Renal microvascular endothelial cell responses in sepsis-induced acute kidney injury

Endothelial cells in the kidney microvasculature have an intrinsic molecular and phenotypic heterogeneity and respond to sepsis-induced acute kidney injury conditions in a segment-specific manner. This Review discusses the roles of these cells and the molecular systems that control endothelial functions in the development of sepsis-induced acute kidney injury. Microvascular endothelial cells in the kidney have been a neglected cell type in sepsis-induced acute kidney injury (sepsis-AKI) research; yet, they offer tremendous potential as pharmacological targets. As endothelial cells in distinct cortical microvascular segments are highly heterogeneous, this Review focuses on endothelial cells in their anatomical niche. In animal models of sepsis-AKI, reduced glomerular blood flow has been attributed to inhibition of endothelial nitric oxide synthase activation in arterioles and glomeruli, whereas decreased cortex peritubular capillary perfusion is associated with epithelial redox stress. Elevated systemic levels of vascular endothelial growth factor, reduced levels of circulating sphingosine 1-phosphate and loss of components of the glycocalyx from glomerular endothelial cells lead to increased microvascular permeability. Although coagulation disbalance occurs in all microvascular segments, the molecules involved differ between segments. Induction of the expression of adhesion molecules and leukocyte recruitment also occurs in a heterogeneous manner. Evidence of similar endothelial cell responses has been found in kidney and blood samples from patients with sepsis. Comprehensive studies are needed to investigate the relationships between segment-specific changes in the microvasculature and kidney function loss in sepsis-AKI. The application of omics technologies to kidney tissues from animals and patients will be key in identifying these relationships and in developing novel therapeutics for sepsis

Development of a Combined Lipid-Based Nanoparticle Formulation for Enhanced siRNA Delivery to Vascular Endothelial Cells

Low transfection efficiency in endothelial cells (EC) is still a bottleneck for the majority of siRNA-based vascular delivery approaches. In this work, we developed a lipid-based nanoparticle (LNP) formulation based on a combination of a permanently charged cationic lipid-DOTAP and a conditionally ionized cationic lipid-MC3 (DOTAP/MC3) for the enhanced delivery of siRNA into EC. Compared with a single DOTAP or MC3-based benchmark LNP, we demonstrated that the DOTAP/MC3 LNP formulation shows the best transfection efficiency both in primary EC in vitro and in endothelium in zebrafish. The high transfection activity of the DOTAP/MC3 LNP formulation is achieved by a combination of improved endothelial association mediated by DOTAP and MC3-triggered efficient siRNA intracellular release in EC. Furthermore, AbVCAM-1-coupled DOTAP/MC3 LNP-mediated siRNARelA transfection showed pronounced anti-inflammatory effects in inflammatory-activated primary EC by effectively blocking the NF-κB pathway. In conclusion, the combination of permanent and ionizable cationic lipids in LNP formulation provides an effective endothelial cell delivery of siRNA

E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells

Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-alpha (TNF-alpha) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.; hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG- Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-alpha activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells

Glomerular Endothelial Cells as Instigators of Glomerular Sclerotic Diseases

Glomerular endothelial cell (GEnC) dysfunction is important in the pathogenesis of glomerular sclerotic diseases, including Focal Segmental Glomerulosclerosis (FSGS) and overt diabetic nephropathy (DN). GEnCs form the first cellular barrier in direct contact with cells and factors circulating in the blood. Disturbances in these circulating factors can induce GEnC dysfunction. GEnC dysfunction occurs in early stages of FSGS and DN, and is characterized by a compromised endothelial glycocalyx, an inflammatory phenotype, mitochondrial damage and oxidative stress, aberrant cell signaling, and endothelial-to-mesenchymal transition (EndMT). GEnCs are in an interdependent relationship with podocytes and mesangial cells, which involves bidirectional cross-talkviaintercellular signaling. Given that GEnC behavior directly influences podocyte function, it is conceivable that GEnC dysfunction may culminate in podocyte damage, proteinuria, subsequent mesangial activation, and ultimately glomerulosclerosis. Indeed, GEnC dysfunction is sufficient to cause podocyte injury, proteinuria and activation of mesangial cells. Aberrant gene expression patterns largely contribute to GEnC dysfunction and epigenetic changes seem to be involved in causing aberrant transcription. This review summarizes literature that uncovers the importance of cross-talk between GEnCs and podocytes, and GEnCs and mesangial cells in the context of the development of FSGS and DN, and the potential use of GEnCs as efficacious cellular target to pharmacologically halt development and progression of DN and FSGS

PLGA-PEG nanoparticles for targeted delivery of the mTOR/PI3kinase inhibitor dactolisib to inflamed endothelium

