are detected and isolated based on single or combined expression of CD34, CD 133 and VEGFR-2, and referred to as endothelial progenitor cells. Here we asked whether CPC subsets defined by single expression of these markers exhibit functional heterogeneity. As functional parameters, we chose the capacity of CPC to differentiate into endothelial cells. Moreover, we studied their role in remodeling by recruitment of inflammatory cells, an aspect that has been little explored. We established an in vivo model in which the intrinsic functional capacity of these human CPC subsets was studied. Human CD34(+) CPC, but not CD133(+) or VEGFR-2(+) CPC, seeded in Matrigel pellets and transplanted subcutaneously in a nude mouse host, contributed little to donor-derived neovascularization. However, host angiogenesis in the Matrigel implant, as demonstrated by the presence of capillaries containing erythrocytes and expressing mouse CD31, was strong in response to implantation of human CD34(+) CPC and significantly lower in response to the other two CPC subsets. Moreover, the CD34(+) CPC subset was significantly superior to CD133(+) CPC and VEGFR-2(+) CPC in the recruitment of host monocytes/macrophages. These three CPC populations were further dissected into seven discrete subsets, based on three-parameter flow cytometry analysis of combined expression patterns of CD34, CD133 and VEGFR-2. In conclusion, in our system, CD34(+) CPC contribute marginally to neovascularization by differentiation but are potent regulators of the host angiogenic and pro-inflammatory response, suggesting a possible role for these cells in the remodeling of vascular lesions. (c) 2006 Elsevier Inc. All rights reserved.

The human cytomegalovirus has found smart ways to exploit the chemokine network in order to subvert immune attack. Chemokines trigger the arrest and firm adhesion of inflammatory cells to the vascular wall. Scavenging of chemokines by viral decoy receptors, such as US28, might prevent arrest of leukocytes to the vascular wall and impair an antiviral immune response. We determined the effect of chemokine scavenging by endothelium-expressed signaling mute US28 (US28R129A) on static monocyte adhesion. Despite the chemokine scavenging capacity of US28R129A, expression of this construct by endothelial cells was insufficient to disrupt leukocyte adhesion to cytokine-activated monolayers. Our results suggest that the concentrations of chemokines that trigger firm leukocyte adhesion are too high to be efficiently scavenged by viral chemokine decoy receptors like US28. From the results of this experimental model a role for US28 in viral immune evasion by chemokine scavenging would appear therefore unlikely. (c) 2005 Elsevier B.V. All rights reserved.

The foreign body response is characterized by enhanced recruitment of inflammatory cells. As the directional movement of cells is controlled by chemokines, disruption of the chemokine network would be an attractive approach to improve biocompatibility of an implanted material. The sequestration of chemokines by cell surface-expressed glycosaminoglycans (GAGS) is vital for in vivo chemokine activity. The myxoma virus encodes a soluble protein, M-T7, that interacts with conserved GAG-binding domains of chemokines to block chemokine-mediated leukocyte recruitment. We hypothesized that M-T7 might also affect the function of other inflammation-associated proteins in addition to chemokines that bind to GAG. In our studies, we focussed on the modulation of the GAG-binding molecules macrophage chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor-164 (VEGF(164)) in the inflammatory reaction against subcutaneously implanted degradable cross-linked dermal sheep collagen discs in AO rats. Genetic delivery of M-T7 delays the influx of macrophages into the collagen discs. In addition, angiogenesis around the implanted material was reduced. The discs revealed reduced levels of rat MCP-1 and rat VEGF(164). This was not due to down regulation of transcription of the genes that encode MCP-1 and VEGF(164). Our in vivo observations suggest that, in addition to chemokines such as MCP-1, M-T7 neutralizes VEGF(164). (C) 2005 Elsevier Ltd. All rights reserved.

