Bernd Denecke

Delivery of MicroRNA-126 by Apoptotic Bodies Induces CXCL12-Dependent Vascular Protection

Apoptotic endothelial cells release microRNA-containing microvesicles to modulate the responses of neighboring cells and reduce atherosclerosis in mice. Sounding the Alarm In addition to its importance in development and homeostasis, apoptotic cell death is implicated in a number of diseases, including atherosclerosis. Apoptotic endothelial cells at atherosclerotic plaques release microvesicles known as apoptotic bodies into the circulation, and their abundance correlates with negative indicators of disease. Zernecke et al. showed that apoptotic bodies from endothelial cells contained microRNA-126 (miR-126). Neighboring vascular cells took up the microvesicles, which allowed miR-126 to reduce the abundance of an inhibitor of the signaling of the chemokine receptor CXCR4, resulting in the increased production of CXCL12, the ligand for CXCR4. CXCL12 mediated the recruitment to atherosclerotic plaques of progenitor cells from the bone marrow, which limited plaque size. Apoptotic bodies isolated from human patients with atherosclerosis reduced the size of plaques in different mouse models of atherosclerosis. Thus, dying endothelial cells send alarm signals in the form of packaged microRNA to neighboring cells to trigger a healing response that reduces atherosclerosis. Apoptosis is a pivotal process in embryogenesis and postnatal cell homeostasis and involves the shedding of membranous microvesicles termed apoptotic bodies. In response to tissue damage, the CXC chemokine CXCL12 and its receptor CXCR4 counteract apoptosis and recruit progenitor cells. Here, we show that endothelial cell–derived apoptotic bodies are generated during atherosclerosis and convey paracrine alarm signals to recipient vascular cells that trigger the production of CXCL12. CXCL12 production was mediated by microRNA-126 (miR-126), which was enriched in apoptotic bodies and repressed the function of regulator of G protein (heterotrimeric guanosine triphosphate–binding protein) signaling 16, an inhibitor of G protein–coupled receptor (GPCR) signaling. This enabled CXCR4, a GPCR, to trigger an autoregulatory feedback loop that increased the production of CXCL12. Administration of apoptotic bodies or miR-126 limited atherosclerosis, promoted the incorporation of Sca-1+ progenitor cells, and conferred features of plaque stability on different mouse models of atherosclerosis. This study highlights functions of microRNAs in health and disease that may extend to the recruitment of progenitor cells during other forms of tissue repair or homeostasis.

Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A.

Fetuins are serum proteins with diverse functions including the regulation of osteogenesis and inhibition of unwanted mineralization. Besides the alpha2-Heremans and Schmid glycoprotein/fetuin-A, the recently identified fetuin-B is a second member of the fetuin family [Olivier, Soury, Risler, Smih, Schneider, Lochner, Jouzeau, Fey and Salier (1999) Genomics 57, 352-364; Olivier, Soury, Ruminy, Husson, Parmentier, Daveau and Salier (2000) Biochem. J. 350, 589-597], which belongs to the cystatin superfamily. We compared the expressions of fetuin-B and fetuin-A at the RNA level and established that both genes are most highly expressed in liver tissue. Like fetuin-A, fetuin-B mRNA is also highly expressed in tongue and placenta tissues. We demonstrated for the first time that fetuin-B is also expressed at the protein level in sera and several organs of mouse, rat and human. We isolated contiguous genomic clones containing both fetuin-B and fetuin-A genes, indicating that these genes are closely linked at the genome level. The close proximity of both these genes may explain our observation that fetuin-B expression was decreased in fetuin-A-deficient mice. Unlike fetuin-A, the amount of fetuin-B protein in human serum varied with gender and was higher in females than in males. Functional analysis revealed that fetuin-B, similarly to fetuin-A, is an inhibitor of basic calcium phosphate precipitation, albeit less active when compared with fetuin-A. Therefore fetuin-B may have a function that is partly overlapping, if not identical, with the function of fetuin-A.

Id1 is a critical mediator in TGF‐β–induced transdifferentiation of rat hepatic stellate cells

Transforming growth factor (TGF)‐β is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF‐β antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)‐induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down‐regulated collagen and α‐smooth muscle actin (α‐SMA) expression. The aim of this study was to further characterize the molecular details of TGF‐β pathways that control the transdifferentiation process. In an attempt to elucidate TGF‐β target genes responsible for fibrogenesis, an analysis of Smad7‐dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7‐dependent inhibition of transdifferentiation. Moreover, knock‐down of Id1 in HSCs interfered with α‐SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF‐β1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co‐induced during fibrogenesis. In conclusion, Id1 is identified as TGF‐β/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. (HEPATOLOGY 2006;43:1032–1041.)

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Myelin debris regulates inflammatory responses in an experimental demyelination animal model and multiple sclerosis lesions

In multiple sclerosis (MS), gray matter pathology is characterized by less pronounced inflammation when compared with white matter lesions. Although regional differences in the cytoarchitecture may account for these differences, the amount of myelin debris in the cortex during a demyelinating event might also be contributory. To analyze the association between myelin debris levels and inflammatory responses, cortical areas with distinct and sparse myelination were analyzed for micro‐ and astrogliosis before and after cuprizone‐induced demyelination in mice. In postmortem tissue of MS patients, leucocortical lesions were assessed for the type and level of inflammation in the cortical and white matter regions of the lesion. Furthermore, mice were injected intracerebrally with myelin‐enriched debris, and the inflammatory response analyzed in white and grey matter areas. Our studies show that the magnitude of myelin loss positively correlates with microgliosis in the cuprizone model. In MS, the number of MHC class II expressing cells is higher in the white compared with the grey matter part of leucocortical lesions. Finally, direct application of myelin debris into the corpus callosum or cortex of mice induces profound and comparable inflammation in both regions. Our data suggest that myelin debris is an important variable in the inflammatory response during demyelinating events. Whether myelin‐driven inflammation affects neuronal integrity remains to be clarified.

