5-Fluorouracil treatment represses pseudouridine-containing miRNA export into extracellular vesicles.

5-Fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. We examined the impact of 5-FU on post-transcriptional small RNA modifications (PTxMs) and the expression and export of RNA into small extracellular vesicles (sEVs). EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. We found that treatment of colorectal cancer (CRC) cells with 5-FU represses sEV export of miRNA and snRNA-derived RNAs, but promotes export of snoRNA-derived RNAs. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and sEV small RNA profiles. In contrast, 5-FU exposure led to increased levels of cellular small RNAs containing a variety of methyl-modified bases. These unexpected findings show that 5-FU exposure leads to altered RNA expression, base modification, and aberrant trafficking and localization of small RNAs.

Structural optimization of siRNA conjugates for albumin binding achieves effective MCL1-directed cancer therapy.

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.

5-Fluorouracil Treatment Represses Pseudouridine-Containing Small RNA Export into Extracellular Vesicles.

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.

Loss of p73 Expression Contributes to Chronic Obstructive Pulmonary Disease.

Rationale: Multiciliated cell (MCC) loss and/or dysfunction is common in the small airways of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Furthermore, the effects of cigarette smoke on p73 transcript and protein expression were examined using in vitro and in vivo models and in studies including airway epithelium from smokers and patients with COPD. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression in vitro and in vivo. Furthermore, tumor protein 73 mRNA expression was reduced in the airways of current smokers (n = 82) compared with former smokers (n = 69), and p73-expressing MCCs were reduced in the small airways of patients with COPD (n = 11) compared with control subjects without COPD (n = 12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.

Anti-Inflammatory Effect of High-Density Lipoprotein Blunted by Delivery of Altered MicroRNA Cargo in Patients With Rheumatoid Arthritis.

OBJECTIVE: High-density lipoprotein (HDL) has well-characterized anti-atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL-miRNA cargo is altered and affects inflammation in RA. METHODS: HDL-microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR-1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte-derived macrophages were treated with miR-1246-loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR-1246 and its effect on interleukin-6 (IL-6). RESULTS: The most abundant miRNA on HDL was miR-1246; it was significantly enriched two-fold on HDL from RA versus control participants. HDL-mediated miR-1246 delivery to macrophages significantly increased IL6 expression 43-fold. miR-1246 delivery significantly decreased DUSP3 1.5-fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR-1246. Unmodified HDL from RA delivered 1.6-fold more miR-1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4-fold higher after RA versus control HDL treatment. CONCLUSION: HDL-miR-1246 was increased in RA versus control participants and delivery of miR-1246 to macrophages increased IL-6 expression by targeting DUSP3. The altered HDL-miRNA cargo in RA blunted HDL's anti-inflammatory effect.

Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy.

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

High-density lipoproteins mediate small RNA intercellular communication between dendritic cells and macrophages.

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.

LDL delivery of microbial small RNAs drives atherosclerosis through macrophage TLR8.

Macrophages present a spectrum of phenotypes that mediate both the pathogenesis and resolution of atherosclerotic lesions. Inflammatory macrophage phenotypes are pro-atherogenic, but the stimulatory factors that promote these phenotypes remain incompletely defined. Here we demonstrate that microbial small RNAs (msRNA) are enriched on low-density lipoprotein (LDL) and drive pro-inflammatory macrophage polarization and cytokine secretion via activation of the RNA sensor toll-like receptor 8 (TLR8). Removal of msRNA cargo during LDL re-constitution yields particles that readily promote sterol loading but fail to stimulate inflammatory activation. Competitive antagonism of TLR8 with non-targeting locked nucleic acids was found to prevent native LDL-induced macrophage polarization in vitro, and re-organize lesion macrophage phenotypes in vivo, as determined by single-cell RNA sequencing. Critically, this was associated with reduced disease burden in distinct mouse models of atherosclerosis. These results identify LDL-msRNA as instigators of atherosclerosis-associated inflammation and support alternative functions of LDL beyond cholesterol transport.

High intraluminal pressure promotes vascular inflammation via caveolin-1.

The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.

Chronic sympathetic driven hypertension promotes atherosclerosis by enhancing hematopoiesis.