Extracellular nucleotides have been identified as important signaling molecules. These nucleotides act on the P2 family of receptors that respond by either forming an ion-channel or by activation of a signal transduction cascade, both of which enable a cellular response. Although a role for P2 receptors in inflammation has been implied, the local expression pattern and kinetics of these receptors at sites of inflammation are not known. Therefore, we have studied the expression of the P2 receptors expressed by inflammatory cells or by cells in the vasculature, with special attention to P2X(1)R, P2X(7)R, P2Y(1)R, and P2Y(2)R. As a suitable model for studying inflammatory reactions, we have employed the foreign body reaction (FBR), a sterile inflammatory reaction induced by implanting degradable cross-linked dermal sheep collagen disks subcutaneously in the rat. We show that, in the vasculature, the expression of P2X(7)R, P2Y(1)R, and P2Y(2)R increase until day 2. The expression of P2X(7)R and P2Y(1)R on macrophages and giant cells increased during the course of the inflammatory reaction which was studied for 21 days. The expression of the P2Y(2)R on macrophages and giant cells inside the foreign body increases with time, whereas the expression on macrophages in the surrounding tissue is maximal at day 5. The expression of P2X(1)R remains at a constant low level. The upregulation of P2X(7)R, P2Y(1)R, and P2Y(2)R over time suggests a regulatory function for these receptors in inflammation.

Background. Immune injury to airway epithelium is suggested to play a central role in the pathogenesis of obliterative bronchiolitis (OB) after clinical lung transplantation. In several studies, a rejection model of murine trachea transplants is used, resulting in obliterative airway disease (OAD) with similarities to human OB. To focus on the role of an immune response specifically against airway epithelium, we transplanted tracheas from transgenic mice expressing human epithelial glycoprotein (hEGP) on epithelial cells. We hypothesized that the immune response against the hEGP-2 antigen would result in OAD in the trachea transplants.Methods. Tracheas from hEGP-2 transgenic and control nontransgenic FVB/N mice were heterotopically transplanted into FVB/N mice and harvested at week 1, 3,6, and 9. Amti-hEGP-2 antibodies were determined in the recipient blood. The trachea grafts were analyzed for cellular infiltration, epithelial cell injury, and luminal obliteration.Results. Recipients of transgenic tracheal grafts gradually developed anti-hEGP-2 antibodies. In the transgenic grafts, the submucosa was infiltrated predominantly by CD4(+) T cells. Epithelial cells remained present but showed progressive abnormality. The tracheal lumen showed a mild degree of obliteration. All these changes were absent in nontransgenic FVB/N trachea transplants.Conclusion. The hEGP-2 antigen on the epithelial cells of transgenic trachea transplants induces specific humoral and cellular immune responses, leading to a mild form of OAD. It provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in human-lung transplantation.

Background. In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of kidney allograft recipients and are often associated with acute rejection and active infections with human cytomegalovirus. In the present study we hypothesized that cEC after kidney transplantation are of donor origin, thus reflecting transplantation-related damage to the allograft endothelium. Methods. Using hydraulic micromanipulation equipment, we isolated single cEC (n=153) from the peripheral blood of nine kidney allograft recipients at various time points after transplantation. We demonstrated the origin of these cells (donor or recipient) by typing their HILA-DRB alleles by single-cell, genomic, nested polymerase chain reaction. Results. The majority (71.8%) of cEC were of donor origin and could be detected up to 141 days after onset of acute rejection episodes. Although less frequent (28.2%), recipient-type cEC were detected in the same time course as donor-type cEC. Conclusion. We conclude that posttransplantational injury to the allograft endothelium is reflected by the presence of donor-derived cEC in the blood.

A wide range of strategies in cancer immunotherapy has been developed in the last decade, some of which are currently being used in clinical settings. The development of these immunotherapeutical strategies has been facilitated by the generation of relevant transgenic animal models. Since the different strategies in experimental immunotherapy of cancer each aim to activate different immune system components, a variety of transgenic animals have been generated either expressing tumor associated, HLA, oncogenic or immune effector cell molecule proteins. This review aims to discuss the existing transgenic mouse models generated to study and develop cancer immunotherapy strategies and the variable results obtained. The potential of the various transgenic animal models regarding the development of anti-cancer immunotherapeutical strategies is evaluated.

