Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of ...small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and ...wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.
Myocarditis can lead to autoimmune disease, dilated cardiomyopathy, and heart failure, which is modeled in the mouse by cardiac myosin immunization (experimental autoimmune myocarditis EAM). Signal ...transducer and activator of transcription 3 (STAT3) systemic inhibition exerts both preventive and therapeutic effects in EAM, and STAT3 constitutive activation elicits immune-mediated myocarditis dependent on complement C3 and correlating with activation of the STAT3-interleukin 6 (IL-6) axis in the liver. Thus, liver-specific STAT3 inhibition may represent a therapeutic option, allowing to bypass the heart toxicity, predicted by systemic STAT3 inhibition. We therefore decided to explore the effectiveness of silencing liver Stat3 and C3 in preventing EAM onset and/or the recovery of cardiac functions. We first show that complement C3 and C5 genetic depletion significantly prevents the onset of spontaneous myocarditis, supporting the complement cascade as a viable target. In order to interfere with complement production and STAT3 activity specifically in the liver, we took advantage of liver-specific Stat3 or C3 small interfering (si)RNA nanoparticles, demonstrating that both siRNAs can significantly prevent myocarditis onset and improve the recovery of heart functions in EAM. Our data demonstrate that liver-specific Stat3/C3 siRNAs may represent a therapeutic option for autoimmune myocarditis and suggest that complement levels and activation might be predictive of progression to dilated cardiomyopathy.
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In experimental autoimmune myocarditis, chronic activation of the STAT3-IL-6 axis in the liver sustains heart inflammation via complement C3/C5 upregulation. Poli and colleagues demonstrate that lipidoid-siRNA nanoparticles triggering liver-specific STAT3 or C3 silencing represent a therapeutic option, potentially bypassing the side effects of systemic STAT3 or complement inhibition.
Inflammation occurs in the context of integrin-mediated adhesive interactions of cells with their extracellular matrix environment. We investigated the role of the collagen binding integrin α1β1 in a ...model of colitis. α1β1 was expressed on lamina propria T cells and monocytes during disease. Both α1 deficiency and anti-α1 mAb treatment (prophylactic and therapeutic) protected against colitis. In vivo α1β1 blockade improved macroscopic and histologic scores, decreased inflammatory cytokine production, and profoundly affected the ability of lamina propria mononuclear cells to proliferate and produce IFN-γ in vitro. Development and α1-mediated inhibition of colitis can be lymphocyte independent, suggesting that activated monocytes also represent a key α1β1-expressing cell type involved in colitis. These results underscore the importance of innate immunity and, specifically, of leukocyte/matrix interactions in regulating local inflammatory responses.
A novel method for the direct synthesis of 5‐arylidene‐2‐thiohydantoins from thioureas and aromatic aldehydes in the presence of base in ethanol was developed. Application of this efficient method ...allowed preparing thiohydantoins, which are difficult to synthesis by traditional methods.
Integrin α1 is a receptor for laminin and collagen which is expressed widely and dynamically in embryogenesis and has been implicated in various developmental processes including establishment of the ...placenta and formation of the central and peripheral nervous system. In the adult it is the sole collagen receptor in smooth muscle and liver and is thought to be important for the stability of these tissues. We have generated a null allele of the α1 gene in the germline of mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutation are viable and fertile and have no overt phenotype, demonstrating that the molecule is not required for development. Embryonic fibroblasts derived from mutant animals are unable to spread on or migrate into substrata of collagen IV and are deficient in spreading on and migrating into laminin. Furtherin vitroanalysis of cell spreading and migration suggests that α1β1 is not required for binding to collagen I and implicates a third receptor, possibly integrin α3β1, in collagen I binding.
BACKGROUND Transforming growth factor β (TGF-β) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-β antibody and ...TGF-β binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-β soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. METHODS The effect of a recombinant TR (TGFβRII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buffered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. RESULTS Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. CONCLUSIONS These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-β is associated with excess collagen accumulation.
Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-beta (TGF-beta) isoforms in vascular repair processes. By en face in situ hybridization, ...proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-beta1, TGF-beta2, TGF-beta3, and lower levels of TGF-beta receptor II (TGF-betaRII) mRNA. Compared with normal endothelium, TGF-beta1 and TGF-beta2, as well as TGF-betaRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-betaRII (TGF-betaR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-betaR:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells. In the vessel wall, TGF-betaR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-betaR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-beta isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.
We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by ...hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
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