microRNA expression signatures and parallels between monocyte subsets and atherosclerotic plaque in humans

Small non-coding microRNAs (miRNAs) have emerged to play critical roles in cardiovascular biology. Monocytes critically drive atherosclerotic lesion formation, and can be subdivided into a classical and non-classical subset. Here we scrutinised the miRNA signature of human classical and non-classical monocytes, and compared miRNA expression profiles of atherosclerotic plaques from human carotid arteries and healthy arteries. We identified miRNAs to be differentially regulated with a two-fold or higher difference between classical and non-classical monocyte subsets. Moreover, comparing miRNA expression in atherosclerotic plaques compared to healthy arteries, we observed several miRNAs to be aberrantly expressed, with the majority of miRNAs displaying a two-fold or higher increase in plaques and only few miRNAs being decreased. To elucidate similarities in miRNA signatures between monocyte subsets and atherosclerotic plaque, expression of miRNAs highly abundant in monocytes and plaque tissues were compared. Several miRNAs were found in atherosclerotic plaques but not in healthy vessels or either monocyte subset. However, we could identify miRNAs co-expressed in plaque tissue and classical monocytes (miR-99b, miR-152), or non-classical monocytes (miR-422a), or in both monocytes subsets. We thus unravelled candidate miRNAs, which may facilitate our understanding of monocyte recruitment and fate during atherosclerosis, and may serve as therapeutic targets for treating inflammatory vascular diseases.

Epigenetic Rejuvenation of Mesenchymal Stromal Cells Derived from Induced Pluripotent Stem Cells

Summary Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns—particularly of DNAm patterns associated with tissue type and aging.

Enhanced blood coagulation and fibrinolysis in mice lacking histidine‐rich glycoprotein (HRG)

Summary.  Histidine‐rich glycoprotein (HRG) is a serum protein belonging to the cystatin superfamily. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain because of a lack of rigorous testing in an animal model. We generated mice lacking the translation start point of exon 1 of the Hrg gene, effectively resulting in a null mutation (Hrg–/–). The mice were viable and fertile but had no HRG in their blood. Antithrombin activity in the plasma of Hrg–/– mice was higher than in the plasma of heterozygous Hrg+/– or wild‐type Hrg+/+ mice. The prothrombin time was shorter in Hrg–/– mice than in Hrg+/– and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg–/– mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg–/– mice was higher than in Hrg+/+ mice. These findings suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivo.

TTC staining of damaged brain areas after MCA occlusion in the rat does not constrict quantitative gene and protein analyses.

In models of ischemic stroke, TTC (2,3,5-triphenyltetrazolium chloride) staining is commonly applied for the fast and reliable visualization of hypoxic brain tissue and for defining the size of cerebral infarction and penumbra. Deciphering molecular processes of pathogenesis within the penumbra is of particular interest for the development of therapeutic strategies. The aim of this study was to assess whether TTC-stained tissues can easily and in a reliable quantitative manner be processed for further molecular and biochemical analyses. We applied phenol-based RNA isolation, protein lysis by conventional RIPA buffer, and combined RNA/protein isolation with NucleoSpinRNA/Protein-Kit. Gene and protein expression analyses were performed by RT-rtPCR and Western-blotting. Middle cerebral arteria occlusion (MCAO) in rats was performed following a standardized experimental procedure. After MCAO, TTC staining revealed massive cell death in cortical and sub-cortical areas. TTC processing did not affect the quality of tissue RNA and protein. The expression of housekeeping and regulatory genes and proteins revealed no difference between control and TTC-stained groups. The expression of known stroke-regulated genes such as TNFalpha and IL1beta revealed similar induction profiles after TTC staining as described in the literature. TTC staining allows the precise delineation of lesioned and primarily non-lesioned brain areas for subsequent dissection of selected tissue pieces for molecular analysis. Our study demonstrates that TTC-stained tissues in stroke animal models can be used for quantitative gene and protein expression analyses without constriction. Pathomechanisms of ongoing tissue damage within the penumbra region can now be investigated in detail.

BLBP-expression in astrocytes during experimental demyelination and in human multiple sclerosis lesions

Several lines of evidence indicate that remyelination represents one of the most effective mechanisms to achieve axonal protection. For reasons that are not yet understood, this process is often incomplete or fails in multiple sclerosis (MS). Activated astrocytes appear to be able to boost or inhibit endogenous repair processes. A better understanding of remyelination in MS and possible reasons for its failure is needed. Using the well-established toxic demyelination cuprizone model, we created lesions with either robust or impaired endogenous remyelination capacity. Lesions were analyzed for mRNA expression levels by Affymetrix GeneChip® arrays. One finding was the predominance of immune and stress response factors in the group of genes which were classified as remyelination-supporting factors. We further demonstrate that lesions with impaired remyelination capacity show weak expression of the radial-glia cell marker brain lipid binding protein (BLBP, also called B-FABP or FABP7). The expression of BLBP in activated astrocytes correlates with the presence of oligodendrocyte progenitor cells. BLBP-expressing astrocytes are also detected in experimental autoimmune encephalomyelitis during the remission phase. Furthermore, highest numbers of BLBP-expressing astrocytes were evident in lesions of early MS, whereas significantly less are present at the rim of (chronic)-active lesions from patients with long disease duration. Transfection experiments show that BLBP regulates growth factor expression in U87 astrocytoma cells. In conclusion, we provide evidence that expression of BLBP in activated astrocytes negatively correlates with disease duration and in parallel with remyelination failure.