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)–associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P

The influence of various adjuvants on the development of influenza virus neutralizing antibodies and distribution of anti-influenza virus IgG isotypes after immunization of mice with influenza A (H3N2) subunit vaccine was investigated. Serum titres of influenza virus neutralizing antibodies and titres of influenza specific IgG isotypes were determined by a neutralization enzyme immunoassay (N-EIA) and a cell-associated antigen enzyme immunoassay (CA-EIA), respectively. Serum antibody titres as measured by the two tests correlated highly (r = 0.82; P < 0.001). N-EIA titres were enhanced by 38- and 34-fold, when L180.5/RaLPS and FCA, respectively, were administered with 1 microgram of vaccine. The adjuvants Q-VAC, L180.5 [W/O/W], L180.5 alone and Montanide ISA 740 were only moderately or not effective in enhancing the immune response to the 1 microgram dose of vaccine. The Q-VAC and L180.5/RaLPS adjuvants favoured IgG2a and IgG2b isotype responses to influenza compared to the other adjuvants. We suggest that N-EIA and CA-EIA may be valuable tools to monitor the effects of adjuvants on the neutralizing antibody and antibody isotype responses after influenza vaccination.

Application of autologous cells is considered for a broad range of regenerative therapies because it is not surrounded by the immunological and ethical issues of allo- or xenogenic cells. However, isolation, expansion, and application of autologous cells do suffer from variability in therapeutic efficacy due to donor to donor differences and due to prolonged culture. One important source of autologous cells is mesenchymal stromal cells (MSCs), which can differentiate toward endothelial-like cells, thus making them an ideal candidate as cell source for tissue vascularization. Here we screened MSCs from 20 donors for their endothelial differentiation capacity and correlated it with the gene expression profile of the whole genome in the undifferentiated state. Cells of all donors were able to form tubes on Matrigel and induced the expression of endothelial genes, although with quantitative differences. In addition, we analyzed the effect of prolonged in vitro expansion on the multipotency of human MSCs and found that endothelial differentiation is only mildly sensitive to expansion-induced loss of differentiation as compared to osteogenic and adipogenic differentiation. Our results show the robustness of the endothelial differentiation protocol and the gene expression data give insight in the differences in endothelial differentiation between donors.

Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction.To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge.Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-alpha and IL-1 beta. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-alpha stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-(DB)-B-0 inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-(DB)-B-0 inhibitor prior to the inflammatory challenge.Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNF alpha, IL-1 beta challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNF alpha exposure was shown to be controlled by NF-(DB)-B-0 signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue repair. Here, we investigate the combined influence of a pro-angiogenic microenvironment and specific extracellular matrix (ECM) components or tissue culture polystyrene (TCPS) on the dynamics of human macrophage polarization. We established that human angiogenically primed macrophages cultured on different ECM components exhibit an M2-like polarization. These M2-like macrophages polarized to M1 and M2 macrophages with classical (LPS and IFN gamma) stimuli and alternative (IL-4 and IL-13) stimuli respectively. Moreover, these M1 and M2 (primary) polarized macrophages rapidly underwent a secondary (re)polarization to M2 and M1 with conditioned media from M2 and M1 primary polarized macrophages respectively. In these initial priming and later (re)polarization processes the soluble factors had a dominant and orchestrating role, while the type of ECM (collagen I, fibronectin, versus tissue culture polystyrene) did not play a crucial role on the polarization of macrophages. (C) 2011 Elsevier GmbH. All rights reserved.