Hypertension is a major, independent risk factor for atherosclerotic cardiovascular disease. However, this pathology can arise through multiple pathways, which could influence vascular disease through distinct mechanisms. An overactive sympathetic nervous system is a dominant pathway that can precipitate in elevated blood pressure. We aimed to determine how the sympathetic nervous system directly promotes atherosclerosis in the setting of hypertension. We used a mouse model of sympathetic nervous system-driven hypertension on the atherosclerotic-prone apolipoprotein E-deficient background. When mice were placed on a western type diet for 16 weeks, we showed the evolution of unstable atherosclerotic lesions. Fortuitously, the changes in lesion composition were independent of endothelial dysfunction, allowing for the discovery of alternative mechanisms. With the use of flow cytometry and bone marrow imaging, we found that sympathetic activation caused deterioration of the hematopoietic stem and progenitor cell niche in the bone marrow, promoting the liberation of these cells into the circulation and extramedullary hematopoiesis in the spleen. Specifically, sympathetic activation reduced the abundance of key hematopoietic stem and progenitor cell niche cells, sinusoidal endothelial cells and osteoblasts. Additionally, sympathetic bone marrow activity prompted neutrophils to secrete proteases to cleave the hematopoietic stem and progenitor cell surface receptor CXCR4. All these effects could be reversed using the ß-blocker propranolol during the feeding period. These findings suggest that elevated blood pressure driven by the sympathetic nervous system can influence mechanisms that modulate the hematopoietic system to promote atherosclerosis and contribute to cardiovascular events.

Nitroxyl (HNO) reduces endothelial and monocyte activation and promotes M2 macrophage polarization.

Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors.

Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation.

The lymphocyte adaptor protein LNK (also known as SH2B3) is primarily expressed in hematopoietic and endothelial cells, where it functions as a negative regulator of cytokine signaling and cell proliferation. Single-nucleotide polymorphisms in the gene encoding LNK are associated with autoimmune and cardiovascular disorders; however, it is not known how LNK contributes to hypertension. Here, we determined that loss of LNK exacerbates angiotensin II-induced (Ang II-induced) hypertension and the associated renal and vascular dysfunction. At baseline, kidneys from Lnk-/- mice exhibited greater levels of inflammation, oxidative stress, and glomerular injury compared with WT animals, and these parameters were further exacerbated by Ang II infusion. Aortas from Lnk-/- mice exhibited enhanced inflammation, reduced nitric oxide levels, and impaired endothelial-dependent relaxation. Bone marrow transplantation studies demonstrated that loss of LNK in hematopoietic cells is primarily responsible for the observed renal and vascular inflammation and predisposition to hypertension. Ang II infusion increased IFN-γ-producing CD8+ T cells in the spleen and kidneys of Lnk-/- mice compared with WT mice. Moreover, IFN-γ deficiency resulted in blunted hypertension in response to Ang II infusion. Together, these results suggest that LNK is a potential therapeutic target for hypertension and its associated renal and vascular sequela.

Increased carotid intima-media thickness and reduced distensibility in human class III obesity: independent and differential influences of adiposity and blood pressure on the vasculature.

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility), structural and functional properties of carotid arteries respectively, are early markers, as well as strong predictors of cardiovascular disease (CVD). The characteristic of these two parameters in individuals with BMI>40.0 kg/m(2) (Class III obesity), however, are largely unknown. The present study was designed to document cIMT and distensibility in this population and to relate these to other factors with established association with CVD in obesity. The study included 96 subjects (65 with BMI>40.0 kg/m(2) and 31, age- and gender-matched, with BMI of 18.5 to 30.0 kg/m(2)). cIMT and distensibility were measured by non-invasive high resolution ultrasonography, circulatory CD133(+)/KDR(+) angiogenic cells and endothelial microparticles (EMP) by flow cytometry, and plasma levels of adipokines, growth factors and cytokines by Luminex immunoassay kits. The study results demonstrated increased cIMT (0.62±0.11 mm vs. 0.54±0.08 mm, P = 0.0002) and reduced distensibility (22.52±10.79 10(-3)kpa(-1)vs. 29.91±12.37 10(-3)kpa(-1), P<0.05) in individuals with BMI>40.0 kg/m(2). Both cIMT and distensibility were significantly associated with traditional CVD risk factors, adiposity/adipokines and inflammatory markers but had no association with circulating angiogenic cells. We also demonstrated, for the first time, elevated plasma EMP levels in individuals with BMI>40.0 kg/m(2). In conclusion, cIMT is increased and distensibility reduced in Class III obesity with the changes predominantly related to conventional CVD risk factors present in this condition, demonstrating that both cIMT and distensibility remain as CVD markers in Class III obesity.

Imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease(2), stroke(3) and heart failure(4). An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species(5). In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers(6) however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration(7-9). When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry(10). Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model(9) which enables real-time analysis of leukocyte-endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter(11) as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.