Responses of retinal and brain microvasculature to streptozotocin induced diabetes revealed by global expression profiling.

This study aims to determine the effects of diabetes in the retinal and brain microvasculature through gene expression profiling. Twelve male Wistar rats were randomly divided into two groups: streptozotocin-induced diabetic rats and time-matched nondiabetic rats. The retinal microvessels (RMVs) and brain microvessels (BMVs) were mechanically isolated from individual rats.

Endothelial Interferon Regulatory Factor 1 Regulates Lipopolysaccharide-Induced VCAM-1 Expression Independent of NFκB.

Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells.

Micromanaging cardiac regeneration: Targeted delivery of microRNAs for cardiac repair and regeneration.

The loss of cardiomyocytes during injury and disease can result in heart failure and sudden death, while the adult heart has a limited capacity for endogenous regeneration and repair. Current stem cell-based regenerative medicine approaches modestly improve cardiomyocyte survival, but offer neglectable cardiomyogenesis. This has prompted the need for methodological developments that crease de novo cardiomyocytes.

Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System.

Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine.

SAINT-liposome-polycation particles, a new carrier for improved delivery of siRNAs to inflamed endothelial cells.

Interference with acute and chronic inflammatory processes by means of delivery of siRNAs into microvascular endothelial cells at a site of inflammation demands specific, non-toxic and effective siRNA delivery system. In the current work we describe the design and characterization of siRNA carriers based on cationic pyridinium-derived lipid 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) (SAINT-C18) and the transfection enhancer protamine, complexed with siRNA/carrier DNA or siRNA only. These carriers, called SAINT-liposome-polycation-DNA (S-LPD) and SAINT-liposome-polycation (S-LP), have a high efficiency of siRNA encapsulation, low cellular toxicity, and superior efficacy of gene downregulation in endothelial cells in vitro as compared to DOTAP-LPD.

Abrupt reflow enhances cytokine-induced proinflammatory activation of endothelial cells during simulated shock and resuscitation.

Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation.

A critical role for Egr-1 during vascular remodelling in pulmonary arterial hypertension.

Aims: Pulmonary arterial hypertension (PAH) is characterized by the development of unique neointimal lesions in the small pulmonary arteries, leading to increased right ventricular (RV) afterload and failure. Novel therapeutic strategies are needed that target these neointimal lesions. Recently, the transcription factor Egr-1 (early growth response protein 1) was demonstrated to be up-regulated early in experimental neointimal PAH.

Synthesis and structure-activity relationships of 4-fluorophenyl-imidazole p38α MAPK, CK1δ and JAK2 kinase inhibitors.

The synthesis and structure-activity relationships of novel 4-(4'-fluorophenyl)imidazoles as selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described. Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5-disubstituted imidazoles. Carboxylate and phosphonate groups were introduced via 'click' reactions.

VCAM-1 specific PEGylated SAINT-based lipoplexes deliver siRNA to activated endothelium in vivo but do not attenuate target gene expression.

In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg.

Syntheses and structure-activity relationships for some triazolyl p38α MAPK inhibitors.

The design, synthesis and biological evaluation of novel triazolyl p38α MAPK inhibitors with improved water solubility for formulation in cationic liposomes (SAINT-O-Somes) targeted at diseased endothelial cells is described. Water-solubilizing groups were introduced via a 'click' reaction of functional azides with 2-alkynyl imidazoles and isosteric oxazoles to generate two small libraries of 1,4-disubstituted 1,2,3-triazolyl p38α MAPK inhibitors. Triazoles with low IC50 values and desired physicochemical properties were screened for in vitro downregulation of proinflammatory gene expression and were formulated in SAINT-O-Somes.

Anti-VCAM-1 SAINT-O-Somes enable endothelial-specific delivery of siRNA and downregulation of inflammatory genes in activated endothelium in vivo.

The pivotal role of endothelial cells in the pathology of inflammatory diseases raised interest in the development of short interfering RNA (siRNA) delivery devices for selective pharmacological intervention in the inflamed endothelium. The current study demonstrates endothelial specific delivery of siRNAs and downregulation of inflammatory genes in activated endothelium in vivo by applying a novel type of targeted liposomes based on the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride). To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1).

Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes.

The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC).

Innovations in studying in vivo cell behavior and pharmacology in complex tissues--microvascular endothelial cells in the spotlight.

Many studies on the molecular control underlying normal cell behavior and cellular responses to disease stimuli and pharmacological intervention are conducted in single-cell culture systems, while the read-out of cellular engagement in disease and responsiveness to drugs in vivo is often based on overall tissue responses. As the majority of drugs under development aim to specifically interact with molecular targets in subsets of cells in complex tissues, this approach poses a major experimental discrepancy that prevents successful development of new therapeutics. In this review, we address the shortcomings of the use of artificial (single) cell systems and of whole tissue analyses in creating a better understanding of cell engagement in disease and of the true effects of drugs.