Endotheelcellen bekleden alle bloedvaten in het lichaam en zijn nodig voor de goede werking van het cardiovasculaire systeem. Het is geen verrassing dat vermindering of verlies van functie van endotheelcellen hand in hand gaat met het ontstaan en ontwikkelen van hart- en vaatziekten. De organen in het lichaam hebben verschillende functies, dus hun endotheelcellen worden blootgesteld aan verschillende omgevingen. Hoewel we ze allemaal ‘endotheliaal’ noemen, zijn ze in feite verschillend in de verschillende delen van het lichaam. Om altijd goed te werken, passen endotheelcellen verschijningsvorm en gedrag aan de locale behoefte. Locaal worden endotheelcellen geprikkeld door biochemische en mechanische omgevingssignalen, die in de cel worden doorgegeven aan het genetisch materiaal. De machinerie van de celkern verandert hierdoor de beschikbaarheid van het genetisch materiaal. We noemen dit epigenetische veranderingen. Epigenetische veranderingen bepalen de verschijningvorm en gedrag van cellen. We weten echter slechts weinig over hoe endotheelcellen de informatie uit hun omgeving doorgeven aan het epigenetische niveau. In dit proefschrift onderzochten we hoe verschillende factoren, in het bijzonder de bloedstroom evenals ontsteking, in de omgeving van endotheelcellen samenwerken om hun gedrag te veranderen. We hebben een epigenetische factor EZH2 geidentificeerd, die wordt gereguleerd door bovengenoemde signalen uit de omgeving van de endotheelcellen. We hebben gevonden dat EZH2 veel genen in endotheelcellen beinvloedt, vooral genen die celdelingen sturen. Onze bevindingen duiden erop dat lage EZH2 niveaus endotheelcellen in een beschermde rusttoestand houden, kenmerkend voor gezonde bloedvaten. In de toekomst zou EZH2 een nieuw doelwit voor de preventie van hart- en vaatziekten kunnen worden.

The antibody response to a previously defined B-cell epitope of Semliki Forest virus (SFV) was investigated in male BALB/c (H-2d) mice. The B-cell epitope, located at amino acid positions 240 to 255 of the E2 protein, was linked to an H-2d-restricted T-helper cell epitope of SFV located at positions 137 to 151 of the E2 protein. Colinearly synthesized peptides, of either T-B or B-T polarity, mixed with different adjuvants (the nonionic block copolymer L 180.5, a water-oil-water [W/O/W] emulsion of L 180.5, Montanide, and Q VAC) were used for immunization. Generally, after one booster immunization, high serum antibody titers were measured against either peptide. With Q VAC and W/O/W L 180.5 as adjuvants, the titers of SFV-reactive (nonneutralizing) antibodies were consistently much higher after immunization with the T-B peptide than with the B-T peptide, which was reflected in a higher vaccine efficacy. With these two adjuvants, the survival ratio in T-B peptide-immunized mice was 82%, compared with 8% in B-T peptide-immunized mice. Intermediate results were obtained with the adjuvant Montanide. L 180.5 alone was ineffective in this study. All immunoglobulin G (IgG) isotypes were induced with either adjuvant, but Q VAC was clearly the most effective in inducing IgG2a and IgG2b isotypes with the T-B peptide as the antigen. Subsequently, monoclonal antibodies (MAbs) of IgM, IgG1, IgG2a, IgG2b, and IgG3 subclasses were prepared against the B-cell epitope. These nonneutralizing but SFV-reactive MAbs protected 40 to 80% of mice against a lethal challenge with SFV. Control mice all died. The availability of those antipeptide MAbs allowed competition binding assays with a previously characterized panel of E2-specific MAbs. Binding of enzyme-labeled antipeptide MAbs was very effectively inhibited by two strongly SFV-neutralizing mutually competitive MAbs, suggesting that the linear B-cell epitope (amino acids 240 to 255) is associated with a major neutralization site of SFV.

Subcutaneously implanted disks of hexamethylenediisocyanate or glutaraldehyde cross-linked sheep collagen (referred to as HDSC and GDSC, respectively) in mice show large differences in degradation rate. Although comparable numbers of macrophages are seen in HDSC and GDSC, phagocytosis of collagen by macrophages occurred only in GDSC. The molecular mechanisms involved in the phagocytosis of collagen by macrophages are essentially unknown. Immunofluorescence and RT-PCR showed that Endo180 was expressed in GDSC only. TissueFaxs showed that Endo180 co-localized with MT1-MMP on F4/80 positive cells, which is likely responsible for the phagocytosis in GDSC. RT-PCR further showed that Endo180 expression correlated with high levels of IFN-gamma mRNA. In vitro, IFN-gamma induced the expression Endo180 and MT1-MMP in murine macrophages cultured on collagen type I (although too high levels of IFN-gamma dampened the expression of Endo180 and MT1-MMP). Moreover, the expression of Endo180 and MT1-MMP induced by IFN-gamma can be inhibited through IL-10. The differences in microenvironment between GDSC and HDSC (high IFN-gamma and low IL-10 levels in GDSC, low IFN- gamma and high IL-10 levels in HDSC) provide an explanation why phagocytosis of collagen by macrophages is only seen in GDSC. In summary, we show for the first time that the IFN-gamma dependent co-expression of Endo180 and MT1-MMP on macrophages coincides with collagen phagocytosis, thus providing evidence that the mechanism of collagen phagocytosis operating in the foreign body reaction by macrophages is comparable with the mechanism of intracellular collagen degradation by fibroblasts seen under physiological conditions.

