Endothelial-specific delivery of siRNA by novel SAINT-based lipoplexes:an in vitro and in vivo study

Short interfering RNA’s (siRNA’s) zijn dubbelstrengs nucleïnezuur moleculen opgebouwd uit een klein aantal basen; ze hebben de bijzondere eigenschap dat ze in de cel met complementaire strengen mRNA kunnen complexeren, die als gevolg daarvan worden afgebroken. Dit mechanisme opent dus de mogelijkheid de vorming van een ongewenst eiwit, bijvoorbeeld een eiwit dat betrokken is bij het ontstaan van een ziekte, te verhinderen en daarmee het ontstaan of verergeren van die ziekte tegen te gaan. Op deze manier zouden siRNA’s als medicijn gebruikt kunnen worden. Het in dit proefschrift beschreven onderzoek richt zich op de ontwikkeling van een op lipiden gebaseerd siRNA dragersysteem dat geschikt is voor efficiënte en selectieve afgifte van siRNA in vivo. Endotheelcellen brengen in ziekteprocessen allerlei eiwitten tot expressie zoals adhesiemoleculen op de celmembraan. Door ons dragersysteem uit te rusten met een moleculaire structuur die een dergelijk adhesiemolecuul herkent wordt het siRNA-drager complex gericht op endotheelcellen in door chronische ontstekingen aangedaan weefsel en zal daar door middel van het ingebouwde specifieke siRNA een ziekte-geassocieerd eiwit kunnen uitschakelen. De onderzoeken die in dit proefschrift zijn beschreven laten zien dat de ontwikkelde siRNA dragersystemen geschikt zijn voor functionele in vitro aflevering van siRNA in “ontstoken” primaire arteriële, microvasculaire en veneuze endotheelcellen. Onderzoek in muizen toonde aan dat, ondanks de hoge opname van het dragersysteem met daarin gen-specifiek siRNA in longweefsel, dit niet resulteert in down-regulatie van het target gen. Vervolgonderzoek zal zich moeten richten op de verbetering van de efficiëntie van in vivo siRNA vrijmaking uit het dragersysteem

Histone deacetylase 3 (HDAC 3) as emerging drug target in NF-kappa B-mediated inflammation

Activation of inflammatory gene expression is regulated, among other factors, by post-translational modifications of histone proteins. The most investigated type of histone modifications is lysine acetylations. Histone deacetylases (HDACs) remove acetylations from lysines, thereby influencing (inflammatory) gene expression. Intriguingly, apart from histones, HDACs also target non-histone proteins. The nuclear factor kappa B (NF-kappa B) pathway is an important regulator in the expression of numerous inflammatory genes, and acetylation plays a crucial role in regulating its responses. Several studies have shed more light on the role of HDAC 1-3 in inflammation with a particular pro-inflammatory role for HDAC 3. Nevertheless, the HDAC-NF-kappa B interactions in inflammatory signalling have not been fully understood. An important challenge in targeting the regulatory role of HDACs in the NF-kappa B pathway is the development of highly potent small molecules that selectively target HDAC iso-enzymes. This review focuses on the role of HDAC 3 in (NF-kappa B-mediated) inflammation and NF-kappa B lysine acetylation. In addition, we address the application of frequently used small molecule HDAC inhibitors as an approach to attenuate inflammatory responses, and their potential as novel therapeutics. Finally, recent progress and future directions in medicinal chemistry efforts aimed at HDAC 3-selective inhibitors are discussed.</p

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

AbstractThe increasing number of patients suffering from chronic obstructive pulmonary disease (COPD) represents a major and increasing health problem. Therefore, novel therapeutic approaches are needed. Class I HDACs 1, 2 and 3 play key roles in the regulation of inflammatory gene expression with a particular pro-inflammatory role for HDAC 3. HDAC 3 has been reported to be an important player in inflammation by deacetylating NF-κB p65, which has been implicated in the pathology of COPD. Here, we applied the pharmacological HDAC 3-selective inhibitor RGFP966, which attenuated pro-inflammatory gene expression in models for inflammatory lung diseases. Consistent with this, a robust decrease of the transcriptional activity of NF-κB p65 was observed. HDAC 3 inhibition affected neither the acetylation status of NF-κB p65 nor histone H3 or histone H4. This indicates that HDAC 3 inhibition does not inhibit NF-κB p65 transcriptional activity by affecting its deacetylation but rather by inhibiting enzymatic activity of HDAC 3. Taken together, our findings indicate that pharmacological HDAC 3-selective inhibition by inhibitors such as RGFP966 may provide a novel and effective approach toward development of therapeutics for inflammatory lung diseases

HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

Chronic obstructive pulmonary disease (COPD) constitutes a major health burden. Studying underlying molecular mechanisms could lead to new therapeutic targets. Macrophages are orchestrators of COPD, by releasing pro-inflammatory cytokines. This process relies on transcription factors such as NF-κB, among others. NF-κB is regulated by lysine acetylation; a post-translational modification installed by histone acetyltransferases and removed by histone deacetylases (HDACs). We hypothesized that small molecule HDAC inhibitors (HDACi) targeting class I HDACs members that can regulate NF-κB could attenuate inflammatory responses in COPD via modulation of the NF-κB signaling output. MS-275 is an isoform-selective inhibitor of HDAC1-3. In precision-cut lung slices and RAW264.7 macrophages, MS-275 upregulated the expression of both pro- and anti-inflammatory genes, implying mixed effects. Interestingly, anti-inflammatory IL10 expression was upregulated in these model systems. In the macrophages, this was associated with increased NF-κB activity, acetylation, nuclear translocation, and binding to the IL10 promoter. Importantly, in an in vivo model of cigarette smoke-exposed C57Bl/6 mice, MS-275 robustly attenuated inflammatory expression of KC and neutrophil influx in the lungs. This study highlights for the first time the potential of isoform-selective HDACi for the treatment of inflammatory lung diseases like COPD

Small molecule inhibitors of histone deacetylases and acetyltransferases as potential therapeutics in oncology

Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by post-translational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues, and histone deacetylases (HDACs) remove acetyl residues from lysines, thereby influencing gene expression. Whereas HAT inhibitors are relatively undeveloped, four HDAC inhibitors (HDACi) reached the clinic for treatment of hematological cancers. HDACi induce apoptosis via inhibition of histone deacetylation and concomitant transcription of aberrantly silenced genes. Nevertheless, other mechanisms have also been described. Intriguingly, HDACs also target nonhistone proteins such as the nuclear factor κB (NF-κB) pathway, which is an important regulator of gene transcription for which a role in cancer has been implicated. Despite the studies that shed more light on the role of HDAC isoenzymes in the NF-κB pathway the challenge remains to develop isoenzymeselective HDACi to target this pathway