Anti-VCAM-1 and anti-E-selectin SAINT-O-Somes for selective delivery of siRNA into inflammation-activated primary endothelial cells.

Activated endothelial cells play a pivotal role in the pathology of inflammatory diseases and present a rational target for therapeutic intervention by endothelial specific delivery of short interfering RNAs (siRNA). This study demonstrates the potential of the recently developed new generation of liposomes based on cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) for functional and selective delivery of siRNA into inflamed primary endothelial cells. To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1) or respectively E-selectin and tested in TNF-α activated primary endothelial cells from venous and aortic vascular beds.

Angiogenesis in synchronous and metachronous colorectal liver metastases: the liver as a permissive soil.

Objective: Resection of a primary colorectal carcinoma (CRC) can be accompanied by rapid outgrowth of liver metastases, suggesting a role for angiogenesis. The aim of this study is to investigate whether the presence of a primary CRC is associated with changes in angiogenic status and proliferation/apoptotic rate in synchronous liver metastases and/or adjacent liver parenchyma.

Methods: Gene expression and localization of CD31, HIF-1α, members of the vascular endothelial growth factor (VEGF) and Angiopoietin (Ang) system were studied using qRT-PCR and immunohistochemistry in colorectal liver metastases and nontumorous-adjacent liver parenchyma.

Targeted siRNA delivery to diseased microvascular endothelial cells: cellular and molecular concepts.

Increased insight in the role of endothelial cells in the pathophysiology of cancer, inflammatory and cardiovascular diseases, has drawn great interest in pharmacological interventions aiming at the endothelium in diseased sites. Their location in the body makes them suitable targets for therapeutic approaches based on targeted drug delivery. Functional heterogeneity of the microvascular bed in normal organ homeostasis has been appreciated for a long time, and more recent studies have revealed heterogeneity in endothelial reactivity to inflammatory stimuli as well.

Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells.

In non-phagocytic cells such as endothelial cells, processing of liposomes and subsequent release of drug content is often inefficient due to the absence of professional processing machinery, which limits pharmacological efficacy. We therefore developed a liposome based drug delivery system with superior intracellular release characteristics. The design was based on long circulating conventional liposomes that were formulated with a cationic amphiphile, 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chlorid (SAINT-C18).

Assessment of liposome-cell interactions.

Liposome-cell interactions have been assessed for over 30 years now by an enormous variety of approaches and methods. In-depth knowledge of liposome-cell interaction is still very relevant since new concepts and applications applying liposomes are being developed every day. This chapter does not aim to give a complete overview on methods to assess liposome-cell interactions but merely gives a handle to approach the assessment of liposome-cell interaction by describing some well-established methods that also allow for modification to adapt them for your specific research questions.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin.

Inhibition of proinflammatory genes in anti-GBM glomerulonephritis by targeted dexamethasone-loaded AbEsel liposomes.

E-selectin-directed targeted drug delivery was analyzed in anti-glomerular basement membrane glomerulonephritis. Liposomes conjugated with anti-E-selectin antibodies (Ab(Esel) liposomes) were internalized by activated endothelial cells in vitro through E-selectin-mediated endocytosis. At the onset of glomerulonephritis in mice, E-selectin was expressed on glomerular endothelial cells, which resulted in homing of Ab(Esel) liposomes to glomeruli after intravenous administration.

Addressing liver fibrosis with liposomes targeted to hepatic stellate cells.

Liver fibrosis is a chronic disease that results from hepatitis B and C infections, alcohol abuse or metabolic and genetic disorders. Ultimately, progression of fibrosis leads to cirrhosis, a stage of the disease characterized by failure of the normal liver functions. Currently, the treatment of liver fibrosis is mainly based on the removal of the underlying cause of the disease and liver transplantation, which is the only treatment for patients with advanced fibrosis.

Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin.

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF-SPLPs and untargeted SPLPs labeled with [3H]cholesteryloleyl-ether were injected into rats. About 87% of the LF-SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h.

A novel lipid-based drug carrier targeted to the non-parenchymal cells, including hepatic stellate cells, in the fibrotic livers of bile duct ligated rats.

In fibrotic livers, collagen producing hepatic stellate cells (HSC) represent a major target for antifibrotic therapies. We designed liposomes with surface-coupled mannose 6-phosphate (M6P) modified human serum albumin (HSA) to target HSC via the M6P receptor. In this study we determined the pharmacokinetics and target specificity of M6P-HSA-liposomes in a rat model of liver fibrosis.

Site-specific inhibition of glomerulonephritis progression by targeted delivery of dexamethasone to glomerular endothelium.

Glomerulonephritis represents a group of renal diseases with glomerular inflammation as a common pathologic finding. Because of the underlying immunologic character of these disorders, they are frequently treated with glucocorticoids and cytotoxic immunosuppressive agents. Although effective, use of these compounds has limitations as a result of toxicity and systemic side effects.