See related article, pages 1058–1065 Cardiovascular complications are a major culprit in the pathogenesis of obesity and diabetes. The increased prevalence of cardiovascular complications in diabetes is attributed to the disturbed balance between increased endothelial injury and hampered endothelial repair processes.1 Retinopathy, a blinding disease, is a major morbidity in diabetic patients. Diabetic retinopathy is initiated by hyperglycemia-induced endothelial cell injury, retinal vessel loss, and the adaptation of an inflammatory phenotype by the endothelium. Secretion of proangiogenic factors and proinflammatory cytokines from the ischemic retina lead to an exacerbated compensatory neovascularization, resulting in abnormal retinal neovascularization.2 Hence, the primary treatment of retinopathy aims to preserve vision through the inhibition of excessive vascularization and reduction of vascular damage. Interestingly, endothelial dysfunction, hampered endothelial repair, and pathological neovascularization during retinopathy reflect similar molecular alterations partially related to a state of mild chronic inflammation, ie, overproduction of inflammatory cytokines and reactive oxygen species, increased leukocyte extravasations, and vascular leakage.3 The traditional role attributed to fat tissue is energy storage; however, a change in perspective has risen since the discovery of the adipokines. Advances in the biology of the adipose tissue has revealed that it is not simply an energy storing organ but also secretes a variety of molecules, termed adipokines, that affect processes beyond metabolic regulation.4 Bioactive substances secreted by adipocytes, the adipokines, include proinflammatory cytokines, such as leptin, angiotensinogen, plasminogen activator inhibitor 1, interleukin (IL)-6, and tumor necrosis factor (TNF)α, and could contribute to the complications of obesity and diabetes through the amplification of inflammatory responses.5 In contrast, the adipokine adiponectin (APN) is unique in its antiinflammatory actions, and plasma levels of APN are paradoxically decreased in obese and diabetic subjects.6 In this issue of Circulation Research …

The foreign body reaction (FBR) is of great importance for the function and turnover of biomaterial scaffolds. The development of biological tools that modulate the FBR will augment scaffold functionality and benefit regenerative medicine. The human cytomegalovirus encodes a functional homolog of the potent anti-inflammatory human cytokine interleukin-10 (cmvIL-10). We hypothesized that cmvIL-10 downmodulates the FBR, impairing degradation of biomaterial. We studied the effect of cmvIL-10 on the FBR to subcutaneously implanted hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) discs in rats. CmvIL-10 impaired macrophage influx, vascularization and ingrowth into the discs up to 21 days. It also impaired the formation of giant cells and the degradation of HDSC. At day 10, deposited fibrin fibers were still present in cmvIL-10 discs. Impaired collagenase activity coincided with the impaired HDSC degradation. These results indicate that cmvIL-10 downmodulates the FBR, impairing the progression of the FBR. This study demonstrates the feasibility of interleukin-10 as a biomolecular tool in biomaterials for regenerative medicine. (C) 2008 Elsevier Ltd. All rights reserved.