Dactolisib (NVP-BEZ235, also referred to as: 'BEZ235' or 'BEZ') is a dual mTOR/PI3 K inhibitor that is of potential interest in the treatment of inflammatory disorders. This work focuses on formulation of BEZ-loaded polymeric nanoparticles composed of a blend of poly(D, L-lactide-co-glycolide) (PLGA) and poly(D, L-lactide-coglycolide)- poly(ethylene glycol)-2000 (PLGA-PEG). The nanoparticles were prepared by an oil/water emulsion solvent evaporation method, and were subsequently characterized for yield, encapsulation efficiency, morphology, particle size, drug-polymer interaction and in vitro drug release profiles. A targeted formulation was developed by conjugation of a S-acetyl-thioacetyl (SATA)-modified mouse-anti human E-selectin antibody to the distal end of PLGA-PEG-SPDP containing nanoparticles. Our results show the successful preparation of spherical PLGA/PLGA-PEG nanoparticles loaded with BEZ. The particle size distribution showed a range from 250 to 360 nm with a high (> 75%) BEZ encapsulation efficiency. Approximately 35% of the loaded BEZ was released within 10 days at 37 degrees C in a medium containing 5% bovine serum albumin (BSA). Evaluation of efficacy of anti E-selectin decorated BEZ-loaded nanoparticles was carried out in tumor necrosis factor-alpha (TNF-alpha) activated endothelial cells. Confocal microscopy analysis showed that cellular uptake of the targeted nanoparticles and subsequent internalization. Cell functional assays, including migration assay and phosphowestern blot analysis of the mTOR and pI3K signaling pathways, revealed that the E-selectin targeted nanoparticles loaded with BEZ had a pronounced effect on inflammation-activated endothelial cells as compared to the non-targeted BEZloaded nanoparticles. In conclusion, E-selectin targeted nanoparticles have a high potential in delivering the potent mTOR/pI3K inhibitor dactolisib to inflamed endothelial cells and are an interesting nanomedicine for anti-inflammatory therapy

Endothelium-targeted delivery of dexamethasone by anti-VCAM-1 SAINT-O-Somes in mouse endotoxemia

Microvascular endothelial cells play a pivotal role in the pathogenesis of sepsis-induced inflammatory responses and multiple organ failure. Therefore, they represent an important target for pharmacological intervention in the treatment of sepsis. Glucocorticosteroids were widely used in the treatment of sepsis but vast evidence to support their systemic use is lacking. The limited effects of glucocorticoids in the treatment of sepsis may be explained by differential effects of drug initiated NF-κB inhibition in different cell types and insufficient drug delivery in target cells. The current study aimed therefore to investigate the effects of an endothelial targeted delivery of dexamethasone in a mouse model of endotoxemia induced by two consecutive i.p. injections of lipopolysaccharide (LPS). To achieve endothelial cell specific delivery of dexamethasone, we modified SAINT-O-Somes, a new generation of liposomes that contain the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl) methyl-pyridinium chloride, with antibodies against vascular cell adhesion molecule-1 (VCAM-1). In LPS challenged mice, the systemic administration of free dexamethasone had negligible effects on the microvascular inflammatory endothelial responses. Dexamethasone-loaded anti-VCAM-1 SAINT-O-Somes specifically localized at VCAM-1 expressing endothelial cells in the microvasculature of inflamed organs. This was associated with a marginal attenuation of the expression of a few pro-inflammatory genes in kidney and liver, while no effects in the lung were observed. This study reveals that, although local accumulation of the targeted drug was achieved, endothelial targeted dexamethasone containing anti-VCAM-1 SAINT-O-Somes exhibited marginal effects on inflammatory endothelial cell activation in a model of endotoxemia. Studies with more potent drugs encapsulated into anti-VCAM-1 SAINT-O-Somes will in the future reveal whether this delivery system can be further developed for efficacious endothelial directed delivery of drugs in the treatment of sepsis

Positron emission tomography (PET) and single photon emission computed tomography (SPELT) imaging of macrophages in large vessel vasculitis:Current status and future prospects

Macrophages are key players in the pathogenesis of large -vessel vasculitis (LW) and may serve as a target for diagnostic imaging of LW. The radiotracer,18F-FDG has proven to be useful in the diagnosis of giant cell arteritis (GCA), a form of LW. Although uptake of 18F-FDG is high in activated macrophages, it is not a specific radiotracer as its uptake is high in any proliferating cell and other activated immune cells resulting in high non-specific background radioactivity especially in aging and atherosclerotic vessels which dramatically lowers the diagnostic accuracy. Evidence also exists that the sensitivity of 18F-FDG PET drops in patients upon glucocorticoid treatment. Therefore, there is a clinical need for more specific radiotracers in imaging GCA to improve diagnostic accuracy. Numerous clinically established and newly developed macrophage targeted radiotracers for oncological and inflammatory diseases can potentially be utilized for LW imaging. These tracers are more target specific and therefore may provide lower background radioactivity, higher diagnostic accuracy and the ability to assess treatment effectiveness. However, current knowledge regarding macrophage subsets in LW lesions is limited. Further understanding regarding macrophage subsets in vasculitis lesion is needed for better selection of tracers and new targets for tracer development. This review summarizes the development of macrophage targeted tracers in the last decade and the potential application of macrophage targeted tracers currently used in other inflammatory diseases in imaging LW. (C) 2018 The Author(s). Published by Elsevier B.V

Serum proteomics reveals hemophagocytic lymphohistiocytosis-like phenotype in a subset of patients with multisystem inflammatory syndrome in children

Children with Multisystem Inflammatory Syndrome in Children (MIS-C) can present with thrombocytopenia, which is a key feature of hemophagocytic lymphohistiocytosis (HLH). We hypothesized that thrombocytopenic MIS-C patients have more features of HLH. Clinical characteristics and routine laboratory parameters were collected from 228 MIS-C patients, of whom 85 (37%) were thrombocytopenic. Thrombocytopenic patients had increased ferritin levels; reduced leukocyte subsets; and elevated levels of ASAT and ALAT. Soluble IL-2RA was higher in thrombocytopenic children than in non-thrombocytopenic children. T-cell activation, TNF-alpha and IFN-gamma signaling markers were inversely correlated with thrombocyte levels, consistent with a more pronounced cytokine storm syndrome. Thrombocytopenia was not associated with severity of MIS-C and no pathogenic variants were identified in HLH-related genes. This suggests that thrombocytopenia in MIS-C is not a feature of a more severe disease phenotype, but the consequence of a distinct hyperinflammatory immunopathological process in a subset of children.</p