Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

Ligand Conjugated Multimeric siRNAs Enable Enhanced Uptake and Multiplexed Gene Silencing.

Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.

Age-dependent and differential effects of Smad7ΔEx1 on neural progenitor cell proliferation and on neurogenesis.

We recently reported that young (3 to 4months old) mice lacking Exon 1 of the Smad7 gene (S7ΔEx1 mice) show enhanced proliferation of neural stem and progenitor cells (NPCs) in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ) of the lateral ventricles. It remained unclear, however, whether this phenotype would persist along aging, the latter typically being associated with a profound decrease in neurogenesis. Analysis of NPCs' proliferation based on the cell cycle marker PCNA in 12month-old S7ΔEx1 mice revealed a reversal of the phenotype. Hence, in contrast to their younger counterparts, 12month-old S7ΔEx1 mice had a reduced number of proliferating cells, compared to wildtype (WT) mice. At the same time, the survival of newly generated cells was enhanced in the aged transgenic animals. 12month-old S7ΔEx1 mice further displayed a reduced level of neurogenesis based on the numbers of cells expressing doublecortin (DCX), a marker for newborn neurons. The reduced neurogenesis in aged S7ΔEx1 mice was not due to a stem cell depletion, which might have occurred as a consequence of hyperproliferation in the young mice, since the number of Nestin and Sox2 positive cells was similar in WT and S7ΔEx1 mice. Instead, Nestin positive cells in the DG as well as primary neurosphere cultures derived from 12month-old S7ΔEx1 mice had a reduced capability to proliferate. However, after passaging, when released from their age- and niche-associated proliferative block, neurospheres from aged S7ΔEx1 mice regained the hyperproliferative property. Further, pSmad2 antibody staining intensity was elevated in the DG and SVZ of 12-month old transgenic compared to WT mice, indicating increased intracellular TGF-beta signaling in the aged S7ΔEx1 mice. In summary, this points toward differential effects of S7ΔEx1 on neurogenesis: (i) a hyperproliferation in young animals caused by a cell autonomous mechanism, and (ii) a TGF-beta dependent modulation of neurogenesis in aged S7ΔEx1 animals that abrogates the cell-intrinsic hyperproliferative properties and results in reduced proliferation, increased stem cell quiescence, and enhanced survival of newly generated cells.

Delivery of siRNA to the mouse lung via a functionalized lipopolyamine.

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.

Smad7 regulates the adult neural stem/progenitor cell pool in a transforming growth factor beta- and bone morphogenetic protein-independent manner.

Members of the transforming growth factor beta (TGF-beta) family of proteins modulate the proliferation, differentiation, and survival of many different cell types. Neural stem and progenitor cells (NPCs) in the adult brain are inhibited in their proliferation by TGF-beta and by bone morphogenetic proteins (BMPs). Here, we investigated neurogenesis in a hypomorphic mouse model for the TGF-beta and BMP inhibitor Smad7, with the hypothesis that NPC proliferation might be reduced due to increased TGF-beta and BMP signaling. Unexpectedly, we found enhanced NPC proliferation as well as an increased number of label-retaining cells in vivo. The enhanced proliferation potential of mutant cells was retained in vitro in neurosphere cultures. We observed a higher sphere-forming capacity as well as faster growth and cell cycle progression. Use of specific inhibitors revealed that these effects were independent of TGF-beta and BMP signaling. The enhanced proliferation might be at least partially mediated by elevated signaling via epidermal growth factor (EGF) receptor, as mutant cells showed higher expression and activation levels of the EGF receptor. Conversely, an EGF receptor inhibitor reduced the proliferation of these cells. Our data indicate that endogenous Smad7 regulates neural stem/progenitor cell proliferation in a TGF-beta- and BMP-independent manner.

A nuclear magnetic resonance biomarker for neural progenitor cells: is it all neurogenesis?

In vivo visualization of endogenous neural progenitor cells (NPCs) is crucial to advance stem cell research and will be essential to ensure the safety and efficacy of neurogenesis-based therapies. Magnetic resonance spectroscopic imaging (i.e., spatially resolved spectroscopy in vivo) is a highly promising technique by which to investigate endogenous neurogenesis noninvasively. A distinct feature in nuclear magnetic resonance spectra (i.e., a lipid signal at 1.28 ppm) was recently attributed specifically to NPCs in vitro and to neurogenic regions in vivo. Here, we demonstrate that although this 1.28-ppm biomarker is present in NPC cultures, it is not specific for the latter. The 1.28-ppm marker was also evident in mesenchymal stem cells and in non-stem cell lines. Moreover, it was absent in freshly isolated NPCs but appeared under conditions favoring growth arrest or apoptosis; it is initiated by induction of apoptosis and correlates with the appearance of mobile lipid droplets. Thus, although the 1.28-ppm signal cannot be considered as a specific biomarker for NPCs, it might still serve as a sensor for processes that are tightly associated with neurogenesis and NPCs in vivo, such as apoptosis or stem cell quiescence. However, this requires further experimental evidence. The present work clearly urges the identification of additional biomarkers for NPCs and for neurogenesis.

A gain-of-function mutation in the PDGFR-beta alters the kinetics of injury response in liver and skin.