Two monoclonal anti-idiotypic antibodies (ab2 MAbs), designated 1.13A112 (immunoglobulin G type 2a [IgG2a]) and 1.13A321 (IgG1), were prepared against Semliki Forest virus (SFV)-neutralizing ab1 MAb UM 1.13. They were identified in hybridoma supernatant fluid by their capacity to block UM 1.13-mediated neutralization of SFV. Although the neutralization-blocking capacities of the ab2 MAbs did not differ, only 1.13A321 evoked SFV-neutralizing ab3 antibodies upon intracutaneous and subcutaneous immunization of BALB/c mice with 1.13A321 chemically cross-linked to keyhole limpet hemocyanin and combined with the adjuvant Quil A. SFV-neutralizing ab3 antibodies appeared in serum within 10 days after primary immunization, and neutralizing antibody titers could be as high as 1/1,000 at day 35. All mice who had developed SFV-neutralizing antibodies upon anti-idiotypic immunization survived an otherwise lethal challenge with virulent SFV. However, induction of SFV-neutralizing ab3 antibodies by ab2 MAb 1.13A321 proved to be genetically restricted to BALB/c mice; even haplotype-identical (H-2d) DBA/2 mice did not respond, and consequently those animals died after infection with virulent SFV.

Semliki Forest virus-(SEV) neutralizing monoclonal antibodies (MoAbs), produced after fusion of spleen cells from BALB/c mice and myeloma cell line P3-X63-AG8. 653 or SP2/0, were used for anti-idiotypic immunization of female BALB/c mice. Two intracutaneous immunizations (2 x 40 micrograms per animal), 3 weeks apart, with keyhole limpet haemocyanin-conjugated MoAbs mixed with the saponin Quil A were sufficient to induce high levels of anti-idiotypic antibodies in the circulation of these mice with the capacity to block specifically in vitro MoAb-mediated virus neutralization. Anti-idiotypic antibodies against SFV-neutralizing MoAbs, either passively transferred or actively acquired by immunization, are also able to abrogate (specifically) passive immunity, mediated by critical protective doses of MoAb, in mice against infection with a lethal strain of SFV. Furthermore we confirmed by intervention with anti-idiotypic serum in vivo that an SFV-neutralizing MoAb exerts its greatest protective effect during the first 2 days of infection.

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites.Neovascularization was studied in C57B1/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels.We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.

Background. Human cytomegalovirus (CMV) uses different strategies to escape from human host defense reactions. Previously we have observed that infection of endothelial cells with CMV in vitro leads to enhanced activity of endothelial ectonucleotidases. These ectoenzymes are responsible for hydrolysis of extracellular adenine nucleotides, resulting in the formation of adenosine. Infection with CMV in vivo therefore may result in local increase of adenosine production, providing an anti- inflammatory and antiaggregatory microenvironment, which may facilitate entry of the virus into the target cell. Methods. The present study focuses on the expression of P2 type purinergic receptors on endothelial cells after infection with CMV. Human endothelial cells were infected with CMV and compared with either uninfected cells or endothelial cells infected with other herpesviruses (herpes simplexvirus [HSV] I or 2) for the expression of P2 receptors such as P2Y1, P2Y2, or P2X7. For comparison, cells stimulated with nonspecific agents were also studied. Results. A strong upregulation of the P2 receptors tested was shown, exclusively in CMV-infected cells. Stimulation with either HSV-1 or HSV2, nonspecific stimulants, or various cytokines did not affect the expression of these P2 receptors significantly. Conclusion. Infection of endothelium with CMV causes significant upregulation of the P2 receptors studied. As these receptors may potentially be able to concentrate nucleotides along the ectonucleotidases of the endothelial cell membrane, rapid local hydrolysis of adenosine triphosphate and adenosine diphosphate may be facilitated by enhanced P2 receptor expression. Such a CMV induced mechanism might enable the virus to escape from an important host defense response, such as local microthrombus formation.

