Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.

The pathophysiology of arteriovenous fistula (AVF) maturation failure is not completely understood but impaired outward remodeling (OR) and intimal hyperplasia are thought to be contributors. This adverse vascular response after AVF surgery results from interplay between vascular smooth muscle cells (VSMCs), the extracellular matrix (ECM), and inflammatory cells. Relaxin (RLN) is a hormone that acts on the vasculature via interaction with RLN/insulin-like peptide family receptor 1 (RXFP1), resulting in vasodilatation, ECM remodeling, and decreased inflammation. In the present study, we evaluated the consequences of RXFP1 knockout ( Rxfp1-/-) on AVF maturation in a murine model of AVF failure. Rxfp1-/- mice showed a 22% decrease in vessel size at the venous outflow tract 14 d after AVF surgery. Furthermore, a 43% increase in elastin content was observed in the lesions of Rxfp1-/- mice and coincided with a 41% reduction in elastase activity. In addition, Rxfp1-/- mice displayed a 6-fold increase in CD45+ leukocytes, along with a 2-fold increase in monocyte chemoattractant protein 1 (MCP1) levels, when compared with wild-type mice. In vitro, VSMCs from Rxfp1-/- mice exhibited a synthetic phenotype, as illustrated by augmentation of collagen, fibronectin, TGF-ß, and platelet-derived growth factor mRNA. In addition, VSMCs derived from Rxfp1-/- mice showed a 5-fold increase in cell migration. Finally, RXFP1 and RLN expression levels were increased in human AVFs when compared with unoperated cephalic veins. In conclusion, RXFP1 deficiency hampers elastin degradation and results in induced vascular inflammation after AVF surgery. These processes impair OR in murine AVF, suggesting that the RLN axis could be a potential therapeutic target for promoting AVF maturation.-Bezhaeva, T., de Vries, M. R., Geelhoed, W. J., van der Veer, E. P., Versteeg, S., van Alem, C. M. A., Voorzaat, B. M., Eijkelkamp, N., van der Bogt, K. E., Agoulnik, A. I., van Zonneveld, A.-J., Quax, P. H. A., Rotmans, J. I. Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.

Local delivery of liposomal prednisolone leads to an anti-inflammatory profile in renal ischaemia-reperfusion injury in the rat.

BACKGROUND: Treatment of inflammatory kidney diseases with systemic high-dose glucocorticoids (GCs) has severe side effects. Liposomal encapsulation could facilitate local delivery of GCs to the inflamed kidney, as liposomes encapsulate their payload until extravasation at sites of inflammation, potentially resulting in local bioactivity. Our aim was to evaluate the ability of liposomes to accumulate locally after renal ischaemia-reperfusion injury in the rat and to study its effect on macrophages. METHODS: In vitro, human macrophages were incubated with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was studied microscopically and treatment effect was assessed by interkeukin 6 (IL-6) enzyme-linked immunosorbent assay. The mechanism of action was evaluated by analysing GC receptor activation by microscopy and quantitative polymerase chain reaction (qPCR). In vivo, rats were subjected to ischaemia-reperfusion injury and were injected intravenously with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was measured by the FLARE camera and the treatment effect by immunohistochemistry for myeloid cells and qPCR for inflammatory markers. RESULTS: In vitro, macrophages internalized liposomes after 8 hours. Prednisolone or liposomal prednisolone treatment reduced IL-6 production and both compounds induced translocation of the GC receptor to the nucleus and upregulation of PER1 messenger RNA (mRNA), indicating a similar mechanism of action. In vivo, fluorescent liposomes accumulated in the inflamed kidney. Liposomal prednisolone treatment increased the presence of ED2-positive anti-inflammatory macrophages and both prednisolone and liposomal prednisolone reduced monocyte chemoattractant protein-1 (MCP-1) mRNA production, indicating a reduced pro-inflammatory profile in the kidney. CONCLUSIONS: Liposomal encapsulation is a promising strategy for local delivery of glucocorticoids to the inflamed kidney.

Recent Advances in Liposomal-Based Anti-Inflammatory Therapy.

Liposomes can be seen as ideal carriers for anti-inflammatory drugs as their ability to (passively) target sites of inflammation and release their content to inflammatory target cells enables them to increase local efficacy with only limited systemic exposure and adverse effects. Nonetheless, few liposomal formulations seem to reach the clinic. The current review provides an overview of the more recent innovations in liposomal treatment of rheumatoid arthritis, psoriasis, vascular inflammation, and transplantation. Cutting edge developments include the liposomal delivery of gene and RNA therapeutics and the use of hybrid systems where several liposomal bilayer features, or several drugs, are combined in a single formulation. The majority of the articles reviewed here focus on preclinical animal studies where proof-of-principle of an improved efficacy-safety ratio is observed when using liposomal formulations. A few clinical studies are included as well, which brings us to a discussion about the challenges of clinical translation of liposomal nanomedicines in the field of inflammatory diseases.

Liposomal Delivery Improves the Efficacy of Prednisolone to Attenuate Renal Inflammation in a Mouse Model of Acute Renal Allograft Rejection.

BACKGROUND: Systemic exposure to high-dose corticosteroids effectively combats acute rejection after kidney transplantation, but at the cost of substantial side effects. In this study, a murine acute renal allograft rejection model was used to investigate whether liposomal-encapsulated prednisolone (LP) facilitates local exposure to enhance its therapeutic effect. METHODS: Male BalbC recipients received renal allografts from male C57BL/6J donors. Recipients were injected daily with 5 mg/kg cyclosporine A and received either 10 mg/kg prednisolone (P), or LP intravenously on day 0, 3, and 6, or no additional treatment. Functional magnetic resonance imaging (fMRI) was performed on day 6 to study allograft perfusion and organs were retrieved on day 7 for further analysis. RESULTS: Staining of polyethylene-glycol-labeled liposomes and high performance liquid chromatography analysis revealed accumulation in the LP treated allograft. LP treatment induced the expression of glucocorticoid responsive gene Fkbp5 in the allograft. Flow-cytometry of allografts revealed liposome presence in CD45 cells, and reduced numbers of F4/80 macrophages, and CD3 T-lymphocytes upon LP treatment. Banff scoring showed reduced interstitial inflammation and tubulitis and fMRI analysis revealed improved allograft perfusion in LP versus NA mice. CONCLUSIONS: Liposomal delivery of prednisolone improved renal bio-availability, increased perfusion and reduced cellular infiltrate in the allograft, when compared with conventional prednisolone. Clinical studies should reveal if treatment with LP results in improved efficacy and reduced side effects in patients with renal allograft rejection.

Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure.

Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cells (VSMCs). Overall, RP105-/- mice displayed a 26% decrease in venous OR. The inflammatory response in RP105-/- mice was characterized by accumulation of anti-inflammatory macrophages, a 76% decrease in pro- inflammatory macrophages, a 70% reduction in T-cells and a 50% decrease in MMP-activity. In vitro, anti-inflammatory macrophages from RP105-/- mice displayed increased IL10 production, while MCP1 and IL6 levels secreted by pro-inflammatory macrophages were elevated. VSMC content in RP105-/- AVFs was markedly decreased. In vitro, RP105-/- venous VSMCs proliferation was 50% lower, whereas arterial VSMCs displayed a 50% decrease in migration, relative to WT. In conclusion, the impaired venous OR in RP105-/- mice could result from of a shift in both macrophages and VSMCs towards a regenerative phenotype, identifying a novel relationship between inflammation and VSMC function in AVF maturation.

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.