Platelet-derived growth factor (PDGF) isoforms stimulate cell proliferation, migration and survival. We recently generated mice carrying a gain-of-function mutation within the activation loop of PDGF beta-receptor (PDGFR-beta D849N). Embryonic fibroblasts derived from these mice show elevated basal phosphorylation and altered kinetics for ligand-induced activation of PDGFR-beta, as well as enhanced proliferation and migration. To investigate the effect of this mutation in vivo, we used carbon tetrachloride-induced liver injury as a model system. We observed a higher basal activation of mutant PDGFR-beta in unchallenged livers; however, the difference in activation upon carbon tetrachloride stimulation was lower than expected, an effect that might be explained by a delayed response of the mutated receptor toward reactive oxygen species. Mutant mice showed enhanced proliferation of nonparenchymal liver cells and activation of hepatic stellate cells, leading to a small increase in early fibrosis formation. Another mouse strain lacking the binding site for phosphatidylinositol-3' kinase in PDGFR-beta showed the reverse phenotype. These results suggest an important role for PDGFR-beta signaling in the early injury-response. We confirmed this hypothesis with a second injury model, cutaneous wound healing, where we observed earlier proliferation and formation of granulation tissue in D849N-mutant mice.

Keratinocyte growth factor protects epidermis and hair follicles from cell death induced by UV irradiation, chemotherapeutic or cytotoxic agents.

Owing to its potent cytoprotective properties for epithelial cells, keratinocyte growth factor (KGF) is successfully used for the treatment of chemotherapy- and radiotherapy-induced oral mucositis in cancer patients. It is therefore of major interest to determine possible clinical applications of KGF in other organs and in different stress situations and to unravel common and organ-specific mechanisms of KGF action. Here we show that KGF protects human keratinocytes from the toxicity of xenobiotics with electrophilic and oxidative properties and reduces the cell death induced by UV irradiation. In contrast to other cell types, cytoprotection of keratinocytes by KGF is not a direct anti-apoptotic effect but requires de novo protein synthesis. The in vitro findings are clinically relevant because KGF protected keratinocytes in organ-cultured human scalp hair follicles from the toxicity of the xenobiotic menadione. Moreover, injection of KGF into murine back skin markedly reduced cell death in the epidermis after UVB irradiation. This activity is dependent on FGF receptor signaling because it was abrogated in transgenic mice expressing a dominant-negative FGF receptor mutant in keratinocytes. Taken together, our results encourage the use of KGF for skin protection from chemical and physical insults.

ADAMTS1 proteinase is up-regulated in wounded skin and regulates migration of fibroblasts and endothelial cells.

The metalloproteinase ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) is induced under inflammatory conditions, and it is also a potent inhibitor of angiogenesis. Due to these properties, we speculated about the role of ADAMTS1 in cutaneous wound repair. Here we have shown up-regulation of ADAMTS1 expression in wounds of normal and particularly of healing-impaired genetically diabetic mice. Immunofluorescence staining identified macrophages as the source of ADAMTS1 in early wounds, whereas keratinocytes and fibroblasts produce this protein at later stages of wound healing. The distribution of ADAMTS1 in the normal and wounded epidermis, its regulation in cultured keratinocytes, as well as the skin phenotype of ADAMTS1 knock-out mice suggests a role of this metalloproteinase in keratinocyte differentiation. Furthermore, we provide evidence for a novel dual function of ADAMTS1 in fibroblast migration; although low concentrations of this protein stimulate fibroblast migration via its proteolytic activity, high concentrations inhibit this process because of binding to fibroblast growth factor-2 and subsequent inhibition of its promotogenic activity. Similar effects were also observed with endothelial cells. Taken together, our results suggest a role of ADAMTS1 in keratinocyte differentiation and migration of fibroblasts and endothelial cells in healing skin wounds.

Activities of the matrix metalloproteinase stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organization in wounded skin.

The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of beta1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing.

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin. Here we show that FGFR1-IIIb is expressed in normal and wounded mouse skin. Reduced expression of this type of receptor was found in wounds of healing-impaired genetically diabetic mice, suggesting that downregulation of FGFR1-IIIb is associated with wound healing defects. To address this possibility, we deleted the IIIb exon of FGFR1 in mice. The lack of FGFR-IIIb did not alter the expression of either FGFR1-IIIc, other FGF receptor genes or of FGFR1-IIIb ligands in normal and wounded skin. Histological analysis of the skin of FGFR1-IIIb knockout animals did not reveal any obvious abnormalities. Furthermore, full-thickness excisional skin wounds in these mice healed normally and no defects could be observed at the macroscopic or histological level. Finally, several genes that encode key players in wound repair were normally expressed in these animals. These data demonstrate that FGFR1-IIIb is dispensable for skin development and wound repair.

Keratinocyte growth factor: effects on keratinocytes and mechanisms of action.

Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, and it can protect these cells from various insults. Due to these properties, it is of particular importance for the repair of injured epithelial tissues, and it is currently therapeutically explored for the treatment of radiation- and chemotherapy-induced mucosal epithelial damage in cancer patients. In this review we summarize the current knowledge on the role of KGF in tissue repair and cytoprotection, and we report on its mechanisms of action in keratinocytes.

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2.

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.