Background. In human renal allografts, recipient-derived cells engrafted in various kidney substructures, have been detected in the long term after transplantation. Here we investigated tubular engraftment and myofibroblast differentiation of recipient-derived cells at short term after experimental kidney transplantation, during a previously described window of regeneration and possible onset of renal interstitial fibrosis. Methods. Fisher (F344, syngeneic) and Dark Agouti (DA, allogeneic) kidneys were transplanted into F344-hPAP transgenic recipient rats, which allowed tracing of recipient-derived cells in nontransgenic donor kidneys. We evaluated tubular engraftment and myofibroblast differentiation of recipient-derived cells on day 14 after kidney transplantation. Results. Kidney transplantation resulted in tubular engraftment of recipient- derived cells. After allogeneic kidney transplantation, 9.7% of tubular cross-sections contained at least one recipient- derived cell, which represented a significant increase in comparison to syngeneic transplantation (4.0%, P

Systemic lupus erythematosus (SLE) is associated with premature and accelerated atherosclerosis. Circulating progenitor cells (CPCs) are circulating bone-marrow derived cells that play an important role in the repair of vascular damage that underlies the development of atherosclerosis. The objective of this study was to determine the number and functionality of CPCs in patients with SLE. The study included 44 female SLE patients in an inactive stage of disease and 35 age-matched female controls. CPC numbers in the circulation were determined by FACS with monoclonals against CD14, CD34 and CD133. Peripheral blood-derived mononuclear cell (PBMNC) fractions were cultured in angiogenic medium. The endothelial-like phenotype was confirmed and the colony forming unit (CFU) capacity, migratory capacity and the potential to form clusters on Matrigel were determined. Expression of apoptosis inhibiting caspase 8L was analyzed in PBMNCs and CPCs by gene transcript and protein expression assays. The number of CD34-CD133 double-positive cells (P

Implanted scaffold materials induce an inflammatory reaction known as the 'foreign body reaction' (FBR). We hypothesized that the observed difference in FBR between rats and mice correlate with different expression dynamics of cytokines and chemokines, which are key orchestrators of the FBR. After implantation of hexamethylene diisocyanate cross-linked dermal sheep collagen, the overall gene expression pattern of IL-1, IL-6, IL-10, TNF alpha, CXCL1/KC, CXCL2/MIP2 and CCL2/MCP1 was roughly similar for the two species. During the onset of the FBR these genes were maximally expressed in rats and mice, after which the expression decreased to basal levels. The expression of CCL3/MIP1 alpha had a similar course, yet it increased after the progression phase of the FBR in both species. The expression of cytokines and chemokines in sham-operated animals was low throughout, showing that the implanted material by itself exerted the changes in gene expression of the invading cells. During the progression, genes encoding the PMN attractants CXCL1/KC and CXCL2/MIP2 were more highly expressed in mice than in rats, which would explain the prolonged presence of PMNs in mice during the FBR. Additionally, the strong induction of IFN gamma in rats coincided with a higher phagocytotic activity by macrophages. Throughout the FBR, the expression of TGF beta was constitutive and high in both species, but increased in mice during the progression phase. This could explain the extensive stroma formation during the murine FBR. Unexpectedly, the stronger expression of TNFa and CCL3/MIP1 alpha in mice, did not result in high macrophage attraction or phagocytosis of the implanted collagen disks. Copyright (c) 2007 John Wiley & Sons, Ltd.

Macrophages are known to play a key role during inflammation in rheumatoid arthritis (RA). Inflammatory macrophages have increased expression of CD64, the high-affinity receptor for IgG. Targeting this receptor through a CD64-directed immunotoxin, composed of an Ab against CD64 and Ricin A, results in effective killing of inflammatory macrophages. In this study, we show elevated levels of CD64 on synovial macrophages in both synovial lining and synovial fluid in RA patients. The CD64-directed immunotoxin efficiently eliminates activated synovial macrophages in vitro, while leaving quiescent, low CD64-expressing macrophages unaffected. To examine whether killing of CD64 macrophages results in therapeutic effects in vivo, we established an adjuvant arthritis (AA) model in newly generated human CD64 (hCD64) transgenic rats. We demonstrate that hCD64 regulation in this transgenic rat model is similar as in humans. After AA induction, treatment with CD64-directed immunotoxin results in significant inhibition of disease activity. There is a direct correlation between immunotoxin treatment and decreased macrophage numbers, followed by diminished inflammation and bone erosion in paws of these hCD64 transgenic rats. These data support synovial macrophages to play a crucial role in joint inflammation in AA in rats and in human RA. Selective elimination of inflammatory macrophages through a CD64-directed immunotoxin may provide a novel approach for treatment of RA.